Programme and abstract - The Purine and Pyrimidine Society

Transcription

Official programme & abstracts for the
Joint 11th International and 9th European Symposium on
Purines and Pyrimidines in Man (PP’03)
9-13 June 2003
Hotel Zuiderduin, Egmond aan Zee, the Netherlands
Editor-in-chief: Prof. Dr. G.J. Peters
Published by: Drukkerij Peters in alliance with VU University Medical Center,
Amsterdam, the Netherlands
May 2003
2
Contents
Welcome
4
Organising committee
5
Contact addresses
6
Support
7
Young Investigator Award
7
Registration fee
8
Venue Hotel Zuiderduin
8
Plan Hotel Zuiderduin floor 0
9
10
Programme PP’03
11
Invited presentations (I01 – I24)
22
Oral presentations (O01 – 046)
50
Poster presentations (P01 – P95)
92
Author index
189
Overview International and European Symposia on Purine and Pyrimidine Metabolism
192
3
Welcome to the Netherlands
This series of congresses on Purine and Pyrimidine metabolism started 30 years ago in Tel Aviv and has since
then moved around the world. It is now coming back to places which have been visited before. Three years ago the
meeting was in Tel Aviv for the second time. The meeting has also been held twice in Austria. You can find an
overview of the meetings on the last page of this abstract book. Twenty-one years ago the meeting was held in the
most southern part of the Netherlands, Maastricht. Now we welcome the international meeting for the 2nd time in
the Netherlands but at the other site with an excellent view on the sea. Usually the weather conditions permit to go
to the beach. Alternatively you can take a walk through the dunes during the afternoon break.
The meeting is planned in such a way that you have some time off after the lunch, enabling to relax or to prepare
for the poster sessions. For these poster sessions we will try a novel concept. Each poster presenter will have a few
minutes to highlight the most important points of his/her research to encourage everybody to come and take a look
at the posters, which are located close to the lecture hall. To compensate for this we continue in the evening with
two exciting sessions. In the methods sessions we are very pleased that we can give you an up to date overview of
analytical and molecular biological techniques. We were encouraged to organize a special Lesch-Nyhan syndrome
session, because molecular biology has provided some new insights in this disease.
The meeting again provides an excellent environment for translational research. For that purpose we have a
session called from “Bench to the Clinic” which is intended to give you some insight, both from a company and an
investigator point of view, how drugs proceed from the test tube to the patient. These meetings on Purine and
Pyrimidine metabolism always provided an excellent forum for so-called horizontal translational research: from
one specialty to the other. In the past we had excellent examples, e.g. from immune deficiency to cancer, now we
have examples from rheumatology to cancer to cardiology and vice versa. These interactions have made this series
of meetings a success in the past and will do similarly in the future. This will be the task of a new generation.
Many of the original participants and organizers retired or are retiring and a new generation is taking over, which
has the challenge to keep this meeting as exciting as ever in a much more competing field. I attended the first
meeting as a Ph.D. student and made friendships with others who are also here as senior investigators. Our current
Ph.D. students should be enabled to proceed similarly. However, they have many other challenges outside and
inside the field. For the last reason we should keep in close contact with others working on nucleosides and
nucleotides, Therefore we decided to publish the proceedings in Nucleosides, Nucleotides & Nucleic Acids.
This meeting would not have been possible without the help of much support. First I have to thank my
collaborators in the oncology laboratory, other colleagues at the VU University Medical Center, my colleagues
from the national organizing committee for their support in planning, and the members of the scientific committee
who suggested sessions and speakers and reviewed the abstracts timely enabling us to make an objective selection
of the abstracts. The excellent support of the conference office was instrumental in enabling this electronic review
and all other logistics. They will be around for any question you may have. As you noticed we did not do any
paper announcement and proceeded entirely electronically. The generous support of the various sponsors is greatly
appreciated. Without their support it would not have been possible to keep the costs of the meeting within limits.
Finally I regret that several colleagues from the Far East (Taiwan, China, Japan) are not able or allowed to travel
because of circumstances outside their control, such as SARS, and I sincerely hope that a next time they will be
able to attend.
I wish everybody a successful meeting
Prof. Dr. Godefridus J. Peters
4
Organising Committee
Local organising committee:
G.J. Peters, Amsterdam, chairman
R.A. De Abreu, Nijmegen
J.W. De Jong, Rotterdam
A.B.P. Van Kuilenburg, Amsterdam
A.P. IJzerman, Leiden
Board of the ESSPPMM:
G.J. Peters (NL) President
I. Sebesta (CZ) Vice-President
G. Van den Berghe (B) Past-President
A.C. Skladanowski (PL) Secretary
A. Griesmacher (A) Treasurer
International Scientific Committee:
J. Balzarini, Belgium
J. Barankiewicz, USA
M.A. Becker, USA
R.I. Christopherson, Australia
R.A. De Abreu, The Netherlands
R.B. Diasio, USA
S. Eriksson, Sweden
M. Fukushima, Japan
V. Gandhi, USA
N. Kamatani, Japan
M. Müller, Austria
D. Perrett, UK
G.J. Peters, The Netherlands
R.L. Sabina, USA
I. Sebesta, Czech Republic
H.A. Simmonds, UK
A.C. Skladanowski, Poland
R. Smolenski, Poland
F.F. Snyder, Canada
O. Sperling, Israel
M. Staub, Hungary
L.F. Thompson, USA
G. Van den Berghe, Belgium
A.B.P. Van Kuilenburg, The Netherlands
G. Weber, USA
ESSPPMM Scientific Committee:
J. Balzarini, Leuven
E.A. Carrey, London
R.A. De Abreu, Nijmegen
S. Eriksson, Uppsala
A. Giacomello, Rome
M.L. Löffler, Marburg
W. Makarewicz, Gdansk
E. Marinello, Siena
V. Micheli, Siena
B. Munch-Petersen, Roskilde
M.M. Müller, Vienna
G.J. Peters, Amsterdam
J.D. Puig, Madrid
I. Sebesta, Prague
H.A. Simmonds, London -Honorary Member
A.C. Skladanowski, Gdansk
R.T. Smolenski, Harefield - Gdansk
O. Sperling, Tel Aviv
M. Staub, Budapest
G. Van den Berghe, Brussels
N. Zöllner, Munich - Honorary Member
5
Contact Addresses
Scientific programme
Prof. Dr. G.J. Peters
Address:
Phone:
Fax:
E-mail:
Dept. Medical Oncology
VU University Medical Center
De Boelelaan 1117
1081 HV Amsterdam
The Netherlands
+31 20 444 2633
+31 20 444 3844
[email protected]
Symposium secretariat
Ms. S. van Geloven
Address:
Phone:
Fax:
E-mail:
Dept. Medical Oncology BR232
De Boelelaan 1117
1081 HV Amsterdam
The Netherlands
+31 20 4442630
+31 20 4443844
[email protected]
[email protected]
Registration and logistics
PAOG Course and Congress Organisation
MF/D-237
Address:
Van der Boechorststraat 7
1081 BT Amsterdam
The Netherlands
Phone:
+31 (0)20 444 8444
Fax:
+31 (0)20 444 8445
E-mail:
[email protected]
6
Support
We would like to thank the following companies/institutes for their support:
Main Sponsor
Taiho Pharmaceutical Co., Ltd.
www.taiho.co.jp/english/
Sponsors
Applied Biosystems
www.applera.com
Eli Lilly & Company
www.lilly.com
Roche Diagnostics Nederland BV
www.roche-diagnostics.com
Royal Netherlands Academy of
Arts and Sciences
www.knaw.nl/english/index.html
Sanofi-Synthélabo
www.sanofi-synthelabo.com
Shire BioChem Inc.
www.shire.com
Contributor
Thermo Electron BV
Young Investigator Award
The Young Investigator Award has been awarded to:
C. Carnrot, Uppsala, Sweden,
Z. Horvath, Vienna, Austria,
Z. Khalpey, Harefield, UK,
S. Mani, Calgary, Canada,
A. Yuen, Harefield, UK.
The Young Investigator Award has been made possible by generous contributions from PP91 and
ESSPPMM '93.
7
Registration fee
The full symposium fee includes the following:
• admission to all sessions
• programme & abstract book
• welcome drinks on Monday, June 9
• coffee and tea during breaks
• lunch from Tuesday, June 10 to Friday, June 13
• dinner at Zuiderduin on Tuesday, June 10 & Wednesday, June 11 (including 1 drink and 1 coffee)
• excursion to Enkhuizen & the Zuiderzee Museum on Wednesday afternoon, June 11
• barbecue at Grand Café Lido on Thursday evening, June 12
• symposium proceedings
Venue Hotel Zuiderduin
Hotel Zuiderduin is perfectly situated within hundred metres of the beach and the town centre of Egmond
aan Zee.
The symposium will be held at the 0-floor of the hotel (see floor plan on the next page). On Monday, June 9,
you are all invited to come to the Van Speykzaal for the welcome reception at 18:00 hrs. From Tuesday, June
10, on all oral presentations will take place at the Zuiderduinzaal. The poster area is situated in the Foyer and
the Le Regal room with are both adjacent to the Zuiderduinzaal. During the symposium, coffee and tea will
be served in Le Regal, and lunch and dinners will take place at the Restaurant of Zuiderduin
Hotel rooms
All rooms are fully equipped with a colour television, telephone, alarm clock and bathroom. The room rates
are including breakfast and taxes. Check-in is from 15:00 hrs and check-out before 10:30 hrs.
Facilities
The hotel is accommodated with a wide range of leisure facilities, including a heated indoor swimming pool
and massage whirlpool (free of charge). For a service charge the following facilities are available: saunas,
Turkish and Japanese baths, solarium, beauty parlour, hairdresser and squash courts. Hotel guests can also
use outdoor tennis courts.
After symposium hours you can relax and have a drink at one of the hotel bars.
Internet facilities are available at Lounge 1 on the 1st floor. The charges are € 1,- per 8 minutes
Finally the hotel has a large parking area, including an indoor parking for cars and bikes.
Opening hours
Restaurant:
• breakfast from 07:00 – 10:30 hrs
• lunch from 12:30 – 14:00 hrs
• dinner from 18:00 – 21:00 hrs
Swimming pool:
• 07:00 – 19:00 hrs
Sauna:
• 10:00 – 23:30
8
9
10
Programme PP’03
Monday, June 9
15:00 - 18:00
18:00 - 19:00
registration (hotel reception)
welcome reception (Van Speykzaal)
Tuesday, June 10
08:30 - 08:45
Welcome (Zuiderduinzaal)
Session 1: Antimetabolites in the treatment of arthritis (Zuiderduinzaal)
Chairs: Kamatani, N; Becker, M.
08:45 - 09:15
I01
09:15 - 09:30
O01
09:30 - 09:45
O02
09:45 - 10:15
10:15 - 10:30
I02a
O04
10:30 - 11:00
Dijkmans, B.A.C.: Antimetabolites in the treatment of arthritis: Current status of the
use of antimetabolites
.
Urano, W.U.: The efficacy and the toxicity of methotrexate in rheumatoid arthritis
patients are associated with polymorphisms in the methylenetetrahydrofolate
reductase gene
Becker, M.A.: Febuxostat, a selective non-purine uric acid production inhibitor, is
safe and decreases serum urate in healthy volunteers
Aarden, L.: Immunosuppressive activities of methotrexate and mycophenolic acid
Koshiba, M.: Modification of cytokine milieu by signaling through A2A adenosine
receptors in rheumatoid arthritis
coffee and tea break (Le Regal)
Session 2: From bench to the clinic (1) (Zuiderduinzaal)
Chairs: Gandhi, V.; Balzarini, J.
11:00 - 11:30
I03
11:30 - 12:00
12:00 - 12:30
I04
I05
12:30 - 15:00
Gandhi, V.: Clofarabine in Acute Leukemias: Clinical success and
pharmacokinetics
Schramm, V. L.: Immucillins as Antibiotics for T-Cell Proliferation and Malaria
Nguyen, B.: ALIMTA (Pemetrexed) from lab bench to clinic.
lunch break (Restaurant) and free time
Session 3: Poster (presentation) session (Zuiderduinzaal & Le Regal/Foyer)
Chairs: Peters, G.J.; Griesmacher, A.
15:00 - 16:30
Poster presentation session (short oral presentations): P30-P37 and P61-P95
16:30 - 17:30
poster session with coffee, tea and soft drinks (all posters)
11
Session 4: From bench to the clinic (2) (Zuiderduinzaal)
Chairs: IJzerman, A.; Balzarini, J.
17:30 - 18:00
18:00 - 18:30
I02b
I06a
18:30 - 18:45
O05
18:45 - 19:00
O06
19:00 – 20:00
Pieters, R.: Prevention of hyperuricemia with rasburicase
Koomen, G.J.: New (1-DEAZA)Purine Derivatives via Efficient C-2 Nitration of
the (1-DEAZA)Purine Ring
Balzarini, J.: 6-[2-(Phosphonomethoxy)alkoxy]pyrimidines: a new class of acyclic
pyrimidine nucleoside phosphonates with antiviral activity
Bergman, A.M.: Antiproliferative activity and mechanism of action of fatty acid
derivatives of gemcitabine in leukemia and solid tumor cell lines and xenografts
dinner (Restaurant)
Session 5: Methodology (Zuiderduinzaal)
Chairs: Perrett, D.; Van Kuilenburg, A.B.P.
20:00 - 20:30
20:30 - 20:45
20:45 - 21:15
I06b
O07
I07
21:15 - 21:45
21:45 - 22:00
I08
O08
Svoboda, M.: LC-MS techniques in clinical applications
Smolenski, R.T.: Nucleotide analysis by liquid chromatography/mass spectrometry
Lutz, V.: Quantitative real-time PCR for thymidylate synthase, thymidine
phosphorylase and dihydropyrimidine dehydrogenase with the Light-Cycler
Wicki, R.: New Levels in Gene Expression Profiling
Hooijberg, J.H.: Online fluorescent method to assess BCRP activity as a function of
cellular folate homeostasis
12
Wednesday, June 11
Session 6: Purines in the cardiovascular system (Zuiderduinzaal)
Chairs: Smolenski, R.T.; De Jong, J.W.
08:30 - 09:00
I09
09:00 - 09:30
I10
09:30 - 10:00
10:00 - 10:15
I11
O09
10:15 - 10:30
O10
10:30 - 11:00
Blackburn, R.: Pulmonary Consequences of Adenosine Overload: Lessons from
Adenosine Deaminase Deficient Mice
Headrick, J.: Species and age-dependent differences in purine metabolism,
adenosinergic signalling, and ischemic tolerance
Struthers, A.: Allopurinol: Will it reduce cardiovascular events in patients?
Gutensohn, W.: Coordinate Regulation of the Expression of Ecto-5'-Nucleotidase
(CD73) and the A2a Adenosine Receptor in a Human B-Cell Line
Yuen, A.H.Y.: Prevention of adriamycin induced heart failure by increase in
endogenous adenosine production
Session 7: Chemotherapy in solid and hematological malignancies (Zuiderduinzaal)
Chairs: Kaspers, G.J.L.; De Abreu, R.A.
11:00 - 11:15
O11
11:15 - 11:30
O12
11:30 - 11:45
O13
11:45 - 12:00
O14
12:00 - 12:15
O15
12:15 - 12:30
O16
12:30 - 12:45
O17
12:45 - 13:00
O18
De Bruin, M.: Role of Platelet derived endothelial cell growth factor / thymidine
phosphorylase in fluoropyrimidine sensitivity and potential role of deoxyribose-1phosphate
Williams, M.V.: Down-Regulation of Human Deoxyuridine Triphosphate
Nucleotidohydrolase (dUTPase) using Small Interfering RNA (siRNA)
Saiko, Ph.: Cytotoxic and apoptotic effects of novel heterodinucleoside dimers
consisting of 5-fluorodeoxyuridine and Ara-c in human cancer cell lines
Mauritz, R.: Increased thymidylate synthase (TS) but unaltered dihydropyrimidine
dehydrogenase (DPD) mRNA levels after administration of 5-fluorouracil (5-FU) to
patients with colon cancer
Cardoen, S.: Effects of 2-chloro-2'-deoxyadenosine on the cell cycle in human
leukemia CCRF-CEM and EHEB cell lines
Verschuur, A.C.: Increased cytotoxicity of 2’,2’-difluoro-2’-deoxycytidine in
human leukemic cell-lines after a preincubation with cyclopentenyl cytosine
Iven, H.: Can whole blood substitute washed red blood cells for the analysis of
TPMT activity and 6-mercaptopurine metabolites?
Giacomello, A.: Potential role of mycophenolate mofetil in the management of
neuroblastoma patients
13:00 - 13:15
delegates should gather in front of the hotel reception for the buses to Enkhuizen and
the Zuiderzee Museum; lunch bags will be handed out.
13:15 - 18:00
excursion to Enkhuizen and the Zuiderzee Museum (open air museum)
18:00 - 19:00
dinner (Restaurant)
13
Session 8: Lesch-Nyhan syndrome (Zuiderduinzaal)
Chairs: Puig, J.G.; Sperling, O.
19:00 - 19:05
19:05 - 19:30
I12
19:30 - 19:55
19:55 - 20:20
I13
I14
20:20 - 20:30
O19
20:30 - 20:40
O20
20:40 - 20:50
O21
Simmonds, A.H.: Tribute to Françoise Roch-Ramel
McCarthy, G.T.: Medical Diagnosis, Management and Treatment of Lesch Nyhan
Disease
Marinaki, A.M.: Lesch-Nyhan Disease
Jinnah, H.A.: Mutations of the HPRT Gene in Lesch-Nyhan Disease and its
Variants
Torres, R.J. : Adenosine transport in HPRT deficient lymphocytes from LeschNyhan patients.
Becker, B.F.: Urate Oxidation in CSF and Blood of Patients With Inflammatory
CNS Disorders
Carrey, E.A.: An unusual pyridine nucleotide accumulating in erythrocytes: its
identity, and positive correlation with degree of renal failure
14
Thursday, June 12
Session 9: New inborn errors; role of mitochondria (Zuiderduinzaal)
Chairs: Eriksson, S.; Loeffler, M.
08:30 - 09:00
I15
09:00 - 09:30
I16
09:30 - 09:45
O23
09:45 - 10:00
O24
10:00 - 10:15
10:15 - 10:30
O25
O26
10:30 - 11:00
Saada-Reisch, A.: New inborn errors; role of mitochondria; Deoxyribonucleoside
kinases in mitochondrial DNA depletion.
Hirano, M.H.: Thymidine Phosphorylase Deficiency Causes MNGIE: An
Autosomal Recessive Mitochondrial Disorder
Bianchi, V.B.: Metabolism of thymidine in mitochondria: role of dNT2, the
mitochondrial deoxyribonucleotidase.
Gattermann, N.: Severe impairment of nucleotide synthesis through inhibition of
mitochondrial respiration
Gojkovic, Z.G.: New class of suicide genes for cancer gene therapy
Snyder, F.F.: Production and Properties of Polyethylene-Glycol Conjugated
Adenosine Phosphorylase: Administration to PNP deficient mice
Session 10: Membrane targets (Zuiderduinzaal)
Chairs: Jansen, G.; Stone T.W.
11:00 - 11:30
11:30 - 11:45
I17
O27
11:45 - 12:15
I18
12:15 - 12:30
O29
12:30 - 12:45
O30
12:45 - 15:00
Cass, C.: Nucleoside transporters
Casado, F.J.: Nucleoside transporters in proliferation in normal and transformed
cells
Borst, P.: Role of Multidrug-resistance associated proteins in resistance to
nucleoside analogs
Gorodeski, I.: Estrogen Attenuates P2X7-R - Mediated Apoptosis of Uterine
Cervical Cells by Blocking Calcium Influx
Snyder, F.F.: Mitochondrial function dependent proliferation assay for the diagnosis
of mitochondrial disorders in human fibroblasts
lunch break (Restaurant) and free time
Session 11: Poster (presentation) session (Zuiderduinzaal & Le Regal/Foyer)
Chairs: Sebesta, I; Micheli, V.
15:00 - 16:30
Poster presentation session (short oral presentations): P1-P29 and P38-P60
16:30 - 17:30
Poster session with coffee, tea and soft drinks (all posters)
15
Session 12: Antimetabolites: parasites and viruses (Zuiderduinzaal)
Chairs: Van den Berghe, G.; Christopherson, R.I.
17:30 - 18:00
18:00 - 18:15
I19
O33
18:15 - 18:30
O34
18:30 - 18:45
O35
18:45 - 19:00
O36
19:30 - 22:30
Christopherson, R.I.: Cloning and expression of malarial pyrimidine enzymes
De Koning, H.P.: Rational design of purine and pyrimidine drugs against protozoan
infections through selective uptake
Deville-Bonne, D.: Improving NDP Kinase for Antiviral Nucleotide Analogs
Activation
McLennan, A.G.: Regulation of dinucleoside polyphosphates by the YgdP and
ApaH diadenosine polyphosphate hydrolases is essential for intracellular invasion by
Salmonella enterica serovar Typhimurium
Slominska, E.M.: The Effect of N-Methyl-2-Pyridone-5-Carboxamide - A
Nicotinamide Catabolite on Poly ADP-Ribosylation and Oxidative Stress Injury in
Endothelial Cells
Barbecue at the beach (Grand Café Lido, Westeinde 1, Egmond aan Zee;
approximately 250 metres walking distance from Hotel Zuiderduin)
16
Friday, June 13
Session 13: Enzymology; Signal transduction (Zuiderduinzaal)
Chairs: Staub, M. ; Sabina R.I.
08:30 - 09:00
09:00 - 09:30
I20
I21
09:30 - 09:45
O37
09:45 - 10:00
O38
10:00 - 10:15
O40
10:15 - 10:30
O31
10:30 - 11:00
Piskur, J.: Diversity and origins of deoxyribonucleoside kinases
Graves, M. : Regulation of Nucleoside Uptake by Protein Kinase Inhibitors and
Mitogenic Signaling.
Sasvari-Szekely, M.: Selective increase of dATP pools upon activation of
deoxycytidine kinase in lymphocytes: implications in apoptosis
Sabina, R.L.: Multi-level regulation of human AMP deaminase isoform E
(AMPD3) by calmodulin
Mani, R.S. Biophysical Studies of ATP, Deoxycytidine and Gemcitabine Binding to
Human Recombinant Deoxycytidine Kinase
Allegrini, S.: Functional studies reveal that cytosolic "High Km" 5'-Nucleotidase
(cN-II) active site has a structure similar to that of HAD superfamily
Session 14: Genetic polymorphisms (1) (Zuiderduinzaal)
Chairs: Peters, G.J; Snyder, F.F.
11:00 - 11:30
I22
11:30 - 12:00
I23
12:00 - 12:15
O42
12:15 – 12:30 O43
12:30 - 14:00
Nishiyama, M.: Transcriptional regulation of DPYD gene expression and
aberrant methylation in the 5'flanking region
Van Kuilenburg, A.B.P.: Pharmacogenetic and clinical aspects of a
dihydropyrimidine dehydrogenase deficiency
Kamatani, N: Identification of 4 unique mutations in exon 4 of uromodulin
(UMOD) gene in 4 separate families with familial juvenile hyperuricemic
nephropathy (FJHN).
Peterson, C.: What role do methylated metabolites play for the cellular effects of
thiopurines?
lunch break (Restaurant)
Session 15: Genetic polymorphisms (2) (Zuiderduinzaal)
Chair: Marinaki A.M.; Nishiyama, M.
14:00 - 14:30
I24
14:30 - 14:45
O44
14:45 - 15:15
O41
15:15 - 15:30
15:30 - 15:45
O45
O46
15:45 - 16:00
O32
16:00 - 16:30
Coulthard, S.: The Clinical Impact of Thiopurine Methyltransferase Polymorphisms
on Thiopurine Treatment
Shobowale-Bakre, E.A.: Pre-treatment TPMT PHE/genotyping: a safe guide for
initial thiopurines drugdosing for specific patient groups
De Jonge, R.: Clinical consequences of polymorphisms in the
methylenetetrahydrofolate reductase gene
Marinaki, A.M.: A mutation in the ITPA gene predicts intolerance to azathioprine.
Taniguchi, A.T.: Identification of two novel mutations in adenine
phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis
Spychala, J.: The association of ecto-5'-nucleotidase (eN), integrin b1, EGFR and
vimentin with invasive breast carcinoma: The role of eN in invasive phenotype
Business Meeting ESSPPMM and closing session (Zuiderduinzaal)
17
Posters
Subject: Gout
P01
Sperling, O.: Modulation of Glycogen Phosphorylase activity affects 5 Phophoribosyl-1Pyrophosphate availability in Rat Hepatocyte Cultures
P02
Stone, T.W.: Purine levels and cytokine release with diuretic therapy in rheumatoid arthritis
P03
Puig, J.G.: The Pathophysiology of Hyperuricemia in Essential Hypertension: A pilot study
P04
Tsutsumi, Z.T.: Serum Adiponectin Concentration in Patients with Primary Gout
P05
Ka, K.: Effect of low purine-containing low malt beer (happo-shu) and regular low malt beer on
purine bases
P06
Ichida, K.I.: SLC22A12 mutation is responsible for most renal hypouricemia in Japan
P07
Yamada, I.Y.: Pharmacokinetics/Pharmacodynamics of Y-700, A novel Xanthine Oxidase Inhibitor,
in Rats and Man
P08
Hoshide, S.H.: PK/PD and safety of a single dose of TMX-67 (febuxostat) in subjects with mild and
moderate renal impairment
P09
Komoriya, K.K.: Pharmacodynamics & pharmacokinetics of TMX-67 (febuxostat), a novel
xanthine oxidase / xanthine dehydrogenase (XOD) inhibitor, in patients with hyperuricemia
Subject: Lesch-Nyhan syndrome
P10
Zoref-Shani, E.: A novel point Mutation (I137t) in the Conserved 5-Phosphoribosyl-1Pyrophosphate Binding Motif of HPRT (HPRTJERUSALEM) in a LNS Variant
P11
Mizunuma, M.M.: Disruption in the hypoxanthine phosphoribosyl-transferase gene caused by
translocation in a patient with Lesch-Nyhan syndrome
P12a Yamada, Y.: Mutations in the hypoxanthine guanine phosphoribosyltransferase gene (HPRT1) in
Asian HPRT deficient families
P12b Sebesta, I.: Highly skewed X-inactivation pattern as a cause of female gout
P13
Micheli, V. : Are allopurinol and metabolites found in HPRT deficient erythrocytes responsible for
increased NAD synthesis?
P14
Torres, R.J.: Effects of hypoxanthine on adenosine transport in human lymphocytes. Implications in
the pathogenesis of Lesch-Nyhan syndrome
P15
Marinello, E.: EPR spin trapping of a radical intermediate in the urate oxidase reaction
P16
Verdu, A.: Cognitive abilities in a series of patients with Hypoxanthine guanine
phosphoribosyltransferase deficiency
P17
Visser, J.E.: The motor features of Lesch-Nyhan disease
P20a McCarthy, G.T.: Lesch Nyhan Disease, essentials of diagnosis and management - a European
perspective
P20b Simmonds, H.A.: Nucleotide degradation products in cerebrospinal fluid (CSF) in inherited and
acquired pathologies
Subject: Inborn errors
P21
Ceballos-Picot, I.C.P.: Genotype and clinical features of adenine phosphoribosyltransferase (APRT)
deficiency in French families
P22
Race, V.: Adenylosuccinate lyase deficiency: study of physiopathologic mechanism(s)
P23
Marinaki, A.M.: Adenylosuccinate lyase deficiency - first UK case
P24
Laurence, A.D.J.: Elevated erythrocyte CDP-choline levels associated with b-thalassaemia in
patients with transfusion independent anaemia
P25
Pawa, S.P.: Nucleotides can afford protection in cirrhosis induced by thioacetamide
P26
Marinello, E.: Serum Folate Level in Mozambique's Children
P27
Marinaki, A.M.: Deoxyuridine accumulation in urine in thymidine phosphorylase deficiency
(MNGIE)
P28
Escuredo, E.; The Genetic Basis of the Interaction Between Pyrimidine 5' Nucleotidase deficiency
and Hemoglobin E
P29
Marinaki, A.M.: Purine nucleoside phosphorylase deficiency: Is there a mitochondrial pathology?
18
Subject: Gene polymorphisms
P30a Salerno, C.: Urinary methylxanthine and autistic disorder: absence of previously reported
correlation
P30b Grunebaum, E.G.: Novel mutations and a 'hot-spot' in Purine Nucleoside Phosphorylase deficient
patients
P31
Taniguchi, A.T.: The frequency and diversity of the haplotypes near adenine
phosphoribosyltransferase (APRT) locus are different between patients with APRT deficiency and
normal controls
P32
Haglund, S.: Pyrosequencing of Thiopurine S-methyltransferase (TPMT) alleles in a Swedish
general population and in IBD-patients
P33a Marinaki, A.M.: Polymorphism in the MTHFR gene affects thiopurine methyltransferase activity
P33b Marinaki, A.M.: Allele frequency of inosine triphosphate pyrophosphohydrolase gene
polymorphisms in a Japanese population
P34
Arenas, M.: Genetic determinants of the thiopurine methyltransferase intermediate activity
phenotype
P35
Calascibetta, A.: Association between Thymidylate Synthase polymorphism with the microsatellite
instability in colorectal cancer
P36
Mauritz, R: Polymorphic tandem repeats in the thymidylate synthase gene and TS protein and
mRNA levels in different tissues of colorectal cancer patients
Subject: Metabolism and enzymology
P38a Loeffler, M.L.: Dihydroorotate dehydrogenase expression analysis in normal and drug-resistant
cells and tissues
P38b Usova, E.V.: Phosphorylitic cleavage of deoxyribonucleosides catalysed by human deoxycytidine
kinase – a side reaction or a function. Preliminary Biochemical and NMR Studies
P39
Staub, M.: The Effect of Signalling Pathway Modulations on the Activity of the Deoxycytidine
Kinase
P40
Krawiec, K.: Unusual phosphate donors for human deoxyrybonucleoside kinases.
P41
Munch-Petersen, B.M.P.: The multisubstrate deoxyribonucleoside kinase from Drosophila
melanogaster can phosphorylate glycerol
P42
Munch-Petersen, B.M.P.: Amino acid residues determining the substrate specificity of
deoxyribonucleoside kinases
P43
Wang, L.W.: Mitochondrial deoxyguanosine kinase/thymidine kinase mutations and mitochondrial
DNA depletion syndrome
P44
Al-Madhoun, A. S.: Biochemical and Biological Evaluation of 3-(Carboranylalkyl) thymidines as
Substrates for Human Thymidine Kinases 1 and 2
P45
Carnrot, C.M.E.: Cloning and biochemical characterisation of thymidine kinase in Ureaplasma
urealyticum
P46
Torres, R.J.: Hypoxanthine effects on cyclic amp levels in human lymphocytes
P47
Kovari, J.: Mechanistic and physiological studies of dUTPases
P48
Rinaldo-Matthis, A.R.M.: Crystal structure of a human deoxyribonucleotidase
P49
Rylova, S.N.: Differential activity of pyrimidine nucleoside kinases in proliferating, resting and
adipocyte differentiated 3T3-L1 cells
P50
Karlsson, S.: Cloning of a mitochondrial UMP/CMP kinase from Drosophila melanogaster
P51
Ipata, P.L.: Purine and Pyrimidine Salvage in Whole Rat Brain: Utilization of ATP-derived Ribose1 Phosphate and 5-phosphoribosyl-1-Pyrophosphate Generated in Experiments with Dialyzed Cellfree Extracts
P52
Higgins, M.J.H.: Colocalization of Human Cytidine Triphosphate Synthetase 1 with Microtubules
P53
Snyder, F.F.: Identification of binuclear zinc coordination for human guanine deaminase by site
directed mutagenesis
P55
Carrey, E.A.: GTP Concentrations are Elevated in Erythrocytes of Renal Transplant Recipients
when Conventional Immunosuppression is replaced by the Inosine Monophosphate Dehydrogenase
Inhibitor Mycophenolic Acid Mofetil (MMF)
P56
Di Pietrantonio, F.: Cyclase and Phosphodiesterase Activity on Pre-T Lymphoid Human Cells,
treated with Dimethyl Sulfoxide (DMSO)
19
P57
P58
P59
P60
Tozzi, M.G. Identification of the 5'-nucleotidase activity altered in neurological syndromes
Khalpey, Z.: Exposure to Human Blood Decreases Swine Endothelial ECTO-5'-Nucleotidase
Activity
Marinello, E.: Liver Transplant: Adenosine Metabolism and Apoptosis
Szydlowska, M.: The immunological and kinetical characteristic of AMP-deaminase from normal
and Hepatocellular carcinoma (HCC) human liver
Subject: Purine metabolism in cardiovascular disease
P61a Salerno, C.: Biochemical and molecular genetic correlations in adenylosuccinate lyase deficiency
P61b Kalsi, K.K. : AMPD1 C34T Mutation Selectively affects AMP-deaminase activity in the Heart
P62
Yuen, A.H.Y.: Association of improved cardiac function in donors with C34T mutation of the AMP
Deaminase 1 gene
P63
Smolenski, R.T.: Purine Metabolism in Pigs and Humans and its Implications for
Xenotransplantation
P64
Osborne, F.: Overexpression of Human ECTO 5' Nucleotidase in Pig Endothelial Cells and its
Implication for Adenosine Production and Xenotransplantation
P65
Kalsi, K.K.: Lidoflazine combined with nucleotide precursors increases ATP content and Adenosine
production in cardiomyocytes
Subject: Gene therapy
P66
Solaroli, S.N.: Investigation of substrate recognition of Drosophila melanogaster nucleoside kinase
by site directed mutagenesis
P67
Marinello, E.: Evaluation of ADA Gene Expression and Transduction Efficiency in ADA/SCID
Patients Underwent Gene Therapy
Subject: Chemotherapy of malignancies
P68
Marinello, E.: Metabolism of Adenosine in Human Colorectal Tumor
P70
Smal, C.: New evidences for regulation of deoxycytidine kinase activity by reversible
phosphorylation
P71
Van Kuilenburg, A.B.P.: Cyclopentenyl cytosine sensitises SK-N-BE(2)c neuroblastoma cells to
cladribine
P72
Van Kuilenburg, A.B.P.: Gemcitabine and cyclopentenyl cytosine: a promising combination for
the treatment of neuroblastoma
P73
Hubeek, I.: Modulation of cytarabine resistance in childhood acute myeloid leukemia
P74
Bergman, A.M.: Antiproliferative activity and mechanism of action of fatty acid derivatives of
arabinofuranosylcytosine in leukemia and solid tumor cell lines
P75
Wehelie, R.: Pyrimidine deoxynucleoside salvage in Ureaplasma urealyticum: nucleoside analogues
acted as potent inhibitors of mycoplasma growth
P76
Horvath, Z.: Synergistic effects of BCNU and Didox combination chemotherapy in 9L glioma cells
P77
Bauer, W.B.: Amidox, an inhibitor of ribonucleotide reductase, potentiates the action of Ara-C in
HL-60 human promyelocytic leukemia cells
P78
Temmink, O.H.: Mechanism of action of trifluorothymidine (TFT) in combination with a thymidine
phosphorylase inhibitor (TPI)
P79
Smid, K.: The effect of different fluoropyrimidines with or without a thymidine phosphorylase
inhibitor (TPI) on the expression of platelet derived endothelial cell growth factor / thymidine
phosphorylase (PD-ECGF/TP)
P80
Temmink, O.H.: Combination studies of trifluorothymidine (TFT) with antifolates and the DNA
synthesis inhibitors irinotecan, oxaliplatin and gemcitabine
P81
Sigmond, J.: Combination studies of 5-fluorouracil with UCN-01 or staurosporine
P82
Razmara, M.: 5’-nucleotidases levels measured in peripheral blood cells from patients with chronic
and acute leukemia
20
P83
P84
P85a
P85b
P86a
P86b
Spoto, G.: Cyclic guanosine monophosphate role in human carcinoma pathogenesis
Giacomello, A.: Cyclic nucleotides and neuroblastoma differentiation
Honma, Y.: Differentiation-inducing Activity of Purine and Pyrimidine Derivatives on
Myelogenous Leukemia Cells: The most suitable analog depends on the leukemia type
Gutsche, S.: 6-mercaptopurine (6-MP) metabolism in man and its fingerprints in erythrocytes (RBC)
Kulikowski, T.: ATP-site directed potential inhibitors of nucleoside triphosphatases
(NTPase)/helicases and polymerases of hepatitis C and other selected Flaviviridae viruses
Shobowale-Bakre, E.A.: Pre-treatment TPMT phenotyping/genotyping : an excellent guide for
initial thiopurine drug dosing
Subject: Methodology
P87
Kotalczyk, A.: A time saving and rapid HPLC-method for the determination of 6-thioguanine and 6
thioguanine (-nucleotides)
P88
Lindqvist, M.: Real time RT-PCR methodology for quantification of TPMT gene expression
P89
Sigmond, J.: Quantitative detection of deoxycytidine kinase by real time light cycler PCR in
comparison with conventional competitive template RT-PCR
P90
Hubeek, I.: Immunocytochemical detection of deoxycytidine kinase
P91
Van Kuilenburg, A.B.P.: Determination of the deoxycytidine kinase activity in cell homogenates
with a non radiochemical assay using reversed-phase HPLC. Identification of a novel metabolite of
2-Chlorodeoxyadenosine
P93
Slominska, E.M.: Application of liquid chromatography - mass spectrometry for identification and
quantitation of metabolic alterations in chronic renal failure
P94
Kaneko, K.: Detection of prothrombin and osteopontin in a renal stone found in a hyperuricemic
patient using 2D-PAGE and LCMS analysis
P95
Noordhuis, P.: A non-radioactive sensitive assay to measure 5-fluorouracil incorporation into DNA
of solid tumors
21
I01
Antimetabolites in the treatment of arthritis: Current status of the use of antimetabolites
B.A.C. Dijkmans, G. Jansen
VU University Medical Center, AMSTERDAM, The Netherlands
Rheumatoid arthritis (RA) is an autoimmune disease characterized by a chronic inflammation of the synovial
joints and infiltration of blood-derived cells. Effective drug treatment of RA patients is aimed at diminishing
the release of pro-inflammatory cytokines (e.g. TNF-α) that play a major role in the pathophysiology of RA.
In daily practise rheumatologists use the antimetabolites methotrexate (MTX) and leflunomide for the
treatment of patients with arthritis. Onother antimetabolite, mycophenolate mofetil, has only a modest place.
The current clinical status (efficacy/toxicity) of these 3 antimetabolites in the treatment of RA will be
discussed.
Methotrexate: Despite its clinical use for many decades, the exact mechanism(s) by which MTX exerts
immunosuppressive or anti-inflammatory effects has not been fully clarified. Most likely a combination of
antifolate effects and anti-purine effects contribute to the therapeutic profile of MTX. At present MTX is
considered as anchor-drug for treatment of RA patients. MTX is used as monotherapy or in combination with
other anti-rheumatics, e.g. sulphasalazine, (hydroxy)chloroquine and corticosteroids. Recently, combinations
of MTX with novel TNF-α antibodies (infliximab, etanercept) demonstrated markedly improved therapeutic
effects for RA patients. MTX treatment can be associated with toxic side effects, including gastrointestinal,
hematological, liver and pulmonary toxicity, but rheumatologists have learned to monitor and influence (e.g.
by folic acid/leucovorin supplementation) toxicity to acceptable proportions.
Leflunomide: Leflunomide is an inhibitor of pyrimidine biosynthesis via inhibition of dihydroorotate
dehydrogenase. Immunosuppressive and antiproliferative effects of leflunomide are illustrated by inhibition
of T-cell proliferation in response to cytokines (IL-2) and inhibition of antibody synthesis. The current
position of leflunomide is that of an established anti-rheumatic drug that is administered after MTX and
sulphasalazine. Adverse effects of leflunomide (diarrhoea, hypertension, rash and increased liver enzymes)
are generally mild and relatively infrequent.
Mycophenolate mofetil (MM): MM is a prodrug, the inactive 2-morpholino ester of mycophenolic acid,
which is an inhibitor of IMP dehydrogenase. The immunosuppressive effects of MM have found widespread
application in the field of organ transplantation. At present some trials are underway in patients with RA and
systemic lupus erythematosus.
Collectively, antimetabolites as single agent or in combinations have proven efficacy for treatment of RA
patients. Further clinical exploitation of combinations of antimetabolites with novel TNF-α blockers is a
challenging option for further clinical evaluation in arthritis treatment.
22
I02a
Immunosuppressive activities of methotrexate and mycophenolic acid
S. De Lathouder1, A.H. Gerards2, E.R. De Groot1, M.G. Valkhof1, L.A. Aarden1
1
Sanquin Research at CLB, AMSTERDAM, The Netherlands
2
Methotrexate (MTX) and mycophenolic acid (MPA) are immunosuppressive drugs used for the treatment
of various immunological disorders. MTX is widely used in the treatment of rheumatoid arthritis (RA).
MPA is used to prevent graft rejection after transplantation and is now experimentally used in RA. MPA is
an inhibitor of inosine monophosphate-dehydrogenase and MTX is a folate antagonist that inhibits
tetrahydrofolate-reductase.
Both drugs inhibit the production of several cytokines (such as IL-4, IFN-γ, TNF-α and GM-CSF) after T-cell
stimulation with anti-CD3/anti-CD28. The mechanism by which inhibition is achieved is different for both
drugs. When T cells are activated in the presence of MTX, the cells enter the cell cycle and progress to S
phase and mitosis. At that point, the cells die by apoptosis. Resting T-cells are not affected. The mechanism of
MPA is different. When resting T-cells are activated in the presence of MPA, some activation markers (CD69
and CD25) are upregulated, but the cells stay small and do not enter S phase. Addition of guanosine and
adenosine can overcome this cell cycle arrest, even after several days.
The observation that MTX cannot prevent T cell activation but induces apoptosis in activated T cells and that
MPA reversibly prevents activation of T cells could explain the immunosuppressive effects both drugs.
23
I02b
Prevention of hyperuricemia with rasburicase
J.M. de Bont, R. Pieters
Erasmus MC - Sophia Children's Hospital, ROTTERDAM, the Netherlands
Tumour lysis syndrome (TLS) is a potentially life-threatening complication in patients with haematological
malignancies1. Massive lysis of tumour cells can lead to hyperuricemia, hyperkalemia, hyperphosphatemia
and hypocalcemia1,2. This can result in renal failure, because of the precipitation of uric acid crystals and
calcium phosphate salts in the kidney1
Until recently the standard prophylaxis and treatment of hyperuricemia consisted of decreasing uric acid
production with allopurinol and facilitating its excretion by urinary alkalinisation and hyperhydration1,2,3. By
inhibiting the enzyme xanthine oxidase, allopurinol blocks the conversion of hypoxanthine and xanthine into
uric acid.
An alternative treatment is the use of urate oxidase, an enzyme which oxidates uric acid into allantoin.
Allantoin is ten times more soluble in urine than uric acid and is therefore excreted easily. Rasburicase, the
recombinant form of urate oxidase has shown to be very effective and is in contrast with the nonrecombinant form, uricozyme, associated with a low incidence of hypersensitivity reactions2,3.
Besides the rapid onset of action rasburicase has several other beneficial features compared to allopurinol2:
• No accumulation of hypoxanthine and xanthine, preventing xanthinenephropathy.
• No interactions with chemotherapeutic agents such as 6-mercaptopurine and cyclophosphamide.
• No further need for urinary alkalinisation, reducing the risk of precipitation of calcium phosphate salts.
• Because the mean plasma terminal half-life of rasburicase is approximately 17-20 hrs, it is sufficient to
administer it once a day.
Compassionate-use programme Rotterdam
In 2001 the Erasmus MC-Sophia Children's Hospital in Rotterdam participated in a multi-centre
compassionate-use programme for rasburicase. The major purpose of this programme was to review efficacy
and safety of rasburicase. Fourteen oncological patients with a high risk of hyperuricemia/TLS (ALL n=11,
ANLL n=2, Wilms tumour stage IV n=1) were given rasburicase (0,2 mg/kg/day) during the first few days of
their induction chemotherapy.
Before therapy was started, 12-24 hrs after the first dose of rasburicase and subsequently daily LDH, WBC,
uric acid, potassium, phosphate, calcium and creatinine were measured.
Before starting therapy none of the patients had obvious clinical signs of TLS. 13 out of 14 patients had a
very good and fast response to rasburicase, leading to levels of uric acid below 0,01 mmol/l within a day.
None of the patients developed metabolic disturbances or acute renal failure during the first phase of
chemotherapy.
To review the safety of rasburicase all adverse events were registered Three adverse events occurred, but
they were most likely complications of the administered chemotherapy and not of rasburicase. No allergic
reactions to rasburicase were seen.
Review of literature
Pui et al.5 administered rasburicase intravenously at 0,15 mg or 0,20 mg/kg/day to 131 children, adolescents
and young adults with newly diagnosed leukaemia or lymphoma. They found a decrease in uric acid levels of
95-99% 4 hours after administration of rasburicase. None of the patients required dialysis. Treatment with
rasburicase was well tolerated, with none of the adverse events having a clear relationship to the
administration of rasburicase.
In a compassionate-use trial6 173 children and 72 adults were treated with rasburicase (0,20 mg/kg/day).
Rasburicase produced a dramatic decrease in uric acid levels in all patients, whether they received it for
prophylaxis (n=79) or treatment (n=166). Uric acid levels had a median decline from 9,7 to 0,6 mg/dL in
children and 11,9 to 0,7 mg/dL in adults. In contrast to the previous (above mentioned) study5, four children
and six adults required hemodialysis, as a result of hyperphosphatemia (n=2), azotemia (n=5) or both (n=3).
The adverse events registered ranged from mild vomiting to skin reactions, fever, myalgia, headache and
oedema.
Goldman et al.3 included 52 paediatric patients with leukaemia or lymphoma in a randomised controlled trial
comparing allopurinol (10 mg/kg/day) to rasburicase (0,20 mg/kg/day). Within four hours after
administration of rasburicase uric acid levels dropped by 86% in the rasburicase arm and by 12% in the
allopurinol arm (p<0,0001). One patient in the allopurinol group required assisted renal support. The adverse
24
events that occurred (fever, pain, mucositis) were mild and most likely secondary to the disease and
chemotherapy.
Rasburicase appears to be superior to allopurinol, but is also far more expensive than therapy with
allopurinol, urinary alkalinisation and hyperhydration. Annemans et al.7,8 conducted an economic evaluation
to assess cost-effectiveness (costs per life-year saved), assuming that rasburicase reduces 100% of the costs
associated with TLS and 90% of the costs associated with hyperuricemia. They concluded that prevention of
hyperuricemia/TLS with rasburicase is highly cost-effective in adults and children and that treatment of
hyperuricemia/TLS with rasburicase is highly cost-effective in adults and cost saving in children. They
calculated that even if the reduction of costs would be only 60%, prevention and treatment of
hyperuricemia/TLS with rasburicase remains cost-effective7. This makes rasburicase not only of great
clinical benefit, but also an economically attractive new option in the management of hyperuricemia.
References
1.
Jeha S. Tumor lysis syndrome. Seminars in hematology 2001; 38(4) suppl 10: 4-8
2.
Brant JM. Rasburicase: An innovative new treatment for hyperuricemia associated with tumor lysis
syndrome. Clinical journal of oncology nursing 2002; 6(1): 12-16
3.
Goldman SC, Holcenberg JC, Finklestein JZ et al. Blood 2001; 97(10): 2998-3003
4.
Easton J, Noble S, Jarvis B. Rasburicase. Paediatric drugs 2001; 3(6): 433-439
5.
Pui CH, Mahmoud HH, Wiley JM et al. Journal of clinical oncology 2001; 19(3): 697-704
6.
Pui CH, Jeha S, Irwin D et al. Recombinant urate oxidase (rasburicase) in the prevention and
treatment of malignancy-associated hyperuricemia in pediatric and adult patients: results of a
compassionate-use trial. Leukemia 2001; 15: 1505-1509
7.
Annemans L, Moeremans K, Lamotte M et al. Pan-European multicentre economic evaluation of
recombinant urate oxidase (rasburicase) in prevention and treatment of hyperuricaemia and tumour
lysis syndrome in haematological cancer patients. Support Care Cancer 2003; 11:249-257
8.
Annemans L, Moeremans K, Lamotte M et al. Incidence, medical resource utilisation and costs of
hyperuricemia and tumour lysis syndrome in patients with acute leukaemia and non-Hodgkin's
lymphoma in four European countries. Leuk Lymphoma 2003;44(1): 77-83
25
I03
Clofarabine in Acute Leukemias: Clinical Success and Pharmacokinetics
V. Gandhi
MD Anderson Cancer Center, HOUSTON, TX, United States of America
Two analogues of deoxyadenosine active in hematologic malignancies, fludarabine and cladribine, differ in
their mechanisms of action and in their spectrum of clinical responses. A hybrid of these drugs, clofarabine,
retains the 2-chloroadenine aglycone of cladribine. Reminiscent of fludarabine, clofarabine is further
derivatized with a fluorine molecule in the arabinosyl configuration at the critical 2'-position of the
carbohydrate. The 2'-fluoro group greatly inhibits cleavage of the glycosidic linkage, a mechanism of
clearance for the parent drugs which also generates potentially toxic halogenated nucleobases. Laboratory
investigations of clofarabine demonstrated that it accumulates as the clofarabine triphosphate after
phosphorylation by deoxycytidine kinase. The triphosphate of the drug is an effective substrate for
replicative DNA polymerases and competes well with normal deoxyadenosine triphosphate, and once
incorporated into DNA disrupts further synthesis. In addition, clofarabine triphosphate inhibits
ribonucleotide reductase, decreasing cellular deoxynucleotides.
Recent phase I dose-escalation clinical investigations identified 40 mg/m2/d as the maximum tolerated dose
of clofarabine when administered daily for 5 days by intravenous one hour infusions to patients with acute
leukemias. In contrast, for patients with solid tumors and indolent leukemias, the maximum tolerated dose
was 2 and 3 mg/m2/d, respectively. For acute leukemias, hepatotoxicity was dose limiting while
myelosuppression was common in solid tumor and indolent leukemias (Ref. 1). Plasma pharmacology
studies done in 25 acute leukemia patients indicate a linear increase in the plasma clofarabine concentration
with increasing doses. At 40 mg/m2, the median plasma clofarabine level was 1.5 µM (range 0.42 to 3.2 µM;
n = 7). Cellular pharmacokinetic studies done at the end of the first clofarabine infusion in 26 patients
appeared dose-proportional, but showed a wide variation in the concentrations of clofarabine triphosphate.
At the maximum tolerated dose, the concentration was a median 19 µM (range 3 - 52 µM). In the majority
of cases, more than 50% of the analog triphosphate was present at 24 hr after infusion. Compared to
clofarabine triphosphate concentration, the endogenous level of dATP was low, resulting in a favorable ratio
of analog triphosphate to normal dNTP for incorporation into DNA. In association with the accumulation of
triphosphate there was a decrease in DNA synthesis. At 40 and 55 mg/m2 doses, the inhibition of DNA
synthesis was maintained to 24 hr (Ref. 2). Consistent with this observation, at the MTD, after 5 days of
therapy, there was a consistent cytoreduction in the WBC count in the peripheral blood of all these patients
(n = 12) with a median decline of 97% (range 68 to 99%). Among 32 patients with acute leukemias in this
phase I study, 2 achieved complete remission and 3 had a marrow CR without platelet recovery. This
encouraging outcome resulted in a phase II study exclusively for patients with acute leukemias.
Sixty-two patients with acute leukemias (including myelogenous leukemia N=31, myelodysplastic
syndrome, MDS; N=8, chronic myeloid leukemia in blastic phase; N=11 and acute lymphocytic leukemia;
N=12) were treated with clofarabine 40 mg/m2 IV over 1 hour daily x 5, every 3 to 6 weeks. Overall, 30
patients achieved complete or partial remission with an overall response rate of 48% (Ref. 3). At the MTD,
clofarabine plasma level was 1.5 µM, which resulted in a median 15 µM triphosphate concentration in the
cells. This intracellular level of clofarabine triphosphate was greater in the circulating leukemia blasts of
responders (median, 18 µM) compared to non-responders (median, 10 µM; p=0.03). Furthermore, this value
increased only in responders (median, 1.8-fold; p=0.008) after the second clofarabine infusion. We conclude
that clofarabine is active in acute leukemias and MDS, and has an acceptable toxicity. The cellular
pharmacokinetic profile indicates a potential for prognostic significance.
References.
1. Kantarjian, H. Gandhi, V., Kozuch, P., et al. Phase I clinical and pharmacology study of clofarabine
(chloro-2’-fluoro-deoxy-9-beta-D-arabinofuranosyladenine) in patients with solid and hematologic
cancers. J Clin. Oncol, 21:1167-1173, 2003.
26
2. Gandhi V, Kantarjian HM, Faderl S, et al. Cellular pharmacokinetics and pharmacodynamics of
clofarabine (2-chloro-2’-fluoro-arabinosyladenine) in acute leukemia blasts during a phase I clinical trial.
Proc. AACR Abstract # LB124. Presented at the 93rd Annual Meeting of the AACR. April 6-10, 2002.
San Francisco.
3. Kantarjian, H., Gandhi, V., Cortes J., et al. Phase II clinical and pharmacology study of clofarabine in
patients with refractory or relapsed acute leukemias. Blood, in press, 2003.
27
I04
Immucillins as Antibiotics for T-Cell Proliferation and Malaria
V.L. Schramm
Albert Einstein College of Medicine, BRONX, NY, United States of America
Human genetic deficiency of purine nucleoside phosphorylase (PNP) causes a specific T-cell
immunodeficiency. Deoxyguanosine (dGuo) accumulates in blood and is converted to toxic levels of dGTP in
T-cells. Control of undesirable T-cell proliferation might be achieved by inhibitors of PNP sufficient to cause
whole-body inhibition. Kinetic isotope effects were used to solve the transition state structure of bovine PNP.
Chemically stable analogues were designed to resemble the transition state and were synthesized. The
Immucills are the most powerful inhibitors known for PNP. Immucillin-H (ImmH) induces apoptosis in
rapidly dividing human T-cells in the presence of dGuo. ImmH is orally available in mice and is effective in
xenogeneic graft-vs-host disease. ImmH has entered human phase I/II clinical trails against T-cell leukemia,
and causes elevated dGuo in humans.
Plasmodium falciparum is a purine auxotroph and known pathways of human and malarial purine salvage all
involve PNP. P. falciparum PNP is inhibited by ImmH and parasites cultured in human RBC without added
hypoxanthine are killed by ImmH. Added hypoxanthine but not nucleosides rescue cultured P. falciparum
from ImmH toxicity.
28
I05
ALIMTA (Pemetrexed) from lab bench to clinic
B. Nguyen
Eli Lilly and Company, INDIANAPOLIS, IN, United States of America
Alimta, which inhibits thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide
formyltransferase, has shown broad activity in a variety of tumors including thoracic, colorectal, pancreas
and breast cancers. The development of antifolates has been challenging because of severe hematological
and gastrointestinal toxicities. Analyses have shown that patients with folate deficiency, as indicated by
homocysteine and methylmalonic acid levels, were at greater risk for Alimta toxicity. Such observations led
to the programmatic intervention in late 1999 for the overall development of Alimta in which all patients
received folic acid and vitamin B12 supplementation.
In a phase III study of Alimta plus cisplatin versus single-agent cisplatin in patients with malignant pleural
mesothelioma, median survivals were 12.1 months versus 9.3 months, respectively (log-rank p=0.020).
Median time-to-progressive disease was significantly longer in the Alimta/cisplatin arm as compared to the
single-agent cisplatin arm (5.7 months vs. 3.9 months, p=0.001), and tumor response rates were 41.3%
versus 16.7%, respectively. The addition of folic acid and vitamin B12 resulted in a significant reduction in
toxicities in the Alimta/cisplatin arm with no detrimental effect on the efficacy of the combination regimen.
A summary of the results from a phase III study of single-agent Alimta versus Taxotere in patients who had
previously failed chemotherapy will also be presented at the meeting. Overall, Alimta showed a significantly
better benefit-risk profile than Taxotere.
29
I06a
New (1-DEAZA)Purine Derivatives via Efficient C-2 Nitration of the (1-DEAZA)Purine Ring
G.J. Koomen
University of Amsterdam, AMSTERDAM, The Netherlands
A couple of years ago, a new selective nitration reaction of substituted purine and deazapurines at the 2position was developed in our lab.1) The resulting nitro groups could be easily exchanged via aromatic
nucleophilic substitution reactions. The nitration reaction, which could also be carried out with purine
nucleosides on solid support 2) turned out to be a combination of an electrophilic and a radical process, as
could be established by 1H, 13C and 15N NMR spectra. Recently, the proposed mechanism was unequivocally
established by observing CIDNP effects during the reaction at low temperature. Reduction of the nitro group
produced the nitroso derivative, which could also be used for a variety of structural modifications, partly via
hetero Diels-Alder reactions.3)
The synthetic approach developed for temozolomide 4) could be used for the development of substituted
benzoyl triazenes with different t1/2 values as precursors for the in vivo production of both diazomethane (as
by temozolomide) and 6-benzylguanosine (O6-BG) as an O6 alkyl-guanine-DNA --transferase (AGT)
inhibitor.
Biological data of the different analogues on Adenosine receptors5), on Plasmodium Falciparum and on
Cancer cell lines will be presented.
NHR1
HN
N
N
H
N
H
N
N
O
N
N
N
NHR1
N
X
N
X
N
N
O2N
X = CH, N
ribose
ribose
R2HN
N
N
ribose
X=N
NHR1
Cl
N
N
O
N
N
N
N
N
O2N
N
N
N
X
NHR1
N
N
ribose
ribose
N
ribose
O
X=N
OCH2Ph
N
N
O
N
N
N
N
N
H
NHR1
NHR1
N
N
N
N
N
N
ribose
CH3
HO
N
N
N
H
N
N
ribose
Y
1.
2.
3.
4.
5.
P.Y.F. Deghati, M.J. Wanner and G.J. Koomen, Tetrahedron Letters, 1291-1295 (2000)
B. Rodenko, M. J. Wanner and G.J. Koomen, J. Chem. Soc. Perkin I, 1245-1252 (2002)
M.J. Wanner and G.J. Koomen, J. Chem. Soc. Perkin I, 1908-1915 (2001)
M.J. Wanner and G.J. Koomen, J. Chem. Soc. Perkin I, 1877-1880 (2002)
M. W. Beukers, M.J. Wanner, J.K. Von Frijtag Drabbe Künzel, E.C. Klaasse, A.P IJzerman and G.J.
Koomen, J. Med. Chem. (2003)
30
I06b
LC-MS techniques in clinical applications
M. Svoboda
Applied Biosystems, DARMSTADT, Germany
LC/MS techniques using combinations of quadrupole(s) and/or time-of-flight analyzers emerged as an
extremely powerful tool for analytical challanges in the clinical arena in the last years. In combination with
soft ionization techniques like Ionspray, Atmospheric Pressure Chemical Ionization and – most recently –
PhotoSpray LC/MS is becoming an indispensable tool for applications like neonatal screening, metabolomics
and drug monitoring. An overview of the current LC/MS instrumentation as well as examples of these
applications will be presented in this talk.
31
I07
Quantitative RT-PCR using the LightCycler System for specific detection of TP (Thymidine
Phosphorylase), DPD (Dihydropyrimidine Dehydrogenase) and TS (Thymidylate Synthase) from fresh
and formalin-fixed, paraffin-embedded tissue
V. Lutz, M. Poignée, T. Ullrich, G. Maass, Roche Diagnostics GmbH, PENZBERG, Germany
The enzymes thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and thymidylate
synthase (TS) metabolize pyrimidines in cells under both normal as well as pathological conditions. In
particular, TP, DPD and TS play a major role in the metabolism of fluorouracil-based chemotherapeutics,
which are widely used as anticancer therapy e.g. for colorectal cancer (CRC) and breast cancer (BC).
Based on preclinical data, the variant levels of TP, DPD and TS in tumor tissue have an impact on the
individual drug response to Xeloda, an oral fluoropyrimidine carbamate with antineoplastic activity, or other
fluoropyrimidines, respectively, but due to various study protocols and methods examining either RNA,
DNA, protein or enzyme activity, the role of TP, DPD and TS in cancer is still unclear.
One important aspect in understanding the molecular basis of carcinogenesis is the quantitation of differential
gene expression of normal and neoplastic cells. However, the availability of fresh pathological tissue is limited
since optimal collection conditions are not easily realizable in clinical routine. In contrast, most pathological
specimens are routinely formalin fixed and paraffin-embedded prior to histological evaluation. We have
therefore developed a reliable method for RNA isolation from paraffin-embedded samples, and a standardized
and quantitative method to assess TP, DPD and TS mRNA expression levels in microdissected, formalin-fixed,
paraffin-embedded tissues using the LightCycler System.
The feasibility of the method has been demonstrated by determining the expression levels and ratios for TP,
DPD, and TS relative to the housekeeping gene G6PDH in several tumor tissues.
Therefore, the development of a reliable method for mRNA isolation from paraffin-embedded samples with
subsequent RT-PCR analysis is a major breakthrough for the acceptance and usefulness of RT-PCR
technology for the analysis of biomarkers in research based and clinical studies.
The aim of this project is to provide in the future specific tests for molecularly targeted cancer drugs,
enabling the more precise prediction of individual clinical outcomes (drug efficacy and safety) and thus more
efficient cancer therapies.
32
I08
New Levels in Gene Expression Profiling
R. Wicki
Applied Biosystems, ROTKREUZ, Switzerland
Gene Expression analysis is a complex mixture of technologies and knowledge that aim at understanding the
mechanisms driving cell life. Improvement of technologies for quantitative measuring of nucleic acid, such
as Real Time PCR, and integration of experimental results with the knowledge about genes, proteins and
functions is the way we are following to take Gene Expression to a new level.
A collection of ready to use Gene Expression Assays-on-Demand(TM) for more than 18.000 human genes
and 9000 mouse genes is available on-line. These assays eliminate the major bottlenecks in using Real time
PCR for Gene Expression studies involving many genes. Features and search possibilities for those assays
will be demonstrated. We will also present our Assays-by-Design(SM) Service.
The new Micro Fluidic Card accessory allows for highly parallel Gene Expression analysis in a low density
array format. From 12 and up to 384 genes can be analysed in parallel on a single run, with only two
pipetting steps per sample. With a total reaction volume of 2ul, more information can be gathered from very
small amounts of samples.
The Celera Discovery System (CDS) is a research platform that provides scientists with simplified online
access to the most comprehensive, up-to-date set of integrated biological data available, incorporated into
one annotated assembly, along with sophisticated visualization and analysis tools. Some of the many
powerful capabilities of CDS for human or mouse Gene Expression analysis will be mentioned.
33
I09
Pulmonary Consequenses of Adenosine Overload: Lessons from Adenosine Deaminase Deficient Mice
R. Blackburn
University of Houston Medical School, HOUSTON, TX, United States of America
Chronic lung diseases are associated with persistent lung inflammation and damage that lead to the progressive
loss of lung function. In contrast to most injury and repair responses, the inflammation seen in these disorders
is chronic and may last throughout the life of the afflicted individual. The mechanisms that are responsible for
the intensity and chronicity of these disorders have not been elucidated. Adenosine is a signaling nucleoside
that can elicit a variety of cellular functions by engaging cell surface adenosine receptors. Many of the actions
of adenosine are cytoprotective or anti-inflammatory in nature, however, studies have shown that adenosine
levels are elevated in the lungs of patients with chronic lung disease suggesting this signaling pathway might be
involved in the exacerbation of these disorders. Support for this hypothesis comes from recent studies in mice
deficient in the enzyme adenosine deaminase (ADA) that controls the levels of adenosine in tissues and cells.
Adenosine levels accumulate in the lungs of these mice and are associated with increased lung inflammation
and histopathologies seen in asthma and chronic obstructive pulmonary disease. Furthermore, lowering
adenosine levels or antagonizing adenosine receptors can reverse many of the lung phenotypes seen. In
addition, pharmacologic and genetic experiments suggest that adenoisne receptor signaling on various cell
types in the inflamed lung play an important role in phenotypes seen in the lungs of ADA deficient mice.
These findings suggest that chronic elevations in adenosine can access signaling pathways that can mediate
aspects of chronic lung disease.
34
I10
Species and age-dependent differences in purine metabolism, adenosinergic signalling, and ischemic
tolerance
J.P. Headrick
Griffith University, SOUTHPORT Q, Australia
Adenine nucleotide and adenosine metabolism are crucial in determining ischaemic tolerance, through
modulating energy state, radical generation, and receptor -mediated cardioprotective actions. Investigating
myocardial 5'-AMP metabolism, adenosine salvage and adenosine responses in hearts from mice, rats, and
guinea pigs, we document increased net myocardial 5'-AMP dephosphorylation as mass-specific metabolic
rate rises or body mass declines (normoxic rates of ~4, 9, and 11 nmol/min/g in guinea pig, rat and mouse,
respectively). Nonetheless, coronary venous adenosine levels are maintained at similar levels in all species
(50-70 nM). Preserved extracellular adenosine levels are accomplished in part by increased salvage vs.
deamination in mouse vs. larger species. Cardiovascular sensitivity to adenosine also differed between
species, with myocardial A1 receptor sensitivity greatest in mouse and coronary A2 sensitivity greatest in
guinea pig. Interestingly, although post-ischaemic loss of adenosine and its catabolites is relatively low in the
mouse vs. other species, myocardial tolerance to ischaemia is poor. Inhibition of either adenosine
deamination (20 µM EHNA) or phosphorylation (5 µM iodotubercidin) enhances ischaemic tolerance in both
mouse and rat. These effects are receptor-dependent. However, simultaneous blockade of both pathways
substantially limits post-ischaemic recovery in both species, revealing a key role for paths of adenosine
salvage in ischaemic tolerance. Blockade of adenosine kinase also reveals an important (receptorindependent) role for phosphorylation in the anti-ischaemic effects of exogenously applied adenosine.
Unfortunately, whilst these metabolic and adenosinergic manipulations prove beneficial in young tissue,
aged myocardium most likely to suffer ischaemic events is insensitive to adenosine-based strategies. Recent
studies in mouse reveal the ischaemia and adenosine intolerant phenotype is manifest well prior to
senescence (being evident within the first 12 months of life), is unrelated to changes in A1 adenosine receptor
transcription or expression, and may involve changes in intracellular signaling and catabolism of nucleotides
and adenosine. Evidence is presented supporting a role for the decline in adenosine-mediated protection in
ischaemic intolerance in aged mammalian myocardium.
35
I11
Allopurinol: Will it reduce cardiovascular events in patients?
A. Struthers, DUNDEE, United Kingdom
36
I12
Medical Diagnosis, Management and Treatment of Lesch Nyhan Disease
G.T. McCarthy
Chailey Heritage Clinical Services, NR LEWES, EAST SUSSEX, United Kingdom
1. Introduction and aims of presentation.
The aim of this presentation is to inform about LND from the point of view of the affected boys and their
families living with the condition from day to day AND to show the importance of research in treating and
managing the disease.
2. What is LND?
Outline enzyme defect, incidence, genetic implications, and importance of family history. History of
investigation into LND.
The genetic defect in LND lies in the deficiency (at <2% of normal) of an enzyme of purine metabolism
Hypoxanthine - Guanine Phosphoribosyl Transferase - abbreviated as HPRT.
Not all boys with HPRT deficiency have LND, there are 3 intermediate forms called LND variants, all of
which have varying but milder neurological symptoms. The fifth is called PARTIAL HPRT deficiency or
Kelley-Seegmiller syndrome (KSS) where no neurological symptoms are found. However, uric acid
overproduction occurs in all five categories.
3. Diagnosis
a) Clinical
pictureÆ in the baby -- neurological signs - developmental delay, hypotonia, irritability, sand in diaper.
Sometimes kidney failure precipitated by infection.
Æ in the older child -- 'athetoid CP' irritability, self injury - biting lips, fingers
b) Biochemical Investigations
Æ importance of metabolic investigations - uric acid in blood and urine. Vital to know that the kidneys of
children clear their uric acid better than adults but urine will show grossly elevated levels. Ratio of uric acid
and creatinine is raised 2-4 fold.
HPRT activity in both disrupted and intact blood cells and sometimes from fibroblasts cultured from skin
biopsy.
c) Radiological Æ ultrasound of kidneys may give the first clue to diagnosis of kidney failure in baby boys
with HPRT deficiency.
4. Early diagnosis is important Æ appropriate management starts from an early age. This benefits the baby
and parents and may prevent the birth of another affected child by promoting genetic counselling for family
members.
5. Management of LND
MOTOR DISORDER Æ evolution from hypotonia to dystonia, rigidity, hypertonia
AFFECTS
Æ trunk, limbs, oral muscles swallowing and speech. Torsion dystonia causes hiatus
hernia and feeding problems - vomiting, pain, bleeding, failure to thrive.
Practical management of posture in lying sitting and standing.
Daily living activities of feeding hand function, dressing and toiletting, play and educational activities.
Speech and communication.
6. SELF INJURIOUS BEHAVIOUR. SIB
Æ description of behaviour, how it differs from SIB in children with severe/profound intellectual disabilities
and management of SIB.
7. BEHAVIOUR & COGNITIVE PROBLEMS an outline of investigation of cognitive ability in boys and
men with HPRT deficiency. Other behavioural problems.
8. Drug treatment in LND
i. Allopurinol. ii. Muscle relaxants Baclofen, Diazepam etc. iii. Anxyolytics Carbamazepine, Valproate,
Gabapentin. iv. L dopa and others.
9. Growth and puberty.
10. Life expectancy.
The Future.
37
I13
Lesch-Nyhan Disease
A.M. Marinaki1, L.D. Fairbanks1, D. Perrett2, H.A. Simmonds1
1
Guy's Hospital, LONDON, United Kingdom
2
St. Bartholomew's Hospital, LONDON, United Kingdom
The relationship between hypoxanthine-guanine phosphoribosyltransferase (HPRT) and the severe neurological
manifestations of Lesch-Nyhan disease (LND) remains an enigma nearly 40 years on since the first case was
reported by Lesch and Nyhan in 1964. Whilst complete deficiency HPRT deficiency is axiomatic for LND the
fact that LND can present neonatally in acute renal to paediatric rather than neurological units can mask
diagnosis initially. At the other end of the spectrum are LND variants presenting in their teens or early
adulthood only with gout or kidney stones (‘partial’ HPRT deficiency, or Kelley-Seegmiller syndrome
described in 1967). Treatment is also problematical. Allopurinol reduces the grossly elevated uric acid in blood
and urine in consequence of lack of feed-back inhibition of purine synthesis in all types of HPRT deficiency
(hence the tendency to gout), but replaces uric acid with the even more nephrotoxic xanthine if the dose is too
high
The dystonia, choreathetosis, spasticity and compulsive self-injurious behaviour chractersitic of LND have
been related to a deficit of dopaminergic neurones in the striatum and will be discussed by Dr McCarthy. GTP
depletion in erythrocytes has been demonstrated which led to the proposals that restriction in supply for GTP
cyclohydrolase in brain could mimic the symptoms in hereditary progressive dystonia. However, such a link
between altered nucleotide metabolism and the observed neuropathology remains speculative.
Recent studies have shown decreased purine and pyridine nucleotide pools in nucleated cells and have related
these findings to altered metabolism of poly-ADP-ribose affecting DNA repair and GTP-dependent
neurotransmitter metabolism. Conversely accumulation of AICA riboside, an intermediate of de novo purine
synthesis, has been proposed to promote apoptosis in a neuroblastoma cell line model.
Analysis of global gene expression profiles of fibroblasts derived from LND patients may provide clues to
other mechanisms
38
I14
Mutations of the HPRT Gene in Lesch-Nyhan Disease and its Variants
H.A. Jinnah1, L. DeGregorio1, J.C. Harris1, W.L. Nyhan2, J.P. O'Neill3
1
Johns Hopkins Hospital, BALTIMORE, MD, United States of America
2
UCSD School of Medicine, LA JOLLA, CA, United States of America
3
University of Vermont, BURLINGTON, VT, United States of America
Introduction. Inherited mutations in the gene encoding hypoxanthine-guanine phosphoribosyl transferase
(HPRT) result in Lesch-Nyhan disease (LND), which is characterized by hyperuricemia combined with
devastating neurological and behavioral abnormalities. Mutations in the HPRT gene may also be associated
with less severe Lesch-Nyhan variants (LNV) that include hyperuricemia with or without varying degrees of
neurological or behavioral dysfunction. The purpose of the current studies was to compare the mutations in
these two populations.
Methods. New and previously reported mutations causing HPRT deficiency were compiled into a database
and grouped into two categories. The first category included those resulting in LND and the second category
included those resulting in LNV. The less severe phenotypes could not be further differentiated because of
inadequate clinical information in many prior reports. Genotype-phenotype correlations were made based on
the locations and types of mutations.
Results.
Mutation
LND (n=206)
LNV (n=64)
NA (n=8)
Total (n=278)
Single base substitution
Missense
Nonsense
Splice error
64
23
28
48
1
7
4
1
0
115
25
31
Deletion
Coding sequences
Splice error
58
4
2
0
3
0
61
4
Insertion
Coding sequences
Splice error
18
1
1
0
0
0
19
1
Others
Duplication
Substitutions
Females
Double
3
2
5
0
3
0
0
2
0
0
0
0
6
2
5
2
Conclusions. There was no apparent correlation for clinical phenotype and mutation location. However,
there was a rough correlation for clinical phenotype and the type of mutation. Specifically, those mutations
predicted to result in severe disruption of enzyme function were more often associated with LND rather than
LNV. This correlation becomes even more striking after excluding unstable and potentially revertible
mutations (splice site and duplication mutations). A detailed examination of the exceptions to this correlation
revealed in most cases unusual molecular mechanisms allowing residual enzyme activity. These results are
consistent with prior enzyme studies showing a correlation between residual activity and disease severity.
Analysis of HPRT mutations may therefore provide some predictive value for early-detected cases.
39
I15
New inborn errors; role of mitochondria; Deoxyribonucleoside kinases in mitochondrial DNA depletion.
A. Saada-Reisch
Shaare Zedek Medical Center, JERUSALEM, Israel
Mitochondrial encephalomyopathies include a heterogeneous group of diseases expressing dysfunction of the
mitochondrial respiratory chain (RC).They are caused by mutations in either the mitochondrial (mtDNA) or
the nuclear genome with maternal or Mendelian inheritance respectively. Many point mutations and single
large-scale deletions in the mitochondrial genome, and several point mutations in the nuclear genome,
involving specific RC complexes have been characterized. Lately attention has focused on a group of
Mendelian disorders represented by impaired intergenomic communication resulting in qualitative (multiple
deletions) and/or quantitative (depletion) defects in mtDNA. Mutations in the genes of mitochondrial adenine
nucleotide translocator, polymerase gamma and helicase have been associated with progressive ophtalmoplegia
with multiple mtDNA deletions. The discovery of mutant thymidine phosphorylase, in mitochondrial
neurogastrointestinal encephalomyopathy associated with multiple deletions and mtDNA depletion, indicated
that imbalances in the mitochondrial deoxynucleotidetriphosphate (dNTP) pools might affect mtDNA integrity.
This was further emphasized by the recent finding, of two enzymes involved the mitochondrial
deoxyribonucleotide salvage pathway, which are mutated in tissue specific forms of mtDNA depletion;
deoxyguanosine kinase (dGK) ,in the hepatoencephalopatic form and thymidine kinase 2 (TK2) in myopathic
form. The pathogenicity of these mutations was confirmed by western blot for dGK and by enzymatic assays
and cloning for TK2.
These findings, together the observation that nucleoside antiviral therapy cause secondary mtDNA depletion,
emphasize the importance of mitochondrial nucleotide metabolism and salvage pathway in the maintenance
and integrity of mtDNA. Many questions remain open including understanding of the mechanism trough with
the defective genes perturb mtDNA replication, why some tissues are spared while others are severely affected,
searching for additional genes in unexplained cases of mtDNA depletion and exploring the possibility of
intervention of mitochondrial nucleotide metabolism as possible treatment.
40
I16
Thymidine Phosphorylase Deficiency Causes MNGIE: An Autosomal Recessive Mitochondrial Disorder
M.H. Hirano, Y. Nishigaki, R. Marti
Columbia University, NEW YORK, NY, United States of America
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused
by mutations in the gene encoding thymidine phosphorylase (TPase). The disease is characterized clinically by
onset between the second to fifth decades, ptosis, progressive external ophthalmoparesis, gastrointestinal
dysmotility, cachexia, peripheral neuropathy, myopathy and leukoencephalopathy. Clinical diagnostic tests
typically reveal leukoencephalopathy, and lactic acidosis whileskeletal muscle biopsies show neurogenic
changes, together with mitochondrial abnormalities such as ragged-red-fibers, ultrastructurally abnormal
mitochondria and decreased cytochrome c oxidase (COX) activity, either in isolation or in association with
multiple respiratory chain enzyme defects. In addition to these signs of mitochondrial impairment, studies of
mtDNA from skeletal muscle have shown depletion and multiple deletions.
In MNGIE patients, TPase activity in buffy coat samples are drastically reduced (2±5 nmol/mg-protein/hour
[mean±standard deviation], n=27) compared to controls (667±205[n=19]). TPase is a cytosolic enzyme
required to maintain nucleoside homeostasis, therefore, loss of TPase function cannot account directly for the
mitochondrial pathology. In addition, skeletal muscle does not express TPase and paradoxically shows
mitochondrial alterations providing further evidence that loss of TPase does not directly alter mtDNA.
As a consequence of loss of TPase function in MNGIE patients, mean plasma levels of thymidine (8.6µM ±3.4
[mean±standard deviation, n=25]) and deoxyuridine (14.2µM ±4.4, n=25) are elevated more than 100-fold
above control values (thymidine <0.05µM, n=23 and deoxyuridine <0.05µM, n=20). We have hypothesized
that loss of TP function leads to increased levels of circulating and intracellular thymidine and deoxyuridine
which cause imbalances of mitochondrial nucleotide pools that, in turn, lead to the mtDNA abnormalities.
In cultured fibroblasts and non-skeletal muscle tissues of MNGIE patients, we identified respiratory chain
defects without depletion or multiple deletions of mtDNA. Therefore, we sequenced mtDNA and demonstrated
the presence of site-specific somatic mtDNA point mutations in tissues and cultured cells from MNGIE
patients.
MNGIE was the first human disorder of mitochondrial nucleoside/nucleotide metabolism to be molecularly
characterized.
41
I17
Nucleoside transporters
C.E. Cass
Cross Cancer Institute, EDMONTON, Canada
42
I18
Role of Multidrug-resistance associated proteins in resistance to nucleoside analogs
P. Borst
Netherlands Cancer Institute, AMSTERDAM, the Netherlands
43
I19
Cloning and expression of malarial pyrimidine enzymes
R.I. Christopherson1, R.I. Menz2, O. Cinquin1, M. Shojaei1
1
University of Sydney, SYDNEY, Australia
2
Flinders University, ADELAIDE, Australia
We have cloned genes encoding three enzymes of the de novo pyrimidine pathway using genomic DNA from
Plasmodium falciparum and sequence information from the Malarial Genome Project. Dihydroorotase
(reaction 3), orotate phosphoribosyltransferase (reaction 5), and OMP decarboxylase (reaction 6) are targets for
development of antimalarial drugs which would be active against drug-resistant parasites. These genes have
been cloned into the plasmid pET 3a or 3d with a thrombin cleavable 9xHis tag at the C-terminus and the
enzymes were expressed in Escherichia coli. We found that the recombinant malarial OMP decarboxylase was
toxic to E. coli and expressed at low levels prior to induction with IPTG. To overcome this problem and the
unusual codon usage of the malarial gene, a hybrid plasmid, pMICO, was constructed which expresses low
levels of T7 lysozyme to inhibit T7 RNA polymerase used in the expression vector, and extra copies of rare
tRNAs. Transformation of E. coli BL21 (DE3) with pMICO and pET-ODC resulted in high level expression of
OMP decarboxylase when induced with IPTG (250 µM). Catalytically-active OMP decarboxylase has been
purified in tens of milligrams by chromatography on Ni-NTA. The enzyme has a turn-over number of 5/s and a
Mr of 38,158 by mass spectroscopy, consistent with removal of the start methionine. The gene encoding
orotate phosphoribosyltransferase includes an extension of 66 amino acids from the N-terminus when
compared with sequences for this enzyme from other organisms. Using our knowledge of the catalytic
mechanism of dihydroorotase, we have designed and synthesized several potent inhibitors, TDHO and HDDP,
which have Ki values of less than 1 µM for interaction with the target enzyme, and IC50 values of less than 10
µM for inhibition of the growth of P. falciparum in erythrocytic culture. The antimalarial drug, atovaquone,
blocks electron transport in the parasite and indirectly inhibits reaction 4 of the pyrimidine pathway catalyzed
by dihydroorotate dehydrogenase. We have shown that atovaquone induces accumulation of pyrimidine
precursors before this step, and there is a major depletion of dTTP but not dCTP, precursors of DNA.
44
I20
Diversity and origins of deoxyribonucleoside kinases
J. Piskur1, M.P.B. Sandrini1, A.K.L. Joergensen1, W. Knecht1, B. Munch-Petersen2
1
Technical University of Denmark, LYNGBY, Denmark
2
Roskilde University, ROSKILDE, Denmark
Deoxyribonucleoside triphosphates, precursors for DNA replication in the cell, are provided by the de novo and
salvage pathways. The key salvage enzymes are deoxyribonucleoside kinases, which phosphorylate
deoxyribonucleosides to the corresponding deoxyribonucleoside monophosphates. In mammals four enzymes,
TK1, TK2, dCK and dGK, with overlapping substrate specificites and different regulation of gene expression
and cellular localisation, can be found. In contrast, insects, like the fruit fly and mosquito, have only a single
TK2-like enzyme, dNK, with a broad substrate specificity. Several other organisms have recently been
examined in our laboratory for the presence of deoxyribonucleoside kinases, the corresponding genes subcloned and the recombinant enzymes analysed for their kinetic parameters, substrate specificity and structurefunction relationship. These findings provide a background to understand the evolutionary history of
deoxyribonucleoside kinases. Apparently, the modern eukaryote TK1 originates from an ancient TK1 enzyme,
which was likely to be present already in the progenitor of bacteria and eukaryotes. On the other hand, in the
eukaryote lineage at least two gene duplication rounds, followed by further specialisation, have taken place to
generate the modern mammalian enzymes, TK2, dCK and dGK, from the common progenitor, TK2/dCK/dGK
kinase. The recently characterised novel kinases are also a valuable tool for development of novel suicide genes
for gene therapy.
45
I21
Regulation of Nucleoside Uptake by Protein Kinase Inhibitors and Mitogenic Signaling
L. Graves, B.S. Mitchell, M. Huang
University of North Carolina, CHAPEL HILL, NC, United States of America
Our laboratory has been studying the regulation of pyrimidine synthesis by protein kinase signaling. The
uptake of pyrimidine nucleosides for salvage synthesis requires specific equilibrative or concentrative
nucleoside transporters. We recently observed that the uptake of pyrimidine nucleosides and nucleoside
analogs (ARA-C, CPEC) in human leukemic K562 cells was prevented by inhibitors of p38 MAPK, EGFR,
BCR-ABL, PKC and CDK's. Comparison of inactive analogs of inhibitors p38 MAPK and PKC suggested
that inhibition of transport occurred independently of kinase inhibition and through direct effects on the
equilibrative transporter ENT1. By contrast, exposure of myeloid 32D or mouse embryo fibroblast cells to
inhibitors of BCR-ABL or SRC suggested that the activity of these kinases regulated the expression and
activity of both the ENT1,2 nucleoside transporters. The ability of protein kinase inhibitors to prevent
nucleoside uptake and the current status of the regulation of pyrimidine nucleotide transporters by protein
kinases will be discussed.
46
I22
Transcriptional regulation of DPYD gene expressoin and aberrant methylation in the 5'flanking region
M. Nishiyama1, K. Ukon1, T. Noguchi1, K. Tanimoto1, K. Hiyama1, M. Fukushima2
1
Hiroshima University, HIROSHIMA, Japan
2
Taiho Pharmaceuticals, HANNO-SHI, Japan
Dihydropyrimidine dehydrogenase (DPD) is the initial key enzyme in 5-fluorouracil (5-FU) catabolism, and
over 80-90% of an administered dose of 5-FU is inactivated via the catabolic pathway. DPD demonstrates
considerable interindividual variations and the activity relates to 5-FU response. Much attention has been
focused on DPD as a predictive marker of individual 5-FU response, and recently a transcriptional
mechanism is considered to be very important to understand DPD phenotype, since DPD activity closely
correlates with the mRNA levels. Though, the detailed mechanisms remain unclear. We cloned a 3kb (2918~+78bp) 5’ flanking region of DPD gene (DPYD) from human placenta genome library, studied the
functions, and found that aberrant methylation of DPYD promotor region strongly influenced cellular
sensitivity to 5-FU through the regulation of DPYD expression. The full length of the cloned region contains
CpG islands as reported by Diasio et al (BBA, 2000), and showed strong promoter activities in MKN45,
HSC42, COLO320DM, 293T, and HepG2 cells. A series of mutants of the region deleted from 5’ site
suggested that several up- and down-regulation sites might exist in the -1154~-64 region in addition to the
previously reported 2 transcriptional regulatory elements just close to the transcription start site. Deletions
from -1154 to -674 and from -453 to -338 resulted in approximately 40~60% of increase of the activity,
whereas deletions from -674 to 453 and from -338 to -64 resulted in approximately 50% loss of the activity.
The promoter activities closely correlated with mRNA expression levels in the cell lines except HepG2.
HepG2 showed significantly high promoter activity, but the DPYD expression was almost undetectable,
suggesting the existence of genetic variation of the region. Nevertheless, no difference was observed in the
nucleotide sequence of the region in genome DNA. We found that methylation of the region played an
important role in the expression regulatory mechanisms. Treatment of HepG2 cells with 5’aza Cytidine for
15 days increased remarkably both the DPYD expression and their sensitivity to 5-FU. Transcriptional
mechanisms controls DPD phenotype at least in part, and aberrant methylation in DPD promotor region can
play an important role in the transcriptional regulation.
47
I23
Pharmacogenetic and clinical aspects of a dihydropyrimidine dehydrogenase deficiency
A.B.P. Van Kuilenburg1, R. Meinsma1, A.H. Van Gennip2
1
Academic Medical Center, AMSTERDAM, The Netherlands
2
Academic Hospital Maastricht, MAASTRICHT, The Netherlands
Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the
pyrimidine bases and it catalyzes the NADPH-dependent reduction of uracil and thymine to 5,6-dihydrouracil
and 5,6-dihydrothymine, respectively. DPD is also responsible for the breakdown of the widely used
antineoplastic agent 5-fluorouracil (5FU). The catabolic route plays a significant role as more than 80% of the
administered 5-fluorouracil is catabolized by DPD. In this light, a pharmacogenetic disorder has been described
concerning cancer patients with a complete or partial deficiency of DPD suffering from severe toxicity,
including death, following the administration of 5FU. Owing to the pharmacogenetic consequences associated
with a DPD deficiency, we have performed an extensive study of the molecular aspects of patients with a DPD
deficiency.
The importance of a DPD deficiency in the etiology of unexpected severe 5FU toxicity is demonstrated by the
high prevalence (61%) of the cases in which a decreased DPD activity can be detected in peripheral blood
mononuclear cells. Patients with a partial DPD deficiency appeared to have a 3.4 fold higher risk of developing
grade IV neutropenia compared to patients with a normal DPD activity. Furthermore, in patients with a low
DPD activity, the onset of toxicity occurred, on average, twice as fast compared to patients with a normal DPD
activity. In patients suffering from severe 5FU-associated toxicity, 12 mutations have been identified in DPYD
including one splice site mutation (IVS14+1G>A); two nonsense mutations (R21X, E386X); 4 missense
mutations (M166V, V335L, I560S and D949V) and 5 polymorphisms (C29R, R21Q, S534N, I543V, V732I).
The IVS14+1G>A mutation proved to be the most common one and could be detected in 28% of all patients
suffering from severe 5FU associated-toxicity. Considering the common use of 5FU in the treatment of cancer
patients, the severe 5FU-related toxicities in patients with a low activity of DPD and the high prevalence of the
IVS14+1G>A mutation, analysis of the DPD activity in PBM cells or screening for the IVS14+1G>A mutation
should be routinely carried out prior to the commencement of the 5FU treatment.
48
I24
The Clinical Impact of Thiopurine Methyltransferase Polymorphisms on Thiopurine Treatment
S. Coulthard
Northern Institute For Cancer Research, NEWCASTLE UPON TYNE, United Kingdom
Acute lymphoblastic leukaemia (ALL) is the most common malignancy of childhood. Although current
treatment results in long term survival in over 70% of cases there is evidence that as many as 50% could
have been cured using a less complex regimen with a lower incidence of long term side effects. In previous
studies it has been found that thiopurines given as part of continuing therapy are key agents in preventing
relapse. However optimal administration during continuing therapy is often not achieved. Variation in the
level of thiopurine methyltransferase (TPMT) activity appears to be a major molecular determinant of the
extent of thiopurine metabolism.
TPMT activity shows a trimodal distribution pattern. A lack of activity is found in approximately one in 300
Caucasians; approximately 11% have intermediate activity and the remaining 89% high activity. Congenital
loss of activity is associated with grossly elevated levels of active drug and profound myelosuppression on
exposure to thiopurine drugs. This loss of activity has been attributed to single nucleotide polymorphisms
(SNPs) within the TPMT gene. The frequency of SNPs is related to ethnicity, with the most common in
Caucasians being TPMT*3A which is characterized by a G to A transition at position 460 with a substitution
of alanine for tyrosine at amino acid 154 (A154Y) and a transition of A to G at nucleotide 719 resulting in a
change of tyrosine to cysteine at position 240 (Y240C). Polymorphisms have also been identified within the
5’ flanking promoter region of the TPMT gene due to a variable number of tandem repeats (VNTR*3-*8).
An overview of the polymorphisms identified to date, their implication on the metabolism of the thiopurine
drugs and therapeutic importance will be discussed.
49
O01
The efficacy and the toxicity of methotrexate in rheumatoid arthritis patients are asociated with
polymorphisms in the methylenetetrahydrofolate reductase gene
W. Urano, A. Taniguchi, H. Yamanaka, E. Tanaka, H. Nakajima, Y. Matsuda, H. Akama, Y. Kitamura, N.
Kamatani
Tokyo Women's Medical University, TOKYO, Japan
A key enzyme involved in folate metabolism, 5,10-Methylenetetrahydrofolate reductase (MTHFR), has two
common polymorphisms that affect enzyme activity. The objective of this study was to examine whether there
was a correlation between the genotype or haplotype of the MTHFR gene and the efficacy or toxicity of
methotrexate (MTX) in the treatment of rheumatoid arthritis (RA).
106 patients with RA who have been treated with MTX were randomly selected and used for a retrospective
study to examine the correlation between genotypes or haplotypes concerning polymorphisms of the MTHFR
gene, and the efficacy or toxicity of MTX. Estimation of the haplotype frequencies was performed by
maximum likelihood estimation based on expectation maximization (EM) algorithm.
Single locus analysis examining each locus separately showed that a higher rate of overall MTX toxicity was
observed in patients with 677T than those without it (p<0.05, RR=1.25, 95%CI=1.05-1.49), while patients with
1298C were receiving significantly lower doses of MTX compared with patients without it (p<0.05, RR=2.18,
95%CI=1.17-4.06). An estimation of haplotype frequencies showed that there was no 677T-1298C haplotype
in the population. Posterior distribution of the diplotype configuration for each individual was concentrated on
a single configuration. Patients with 677T-1298A had a higher frequency of side effects from MTX (p<0.05,
RR=1.42, 95%CI=1.11-1.82), while subjects with the 677C-1298C haplotype were receiving lower doses of
MTX than those without it (p<0.05, RR=2.14, 95%CI=1.13-4.07).
Both single locus and haplotype analyses have suggested that polymorphisms within the MTHFR gene are
associated with both the efficacy and toxicity of MTX in RA patients.
50
O02
Febuxostat, a selective non-purine uric acid production inhibitor, is safe and decreases serum urate in
healthy volunteers
M.A. Becker1, J.C. Kisicki2, R. Khosravan3, J. Wu3, D. Mulford3, B. Hunt3, P. MacDonald3, N. Joseph-Ridge3
1
The University of Chicago Hospital, CHICAGO, IL, United States of America
2
MDS Harris, LINCOLN, NE, United States of America
3
TAP Pharmaceutical Products Inc., LAKE FOREST, IL, United States of America
Introduction: Febuxostat is a selective non-purine uric acid production inhibitor of the xanthine
oxidase/xanthine dehydrogenase (XOD) enzyme. Febuxostat is under evaluation for the treatment of
hyperuricemia associated with gout. In in vivo animal studies, febuxostat was more potent than allopurinol in
lowering serum urate and urine uric acid concentrations. A Phase I dose-escalation clinical trial was conducted
to assess the safety of febuxostat, and to determine pharmacokinetic and pharmacodynamic profiles of
febuxostat during oral administration of repeated daily doses over a range of doses and regimens (e.g., QD
and/or BID) to healthy subjects.
Methods: This was a double blind, placebo-controlled, dose escalation study conducted in 154 healthy subjects,
ages 19 to 54 years. In each dose group, 12 subjects were randomized in a 5:1 ratio to febuxostat or placebo.
Subjects received orally administered febuxostat, with doses ranging from 10 to 240 mg QD and 30 mg BID.
Results: Febuxostat effectively decreases serum urate concentration in a nearly dose-proportional manner up to
120 mg/day and with little further decrease noted between 120 and 240 mg/day. The reduction of serum urate
concentration was accompanied by corresponding increases in serum xanthine and hypoxanthine
concentrations, confirming that febuxostat lowers serum urate concentrations by means of inhibition of XOD
activity. Febuxostat was safe and well tolerated. The most frequently reported adverse events included
headache, nausea, dizziness, vasodilatation (flushing, feeling of warmth) and abdominal pain. No clear dosedependence was noted for the incidence of most adverse events. However, incidence of vasodilatation
(flushing, feeling of warmth) was higher at the 160, 180 and 240 mg QD doses (40-50%) as compared to the
lower dose groups (0-25%). The majority of adverse events were mild in severity and self-limiting. There were
no significant changes in laboratory values, electrocardiograms or physical examinations. There were nine early
terminations due to adverse events and no serious adverse events or deaths reported. No dose- limiting toxicity
was observed.
Conclusions: Treatment with febuxostat is safe, well tolerated, and effectively decreases serum urate
concentrations in a nearly dose-proportional manner with once a day dosing in healthy volunteers.
51
O04
Modification of cytokine milieu by signaling through A2A adenosine receptors in rheumatoid arthritis
M. Koshiba, Y. Nakamachi, H. Kosaka, T. Nakazawa, G. Tsuji, S. Kumagai
Kobe University, KOBE, Japan
Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF) are considered to be responsible with
various human diseases including rheumatoid arthritis and endotoxin shock. The anti-rheumatic effect of
methotrexate is reported to be mediated by the increase in the extracellular adenosine at the inflammatory
sites, but the detailed mechanisms of action of adenosine are yet to be clarified. We investigated the effects
of A2A adenosine receptor and beta-adrenergic receptor signaling on the cytokine production of LPSstimulated human CD14+ cells (monocytes/macrophages) derived from peripheral blood and rheumatoid
synovial fluids. TNF production of LPS-stimulated peripheral CD14+ cells was inhibited in a concentration
dependent manner by A2A receptor agonist. Co-administration of beta2-adrenergic agonist enhanced the
inhibition of the TNF production by A2A agonist. Similarly on LPS-stimulated CD14+ cells from rheumatoid
synovial fluids, observed was the concentration-dependent inhibition of TNF and IL-12 but not IL-1beta and
IL-6 production via A2A signaling. These results suggest that the anti-rheumatic effect of extracellular
adenosine is, at least in part, mediated via A2A receptor signaling by the modification and normalization of
Th1-shifted cytokine milieu in the rheumatoid arthritis.
52
O05
6-[2-(Phosphonomethoxy)alkoxy]pyrimidines: a new class of acyclic pyrimidine nucleoside phosphonates
with antiviral activity
J. Balzarini1, C. Pannecouque1, L. Naesens1, G. Andrei1, R. Snoeck1, E. De Clercq1, S. Aquaro2, C.-F. Perno2,
H. Egberink3, I. Votruba4, A. Holý4
1
Rega Institute for Medical Research, LEUVEN, Belgium
2
University of Rome 'Tor Vergata', ROME, Italy
3
Institute of Virology, UTRECHT, The Netherlands
4
Institute Org. Chem. Biochem., PRAGUE, Czech Republic
A new class of acyclic pyrimidine nucleoside phosphonate analogues have been designed, in which the base
consists of a pyrimidine containing an amino or hydroxy group at C-2 and C-4 and a 2(phosphonomethoxy)ethoxy (PMEO) or a 2-(phosphonomethoxy)propoxy (PMPO) group at C-6. The 6PMEO-2,4-diaminopyrimidine (PMEO-DAPY), the 6-PMEO-2-amino-4-hydroxypyrimidine (PMEA-AHPY)
and the 6-PMPO-2,4-diaminopyrimidine (PMPO-DAPY, R-enantiomer) were inhibitory against DNA viruses
[i.e. herpes simplex virus type 1 (HSV-1), HSV-2 and varicella-zoster virus (VZV)], but also against
retroviruses [i.e. human immunodeficiency virus type 1 (HIV-1), HIV-2, feline immunodeficiency virus (FIV)
and Moloney murine sarcoma virus (MSV)] in cell culture (50% effective concentration between 0.002 µg/ml
and 1.9 µg/ml, depending the virus strain and cell line investigated). PMEO-DAPY and PMPO-DAPY (Renantiomer) were also highly effective against MSV-induced tumor formation in newborn NMRI mice. In this
respect they were equally active as PMEA (adefovir) and (R)-PMPA (tenofovir), which are now approved for
treatment of hepatitis B virus and HIV infection, respectively. Substitution of a bromine at the 5-position of the
pyrimidine ring further increased the selectivity. The novel 6-PMEO and 6-PMPO pyrimidine derivatives
closely mimic the corresponding 2,6-diaminopurine nucleoside phosphonates in that the 2,4-diaminosubstituted pyrimidine ring may be considered as analogous to the pyrimidine ring in the acyclic 2,6diaminopurine nucleoside phosphonate derivatives. Hence, these novel PMEO and PMPO pyrimidine
derivatives retain the most important part of the purine base for recognition by the phosphorylating (activating)
cellular enzymes, with the viral reverse transcriptase being the most likely target enzyme for their antiretroviral
activity. The antiviral activity of PMEO-DAPY, unlike that of and PMPO-DAPY, unlike that of zidovudine
(AZT) and lamivudine (3TC), cannot be reversed by natural nucleosides or nucleobases. The PMPO
enantiospecificity (preference of (R) over (S) in the PMPO-series) has also been found for the PMPA series of
drugs, again pointing to a striking similarity in the activation pattern and mechanism of anti(retro)viral action
between the new pyrimidine derivatives and the established purine derivatives. The molecular bases of the
antiretroviral activity and intracellular metabolism of the PMEO and PMPO derivatives is under current
investigation.
53
O06
Antiproliferative activity and mechanism of action of fatty acid derivatives of gemcitabine in leukemia
and solid tumor cell lines and xenografts
A.M. Bergman1, P. Noordhuis1, E.M. Comijn1, J. Balzarini2, F. Myhren3, R. Tuen4, Ø. Fodstad4, H.R.
Hendriks5, M.L. Sandvold3, G.J. Peters1
1
2
Rega Institute for Medical Research, LEUVEN, Belgium
3
Clavis Pharma, PORSGRUNN, Norway
4
The Norwegian Radium Hospital, OSLO, Norway
5
EORTC-NDDO, AMSTERDAM, The Netherlands
Gemcitabine is a deoxycytidine (dCyd) analog, with antitumour activity in solid tumours and some hematologic
malignancies. Gemcitabine requires phosphorylation by deoxycytidine kinase (dCK) to allow formation of its
active phosphate dFdCTP. It can be deaminated by deoxycytidine deaminase (dCDA). In addition to dCK
deficiency altered membrane transport over the cell membrane is a mechanism of drug resistance for
gemcitabine. In order to facilitate gemcitabine uptake and prolong retention in the cell, lipophillic pro-drugs
were synthesized. An elaidic fatty acid group was esterified at the 5´ position on the sugar moiety of gemcitabine
(CP-4126), or acylated on the 4-amino group (CP-4125), which might reduce deamination. The compounds were
tested in four pairs of either ara-C (the murine leukemia L5 and L4A6 and the rat leukemia BCLO and Bara-C)
or gemcitabine (the human ovarian cancer A2780 and AG6000 and murine colon C26-A and C26-G) resistant
cell lines. L4A6, Bara-C and AG6000 have varying degrees of decreased dCK activity. In the parent cell lines
gemcitabine was more active, but in the various resistant variants the prodrugs were more active, while the
degree of cross-resistance varied. No difference in activity of the 4-amino acylated CP-4125 or the 5´ esterfied
CP-4126 was found. In the malignant melanoma THX cells the nucleoside transport inhibitors NBMPR and
dipyridamole increased the IC50 for gemcitabine 56 and 115-fold, respectively, while the IC50 for CP-4126 was
increased 1.6 and 1.3-fold, respectively. Similar results were found in the human T-cell leukemia MOLT4 cells
suggesting a nucleoside transport independent passage of CP-4126 over the cell membrane. In contrast to
dFdCTP formed after gemcitabine exposure, dFdCTP pools after CP-4125 exposure continued to increase after
drug removal in C26-A cells. CP-4125 induced inhibition of DNA synthesis effectively in C26-A and C26-G
cells. The retention of inhibition was longer for CP-4125 than for gemcitabine in C26A cells. In vivo evaluation
of a panel of 3 xenografts showed a similar activity for gemcitabine and CP4125 and CP4126. In conclusion,
based on in vitro and in vivo data, cytotoxic and metabolic effects of CP-4125 and CP-4126 are different and
longer lasting than for the parent drugs. Both CP-4125 and CP-4126 seem to be promising new prodrugs of
gemcitabine.
54
O07
Nucleotide analysis by liquid chromatography/mass spectrometry
R.T. Smolenski1, E.M. Slominska2, H. Dziewit2, E.A. Carrey3, H.A. Simmonds3, M.H. Yacoub1
1
Imperial College, London, United Kingdom
2
Medical University of Gdansk, Gdansk, Poland
3
Guy’s Hospital, London, United Kingdom
High Performance Liquid Chromatography with UV detection has its limitations, especially with
identification of unknown metabolites and quantitative analysis of chromatographically unresolved
peaks, even using in-line diode array detectors. Application of mass detection with HPLC has the
potential for not only positive identification and quantitation of chromatographically unresolved peaks,
but also for retrieval of structural information of unknown metabolites and isotopomer analysis.
However, before it can become a routine technique for nucleotide analysis, some difficulties have to
be resolved such as matching optimal chromatographic separation with optimal conditions for
ionization. We developed an anion exchange chromatographic system using an amino column
whereby high inorganic salt concentration in the mobile phase has been replaced by an ion-pairing
agent at moderately elevated pH to elute highly polar compounds such as triphosphates. This system
has been found to be superior to ion pairing due to lack of sample effect on separation and better
definition of compound class retained on the column, to the anion exchange system with alkaline
elution due to less in source fragmentation of triphosphates to diphosphates and to reversed-phase
separation due to better chromatographic separation and efficiency of ionization. With our procedure
we have been able to separate chromatographically and quantitate di- and triphosphates of adenosine,
guanosine, uridine and cytidine. Perchloric acid and trichloroacetic acid extracts of heart or other cells
have been run with separation quality identical to standards. Analysis of red blood cell extracts from
patients with renal failure in addition to the presence of known nucleotides allowed identification of
hitherto unidentified components such as 2-pyridone-5-carboxamide ribonucleoside triphosphate.
Analysis of heart extracts allowed identification and quantitation of diadenosine polyphosphates. In
conclusion, application of mass spectrometry in nucleotide analysis opens new opportunities for
studies of nucleotide metabolism. It may not only be limited to analysis of static concentration but also
for analysis of metabolic fluxes by isotopomer analysis, but practical evaluation of this possibility
requires further studies.
55
O08
Online fluorescent method to assess BCRP activity as a function of cellular folate homeostasis
J.H. Hooijberg1, G.J. Peters1, G.J.L. Kaspers1, N. De Vries1, I. Kathmann1, R. Pieters2, G. Jansen1
1
2
Sophia Childrens Hospital, ROTTERDAM, The Netherlands
The use of the antifolate methotrexate (MTX) plays an important role in the treatment of acute lymphoblastic
childhood leukemia (ALL), and several other diseases. Recently, the Breast Cancer Resistance Protein
(ABCG2/MXR) was reported to confer cellular resistance to MTX. This observation opens the possibility of a
role for BCRP in the homeostasis of natural folates as well. In theory, BCRP mediated transport of cellular
folates might competitively inhibit MTX efflux, and thus influence cellular sensitivity to MTX.
In this study we report effects of differences in cellular folate status on the transport activity of BCRP. We
measured the cellular efflux of the BCRP substrate Hoechst 33342 in the human myeloid cell line RPMI
8226(s) and its mitoxantrone resistant and BCRP overexpressing subline 8226/MR. A new method was
developed by which the cellular efflux of fluorescent compounds from suspension cells can be monitored
online. This method uses a two-compartment cuvette system, divided by a semi-permeable membrane, and can
be used with a relatively small amount of cells (~ 50.000). The influence of cellular folate status on BCRP
mediated efflux of Hoechst 33342 was determined by culturing cells under folate-rich (2.3 microM folic acid,
i.e. standard culture medium), or folate-restricted conditions (i.e. in folate free medium). When grown in
medium containing 2.3 microM folic acid, the efflux rate of Hoechst 33342 in 8226/MR cells was 2-fold higher
(~ 0.64 pmol/min/106 cells) than in wild type 8226(s) cells (~ 0.30 pmol/min/106 cells). When 8226/MR cells
were cultured for 48 hours in folate-free medium Hoechst 33342 efflux was decreased to a comparable level as
in 8226(s) cells (~ 0.28 pmol/min/106 cells). As determined by Western blotting the expression of BCRP in
both these cell lines did not alter upon changes in cellular folate status. In conclusion, cellular folate
homeostasis appears to influence BCRP mediated transport activity. These results are relevant to understand the
role of BCRP in drug resistance.
Supported by the Dutch Cancer Society, grant VU-2000-2237.
56
O09
Coordinate Regulation of the Expression of Ecto-5'-Nucleotidase (CD73) and the A2a Adenosine
Receptor in a Human B-Cell Line
W. Gutensohn, R. Napieralski, B. Kempkes
University of Munich, Munich, Germany
A human B-cell line (P493-6) was used as a model system to study the regulation of the expression ecto-5´nucleotidase (CD73) and one of the adenosine receptors. In P493-6 cells the activity of two different
mitogenic signals, the Epstein-Barr virus nuclear antigen 2 (EBNA2) and myc, can be independently
regulated. A stepwise shut off of EBNA2 and myc leads to a change from a more lymphoblastoid phenotype
to a resting state. Shut off of EBNA2 – either in the presence or absence of myc activity – causes a
significant increase in enzymatic activity and surface expression of CD73 as well as an increased adenosine
receptor response in cyclicAMP formation. The adenosine receptor in question is identified as the A2a
subtype by using specific agonists and inhibitors and by measuring mRNA levels. Shut off of myc has an
additional positive effect on CD73 expression but not on the adenosine receptor. Upon turn on of EBNA2
and myc both CD73 and the A2a receptor are repressed. Induction and repression of both components follow
a rather slow time course in the order of days. The signaling pathways involved in this regulation are
unknown sofar and a direct connection with regulatory elements described in the promotor region of the
human CD73 gene is not evident. A coordinated up-regulation of the ecto-enzyme and an A2 adenosine
receptor could reflect the establishment of a positive feed back loop for autocrine stimulation in the presence
of extracellular nucleotides.
57
O10
Prevention of adriamycin induced heart failure by increase in endogenous adenosine production
A.H. Yuen1, M. Boscoe1, R. Lango2, K. Suzuki1, E.M. Slominska2, M.H. Yacoub1, R.T. Smolenski1
1
Imperial College, LONDON, United Kingdom
2
Medical University of Gdansk, GDANSK, Poland
Adenosine (Ado) triggers several protective mechanisms that may attenuate development of heart failure. We
developed a procedure allowing sustained increase in endogenous Ado production by the combined application
of Ado metabolism inhibitors and nucleotide precursors. This study aimed to evaluate whether this intervention
will attenuate development of experimentally induced heart failure.
Blood adenosine and other purine metabolites concentration was evaluated following administration of
inhibitor/substrate solution containing Ado deaminase inhibitor - deoxycoformycin, Ado kinase inhibitor - 5'aminoadenosine and nucleotide precursors - adenine and ribose. After these preliminary studies, in two
experimental groups (AC, AT), heart failure was induced by repeated intraperitoneal injections of adriamycin
for two weeks while in two other groups saline was injected (CC, CT). Subsequently in the following four
weeks an inhibitors/substrate solution was administered in treated groups (AT, CT) in the form of daily
intraperitoneal injections while saline was given to controls (AC, CC). Cardiac function was monitored using
transthoracic echocardiography at weekly intervals and is expressed as left ventricular ejection fraction
(EF±S.E.M).
Administration of inhibitors/substrates induced prolonged elevation of blood adenosine concentration for up to
6 h with its peak, about 1 µM after 30 min following administration. Some other changes such as elevated
hypoxanthine or uric acid concentrations were also observed. There were no differences in cardiac function at
the beginning of the experiment between all four groups (EF=68.2±1.9, 71.4±1.1, 72.4±1.4 and 73.4±1.9% for
AC, AT, CC and CT respectively). At the end of the experiment, after four weeks of treatment with
inhibitors/substrates cardiac function was singnificantly improved in adriamycin and inhibitors/substrates group
(AT, EF=68.2±3.5%) as compared to adriamycin group (AC, EF=58.3±1.8, p=0.022). There were no
differences in cardiac function between CC and CT (EF=78.7±1.1 and 79.3±1.2% in CC and CT respectively).
Increase in endogenous Ado production by inhibition of Ado metabolism and nucleotide precursor supply
resulted in attenuation of development of heart failure induced by adriamycin. Regulation of adenosine
production is protective not only during ischemia/reperfusion injury or allograft rejection as we have previously
shown, but this may also be applicable in heart failure.
58
O11
Role of Platelet derived endothelial cell growth factor / thymidine phosphorylase in fluoropyrimidine
sensitivity and potential role of deoxyribose-1-phosphate
M. De Bruin1, T. Van Capel1, A.C. Laan1, K. Smid1, M. Fukushima2, K. Hoekman1, H.M. Pinedo1, G.J. Peters1
1
2
Platelet derived endothelial cell growth factor (PD-ECGF) / thymidine phosphorylase (TP) catalyses the
reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate and is involved the
metabolism of fluoropyrimidines. It can activate 5'-deoxyfluorouridine (5’DFUR) and possibly 5-fluorouracil
(5FU) but inactivates trifluorothymidine (TFT). We studied the contribution of TP activity to the sensitivity for
these fluoropyrimidines by modulating its activity and/or expression level in colon and lung cancer cells using a
specific inhibitor of TP (TPI) or via stable transfection of TP. Expression was analysed using competitive
template-RT-PCR (CT-RT-PCR), western blot and an activity assay. To determine the role of TP in the
sensitivity to 5FU, 5'DFUR and TFT, cells were cultured with the various fluoropyrimidines with or without
TPI and differences in IC50s were established. TPI modulated 5’DFUR, increasing the IC50s 2.5 to 1396-fold
in WiDR and Colo320TP1, respectively. 5FU could be modulated by inhibiting TP but to a lesser extent than
5'DFUR: IC50s increased 1.9 to 14.7-fold for WiDR and Colo320TP1, respectively. There was no effect on
TFT. There appears to be a threshold level of TP activity to influence the 5'DFUR and 5FU sensitivity, which is
higher for 5FU. In the metabolism of fluoropyrimidines the levels of dR-1-P might play an important role eiter
as inhibitor of the activation of 5’DFUR or as co-substrate for activation of 5FU. We therefore determined the
levels of dR-1-P, using a purine nucleoside phosphorylase based assay and suprisingly it was found that TdR
incubation did not increase dR-1-P accumulation in Colo320 and its PD-ECGF/TP transfected variant
Colo320TP1. There was a huge discrepancy between thymine production and the measured dR-1-P level,
0.05% of the expected value for dR-1-P was found, indicating that there was a rapid disappearance of dR-1-P.
However, in cellular extracts, TdR incubation increased dR-1-P, measurable by trapping, which was inhibited
by a TPI. dR-1-P directly added to cellular extracts, disappeared. In conclusion, large amounts of dR-1-P are
produced by Colo320TP1 cells, which rapidly disappear thus not resulting in a net accumulation of dR-1-P in
these cells. dR-1-P appears to be the limiting factor in 5FU activation by PD-ECGF/TP.
59
O12
Down-Regulation of Human Deoxyuridine Triphosphate Nucleotidohydrolase (dUTPase) using Small
Interfering RNA (siRNA)
M.V. Williams, A.W. Studebaker
The Ohio State University, COLUMBUS, OHIO, United States of America
Human dUTPase is a member of the homotrimeric family of dUTPases that include enzymes from most
eukaryotes, prokaryotes and RNA viruses. dUTPase is the enzyme responsible for hydrolyzing dUTP to
dUMP and pyrophosphate, which prevents dUTP from being incorporated into DNA and simultaneously
provides a precursor for dTMP synthesis. It has been suggested that dUTPases could be used as a target for
the development of specific agents useful for treatment of infections caused by certain viruses and also for
cancer chemotherapy, however, it has not been possible to develop agents that selectively inhibit dUTPase
activity in vivo. Furthermore, studies to determine the role(s) of dUTPase in cellular metabolism in human
cells have been hampered because cells deficient in dUTPase activity have not been isolated or constructed.
Structural and functional studies have demonstrated that homotrimeric and monomeric dUTPases contain
five conserved amino acid domains that contribute to the formation of the catalytic site. Since recent studies
have demonstrated that gene expression can be down-regulated using small interfering RNA (siRNA). To
determine whether such an approach could be used to decrease dUTPase activity, we synthesized a doublestranded siRNA targeted against domain 3 (nucleotides 339 to 357) of human dUTPase and used this siRNA
to determine its effect on dUTPase activity in SW620, HT29 and HeLa cells. Cells were transfected with 1 to
4 µg of siRNAdut3 and examined 48 hrs after transfection for residual dUTPase activity. There was a
significant decrease in dUTPase activity in cells transfected with siRNAdut3 when compared to nontransfected controls. dUTPase activity was decreased approximately 50 ± 0.9% in HeLa and HT29 cells and
approximately 27 ± 11% in SW620 cells. This difference probably reflects not only differences in rate of
transcription of dut gene, and stability of dUTPase specific mRNA in the cell lines but also differences in
transfection efficiencies of the cells. There was no effect on uracil-DNA glycosylase activities in transfected
cells when compared to non-transfected controls. These results demonstrate that a siRNA can be used to
decrease expression of dUTPase in human cells and that such an approach could be useful either alone or in
combination with established therapies for anti-viral and/or cancer chemotherapy.
60
O13
Cytotoxic and apoptotic effects of novel heterodinucleoside dimers consisting of 5-fluorodeoxyuridine
and ara-c in human cancer cell lines
Ph. Saiko1, W. Bauer1, Z. Horvath1, Th. Hoechtl1, M. Grusch1, G. Krupitza1, R. Mader1, H. Schott2, M. FritzerSzekeres1, T. Szekeres1
1
University of Vienna - General Hospital, VIENNA, Austria
2
University of Tuebingen, TUEBINGEN, Germany
5-fluorinated pyrimidines have become very useful in the treatment of human solid tumors, including breast
cancer, gastrointestinal adenocarcinomas and squamous cell carcinomas arising in the head and neck.
Moreover, they have invoked interest because of their synergistic interaction with other antitumor agents. One
of them, ara-C, has only been used in the treatment of leukemia so far, since the active form of the drug does
not reach solid tumors.
In search for possible alternative chemotherapeutic agents we tested a number of recently synthesized
heterodinucleoside phosphate dimers consisting of 5-FdU and ara-C, which had already exhibited cytotoxic
activity in leukemia and prostate cancer cell lines. These dimers enter the tumor cells due to their lipophilic
character which also enables the transport of ara-C to the tumor site. After cellular uptake, the 5´-phosphatederivatives of 5-FdUrd and ara-C can be released via enzymatic cleavage.
In our present study, we investigated the cytotoxic effects of several 5-FdU-ara-C-Dimers in HT29 colon
carcinoma cells as well as in ara-C sensitive and resistant H9 lymphoma cells. Moreover, we analyzed the
induction of apoptosis in HT29 cells.
Cytotoxic activity was determined by growth inhibition and clonogenic assays, while induction of apoptosis
was detected employing a cell staining method using hoechst/propidium iodide. We can show that all dimers
tested inhibited the number of colonies of HT29 human colon cancer cells with IC50 values ranging from 35 to
100nM. While examining the two different H9 cell lines, no significant difference in cytotoxicity could be
observed between ara-C sensitive and resistant cells, indicating that these compounds might be used in the
treatment of ara-C resistant tumors. Growth inhibition assays resulted in IC50 values ranging from 250 to
300nM. Furthermore, the investigated dimers induced dose and time dependent apoptosis in HT29 cells.
We therefore conclude that these novel heterodinucleoside dimers exhibit in vitro cytotoxic activity against
human colorectal cancer cells and human lymphoma cells. They are able to circumvent ara-C resistance in H9
cells and might therefore offer an additional option for the treatment of human tumors.
61
O14
Increased thymidylate synthase (TS) but unaltered dihydropyrimidine dehydrogenase (DPD) mRNA
levels after administration of 5-fluorouracil (5-FU) to patients with colon cancer
R. Mauritz, C.J. Van Groeningen, K. Smid, C.H. Smorenburg, H.M. Pinedo, G.J. Peters
Clinical resistance to 5-FU is associated with upregulation of its target enzyme, TS or the rate-limiting catabolic
enzyme DPD. In vitro studies have demonstrated that increased TS enzyme levels following exposure to 5-FU
result from a disrupted autoregulation of TS mRNA translation. This study explores the effect of 5-FU
exposure on TS mRNA levels in primary tumors and metastases of colon cancer patients. TS mRNA levels
were determined in biopsy specimens from patients who were either not exposed to 5-FU or given one presurgery bolus of 5-FU (500 mg/m2). Tumor biopsy specimens were obtained 1 to 48 hours after 5-FU
administration. TS mRNA expression was measured by competitive template RT-PCR using ß-actin as an
internal standard. In both patient groups a wide variation in intratumoral TS mRNA levels was observed. No
difference was observed between TS mRNA levels in colon tumors (n = 19; median TS/ß-actin 0.96 × 10-3,
range 0.11 – 46.4 × 10-3) and in liver metastases (n = 53; median TS/ß-actin 1.01 × 10-3, range 0.068 – 16.8 ×
10-3) of unexposed patients. However, TS mRNA levels were significantly higher both in colon tumors and in
liver metastases of patients that were pretreated with 5-FU compared to samples of patients not exposed to 5FU: median TS mRNA expression in colon tumors of exposed patients (n = 14; median TS/ß-actin 2.87 × 10-3,
range 1.03 – 69.8 × 10-3) was 3.0-fold higher than in unexposed patients (p = 0.01), while in liver metastases of
exposed patients (n = 16; median TS/ß-actin 5.38 × 10-3, range 0.67 – 38.5 × 10-3) TS mRNA levels were 5.3fold higher than in unexposed patients (p < 0.001). DPD mRNA levels did not differ significantly between
colon tumors and liver metastases. In addition, no effect of 5-FU exposure on DPD mRNA expression was
observed in both patient groups. In conclusion, 5-FU administration did not alter DPD mRNA levels but was
associated with increased TS mRNA expression both in primary colon tumors and in liver metastases.
(Supported by the Netherlands Organization for Scientific Research).
62
O15
Effects of 2-chloro-2'-deoxyadenosine on the cell cycle in human leukemia CCRF-CEM and EHEB cell
lines
S. Cardoen1, E. Van den Neste1, C. Smal1, J.-F. Rosier2, A. Ferrant3, G. Van den Berghe1, F. Bontemps1
1
ICP and Catholic University of Louvain, BRUSSELS, Belgium
2
C.H. Jolimont-Lobbes, LA LOUVIèRE, Belgium
3
Clinic St. Luc, BRUSSELS, Belgium
The human leukemia cell line EHEB, established from a patient with B-chronic lymphocytic leukemia, was
previously found to be less sensitive (10- to 1000-fold) to the nucleoside analogue 2-chloro-2’-deoxyadenosine
(CdA) than other human lymphoblastic cell lines. Moreover, DNA synthesis, measured by thymidine
incorporation into DNA, was unexpectedly increased in EHEB cells, up to 2-fold, after a 24 h-incubation with
10 µM CdA (Cardoen et al., Clin. Cancer Res. 7, 3559-66, 2001). Analysis by flow cytometry, using double
labelling with propidium iodide and bromodeoxyuridine, showed that CdA provoked an increase in the
proportion of cells in S phase, synthesizing actively DNA. These results contrast with those reported in other
leukemic cell lines sensitive to CdA, like CCRF-CEM cells, in which CdA inhibits DNA synthesis and
provokes an accumulation of most cells in either early S phase or at the G1-S border. Kinetics and
synchronisation experiments showed that CdA stimulates the progression of EHEB cells from G1 to S phase,
rather than blocking them in S phase. This led us to study the effect of CdA on proteins regulating the G1/S
checkpoint of the cell cycle, and firstly on the phosphorylation of the retinoblastoma (Rb) protein, which is
increased during the G1/S transition. We observed that CdA enhances the phosphorylation of Rb in EHEB
cells, whereas it decreases it in CCRF-CEM cells. The p53 status of this cell line was determined and found
unmutated. Additional preliminary experiments showed that CdA decreases p21 expression in EHEB cells. In
conclusion, we show a new mode of cellular response to CdA, implying modification of the cell cycle
regulation leading to enhanced DNA synthesis. We propose that this peculiar effect might be implied in some
types of yet unexplained resistance of leukemic cells to CdA.
63
O16
Increased cytotoxicity of 2’,2’-difluoro-2’-deoxycytidine in human leukemic cell-lines after a
preincubation with cyclopentenyl cytosine
A.C. Verschuur1, A.H. Van Gennip2, R. Leen3, A.B.P. Van Kuilenburg3
1
Emma Children's Hospital, AMSTERDAM, The Netherlands
2
3
Introduction
The cytotoxicity of 2’,2’-difluoro-2’-deoxycytidine (dFdC, gemcitabine) depends amongst others on the
phosphorylation into dFdCTP and on the incorporation of dFdCTP into DNA. dCTP may inhibit the
phosphorylation of dFdC by feedback inhibition of deoxycytidine kinase and may decrease the incorporation of
dFdCTP into DNA by competition for DNA polymerase. Cyclopentenyl cytosine (CPEC) inhibits CTP
synthetase inducing a depletion of CTP and dCTP. We hypothesized that in leukemic cell-lines the cytotoxicity
of dFdC could be enhanced by a preincubation with CPEC.
Materials and methods
MOLT-3 and HL-60 cells were preincubated for 24h with 6.25 and 12.5 nM of CPEC (kindly provided by the
NCI, U.S.A.). Subsequently, 0-50 nM [3H]dFdC was added for 4h and [3H]dFdC metabolites were quantified
by anionic exchange HPLC and in DNA. Apoptosis and necrosis were assessed by flowcytometry with FITClabeled-Annexin V and propidium iodide.
Results
In MOLT-3 cells no alterations were observed in concentrations of dFdCMP, dFdCDP or dFdCTP after a
preincubation with CPEC. However, the incorporation of dFdCTP into DNA was significantly increased in the
CPEC pretreated samples by 99 ± 4% (mean relative increase ± SD) and 57 ± 2% using 3 and 32 nM of dFdC,
respectively. The percentage of apoptotic and necrotic cells increased relatively by 17, 34 and 27% after 37.5
nM of CPEC and 3, 6 and 12 nM of dFdC respectively compared to the cumulative effect of both single drugs.
In HL-60 increased concentrations of dFdCMP, dFdCDP and dFdCTP of 65, 42 and 43% were observed in the
samples (pre)treated with 12.5 nM of CPEC and 3 nM of dFdC as compared with dFdC treated samples. The
incorporation of dFdCTP into DNA was significantly increased by 773 ± 38%. The percentage of apoptotic
and/or necrotic cells increased relatively by 278 and 67% after a preincubation with 12.5 nM of CPEC using 6
and 12 nM of dFdC.
Discussion
A preincubation with low concentrations of CPEC increases the incorporation of dFdCTP into DNA in Tlymphocytic or myeloid leukemic cells, which is paralleled by an increase of apoptosis and/or necrosis.
64
O17
Can whole blood substitute washed red blood cells for the analysis of TPMT activity and 6mercaptopurine metabolites?
H. Iven, A. Kotalczyk, S. Gutsche
Medical University of Luebeck, LUEBECK, Germany
6-Mercaptopurine (6-MP) and its prodrug azathioprine are used to treat numerous diseases including acute
lymphoblastic leukaemia (ALL) and chronic inflammatory bowel disease (CIBD). 6-MP undergoes three
competing metabolic pathways of which one is linked to the enzyme Thiopurine S-methyltransferase (
TPMT ) for which a genetic polymorphism was demonstrated. Numerous groups have tried to link clinical
response and/or toxicity to the concentrations of 6-thioguanine-nucleotides (6-TG), 6-methylmercaptopurinenucleotides (6-MMP) and inversely to TPMT activity determined in peripheral washed red blood cells
(RBC). Recently Pike et al. (J Chromatog B 757 (2001), 1 – 9) showed that whole blood could substitute
RBC to monitor 6-TG and 6-MMP concentrations. Since this conclusion is based on results obtained from 8
blood samples from patients with CIBD, we reevaluated this proposal and extended it to TPMT and 6methylthioguanine-nucleotides (6-MTG).
Methods: From 95 consecutive, unselected EDTA blood samples from children with ALL and patients with
CIBD, 0.3 ml were diluted and haemolysed with 0.9 ml dest. water. The rest of the sample was centrifuged,
packed RBC washed once with ice-cold saline and then haemolysed with 5 volumes of dest. water. The
haemoglobin concentration in the haemolysed samples was determined and they were kept at –80° C until
analysis. TPMT activity was measured in duplicate, 6-TG, 6-MMP, and 6-MTG after acid hydrolysis as
single determinations by established HPLC methods. Both, RBC and whole blood samples were always
analysed in parallel in the same series. Intraindividual differences and the % difference from RBC
activity/concentrations were calculated and used for statistics.
Results: TPMT activity in RBC ranged between 0.1 and 75.8 ng MTG/gHb/h (mean: 47.6). TPMT activity
in whole blood was 9.3 % higher (range –25,2 to +46.5; p<0.0001). One patient with TPMT deficiency could
be equally well identified. In RBC, 6-TG ranged between 0 (i.e. TPMT before treatment) and 9533 ng/gHb
(i.e. ALL patient on 6-TG treatment). In whole blood 6-TG was 8.3% higher ( range –100 to +107;
p=0.0272, n=71). Major differences occurred at low concentrations.
6-MMP in RBC ranged between 0 and 162402 ng/gHb. In whole blood of some patients with CIBD 6-MMP
could not be quantified because of an interfering compound. In 58 sample pairs the difference was +1.74 %
(range –71.2 to + 46.5; p = 0.5748). 6-MTG in RBC ranged between 0 and 7229 ng/gHb. The mean
difference in whole blood was +9.5% (range –54.1 to + 61.9; p=0.0003).
Conclusions: Whole blood can substitute RBC for monitoring TPMT activity and the concentrations of 6TG, 6-MTG, and 6-MMP, giving results approx. 10% higher than in RBC.
Department of Pharmacology, *Center for Pediatrics , Medical University of Luebeck, Ratzeburger Allee
160. D-23538 Luebeck, FRG .
These studies were supported by the Deutsche José Carreras Leukaemie Stiftung.
Note: To convert ng/gHb into pmol/8x108 RBC divide by 7 for 6-TG and 6-MMP and 7.59 for 6-MTG
(based on a MCH of 30 pg).
65
O18
Potential role of mycophenolate mofetil in the management of neuroblastoma patients
A. Giacomello, E. Messina, L. Barile, F. Lupi
University of Rome 'La Sapienza', ROME, Italy
In human neuroblastoma cell lines (LAN5, SHEP and IMR32), mycophenolic acid (MPA), at concentrations
(10-7 - 10-6M) readily attainable during immunosuppressive therapy with mycophenolate mofetil (Cellcept),
induces guanine nucleotide depletion leading to cell cycle arrest and apoptosis through a p53 mediated
pathway (up-regulation of p53, p21 and bax and down-regulation of bcl-2 and survivin). MPA-induced
apoptosis is also associated to a marked decrease of p27 protein. In the same cell lines MPA, at lower
concentrations (50 nM), corresponding to the plasma levels of the active free drug during Cellcept therapy,
induces differentiation toward the neuronal phenotype by causing a partial chronic guanine nucleotide
depletion. MPA-induced differentiation is not associated to p27 accumulation as occurs using retinoic acid.
At a fixed concentration of MPA a higher percentage of apoptotic or differentiated cells is obtained when
non dialysed serum substitutes for the dialysed one, due to the higher hypoxanthine concentration in the
former (about 10 µM) leading to competition on HPRT-mediated salvage of guanine. At hypoxanthine or
oxypurinol concentrations higher than 1 µM (up to 100µM) no further enhancement of MPA effects was
obtained, in agreement with the recently described safety of the allopurinol-mycophenolate mofetil
combination in the treatment of hyperuricemia of kidney transplant recipients. The effect of the UDPglucuronosyltransferases (UGT, having a major role in MPA inactivation) inhibitors curcumin and niflumic
acid (NA) on MPA-induced apoptosis was studied in the neuroblastoma cell lines. A slight increase of
cytotoxic activity was obtained with 10 mM NA. At concentrations higher than 1 µM curcumin was
cytotoxic even when employed in the absence of MPA leading to cell death by apoptosis (almost all cells
died after 24 h of incubation in the presence of 30 µM curcumin). In conclusion : 1. Cellcept, at the doses
employed in immunosuppressive regimen, appears to have a potential role in the management of
neuroblastoma. 2. The apoptotic and differentiating effects of his active metabolite MPA cannot be increased
in patients by combination with allopurinol. 3. The natural compound curcumin is a strong apoptosisinducing agent in neuroblastoma cells and could improve therapy and assist in the control of this common
and aggressive childhood solid tumor.
66
O19
Adenosine transport in HPRT deficient lymphocytes from Lesch-Nyhan patients
R.J. Torres, I. De Antonio, C. Prior, J.G. Puig
La Paz Hospital, MADRID, Spain
In previous studies, we have found that elevated concentrations of hypoxanthine reduce adenosine transport in
human lymphocytes in culture (PBL-C). The aim of the present study is to analyse adenosine transport in
HPRT deficient lymphocytes (PBL-LN) obtained from Lesch-Nyhan patients.
Methods: [2-3H] adenosine transport, and [3H] NBTI binding assays were determined in basal conditions and
after 24h incubation with 25 uM hypoxanthine.
Results:
1. Adenosine transport displays a sigmoid curve in PBLLN versus the hyperbolic normal curve. Adenosine
transport was significantly decreased in PBLLN with respect to PBLC at adenosine concentrations of 5 and 7
uM.
2. NBTI binding sites were significantly decreased in PBLLN versus PBLC at 10 and 15uM NBTI
concentrations. The binding of [3H] NBTI quantified a number of 6,782 +- 395 high affinity sites per cell
(Bmax) in PBLLN, versus 9,500 +- 9 in PBLC; p< 0.05. A dissociation constant (Kd) of 3,7+- 0,2 nM in
PBLLN was observed versus a Kd of 1,25+- 0,11 nM in PBLC; p<0.05.
3. After 24 h incubation with 25 uM hypoxanthine, adenosine transport is decreased in PBLLN with respect to
basal transport, and the sigmoidal kinetics changes to Michaelis Menten saturation kinetics. However, NBTI
binding in PBLLN was not decreased with hypoxanthine addition.
Conclusions:
This study shows that adenosine transport is markedly abnormal in HPRT deficient lymphocytes, and that
hypoxanthine concentration influences adenosine transport in these cells. Further studies are necessary to fully
characterize adenosine transport in HPRT deficient cells.
67
O20
Urate Oxidation in CSF and Blood of Patients With Inflammatory CNS Disorders
B.F. Becker1, S. Kastenbauer2, U. Koedel2, D. Kiesl2, H.-W. Pfister2
1
University of Munich, MUNICH, Germany
2
LMU, MUNICH, Germany
Several disorders of the central nervous system (CNS), including bacterial meningitis, Guillain-Barré
Syndrome (GBS) and multiple sclerosis (MS), are linked to inflammatory processes. Inflammation should give
rise to enhanced oxidative stress, changing the levels of antioxidants and characteristic degradation products in
the cerebro-spinal fluid (CSF). Urate is considered to be an important neuroprotective antioxidant in man, a
specific product of oxidation being allantoin. To evaluate cerebral oxidative stress, the levels of urate and
allantoin were compared quantitatively by HPLC in CSF and serum of controls (39), and patients suffering
from meningitis (15), GBS (18) or MS (18). The latter has been associated with low levels of urate. Both
meningitis and GBS are accompanied by disturbances of the blood-brain barrier (BBB).
The distribution of urate between serum and CSF was distinctive: low CSF values in controls and MS (about
20µM, i.e., 10-15% of the plasma levels), moderately elevated levels in GBS (about 40µM), and very high
levels, almost at equillibrium with serum, in meningitis. However, 5/15 patients with meningitis had CSF urate
levels exceeding those of serum. Thus, at least some of the CSF urate had to be of central origin. This is
supported by the absence of any correlation between CSF urate and serum urate in all groups, which, in
meningitis and GBS, also demonstrates that disruption of the BBB alone is insufficient to cause increase of
CSF urate. Neither the serum nor CSF urate level of MS patients was lower than in controls. Significantly
higher levels of allantoin were found in serum and CSF of patients with meningitis (53 and 41µM, resp.) than
in the three other groups (serum 20-30µM, CSF 5-10µM). The distribution ratio of allantoin between CSF and
serum increased significantly from 0.35 (controls) to 0.61 in meningitis, suggesting greater intracerebral
enhancement of oxidative processes.
In conclusion, the brain relies relatively little on uric acid to protect itself from oxidative stress under normal
conditions. In the special case of meningitis, and perhaps also GBS, additional uric acid is recruited to subserve
as antioxidant. We were unable to substantiate the occurrence of low urate levels in MS.
68
O21
An unusual pyridine nucleotide accumulating in erythrocytes: its identity, and positive correlation with
degree of renal failure
E.A. Carrey1, R.T. Smolenski2, S.M. Edbury1, A.D.J. Laurence3, A.M. Marinaki1, J.A. Duley1, L. Zhu4, D.J.A.
Goldsmith1, H.A. Simmonds1
1
2
University of Gdansk, GDANSK, Poland
3
University College Hospital London, LONDON, United Kingdom
4
London Metropolitan University, LONDON, United Kingdom
We report the identification of an unusual nucleotide that accumulates, with precursors, in the erythrocytes of
patients in uraemia. The nucleotide was identified, using liquid chromatography and mass spectrometry, as
2-pyridone-5-carboxamide ribonucleoside triphosphate (2PyTP). It is related chemically to the NAD
breakdown product, N1-methyl-2-pyridone-5-carboxamide (M2Py), found in high concentrations in the
plasma of uraemic patients.
Concentrations of 2PyTP and M2Py were measured in four categories of patients: mild renal failure (CRF),
end-stage renal failure (ESRF), haemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD); also
in healthy controls, and patients after successful kidney transplantation (Post-Tx). In the patient groups,
mean 2PyTP concentrations are lowest in CRF (21.8 µmol/l), increasing with degree of renal failure through
ESRF (55.1 µmol/l) and HD patients (70.9 µmol/l), being highest in CAPD patients (216.7 µmol/l). The
greatly elevated concentrations of 2PyTP in CAPD patients reflect accumulation during a longer half-life for
erythrocytes than in HD patients. Since plasma concentrations of M2Py correlate with 2PyTP in the
erythrocytes in all renal failure groups, and concentrations of both fall to the control range following
successful transplantation, we infer that either M2Py or 2PyTP may be a toxin implicated in the diverse
symptoms of uraemia.
69
O23
Metabolism of thymidine in mitochondria: role of dNT2, the mitochondrial deoxyribonucleotidase
V.B. Bianchi, P. Ferraro, L. Gallinaro, G. Pontarin, C. Rampazzo, K. Crovatto, P. Reichard
University of Padova, PADOVA, Italy
Maintenance of mitochondrial (mt) DNA requires an appropriate supply of thymidine triphosphate (dTTP).
Which is the origin of mitochondrial dTTP and how is its concentration regulated to support accurate mtDNA
synthesis? Mitochondria contain a constitutive thymidine kinase, TK2, that phosphorylates thymidine imported
from the cytoplasm also in terminally differentiated cells. The importance of TK2 is demonstrated by the
mtDNA depletion phenotype associated with its inactivation by mutation. Depletion and multiple deletions of
mtDNA arise when TK2 is presented with excess thymidine substrate as in the case of MNGIE, a
mitochondrial pathology caused by mutation of cytosolic thymidine phosphorylase. Normally the
phosphorylation of thymidine by TK2 is counterbalanced by the dephosphorylation of dTMP by dNT2, the
mitochondrial deoxyribonucleotidase discovered in our laboratory. Its 3D structure, recently solved, accounts
for its specificity for dUMP and dTMP. dNT2 together with TK2 gives rise to a substrate cycle that mirrors the
cytosolic TK1/dNT1 cycle regulating thymidine phosphates in the cytosol. To experimentally assess the role of
dNT2 in the control of mitochondrial dTTP, we have constructed inducible cell lines that overproduce dNT2 in
graded amounts. Some of them lack cytosolic TK1 and phosphorylate thymidine exclusively by mitochondrial
TK2. In this system we measure the specific activity of the cytosolic and mitochondrial dTTP pools by
incubating whole cells with tritiated thymidine. Alternatively, we run similar experiments with isolated
mitochondria. The results obtained so far clearly show that the dTTP produced by mitochondrial
phosphorylation of thymidine reaches a steady-state within mitochondria, is incorporated into mtDNA and is
continually exported to the cytosol from which it feeds nuclear DNA synthesis. In pulse-chase experiments the
labeled mitochondrial dTTP pool is replaced by unlabeled dTTP. In cells overproducing dNT2 incorporation
of radioactive thymidine is decreased resulting in lower specific activity of the dTTP pool. At high level of
dNT2 overproduction the size of this pool is also decreased. We are currently working at the downregulation of
dNT2 by RNA interference with the aim of studying the effects of reduced dNT2 activity on thymidine
phosphorylation by TK2. (Supported by the European Commission, Telethon and AIRC)
70
O24
Severe impairment of nucleotide synthesis through inhibition of mitochondrial respiration
N. Gattermann1, G. Hofhaus2, M. Dadak1, M. Wulfert1, M. Berneburg1, M.L. Loeffler3, H.A. Simmonds4
1
Heinrich-Heine University, DüSSELDORF, Germany
2
Institute of Biochemistry, DüSSELDORF, Germany
3
Philipps-University Marburg, MARBURG, Germany
4
Leflunomide and Brequinar inhibit de novo pyrimidine synthesis at the level of dihydroorotic-acid
dehydrogenase (DHODH) which is located in the inner mitochondrial membrane, and is coupled to the
mitochondrial respiratory chain (RC). DHODH uses ubiquinone as the proximal and cytochrome c oxidase as
the ultimate electron transfer system. Therefore, RC dysfunction should impair pyrimidine biosynthesis. This is
supported by the fact that rho-0-cells, lacking mitochondrial DNA and thus a functioning respiratory chain, are
pyrimidine auxotrophs requiring uridine for growth. Our interest in the consequences of RC dysfunction stems
from the finding of acquired clonal mitochondrial DNA mutations in bone marrow of patients with
myelodysplastic syndromes (MDS, preleukemias). Thus mitochondrial defects, with concomitant impairment
of nucleotide biosynthesis, may shed some light on a hitherto cryptic pathophysiology, which includes
megaloblastic changes.
To investigate this, we used specific RC inhibitors, Antimycin A and Rotenone, to treat primary human
keratinocytes and fibroblasts in culture. This resulted in severe impairment of nucleotide synthesis.
Interestingly, the effects were not restricted to pyrimidine synthesis, but concerned purine synthesis, too. The
effect of Rotenone did not differ significantly from that of Antimycin A. This was surprising since Rotenone
inhibits complex I, which is upstream of ubiquinone where DHODH interacts with the respiratory chain. In
order to avoid any unspecific effects of Antimycin A or Rotenone, we examined the consequences of two
mitochondrial DNA point mutations, each causing a specific defect of complex I. These mutations were
introduced into rho-0 cells and compared with wildtype mtDNA.
Our results indicate that drastic inhibition of mitochondrial RC activity severely impairs biosynthesis of purine
as well as pyrimidine nucleotides. The lack of specificity for de novo pyrimidine synthesis indicates that
diminished ATP regeneration may play a major role. We are currently investigating whether more subtle RC
defects in MDS lead to an unbalanced production of pyrimidine and purine nucleotides.
References:
N Gattermann (2000) From sideroblastic anemia to the role of mitochondrial DNA mutations in
myelodysplastic syndromes. Leukemia Res. 24:141-151.
M Löffler et al. (1997) Dihydroorotate-ubiquinone oxidoreductase links mitochondria in the biosynthesis of
pyrimidine nucleotides. Mol Cell Biochem 174: 125-129.
71
O25
New class of suicide genes for cancer gene therapy
Z. Gojkovic1, M.P.B. Sandrini1, M. Willer2, P. Kristoffersen2
1
2
ZGene A/S, LYNGBY, Denmark
Suicide genes code for enzymes that convert nontoxic prodrugs into toxic products. Among more than 30
different suicide genes described, so far the most used is Herpes simplex virus thymidine kinase (HSV-TK) in
combination with the nucleoside analog gancyclovir. Nucleoside analogs require activation by phosphorylation.
This activation, catalyzed by deoxyribonucleoside kinase, is the rate limiting process and severely inhibits the
potential of many drugs in cancer chemotherapy.
We have isolated and tested a variety of deoxyribonucleoside kinases with the aim to develop a wide range of
potent suicide systems. This approach, called ZAS (ZGene Activation System) can benefit potential patients
either by significantly increasing the therapeutic effect and shortening the course of the therapy (because of
faster activation) or eliminating side effects and toxicity (because of the lower drug dose which can be used).
The corresponding genes from different organisms were subcloned and tested for phosphorylation activity on
different, FDA approved, anti-cancer and anti-viral nucleoside analogs. ZAS suicide genes exhibited much
higher activation rates than HSV-TK and were able to phosphorylate a large number of analogs. ZAS was
tested in several cancer cell lines and showed an increased activation of Cladribine, AZT, Gemcitabine,
Fludarabine, Cytarabine and Vidarabine. This synergy between the ZAS gene therapy and chemotherapy opens
novel possibilities for enhanced therapy.
The major obstacle of currently used suicide cancer therapies is that prodrug has to be forced to different
cancers and cell types. Prodrugs exhibit great differences in affinity, biodistrubution or stability depending on
cell types. Therefore, our approach to develop activator genes and adapt them to drugs, which are already
effective against certain cancer types, is fundamentally different from the existing suicide methods. Because of
the fairly small size (550-1100 bp), the ZAS genes are applicable for a wide range of delivery systems. The
size of the genes and multidrug activation makes it possible to use cocktail treatment regimes and even further
improve the therapy. All this makes ZAS genes both powerful safety mechanism and suicide system for cancer
gene therapy.
72
O26
Production and Properties of Polyethylene-Glycol Conjugated Adenosine Phosphorylase:
Administration to PNP deficient mice
F.F. Snyder, L.J. Brewerton, E. Fung
University of Calgary, CALGARY, ALBERTA, Canada
Purine nucleoside phosphorylase was engineered to accept 6-amino substituted purine nucleosides by two
active site substitution, Asn243Asp;Lys244Gln. The enzyme preferentially accepts adenosine nucleosides and
is referred to as adenosine phosphorylase (AP). Recombinant AP has been conjugated to branched
polyethylene glycol polymers of approximately 42.5 kDa. SDS PAGE and matrix assisted laser
desorption/ionization characterization of the product indicated the conjugation of as many as 4 PEG molecules
per AP subunit. The PEG conjugated enzyme retained greater than 90% of the native catalytic activity. Purine
nucleoside phosphorylase deficient B6-NPG, mice were injected intraperitoneally with a single bolus of native
and PEG-AP enzyme. Peak plasma activities were obtained at 4 and 8 hours for the native and PEGconjugated enzymes and were 10-fold greater for PEG-AP as opposed to native AP injected mice. The native
AP activity in plasma declined quickly and was essentially negligible by 10 hours having a half-life of 68
minutes. The PEG-AP enzyme persisted in plasma for greater than 5 days having a half-life of 65 hours.
Tissues from PEG-AP treated mice showed only minimal levels of activity and there was no evidence of loss of
AP activity in urine samples. Although engineered AP has a preference for adenosine, substantial activity has
been retained with inosine. As hypouricemia is a recognized feature of PNP deficiency in man, the
consequence of PEG-AP administration on urate levels of PNP deficient mice was examined. Urine urate was
increased 2.7-fold for voids between 22 and 58 hours and 1.8-fold between 94 and 154 hours for PEG-AP
treated mice providing evidence for in vivo function of the administered enzyme. The ability of AP to protect
human CEM lymphoblasts in culture from the toxic effects of deoxyadenosine, 0.010 mM, plus the adenosine
deaminase inhibitor, EHNA, was demonstrated by the addition of AP to the culture media. These studies
provide evidence for consideration of PEG-AP as an alternative enzyme therapy for the inherited deficiency of
adenosine deaminase.
Supported by the Canadian Institutes of Health Research.
73
O27
Nucleoside transporters in proliferation in normal and transformed cells
F.J. Casado, R. Valdés, S. Duflot, M. Molina, S. Fernández-Veledo, M. Pastor-Anglada
University of Barcelona, BARCELONA, Spain
The concentrative nucleoside transport systems of the CNT family are tightly regulated by the differentiation
and the proliferation status of the cell. We showed that in two different experimental hepatocarcinoma
models, the expression of rCNT1 and rCNT2 are lost in the early phases of tumor progression. The activity
of these transporters is related to cell cycle progression, apparently as a consequence of changes in
nucleotide metabolism, since ribonucleotide reductase inhibition leads to rCNT1 induction. The relationship
between proliferation, differentiation and nucleoside transporter expression has been recently elucidated
using primary cultures of murine bone marrow macrophages. MCSF-induced proliferation only up-regulated
the equilibrative nucleoside transporter ENT1, while activation mediated by IFN-gamma decreased ENT1
expression and induced CNT1 and CNT2 by a STAT-1 independent mechanism. All these observations may
be relevant to the pharmacological properties of these transporters, since most drugs currently used in
antitumoral and antiviral therapy are structurally related to nucleosides. Evidence suggests that transporter
mRNA levels may not reflect protein expression. Indeed, cells from chronic lymphocytic leukemia patients
showed no clear relationship between NT mRNA amounts and the ex-vivo fludarabine citotoxicity, whereas
a significant correlation was found between equilibrative transport activity and fludarabine cytotoxic action.
Although for some NT proteins, as we recently reported for hCNT1, intracelullar location is also found, the
analysis of nucleoside transporter patterns in human biopsies may require immunohistochemistry. With this
aim antibodies suitable for immunohistochemistry analysis on paraffin embedded tissues were raised and
characterized. Recently a tissue array of gynaecological tumors was screened for hCNT1, hENT1 and
hENT2 expression. According to what the cell biology of these transporters could anticipate, almost all
tumor samples retained a certain expression of equilibrative transporters but a much higher variability in
hCNT1 expression was found.
74
O29
Estrogen Attenuates P2X7-R - Mediated Apoptosis of Uterine Cervical Cells by Blocking Calcium Influx
I. Gorodeski
CWRU School of Medicine, CLEVELAND, OH, United States of America
We have previously shown that estrogen blocks apoptosis of cultured human ectocervical cells (hECE), but the
mechanism of estrogen action is unknown. Activation of P2X7-R in hECE cells stimulates Cai increase and
augments apoptosis. In this study we determined the degree of which estrogen can modulate P2X7-R – related
apoptosis. The first experiment studied the effect of P2X7-R – induced Cai-increase on apoptosis. The
experiment utilized cultured hECE cells on filters. Cells were bathed either in normal Cao (1.2 mM) or low
Cao (<0.1 mM), and treated with 100 mM BzATP (P2X7-R – agonist) for up to 9 hrs. Changes in Cai and
influx of ethidium bromide (EB) were determined fluoroscopically in attached cells; apoptosis was determined
as DNA fragmentation in [3H]thymidine-labeled cells, and was calculated as % solubilized DNA. In cells
bathed in normal Cao BzATP induced time-dependent increase in Cai and EB influx, and augmented apoptosis.
Incubation in low Cao blocked BzATP-induced Cai-increase and apoptosis, but not EB influx (i.e. P2X7 pore
formation), suggesting BzATP-induced apoptosis is mediated by augmented calcium influx. The second
experiment studied the degree of which estrogen can regulate calcium influx via activated P2X7-R – pores.
Experiments utilized steroid-deprived hECE cells treated with either the vehicle or with 10 nM 17b-estradiol.
Fura-2 – loaded cells attached on filters were shifted to low Cao and treated for 30 min with 100 mM BzATP.
Addition of CaCl2 (to increase Cao to 1.2 mM) resulted in a rapid increase in Cai, and the effect was smaller in
estrogen-treated cells than in estrogen-deprived cells. In parallel experiments pre-treatment with estrogen
attenuated BzATP-augmented apoptosis, suggesting that the apoptosis-sparing effect of estrogen is mediated in
part by blocking Cai increase. Since ATP is present in the extracellular medium at concentrations that could
activate P2X7-R – pores, the present results suggest a physiological role for estrogen in the cervix as an antiapoptotic factor. Support: NIH AG15955.
75
O30
Mitochondrial function dependent proliferation assay for the diagnosis of mitochondrial disorders in
human fibroblasts
F.F. Snyder, S.D. Hodges
Mitochondrial disorders are difficult to diagnose due to confounding factors including maternal transmission,
heteroplasmy and the contribution of nuclear and mitochondrial gene products to organelle function. A
fibroblast proliferation assay has been developed which compares cellular proliferation in a mitochondrial
function sparing media versus a media, which requires mitochondrial function. Fibroblasts are harvested and
plated at equal density in replicate flasks in Dulbecco's minimum essential media with 10% serum for
comparison. The mitochondrial function sparing media contains glucose, pyruvate, glutamine and uridine.
Uridine relieves the pyrimidine de novo synthetic requirement, which encompass the mitochondrial enzyme
step catalyzed by dihydroorotate dehydrogenase. The mitochondrial function dependent media lacks glucose
and uridine. Glutamine is provided as an energy source, which is dependent upon oxidative phosphorylation.
Inosine is provided as a required pentose phosphate source precursor. Cells are photographed daily during a 5
to 8 day proliferation phase. Cells with normal mitochondrial function show essentially normal rates of
proliferation in both media. A number of cell lines with known mitochondrial defects have been shown to
proliferate to confluence in the mitochondrial function sparing media whereas they exhibit severely restricted
proliferation and/or cell death in the mitochondrial function dependent media. These include cell lines with the
following defects: complex I deficiency, complex IV deficiency, cytochrome C oxidase deficiency, NARP
8993 (T-G), and Surf. Putative evidence for fibroblast populations exhibiting heteroplasmy have also been
observed where the major fraction of the population is unable to proliferate while a subset shows outgrowth
indicative of a clonal population. We propose the mitochondrial dependent proliferation assay to be a useful
tool in screening fibroblast lines from patients for whom a mitochondrial disorder is being considered as part of
the differential diagnosis.
Supported by the Canadian Institutes of Health Research and Alberta Children's Hospital Foundation
76
O31
Functional studies reveal that cytosolic "High Km" 5'-Nucleotidase (cN-II) active site has a structure
similar to that of HAD superfamily
S. Allegrini1, M.G. Careddu1, G. Cuccu2, R. Pesi2, A. Scaloni3, M.G. Tozzi2
1
University of Sassari, SASSARI, Italy
2
University of Pisa, PISA, Italy
3
IABBAM - CNR, NAPELS, Italy
cN-II is an ubiquitous cytosolic mononucleotide phosphatase, acting also as phosphotransferase. Even though
its physiological role is not yet well understood, it is generally believed that the enzyme participates, along with
other six 5’-nucleotidases, one membrane bound, one mitochondrial and four cytosolic, in the nucleotide pools
regulation. cN-II activity seems to play its major role regulating the intracellular IMP concentration. cN-II is
also involved in the metabolism of purine prodrugs, furthermore a cN-II hyperactivity has been related to
severe paediatric neurological syndromes, including Lesch-Nyhan syndrome (1). cN-II acts through the
formation of a phosphorylated enzyme intermediate. The phosphorylation occurs on Asp52 (2) that is part of a
conserved motif – DXDX(T/V) - common to a big family of phosphotransferases named HAD superfamily.
This motif is present in at least four among the other 5’-nucleotidases. Recently the structure of mitochondrial
deoxyribonucleotidase (dNT-2) has been published (3). From a comparison with the structure of phosphoserine
phosphatase, another member of HAD superfamily, the authors could show the high homology between their
active site (composed of three separate motives). Computer alignments of cN-II with other members of HAD
superfamily, included the four cytosolic 5’-nucleotidases and dNT-2, allowed us to identify a number of amino
acids which could constitute cN-II active site. Point mutants of these amino acids, T249, S251, K292, D351,
D356 were prepared. For each of them, both a conservative and a non-conservative mutation were obtained
trying, in this second case, to keep the physical dimension of the mutated amino acid as similar as possible to
that present in the wild type protein. Modification of the kinetic parameters of cN-II caused by the substitutions
are in good agreement with those described for other enzymes of this superfamily (4), suggesting a similar role
in the catalytic activity. Other mutants were prepared for M53 and T56 ( motif I ) and the modification of their
kinetic properties were determined.
1.Pesi et al. (2000) NeuroReport 11, 1827-1831
2.Allegrini et al. (2001) JBC 276 (36), 33526-33532
3.Matthis et al. (2002) Nat. Stuct. Biol. 9 (10), 779-787
4.Collet et al. (1999) JBC 274 (48), 33985-33990
77
O32
The association of ecto-5'-nucleotidase (eN), integrin b1, EGFR and vimentin with invasive breast
carcinoma: The role of eN in invasive phenotype
J. Spychala1, A. Ostapkowicz1, N. Hironobu2, N. Sakon2, F. Masaru2, K. Inai2
1
2
Fukui Medical University, FUKUI, Japan
Breast carcinoma is a heterogeneous disease and expression profiling has identified several subtypes that may
be useful in determining more effective individualized treatment regimens in the clinic. Previously we have
found ecto-5'-nucleotidase overexpression in ER negative breast cancer. In order to associate the expression of
eN with specific cell phenotype, we have performed expression profiling at the level of protein in several breast
cancer cell lines. Our focused analysis of membrane and cytoskeletal proteins revealed that eN is specifically
co-expressed with a number of membrane proteins, such as EGFR, CD44, N-cadherin, OB-cadherin, caveolin,
integrin b1 and integrin a5. Among cytoskeletal proteins eN co-expresses with vimentin, merlin, fascin, moesin
and spectrin. Also several signaling molecules, such as tyrosine kinase Lyn, trimeric Gai and PKC tightly coexpress with eN. This expression profile is characteristic for normal fibroblasts and was also found in more
aggressive and tumorigenic breast cancer cell lines that have undergone Epithelial to Mesenchymal
Transdifferentiation (EMT). Since eN and several indentified membrane proteins, that either co-express or have
exclusive expression pattern, are components of lipid rafts, these results suggests that there is a significant
remodeling of this membrane microdomain in EMT. The co-expression of eN with vimentin also correlated
with the responsiveness to ConcanavalinA in more aggressive breast cancer cell lines suggesting functional
association. Preliminary survey of clinical samples from breast cancer patients show coexpression of eN with
Integrin b1 and vimentin at the invasive edge of the tumor. Thus, these data suggest that elevated expression of
eN and increased potential to generate extracellular adenosine may have specific functions related to cell
migration and strengthen the significance of this protein as a novel marker for invasive and metastatic breast
carcinoma.
78
O33
Rational design of purine and pyrimidine drugs against protozoan infections through selective uptake
H.P. De Koning, L.J.M. Wallace, M.I. Al-Salabi, D. Candlish, J.S. Burchmore, S.A. Baldwin
University of Glasgow, GLASGOW, United Kingdom
Purine and pyrimidine analogues have been hugely successful as antiviral and antineoplastic agents, but no
coherent strategy has been developed for exploiting this class of compounds for the treatment of parasitic
disease. A major challenge in any such effort is the potential for toxic side-effects. By comparing the structural
basis for substrate recognition of nucleoside and nucleobase transporters in protozoan and mammalian cells,
analogues can be selected which will be efficiently accumulated by the parasite but not by the host cells. We
have developed a technique for the construction of quantitative models of substrate-transporter interactions.
Using this approach, models were constructed of the substrate binding by purine and pyrimidine transporters,
including Trypanosoma brucei hypoxanthine transporters H2 and H4, and nucleoside transporters P1 and P2,
Leishmania major nucleobase transporter NBT1 (responsible for allopurinol uptake), Toxoplasma gondii
nucleoside transporter TgAT2, and the common human facilitative nucleobase transporter hFNT1
(characterised in erythrocytes). We have shown that these models have predictive value for the selective uptake
of purine analogues. In addition, these models also provide important insights in the structural basis for
substrate recognition by solute transporters and highlight putative structural similarities between transporters of
related species. It was shown that hFNT1 binds the purine ring in a very different way from the protozoan
nucleobase transporters, but hat, in particular, T. brucei H2 and L. major NBT1 form almost the same
interactions with purines, and of similar strength. Likewise, the T. brucei P1 purine nucleoside transporter
bound nucleosides in a very similar way as T. gondii AT2, which displays an equally high affinity for most
pyrimidine nucleosides as for purine nucleosides. We are currently proceeding to identify the amino acids
responsible for particular bonds, using functional mutants, site-directed mutagenesis and modelling of protein
folding. The genes encoding the T. b. brucei nucleoside transporter have been described and we recently cloned
the first protozoan nucleobase transporter gene, TbNBT1, which encodes the H4 transporter. This gene was
expressed in yeast and Xenopus oocytes, and encodes a high affinity purine nucleobase transporter of the ENT
family, with low affinity for inosine and adenosine.
79
O34
Improving NDP Kinase for Antiviral Nucleotide Analogs Activation
D. Deville-Bonne1, S. Gallois-Montbrun1, B. Schneider1, V. Giacomoni-Fernandes1, M. Véron1, Y. Chen2, S.
Morera2, J. Janin2
1
Pasteur Institute, PARIS, France
2
LEBS, GIF-SUR-YVETTE, France
Nucleoside analogs (ddC, AZT, d4T) used in anti-AIDS therapies are devoid of the 3'OH in the ribose
moiety preventing viral DNA elongation. Nucleoside analogs are phosphorylated into triphospho-derivatives
by cellular kinases of the salvage pathway with Nucleoside Diphosphate (NDP) kinase as the last enzyme.
The phosphorylation efficiency of antiviral drugs by human NDP kinase is drastically reduced. Our previous
work demonstrated a crucial role for the 3’OH of natural nucleotides involved in a H-bond network with the
substrate phosphate and two active site residues.
In order to improve the processing of nucleoside prodrugs, we have engineered a NDP kinase variant with an
enhanced activity for nucleotide analogs. An hydroxyl group was introduced in the enzyme, substituting the
missing 3'OH of the analogs. The X-ray structure of the N115S mutant complexed with a triphosphate
derivative of AZT indicates that the increased activity reflects an improved geometry of binding. The N115S
and L55H mutations were combined in NDPK. The catalytic efficiency of the L55H-N115S double mutant
was more than 80 fold improved for the triphospho form of AZT, d4T, ddT and acyclovir derivatives, whilst
it was 5 times decreased for natural nucleotides as compared to the wild type enzyme. Such a mutant could
be used as a suicide enzyme in cell therapies to improve the concentration of activated nucleoside analogs.
80
O35
Regulation of dinucleoside polyphosphates by the YgdP and ApaH diadenosine polyphosphate
hydrolases is essential for intracellular invasion by Salmonella enterica serovar Typhimurium
A.G. McLennan, T.M. Ismail, C.A. Hart
University of Liverpool, LIVERPOOL, United Kingdom
Salmonella enterica is a common and potentially dangerous pathogen, causing a variety of illnesses from food
poisoning to septicaemia and typhoid fever. It has the ability to avoid host defence systems by invading and
multiplying within host cells, such as the intestinal epithelium. The spread of antibiotic resistance requires the
development of new anti-virulence drugs. The ygdP and apaH genes of Salmonella enterica serovar
Typhimurium encode asymmetrically- and symmetrically-cleaving dinucleoside polyphosphate (NpnN)
hydrolases respectively. For example, YgdP, a Nudix hydrolase, cleaves diadenosine tetraphosphate (Ap4A)
producing AMP and ATP, with Km and kcat values of 18 µM and 18 s-1,while the unrelated ApaH cleaves
Ap4A producing 2ADP, with Km and kcat values of 37 µM and 37 s-1. Disruption of ygdP reduced
intracellular invasion of human Hep-2 epithelial cells by S. Typhimurium by 9-fold, while disruption of apaH
reduced invasion 250-fold and resulted in filamentous growth. Disruption of both genes led to a 3000-fold
reduction in invasion. Adhesion of the mutants to both HEp-2 and U-937 macrophage-like cells was greatly
reduced compared to the wild type. Invasive capacity of both single mutants was restored by
transcomplementation by a plasmid expressing the ygdP gene, suggesting that loss of invasion was due to
increased intracellular NpnN. The wild type level of 3 µM adenylated NpnN (ApnN) was increased 1.5-, 3.5and 10-fold in the ygdP, apaH and double mutants respectively. Expression of the putative ptsP virulence gene
immediately downstream of ygdP was not affected in the ygdP mutant. Analysis of 19 metabolic enzyme
activities and the ability to use a range of carbohydrate carbon sources revealed a number of differences
between the mutants and wild type. It is proposed that the increase in intracellular NpnN in the mutants leads
to changes in gene expression that limit the ability of S. Typhimurium to adhere to and invade mammalian
cells. These enzymes may represent new targets for anti-virulence drugs. Preliminary data with effective
synthetic Ap4A analogues will be presented.
81
O36
The Effect of N-Methyl-2-Pyridone-5-Carboxamide - A Nicotinamide Catabolite on Poly ADPRybosylation and Oxidative Stress Injury in Endothelial Cells
E.M. Slominska1, R.T. Smolenski2, F. Osborne2, H. Dziewit1, J. Swierczynski1, M.H. Yacoub2
1
2
The poly(ADP-ribosyl)-ation is essential mechanism of DNA repair but if excessively stimulated may impair
cellular viability by depletion of NAD pool and induction of apoptosis. Therefore some studies demonstrated
deleterious effects of inhibitiors of poly(ADP-ribose) polymerase (PARP) while the other have shown that it
may be protective during clinical conditions associated with ischemia reperfusion injury. We studied whether
2PY (N-methyl-2-pyridone-5-carboxamide) which is a product of nicotinamide metabolism excessively
accumulating in chronic renal failure (CRF) could inhibit PARP. Subsequently, we have analysed the effect of
2PY on oxidative stress injury in endothelial cells. Effect of 2PY and nicotinamide (NA) on poly(ADPribosyl)-ation have been evaluated with isolated PARP and endothelial cell lysates. Cellular effects were
studied with cultured human endothelial line (PIEC) exposed to 100 uM peroxynitrite for 60 min followed by
measurement of cellular ATP, NAD and lactate dehydrogenase release. 2PY or NA was added at 0.3, 1 and 3
mM concentration while 3 mM imidazole was given to controls. Half-maximal concentrations for the enzyme
inhibitory effect of 2PY was 0.53 mM and at 0.75 mM with the isolated PARP and endothelial cell
homogenate, respectively. Cultured endothelial cells ATP decreased from 16.1±2.1 to 2.3±0.6 nmol/mg cellular
protein after peroxynitrite exposure in controls. Addition of 2PY or NA protected from peroxynitrite induced
ATP decrease already at 0.3 mM concentration. Maximal effect was observed at 3 mM 2PY or NA, with ATP
maintained at 10.2±1.7 and 13.1±1.5 in cells treated with 2PY and nicotinamide respectively (p<0.001 vs.
control). 2PY and NA attenuated peroxynitrite induced endothelial lactate dehydrogenase release and NAD
pool decrease.
Conclusion: 2PY inhibits poly(ADP-ribose) polymerase in sub-milimolar concentrations. Endogenous
concentration in healthy subjects (about 1 µM) may be too low to exert any effects but inhibition of PARP is
likely in CRF where 2PY concentration is elevated up to 20 times. 2PY effectively prevented oxidative stress
injury in endothelial cells pointing out to important beneficial effect of transient elevation of 2PY
concentration. However, analysis whether prolonged elevation of 2PY concentration which occurs in CRF is
beneficial or deleterious require further studies.
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O37
Selective increase of dATP pools upon activation of deoxycytidine kinase in lymphocytes: implications in
apoptosis
M. Sasvári-Székely, G. Keszler, T. Spasokoukotskaja, Z.C. Csapó, S. Virga, M. Staub
Semmelweis University, BUDAPEST, Hungary
Stimulation of the activity of deoxycytidine kinase (dCK), the principal nucleoside salvage enzyme, has been
recently considered as a protective cellular response to a wide range of agents interfering with DNA repair and
apoptosis. In light of this, the potential contribution of dCK activation to apoptosis induction - presumably by
supplying dATP or its analogues for the apoptosome deserves consideration.
Two-hour exposure of human tonsillar lymphocytes to 2-chloro-deoxyadenosine (CdA), to the DNA
polymerase inhibitor aphidicolin as well as to the topoisomerase II inhibitor etoposide led to a 2 - 2.5 fold
activation of dCK. When the dNTP pools were determined, both deoxypyrimidine triphosphate pools were
reduced after the treatments, the dGTP levels were roughly constant, while dATP levels elevated by 60 %, 100
% and 50 % in the CdA, aphidicolin and etoposide-treated cells, respectively. We assume that dCK activation
elicited by cellular damage might be a proapoptotic factor in terms of generating dATP well before the release
of cytochrome c and deoxyguanosine kinase from mitochondria.
As far as the molecular basis of dCK activation is concerned, phosphorylation of the dCK protein by PKC or
downstream members of the PI(3)K signalling pathway has previously been suggested. However, neither the
PKC activator PMA and the PKC inhibitor bisindolyl-maleimide, nor the PI(3)K inhibitor wortmannin
influenced either basal or stimulated dCK activity. Results of native western blotts clearly indicated parallel
changes between the enzyme activity and protein levels of dCK. Moreover, the more intensive bands
corresponding to CdA- and aphidicolin-treated samples exhibited a bit higher mobility as compared to the less
intensive bands of the untreated cell extracts, probably due to the presence of negatively charged group(s) or to
other changes of aminoacid residues resulting from post-translational modification during enzyme activation.
These results show a conformational change of the dCK protein upon activation that probably renders the Cterminal epitope more accessible to the antibody.
This work was supported by the following grants: Hungarian National Grant ETT 273/2001, OTKA T035203
and EC-BMH-CT98/3079.
83
O38
Multi-level regulation of human AMP deaminase isoform E (AMPD3) by calmodulin
R.L. Sabina, D.K. Mahnke-Zizelman
Medical College of Wisconsin, MILWAUKEE, WISCONSIN, United States of America
A recently proposed regulatory mechanism for human AMP deaminase isoform E (AMPD3) involves its
reversible association with the cytoplasmic membrane (J Biol Chem 277:42654, 2002). Membrane-bound
isoform E is relatively inactive due to potent noncompetitive inhibition by phosphatidylinositol 4,5bisphosphate (J Biol Chem 274:25701, 1999). Protonation of histidine residues in the AMPD3 polypeptide
promotes membrane recruitment of isoform E under conditions of metabolic acidosis. Herein, we describe a
Ca2+-dependent protein-protein interaction with calmodulin (CaM) that contributes to this mechanism for
isoform E regulation. Ca2+-CaM has several effects on enzyme behavior, including: 1) displacement of
membrane-bound enzyme, 2) antagonism of membrane binding by the soluble enzyme, and 3) modest
activation of the soluble enzyme through a Km effect (Km [-ATP], mM [n=3]: control, 1.8±0.1; Ca2+-CaM,
1.0±0.2*; CaM only, 1.6±0.3; Ca2+ only, 1.7±0.2. *, p<0.05 when compared to other conditions in two-tailed ttests). We now further propose that the association between CaM and isoform E plays a counter-regulatory
role to the previously described enzyme-lipid interaction. This working model may also be related to the
indirect stimulatory effect that calcium has on erythrocyte AMPD (FEBS Lett 244:417, 1989). Currently, we
are attempting to identify a CaM-binding domain in isoform E and have preliminary evidence that critical
determinants are located within residues 65-89 of the AMPD3 polypeptide.
84
O40
Biophysical Studies of ATP, Deoxycytidine and Gemcitabine Binding to Human Recombinant
Deoxycytidine Kinase
R.S. Mani1, E.V. Usova2, S. Eriksson2, C.E. Cass3
1
University of Alberta, EDMONTON, ALBERTA, Canada
2
Swedish Univ. of Agricultural Sciences, UPPSALA, Sweden
3
Cross Cancer Institute, EDMONTON, Canada
Deoxycytidine kinase (dCK), a cytosolic enzyme with broad substrate specificity, activates a number of
medically important nucleoside analogs by their 5’-phosphorylation. The enzyme exhibits different affinities
for various substrates, implying that it can assume different conformational states. For this reason, in this
study, the binding of phosphate donors (ATP and UTP), and the phosphate acceptors (deoxycytidine (dCyd)
and gemcitabine (Gem), a drug often used in the treatment of solid tumors) to dCK was studied by
spectroscopic methods. Biophysical studies were conducted with purified recombinant human dCK. The Mr
determined by low-speed sedimentation equilibrium under nondenaturing conditions was 60,250 ± 1,000,
indicating that dCK, which has a predicted Mr of 30,500, exists in solution as a dimer. Analysis of circular
dichroism spectra revealed the presence of two negative dichroic bands located at 222 and 209 nm with
ellipticity values of –11,900 ± 300 and –12,500 ± 300 deg.cm2.dmol-1, respectively, indicating the presence
of approximately 40% α-helix and 50% β-structure. Circular Dichroism studies in the aromatic and farultraviolet range and UV difference spectroscopy indicated that binding of ATP, UTP, dCyd and Gem to
dCK reduced its α-helical content and perturbed tryptophan and tyrosine. Steady-state fluorescence
demonstrated that dCyd (or Gem) and ATP bound to different sites on dCK and fluorescence quenching
revealed bimodal binding of dCyd and Gem and unimodal binding of ATP and UTP. We have also
demonstrated the formation of a ternary complex involving dCK, dCyd (or Gem), and ATP (or UTP).
Spectroscopic studies revealed changes in the secondary and tertiary structures of dCK upon binding of
substrates. The nature and extent of these changes differed with the substrate, with the result that dCK
exhibited different affinities for different substrates.
85
O41
Clinical consequences of polymorphisms in the methylenetetrahydrofolate reductase gene
R. De Jonge
Erasmus MC, ROTTERDAM, The Netherlands
The enzyme 5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of
5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the major circulatory form of folate. The
enzyme plays a pivotal role in cellular folate metabolism because it is located at a metabolic branch point
directing one-carbon fragments towards homocysteine remethylation at the expense of purine and pyrimidine
synthesis. Severe MTHFR deficiency results from rare mutations and patients present in infancy or
adolescence with homocystinuria and neurological and vascular complications. Mild MTHFR deficiency
results from two common polymorphisms in the MTHFR gene (allele frequencies ∼ 33%): 677 C>T and
1298 A>C. The central role of MTHFR in cellular folate distribution is demonstrated by the observation that
reduced MTHFR activity (e.g., MTHFR 677 TT subjects) alters normal intracellular folate distribution:
folates committed to purine and pyrimidine synthesis accumulate at the expense of 5-methyltetrahydrofolate
(the most abundant form in wild type MTHFR 677 CC subjects).
The relationship between these polymorphisms and disease might be explained by several
mechanisms. First, the MTHFR 677 C>T and 1298 A>C polymorphisms in interaction with poor B-vitamin
and folate status result in mildly elevated plasma homocysteine levels. Hyperhomocysteinemia is a wellestablished independent risk factor for cardiovascular disease and has been linked to other diseases such as
pregnancy complications, Down’s syndrome and Alzheimer’s disease. Second, reduced MTHFR activity
leads to reduced availability of 5-methyltetrahydrofolate required for S-adenosylmethionine (SAM)
biosynthesis and consequently results in DNA hypomethylation. Third, low availability of folates for
pyrimidine and purine synthesis may lead to uracil misincorporation into DNA leading to double-strand
breaks and chromosomal damage. DNA hypomethylation and uracil misincorporation are considered
important factors in carcinogenesis. Thus, MTHFR 677 TT subjects have a decreased risk of colorectal
cancer possibly due to increased folate availability for RNA and DNA synthesis. Furthermore, both the
MTHFR 677 C>T and 1298 A>C polymorphisms have been linked to a reduced risk of lymphoid leukemias
but not myeloid leukemias.
In this presentation, the correlation between MTHFR 677 C>T and 1298 A>C polymorphisms and
several disease entities such as osteoporosis, acute lymphoid leukemia and pre-eclampsia will be discussed.
86
O42
Identification of 4 unique mutations in exon 4 of uromodulin (UMOD) gene in 4 separate families with
familial juvenile hyperuricemic nephropathy (FJHN)
N. Kamatani1, E. Kudo2, M. Moritani2, T. Yamaoka2, A. Taniguchi1, H. Yamanaka1, M. Itakura2
2
The University of Tokushima, TOKUSHIMA, Japan
1
Scolari et al. reported a locus for medullary cystic kidney disease (MCKD2) on chromosome 16p12. Our
independent genome-wide linkage study of a large Japanese family identified a locus for familial juvenile
hyperuricemic nephropathy (FJHN) at the same region. Subsequent studies have suggested that the 2 diseases
(MCKD2 and FJHN) could be allelic variants. Recently, Hart et al. for the first time found that mutations in
uromodulin (UMOD) gene were responsible for the 2 conditions. They found 4 independent mutations in 3
families with FJHN and one family with MCKD2. We examined DNA from 5 separate families whose
diagnosis is compatible with that of FJHN. Including the large family we reported previously, 4 of the 5
families had different unique missense mutations in exon 4 of UMOD gene. The mutations segregated along
with the FJHN phenotype. In 3 of the 4 families, other amino acids were substituted for conserved cysteine
residues (Cys). In the large family, we found a missense mutation that substituted leucine for conserved proline
residue. Our findings further confirmed that UMOD gene is responsible for most of the families with FJHN.
The facts that 3 of the 4 families of us and 2 of the 3 families of Hart et al. had missence mutations involving
Cys residue strongly suggest that structural changes of UMOD molecule due to impaired formation of intra- or
intermolecular disulphide bond or substitution of proline residue may be responsible for the 2 disorders. The
domain encoded by exon 4 probably has critical importance in functions of UMOD molecule, because all 8
mutations found in UMOD gene were in exon 4. The fact that 8 separate families so far reported with
MCKD2/FJHN had different responsible mutations is compatible with the theories of population genetics.
Thus, diseases with autosomal dominant inheritance, with high penetrances and with considerable disability are
not likely to be caused by common mutations with common origins.
87
O43
What role do methylated metabolites play for the cellular effects of thiopurines?
C. Peterson, M. Lindqvist, S. Rahman, S. Almer
University of Linköping, LINKÖPING, Sweden
Thiopurines (azathioprine, 6-mercaptopurine, 6-thioguanine) are old drugs but still cornerstones in the
treatment of inflammatory bowel disease and childhood leukemia. The drugs undergo deamination, methylation
and phosphorylation to form identical terminal thioguanine nucleotides. Weinshilboum showed many years ago
that thiopurinemethyltransferase (TPMT) is a polymorphic enzyme. Several mutations have been identified and
about 10% of Caucasians have reduced enzyme activity. Methylation and phosphorylation can be regarded as
competing metabolic pathways. Me-MP lacks cytotoxicity and evidence has been presented for a correlation
between the formation of thioguanine nucleotides (TGN) and clinical effects. We have determined erythrocyte
TGN and me-TIMP concentrations in more than 2500 blood samples from IBD-patients and confirmed
previous results showing marked differences in metabolite pattern after treatment with TG and AZA or 6-MP at
doses equitoxic to the bone marrow. TGN levels are severalfold higher after TG, and after 6-MP the
concentration of metylated TIMP is much higher than that of TGN.
Aim: To elucidate the cellular effects of thiopurine metabolites, we compared the cytotoxic effects of TG and
6-MP and metabolites in a human T-cell acute lymphoblastic cell line (Molt-4) and in sublines resistant to 6MP and TG.
Methods: Resistant sublines were produced by stepwise increase in 6-MP and TG concentrations to the Molt-4
cells during several months. Cytotoxicity was determined by the colorimetric MTT-assay of viable cells. The
drug concentration causing a 50% reduction in the number of viable cells during exposure for 72 hours was
determined (IC50).
Results: In the parental cell line, the cytotoxicties of 6-MP, TG and TIMP were similar with IC50 values of 7.2,
7.7 and 7.9 nM, respectively. Me-TIMP and me-MPR were more cytotoxic with IC-50-values of 1.7 and 1.4
nM whereas me-MP and me-TG were non-toxic. As expected, the resistant cell lines showed low sensitivity to
the parent compounds. However, me-TIMP and me-MPR had markedly higher cytotoxicity on the resistant cell
lines as compared to the parental line with IC50-values of 0.3 and 0.4 nM.
Conclusion: Certain methylated thiopurine metabolites have the potential to contribute to the therapeutic and
toxic effects of thiopurines.
88
O44
Pre-treatment TPMT PHE/genotyping: a safe guide for initial thiopurines drugdosing for specific
patient groups
E.A. Shobowale-Bakre, A. Ansari, A.M. Marinaki, L.D. Fairbanks, J.A. Duley, J.D. Sanderson
Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme. Deficiency of the
enzyme is predictive of thiopurine drugs (i.e. azathioprine, mercaptopurine and
thioguanine)intolerance. In Caucasian and African populations, 11% are carriers while 0.3% of the
individuals are totally TPMT deficient. Two-thirds of these carriers have a high risk of drug induced
side-effects on a full dose and are typically withdrawn from therapy when bone marrow depression
sets in. To test the hypothesis that carriers should tolerate half the normal dose, we identified 28
TPMT carriers who had pre-therapy TPMT phenotyping done in our laboratory mainly from
gastroenterology and dermatology clinics(2 patients had autoimmune hepatitis, 6 skin and or oral
ulceration and 3 eczema). Each patient was given azathioprine(aza) at a reduced dose of 1mg/kg
(initially 0.5mg/kg) as a steroid-sparring agent according to departmental guidelines. Clinical notes
were reviewed after one year of treatment for adverse effects and clinical response (defined as
withdrawal of steroids :- complete, partial or non-response). Phenotype results were confirmed for
all patients by genotyping for the three most common TPMT allelic variants-: TPMT *3A (G460AAla154Thr and A719G-Tyr240Cys; 10% in Caucasians), *3B (G460A-Ala154Thr),*3C (A719Gtyr240Cys; 10-14% in Africans and 2.5% in South Asians) considering the inter-ethnic variations of
TPMT genotypes and the racial mix of the UK population. 6 of the 28 patients experienced sideeffects requiring drug withdrawal (2 pacreatitis, 4 nausea/malaise). The 4 patients that experienced
nausea on aza were switched onto low dose mercaptopurine. No additional episodes of
myelotoxicity or other side effects were observed over the treatment period. 24 of the 26 carriers
who remained on drug therapy responded favourably. Others factors must be considered for the 2
patients that did not respond to therapy. In Conclusion, pre-therapy TPMT testing is a very costeffective and useful tool for guiding against drug over-dosing in TPMT carriers.
89
O45
A mutation in the ITPA gene predicts intolerance to azathioprine
A.M. Marinaki1, J.A. Duley2, M. Arenas1, S. Sumi1, A. Ansari1, J.D. Sanderson1, C.M. Lewis1, E.A.
Shobowale-Bakre1, L.D. Fairbanks1
1
2
University of Queensland, QUEENSLAND, Australia
Introduction. Adverse drug reactions to AZA or 6-mercaptopurine (6-MP) occurs in 20-30% of inflammatory
bowel disease (IBD) patients and often necessitates withdrawal of therapy. Polymorphism in the TPMT gene
predicts intolerance in a minority of these patients. Inosine triphosphate pyrophosphatase (ITPase) deficiency is
a benign polymorphism in Caucasian populations and is characterized by accumulation of the nucleotide ITP in
erythrocytes. As 6-MP is activated through a thio-IMP intermediate, we predicted that the abnormal nucleotide
thio-ITP would accumulate in ITPase deficient patients treated with thiopurine drugs, resulting in toxicity. We
have reported (Hum Genet (2002) 111 :360–367) the genetic basis of ITPase deficiency; a 94C>A missense
mutation (Pro32 to Thr) in the ITPA gene results in complete deficiency in homozygotes and approximately
25% activity in heterozygotes. Homozygotes for an intron 2 mutation (IVS2+21A>C) averaged 60% of
controls.
Methods. ITPA and TPMT genotypes and TPMT phenotypes were determined retrospectively in 62 IBD
patients with adverse drug reactions to AZA therapy. The adverse drug reaction cohort was compared to 68
patients who did not experience an adverse reaction to AZA therapy.
Results. Variant TPMT genotype was not significantly associated with adverse drug reactions overall, but the
association of TPMT heterozygosity with side effects was significant in a subset of 14 patients with nausea and
vomiting (OR 5.5, CI 1.4-21.3, p=0.0206). Overall, the ITPA 94C>A mutation was significantly associated
with adverse drug reactions (OR 4.2, CI 1.6-11.5, p=0.0033) which sub-divided into: flu-like symptoms (OR
4.7, CI 1.2-18.1, p=0.0308), pancreatitis (OR 6.2, CI 1.1-32.6, p=0.0485) and rash (OR 10.3, CI 4.7-62.9,
p=0.0190). Bone marrow suppression, myalgia and hepatitis were not predicted by ITPA nor TPMT genotypes.
The ITPA intron mutation did not predict adverse drug reactions.
Conclusion. The ITPA 94C>A mutation predicts intolerance to AZA. Thioguanine therapy may benefit AZA
intolerant patients with ITPase deficiency.
90
O46
Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8dihydroxyadenine urolithiasis
A. Taniguchi, H. Yamanaka, S. Tsuchida, S. Kuno, N. Kamatani
Adenine phosphoribosyltransferase deficiency is associated with 2.8-dihydroxyadenine (DHA) urolithiasis. The
largest number of patients has been reported in Japanese. So far, four mutations in the adenine
phosphoribosyltransferase (APRT) gene have been described in Japanese. The most common mutation is
M136T. Three other mutations, a nonsense (W98X), a frameshift caused by CCGA insertion in exon 3 and a
termination failure at stop codon (X186S). We now report two new mutations in patients with 2,8dihydroxyadenine urolithiasis.
<Method> We examined 3 patients (patient A, B, C) with 2.8-dihydroxyadenine urolithiasis. The five exons
and splice-donor sites were amplified with several sets of primers and the sequence analysis of PCR products
were performed on automated DNA sequencer.
<Results and discussion> The patient A is homozygous for G1359A leading to the amino acid substitution
R67Q which have been already reported in USA. The patient B was shown to be homozygous for a single base
substitution G1831A leading to amino acid substitution G133D. Patient C was found to be compound
heterozygous for two mutations: a W98X which is one of the common mutations in Japanese population and a
G1409A transition which is a new missense mutation leading V to M substitution in codon 84. These mutations
were confirmed by restriction endonucleases. Based on the structure of the enzyme from Leishmania donovani,
Saccharomyces cerevisiae and Giardia lumblia, R67 and G133 are located in the PRPP binding site and V 84
are supposed to be located at the dimer interface, suggesting these mutation are expected to affect the
enzymatic activity.
91
P01
Modulation of Glycogen Phosphorylase activity affects 5-Phophoribosyl-1-Pyrophosphate availability in
Rat Hepatocyte Cultures
P. Boer, O. Sperling
Tel-Aviv University, TEL-AVIV, Israel
The effect of modulation of the rate of glycogenolysis on the availability of 5-phosphoribosyl-1pyrophosphate (PRPP) was investigated in rat hepatocyte cultures. dbcAMP, forskolin and glucagon,
activating glycogen phosphorylase through activation of protein kinase A (PKA), were found to raise PRPP
availability by 44%-56%. Arg-vasopressin and phenylephrine, activating glycogen phosphorylase through
the phosphoinositide cascade, and the protein kinase C (PKC) activators 1,2-dioctanoyl-sn-glycerol (DOG)
and phorbol 12-myristate β-acetate (PMA), did not affect PRPP availability. dbcAMP, but not
phenylephrine, was also shown to increase the degradation of pre labeled glycogen in these cells by 57%.
Caffeine and CP-91149, inhibitors of glycogen phosphorylase, decreased PRPP availability by 33% and
43%, respectively. The finding that induction of glycogenolysis enhances, and inhibition of glycogenolysis
decelerates PRPP generation suggests that glycogenolysis is a major contributor to PRPP generation in liver
tissue in the basal (postabsorptive) state.
92
P02
Purine levels and cytokine release with diuretic therapy in rheumatoid arthritis
T.W. Stone1, C.M. Forrest1, G. Harman2, R.B. McMillan1, C. Rana1, S. Shaw2, N. Stoy2, L.G. Darlington2
1
University of Glasgow, GLASGOW, United Kingdom
2
Epsom General Hospital, EPSOM, United Kingdom
Treatment with diuretics has been reported to increase blood urate levels, and urate is a potent free radical
scavanger. Since free radicals are implicated in rheumatoid arthritis and since it is rare to find a patient with
both gout and RA, we examined the effects of treating rheumatoid arthritic patients with bumetanide to try to
improve their arthritic control. We measured blood levels of urate, other major purine metabolites, lipid
peroxidation products and the cytokines tumour necrosis factor-a (TNFa), interleukin-1b (IL-1b) and
interleukin-6 (IL-6). In addition, we examined the ability of the adenosine receptor agonist 5'Nethylcarboxamido-adenosine (NECA) to modify cytokine release in cultures of whole blood taken from the
patients. Seventy patients, aged 18-75 years, were recruited from routine rheumatology clinics and rheumatoid
arthritis was diagnosed using the American Rheumatism Association revised criteria. Patients were excluded if
they were taking any drug which would affect uric acid levels in the blood. Patients were randomised to
receive bumetanide 4mg/day or placebo. Results showed increased levels of urate, but not of other purines, in
the blood of drug-treated patients compared with placebo-treated controls. There were no significant changes
in clinical measurements of disease activity or in ESR or CRP levels. There were no over all differences in the
blood levels of the cytokines, nor in the basal or stimulated production of cytokines from the blood cultures.
However, while NECA depressed the release of TNFa, it failed to depress the release of IL-1b or IL-6, a
difference from earlier studies of healthy control subjects and, thus, a difference which may contribute to the
disease activity.
93
P03
The Pathophysiology of Hyperuricemia in Essential Hypertension: A pilot study
J.G. Puig1, R.J. Torres1, L.M. Ruilope2, C. Campo2, T. Sancho1, I. Bernardino1
1
2
Hospital 12 de Octubre, MADRID, Spain
Background: Hyperuricemia in essential hypertension has been related to renal uric acid underexcretion. This
impaired uric acid excretion may be linked to an increased tubular sodium reabsorption mediated by insulin. In
this pilot study we examined whether insulin influences the renal handling of urate.
Patients and Methods: Ten non-obese (body mass index, 28,22 Kg/m2), non-diabetic male patients with
essential hypertension (mean age+-SD, 54+-7 years) and normal glomerular filtration rate were submitted to
and oral glucose tolerance test (OGTT, 75 g). Serum glucose, insulin, urate and urinary uric acid to creatinine
ratio were measured at baseline and 120 min following the OGTT.
Results: Two patients had impaired fasting glucose (>110 and <126 mg/dL) and only one patient showed
glucose intolerance (141 mg/dL, 120 min after the OGTT). Serum insulin increased from a mean baseline value
of 16 mcU/mL to 72 mcU/mL (mean increase, 4.5 fold). Mean baseline glucose was 95 mg/dL and 120 min
after the OGTT, 105 mg/dL. Mean serum urate remained stable (8.1 mg/dL). Urinary uric acid to creatinine
ratio decreased from a mean baseline value of 0.44+-0.18 mg/mg to 0.28+-0.12 mg/mg (p<0.05) after the
OGTT. In 5 patients in whom urinary sodium to creatinine ratio was determined, sodium excretion was
markedly reduced (from 1.23+-0.61 at baseline to 0.70+-0.26 mmol/mg creatinine following the OGTT;
p<0.05).
Conclusion: This pilot study shows that acute endogenous insulin secretion is accompanied by a reduction in
uric acid excretion that may be mediated by the sodium-retaining effect of insulin.
94
P04
Serum Adiponectin Concentration in Patients with Primary Gout
Z. Tsutsumi, T. Yamamoto, S. Takahashi, Y. Moriwaki, T. Ka, H. Toshikazu
Hyogo College of Medicine, NISHINOMIYA, Japan
Objectives: Adiponectin is an adipose tissue derived protein. It was suggested that decreased adiponectin
concentration in serum is a possible risk factor for atherosclerosis. Clinical studies have shown that obesity is
closely related to the decreased adiponectin concentration in serum. Atherosclerosis and obesity have been
frequently observed in patients with gout. Therefore, we investigated adiponectin concentration in serum,
together with abdominal fat area, serum insulin level, urinary excretion of c-peptide, as well as markers of urate
metabolism in patients with gout.
Subjects and Methods: Age- and body mass index-matched male patients with primary gout (n=88) and healthy
adult male (n=58) were included in the study. Primary gout was diagnosed on the basis of criteria advocated by
ARA. Adiponectin was measured by a radio-immunoassay method using a commercially available kit (Human
adiponectin RIA, Linco Research Inc. MO, USA). Abdominal fat area was measured by a computed
tomography at the level of umbilicus . Other biochemical parameters were measured in our laboratory.
Results: Serum adiponectin concentration was not different between gout patients and control subjects (9.8 +/1.4 vs 8.3+/- 0.5 microg/mL). Serum adiponectin level was negatively correlated with body mass index,
visceral fat area, subcutaneous fat area, serum insulin level, and urinary excretion of c-peptide, while it was
positively correlated with serum HDL-cholesterol level. However, there were no significant relationships
between adiponectin and any markers of urate metabolism. Multivariate analysis demonstrated that the
strongest contributor to serum adiponectin concentration was body mass index, followed by subcutaneous fat
area.
Conclusion: Obesity contributes to decreased serum concentration of adiponectin, irrespective of gout or not.
However, decreased serum concentration of adiponectin was correlated with various elements of metabolic
syndrome. Further investigations on the relationship between adiponectin and atherosclerosis in gout will be
required.
95
P05
Effect of low purine-containing low malt beer (happo-shu) and regular low malt beer on purine bases
K. Ka, T. Yamamoto, Y. Moriwaki, S. Takahashi, Z. Tsutsumi, M. Fukuchi, H. Toshikazu
Hyogo College of Medicine, NISHINOMIYA, Japan
Background: Regular beer contains large amounts of purines, which may increase the plasma concentration of
uric acid. Low malt beer also contains considerable amounts of purines, though low molt beer less contains
purines than regular beer. On the other hand, purines in low purines-containing low malt beer are less than 10%
of purines in regular beer. Therefore, we tested regular and low purine-containing low malt beer in order to
determine whether they caused an increase in plasma concentration and urinary excretion of purine bases
(hypoxanthine, xanthine, and uric acid).
Subjects and Methods: Five healthy males were given regular low malt beer (10 ml/kg of body weight) and low
purine-containing low malt beer (10 ml/kg of body weight). Blood and urine sampled were collected before and
after regular beer and low purine-containing low malt beer.
Results: The plasma concentrations of uric acid, hypoxanthine, and xanthine increased by 1.10-fold (P<0.05),
4.23- (P<0.05), and 9.723-fold (P<0.05), respectively, 90 minutes after beginning regular beer ingestion. On the
other hand, the plasma concentrations of xanthine increased by 5.77-fold (P<0.05), respectively, 90 minutes
after beginning low purine-containing low malt beer ingestion, though those of uric acid or hypoxanthine did
not increase. The 1-hour urinary excretion of hypoxanthine and xanthine increased by 4.31- (P<0.01), and
11.15-fold (P<0.01), respectively, when measured 2 hours after beginning regular happo-shu ingestion, though
that of uric acid or uridine did not increase. On the other hand, the 1-hour urinary excretion of xanthine
increased by 4.52-fold (P<0.05), when measured 2 hours after beginning purine-free happo-shu ingestion,
though that of uric acid, hypoxanthine, or uridine did not increase, when measured 2 hours after ingestion.
Conclusions: These results suggest that the purines contained in regular happo-shu contribute to an increase in
the plasma concentration of uric acid due to purine degradation and the increase in plasma concentration of uric
acid may be significantly less with purine-free happo-shu than with regular happo-shu. In addition, uridine
contained in regular happo-shu seems to contribute to an increase in the plasma concentration of uridine along
with ethanol.
96
P06
SLC22A12 mutation is responsible for most renal hypouricemia in Japan
K. Ichida1, M. Hosoyamada2, I. Hisatome3, H.E. Endou2, T. Hosoya1
1
Jikei University School of Medicine, TOKYO, Japan
2
Kyorin University School of Medicine, TOKYO, Japan
3
Tottori University, YONAGO, Japan
Renal hypouricemia is an inherited and heterogeneous disorder characterized by increased urate clearance
(CUA). We recently established that urate was reabsorbed via URAT1 on the apical membrane in the proximal
tubules, and that mutations in SLC22A12 encoding URAT1 cause renal hypouricemia.
We elucidated a URAT1 function for renal urate handling in vivo by examining 32 unrelated patients with
idiopathic renal hypouricemia (18 males and 14 females).
Serum urate levels of all the patients were below 2 mg/dl and averaged 0.93 ± 0.49 mg/dl (n=32). Serum urate
levels of the male and female patients were 0.77± 0.31 mg/dl (n=18) and 1.14 ± 0.61 mg/dl (n=14),
respectively. Mean CUA/Ccr (0.584 ± 0.264; n=32) and urinary urate excretion (704.6 ± 233.0 mg/day; n=31)
were increased. CUA/Ccr and urinary urate excretion of the male and those of female patients were 0.668 ±
0.202 (n=18) and 747.7 ± 242.1 mg/day (n=17), and 0.476 ± 0.301 (n=14) and 655.9 ± 226.9 mg/day (n=12),
respectively. Three male patients had exercise-induced acute renal failure (9.4%) and 4 patients two of whom
were male urolithiasis (12.5%). The frequency of exercise-induced acute renal failure in patients with renal
hypouricemia was higher than initially perceived. We identified 8 new mutations and 2 previous reported
mutations that result in loss of function. Thirty patients had SLC22A12 mutations, 24 homozygotes and 6
heterozygotes. Mutation G774A dominated SLC22A12 mutations (74.1 % in 54 alleles). Serum urate levels
were significantly lower and CUA/Ccr significantly higher in heterozygotes as compared to healthy subjects,
and these changes were even more significant in homozygotes. These CUA/Ccr relations demonstrated a gene
dosage effect, corresponding with the difference in serum urate levels. In contrast to healthy subjects, the
CUA/CCr of patients with homozygous SLC22A12 mutations was unaffected by the anti-uricosuric drug
pyrazinamide and the uricosuric drugs benzbromarone and probenecid.
Our findings indicate that URAT1 is the main reabsorptive urate transporter and SLC22A12 was responsible
for most renal hypouricemia. Furthermore, we established that pyrazinamide, benzbromarone, and probenecid
exert anti-uricosuric and uricosuric effects by acting on URAT1 in vivo.
97
P07
Pharmacokinetics/Pharmacodynamics of Y-700, A novel Xanthine Oxidase Inhibitor, in Rats and Man
I. Yamada*, A. Fukunari*, T. Osajima, M. Kamezawa, H. Mori, J. Iwane
Mitsubishi Pharma Corporation, CHIBA, Japan
Objectives
Y-700, 1-(3-Cyano-4-neopentyloxyphenyl)pyrazole-4-carboxylic acid, has been introduced as a novel
xanthine oxidase inhibitor, which bears no structural relationship to the known inhibitor, allopurinol (1). The
present study was conducted to evaluate the pharmacokinetics and pharmacological actions of Y-700 in rats
and healthy adult male volunteers.
Design and Methods
Male Sprague-Dawley rats were used for animal study. A rat model of hyperuricemia was established by
repeated treatment of the animal with the uricase inhibitor, potassium oxonate (250 mg/kg, s.c.) . Plasma uric
acid (UA) levels were determined by the phosphotungstic acid colorimetry. 14C - labelled Y-700 (504.9
kBq/mg) was used for the study of absorption, metabolism and excretion in rats, and the concentrations of
radioactivity in samples were determined by LSC or HPLC equipped with a radio-detector. In a clinical
study, single doses (5, 20 or 80 mg) of Y-700 or placebo was administered orally to healthy Japanese
volunteers. Serum UA levels were determined by the uricase method. Plasma and urinary concentrations of
Y-700 and urinary amounts of xanthine and hypoxanthine were detected by a validated HPLC method.
Results
Y-700 was absorbed rapidly in both species and was eliminated with t1/2 of 2.7-5.0 h for rats and 27.6-40.2
h for humans. In rats and humans, Cmax and AUC of oral Y-700 were increased dose-dependently.
Bioavailability was 84.1% (1 mg/kg) in rats. Only Y-700 was detected in rats and humans plasma. Total
radioactivity in urine and feces accounted for 21.8% and 79.8% of the dose, respectively. Urinary excretions
of Y-700 in rats and humans were 1.1% and 1.5%, respectively. In hyperuricemic rats, oral Y-700 (0.3 – 3
mg/kg) showed hypouricemic action in a dose-dependent manner, and was more potent and a longer lasting
than allopurinol. In humans, hypouricemic action at doses of 20 and 80 mg were statistically significantly
different from the placebo. Emax of change in serum UA level at doses 20 and 80 mg were -1.01 and 2.66
mg/dL, respectively. No direct relationship between tmax(2.2-3.0 h) and Etmax(18.7-36.0 h) was recognized.
The hypouricemic action of Y-700 was maintained throughout the post dose. Urinary excretion (0-72 h) of
xanthine and hypoxanthine were significantly increased compared with placebo group.
Discussion
The present study demonstrated that Y-700 is a new effective inhibitor of XO with a potent and a longlasting hypouricemic action in rats and healthy human volunteers. The data suggest that Y-700 is likely to
possess clinical efficacy in the patients. Another important finding was that Y-700 is a drug of non-renal
excretion type. The distinctive elimination route of Y-700 is expected to provide a beneficial property as a
new drug for the treatment of gout and hyperuricemia. In other words, unlike the case for allopurinol, it may
not be necessary to adjust the dosage of Y-700 in patients according to their degree of renal function.
(1) Ishibuchi, S.; Morimoto, H.; Oe, T.; Ikebe, T.; Inoue, H.; Fukunari, A.; Kamezawa, M.; Yamada, I.;
Naka, Y. Synthesis and structure-activity relationships of 1-phenylpyrazoles as xanthine oxidase inhibitors.
Bioorg. Med. Chem. Lett. 2001, 11, 879-882.
* These authors equally contributed to this work.
98
P08
PK/PD and safety of a single dose of TMX-67 (febuxostat) in subjects with mild and moderate renal
impairment
S. Hoshide1, Y. Takahashi1, T. Ishikawa1, J. Kubo1, M. Tsuchimoto1, K. Komoriya1, I. Ohno2, T. Hosoya2
1
Teijin Limited, TOKYO, Japan
2
Jikei University School of Medicine, TOKYO, Japan
Objective: Decreased renal urate clearance associated with renal impairment causes enhancement of
hyperuricemia. In patients with decreased renal clearance, it is well recognized that plasma oxypurinol, an
active metabolite of allopurinol, shows a prolonged half-life due to its purine-like structure. To investigate the
effects of renal impairment on PK/PD and safety of TMX-67, a novel XOD inhibitor with non-purine structure,
an open label, single dose clinical study was conducted in normal or renally compromised subjects.
Methods: Fifteen subjects (13 males/2 females) were assigned to three groups of 5 subjects based on creatinine
clearance (Ccr, mL/min); group 1: Ccr>80; group 2: 50<Ccr<79; group 3: 30<Ccr<49. TMX-67 (20 mg) were
orally administered and blood samples were collected at timed schedule after dose while urine samples were
collected for 2 hrs pre-dose and up to 48 hrs post-dose. Plasma and urinary concentrations of TMX-67, its
metabolites, uric acid and xanthine were determined by HPLC or LC-MS/MS.
Results: T1/2 of TMX-67 delayed in moderate groups, coinciding with slight increase in AUC for moderate
groups. Cmax of TMX-67 and its oxidative metabolites revealed no significant correlation with Ccr. The
maximum reduction of plasma urate attenuated slightly, but not significantly in renally compromised subjects.
Along with the delay in T1/2 of TMX-67, reduced plasma urate showed a moderate recovery to its pre-dose
level in renally compromised subjects. Simulation of plasma TMX-67 in multiple doses, 20 mg QD for 7 days,
predicted little systemic accumulation in subjects with moderate renal impairment. Five cases of adverse events
(mild) were reported in five subjects: Gastric ulcer, constipation, nausea, acute upper respiratory inflammation,
and headache.
Conclusions: T1/2 of TMX-67 slightly delayed in renal impairment, but PK simulation predicted that the delay
observed in single dose would have less impact on plasma TMX-67 in multiple doses. The present study
indicated that TMX-67 would be a safely prescribed drug to patients with mild or moderate renal impairment.
99
P09
Pharmacodynamics & pharmacokinetics of TMX-67 (febuxostat), a novel xanthine oxidase / xanthine
dehydrogenase (XOD) inhibitor, in patients with hyperuricemia
K. Komoriya1, K. Takeda1, H. Kobayashi1, J. Kubo1, M. Tsuchimoto1, T. Nakachi2, H. Yamanaka3, N.
Kamatani3
1
Teijin Limited, TOKYO, Japan
2
TOCROM, TOKYO, Japan
3
Introduction and Objective: TMX-67, a novel XOD inhibitor, is a drug under clinical development for the
treatment of hyperuricemia and gout. A study was conducted to investigate the pharmacokinetics (PK) and
pharmacodynamics (PD) as diurnal changes in the serum urate (sUA) level before and after repeated
administration of TMX-67 to hyperuricemic patients.
Methods: Ten hyperuricemic patients (sUA level 8.0 – 13.7 mg/dL) received a 10 mg once daily (QD) dose of
TMX-67 for 2 wks, followed by a 20 mg QD dose for 4 wks. On the day before TMX-67 administration (Day
-1) and the day of the last administration (Day 41), each patient was admitted to the hospital for laboratory tests
and PK/PD evaluation. Serum urate levels were determined at 0, 2, 4, 6, 8, 12, and 24 hrs. The time course of
plasma drug concentrations was also determined on the day of the last administration.
Results: Before TMX-67 administration (Day -1), diurnal changes of sUA were small (range of 8.4-9.1 mg/dL)
and the mean sUA concentration for 24 hrs (sUA0-24) was 8.7 mg/dL. After the last TMX-67 administration
(Day 41), a small mean sUA decrease from 6.0 (0 hr) to 5.5 mg/dL (6 and 8 hrs) was observed and then the
mean sUA gradually elevated to 6.4 mg/dL (24 hrs). The mean sUA0-24 was 5.8 mg/dL which corresponds to
the mean sUA value approximately 2 hrs after administration, respectively. The pharmacokinetic profiles for
TMX-67 and its oxidative metabolites were nearly the same as that in healthy volunteers in a previous phase I
study. Adverse events were minimal.
Conclusions: The pharmacodynamic profile after repeated oral administration of TMX-67 suggests that the
mean sUA 2 hrs after drug administration may accurately represent the sUA for the clinical evaluation of
efficacy of TMX-67 at a 20 mg QD dose.
100
P10
A novel point Mutation (I137t) in the Conserved 5-Phosphoribosyl-1-Pyrophosphate Binding Motif of
HPRT (HPRTJERUSALEM) in a LNS Variant
E. Zoref-Shani1, O. Sperling1, Y. Bromberg1, J. Hirsch1, S. Feinstein2, Y. Frishberg2
1
Tel-Aviv University, TEL-AVIV, Israel
2
Shaare Zedek Medical Center, JERUSALEM, Israel
We have reported before a case of partial HPRT deficiency, characterized (in fibroblast lysates) by reduced affinity of
HPRT for 5-phosphoribosyl-1-pyrophosphate (PRPP), manifested in low activity at low (physiological), but not at
high PRPP concentrations, and with normal Km for hypoxanthine [1]. Here we report that we identified in this patient
a novel point mutation (I137T) in the gene encoding HPRT. The mutation, ATT to ACT, resulting in substitution of
isoleucine to threonine, occurred at codon 137 (exon 6), which is within the polypeptide segment (130-141) encoding
the binding site for PRPP. The structure of this sequence is comprised of beta strand β6-β6’ and a turn, which is
followed immediately by the beginning of alpha helix α5. Residue 137, an isoleucine in the wild type protein,
stabilizes the kinked beta strand β6-β6’ and turn that have direct interaction with PRPP. The trajectory and
stereochemistry of the main chain in this area and its stability is clearly essential to the binding of the 5’ phosphate
moiety of the PRPP. We suggest that perturbation by substitution to threonine, a polar residue versus the apolar and
bulky isoleucine, may destabilize the trajectory of the polypeptide affecting the affinity to PRPP. Possible effects on
the binding of the base, guanine or hypoxanthine, might well be masked due to the sequential nature of substrate
binding, since it appears that the PRPP plays a role in forming the binding pocket of the base.
[1] Zoref-Shani, E., Feinstein, S., Frishberg, Y., Bromberg, Y. and Sperling, O., Kelley-Seegmiller
syndrome due to a unique variant of hypoxanthine-guanine phosphoribosyltransferase: reduced affinity
for 5-phosphoribosyl-1-pyrophosphate manifested only at low, physiological substrate concentrations.
BBA, 1500, 197-203, 2000
101
P11
Disruption in the hypoxanthine phosphoribosyl-transferase gene caused by translocation in a patient
with Lesch-Nyhan syndrome
M.M. Mizunuma1, Y. Yamada2, K. Yamada2, S. Sonta2, N. Wakamatsu2, K. Kaneko3, N. Ogasawara2, S.
Fujimori3
1
Laboratory of Purine Metabolism, TOKYO, Japan
2
Àichi Human Service Center, AICHI, Japan
3
Teikyo University School of Medicine, TOKYO, Japan
Germline mutations at hypoxanthine phosphoribosyltransferase gene (HPRT1) cause HPRT deficiency
responsible for heritable Lesch-Nyhan (LN) syndrome. More than 200 different mutations associated with LN
syndrome have been reported. Most of the mutations (~85%) are single point mutations causing missense,
nonsense and abnormal splicing, and the remaining of the mutations are concerned with large genomic
alterations such as total or partial gene deletions and duplications. However the structure of bareakpoint
junctions in large genomic alterations have been defined for only 11 mutations.
In this study, we have identified the structure of the large alteration mutation in the HPRT1 in LN patient
(HRT-25). Since the full-length HPRT1 cDNA product of HRT-25 was not amplified by RT-PCR, we
attempted to amplify cDNA using 3?RACE method. Sequencing of the PCR products by 3?RACE revealed
intron 3 (137-bp) and unknown sequence (1008-bp) in the exon 3 downstream region of HRT-25 cDNA. We
next examined for this unknown sequence using the genome database, it was shown that unknown sequence
was in agreement with the sequences 0.5 - 0.6 Mb telomere domain away from HPRT1 gene. This result
suggests that the HPRT was disrupted in the region surrounding exon 4 by translocation or inversion between
HPRT1 allele and different allele in X chromosomes. This translocation mutation is the first report for the
mutant HPRT1 associated with LNS. It is thought that the characterization of the other macro-alterations will
complete the picture of how these types of germinal mutations occur in human.
102
P12a
Mutations in the hypoxanthine guanine phosphoribosyltransferase gene (HPRT1) in Asian HPRT
deficient families
Y. Yamada, K. Yamada, S. Sonta, N. Wakamatsu, N. Ogasawara
Àichi Human Service Center, AICHI, Japan
Inherited mutation of a purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT,
MIM308000), gives rise to Lesch-Nyhan syndrome (MIM300322) or HPRT-related gout (MIM300323). We
have identified 33 mutations in 27 Japanese, 7 Korean, and 1 Indian families with the patients manifesting
different clinical phenotypes, including two rare cases in female subjects, by the analysis of all nine exons of
HPRT gene (HPRT1) from the genomic DNA and reverse transcribed mRNA using polymerase chain
reaction (PCR) technique coupled with direct sequencing. Moreover, prenatal gene diagnoses were carried
out in 12 fetuses in 9 families by PCR-RFLP method and direct sequencing using both mRNA and genomic
DNA from chorionic villi or amniotic fluid cells. In the two partial HPRT deficient patients with
hyperuricemia and gout, two missense mutations (V188A and Y195C) in the exon 8 of HPRT1 were
identified. Three independent missense mutations (Y72C, L147P, and L159E) were detected from the patient
manifested with hyperuricemia and mild neurological symptoms, such as ataxia and spasticity. In a severe
form of partial HPRT deficiency referred as Lesch-Nyan syndrome without self-mutilation, a single
nucleotide substitution (532+2T>C) resulted in splicing error and markedly decreased expression of normal
mRNA. By the analysis of HPRT1 from classical Lesch-Nyhan patients, we have defined lots of mutations, 2
nonsense mutations (R51X in 3 families and Q109X in a family), 4 missense mutations (A64P, L65P, G70E
and L78Q), 6 single base substitutions causing splicing error (27+1G>T, 28-1G>C, 318-1G>T, 403-2A>G,
533-9T>A, and 610-1G>A), 3 insertions (212insG, 330insA, and 435insTTTG), 9 deletions (50delA,
631delA, 289delGT, 316delGT, 318delAATG, 532+1delGT, 609-16del74-bp, 1-368del353-bp, and a total
gene deletion), a translocation, and an abnormal methylation. Furthermore, we have identified recently a
large genomic deletion (~15 kb) including the promoter region and the whole exon 1 of HPRT1, by the
Southern analysis and PCR methods referring the nucleotide sequences from human genome database.
103
P12b
Highly skewed X-inactivation pattern as a cause of female gout
I. Sebesta1, L. Dvorakova1, J. Hujova1, R. Dobrovolny1, L. Stolnaja1, E. Tietzeova1, M. Hrebicek1, J.A. Duley2
1
Charles University, PRAGUE, Czech Republic
2
Gouty arthritis in females is unusual and patients with such clinical presentation are suspicious for purine
genetic defects. Partial deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT), described as
Kelley-Seegmiller syndrome is an X-linked inborn error of purine metabolism associated with gout and
urolithiasis and has been reported exclusively in males. Female carriers have somatic cell mosaicism of
HPRT activity, and are healthy. Genetic studies were performed in a girl suffering from gouty arthritis of
big toe. Hyperuricemia and affected joint were found at the age of 9 years, when therapy with allopurinol
started. No evidence of neurological involvement was found. The absence of HPRT activity, together with
raised activity of adenine phosphoribosyltransferase in lysed erythrocytes and raised concentration of
nicotinamide adenine dinucleotide in erythrocytes confirmed partial HPRT deficiency at the age of 16 years.
Deficient HPRT activity was found also in the patient´s father, who presented with renal colic and gout since
the age of 18 years. The sister of the proband is healthy and her HPRT activity is within normal limits.
Mutation analysis using direct sequencing revealed that both sisters inherited from their father a previously
described mutation in the 3rd exon of HPRT gene, c.158>C (V53A). The extreme skewing of the Xinactivation was found. The symptomatic girl has predominantly active the paternal mutated chromosome,
while her asymptomatic sister has almost exclusively active maternal non-affected chromosome. In
conclusion: the point mutation and skewed X-inactivation ratio in favour of mutant allele were responsible as
genetic basis for clinical expression in this first described female patient with partial HPRT deficiency. This
condition should be taken to differential diagnosis of unexplained hyperuricemia in girls or women.
(Supported by grants MSM-11110005 of the Czech Ministry of Education and NE 6557-3-01 of the Czech
Ministry Health ).
104
P13
Are allopurinol and metabolites found in HPRT deficient erythrocytes responsible for increased NAD
synthesis?
V. Micheli, G. Jacomelli, S. Sestini, L. Notarantonio, B. Cerboni, L. Peruzzi, G. Pompucci
University of Siena, SIENA, Italy
INTRODUCTION: Inborn deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) causes
hyperuricemia, gout and different degrees of neurological disturbances, the most serious of which is LeschNyhan Syndrome [1]. HPRT-deficient erythrocytes present associated biochemical alterations: GTP depletion,
increased concentrations of ZTP, phosphoribosylpyrophosphate (PPribP), NAD and UDP sugars [2], increased
activity of adenine phosphoribosyltransferase (APRT), IMPdehydrogenase, UMPsynthetase [3], 5’nucleotidase
[4], nicotinate phosphoribosyltransferase (NAPRT) and NAD synthetase (NADs) [5,6].
Aim of this study was to ascertain whether allopurinol, usually administered to such patients, and its metabolite
oxypurinol, or metabolites abnormally increased in HPRT deficient erythrocytes (NAD, PPribP) could directly
affect NAPRT and NADs in normal erythrocytes and thus be responsible for their increase in patients. Since
allopurinol is known to affect orotate phosphoribosyltransferase (OPRT)- OMPdecarboxylase (ODC) activities
[3], these enzymes were used as control.
MATERIALS AND METHODS: Specific activities of NAPRT, NADs, APRT, HPRT, OPRT and ODC were
measured in erythrocyte lysates by previously described HPLC-linked methods [5,6] in experimental
conditions mimicking the metabolic situation of HPRT deficient erythrocytes. Conditions included addition of
allopurinol, oxypurinol, NAD, PPribP to the assay mixtures; incubation of erythrocytes before lysis with
PPribP generating medium either alone or plus nicotinate (increasing NAD production), plus allopurinol or
oxypurinol. The endogenous nucleotide pattern was also measured in pre-incubated erythrocytes.
RESULTS: No remarkable increase of NAPRT and NADs activities in the above conditions mimiking HPRT
deficient erythrocytes was found. Direct effect by the mentioned metabolites was ruled out (only a mild
protection of PPribP on NAPRT activity). Nucleotide pattern did not show any alteration following incubations
(except for NAD increase with NA, and for respective nucleoside monophosphates appearing with allopurinol
or oxypurinol), nor were enzyme activities increased, except OPRT and ODC as expected after allopurinol and
oxypurinol incubation. Data were confirmed in allopurinol-treated patients.
1) M.Lesch , W.L. Nyhan, Am. J. Med., 36 561-570 (1970)
2) H.A. Simmonds et al., Clin. Chim Acta 171 197-210 (1988)
3) T.D. Beardmore et al. J.Lab.Clin. Med., 81 43-52 (1973)
4) R.Pesi et al. Neuroreports, 11,9, 173-177 (2000)
5) V.Micheli et al., Life Sci, 64,26,2479-2487 (1999)
6) V.Micheli et al., Bioch.Biophys Acta,1587, 45-52 (2002)
105
P14
Effects of hypoxanthine on adenosine transport in human lymphocytes. Implications in the pathogenesis
of Lesch-Nyhan syndrome
Introduction: We postulate that hypoxanthine excess could be implicated in the pathogenesis of the
neurological symptoms of Lesch-Nyhan patients. This hypoxanthine excess could affect adenosine function by
altering adenosine transport or adenosine receptor binding.
The purpose of this study is to examine the effect of hypoxanthine on adenosine transport and receptormediated adenosine action in human cells. To this approach, we have studied the effect of hypoxanthine on
adenosine transport in peripheral blood lymphocytes (PBL) cultures.
Methods: [2-3H] adenosine transport, and [3H] NBTI binding assays were determined in basal conditions and
after 24h incubation with hypoxanthine at different concentrations.
Results:
1. Hypoxanthine at concentrations ranging from 1 to 50 uM concentrations caused a dose-dependent decrease
on adenosine transport after 24 hours incubation-time.
2. Hypoxanthine at 25 uM originates a significant decrease, versus control cells, of the Vmax for the adenosine
transport in PBL cultures (9 +- 0,11 vs. 19 +- 0,5 pmol/106 cells/min; p<0.001).
3. Hypoxanthine originates a dose-dependent reduction, in the number of [3H] NBTI binding sites per cell in
PBL cultures.
4. Hypoxanthine at 25 uM originates a significant decrease of the Bmax for NBTI with respect to their
respective control cells (7,880 +- 322 vs. 9873 +- 404 high affinity sites per cell; p<0.001) in PBL cultures.
Conclusions: This study shows that an excess of hypoxanthine reduces adenosine transport in cultured PBL.
106
P15
EPR spin trapping of a radical intermediate in the urate oxidase reaction
E. Marinello, E. Busi, L. Terzuoli, R. Basosi, B. Porcelli
Urate oxidase is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin, occupying a pivotal
position in the chain of enzymes responsible for the metabolism of purines. The end product of purine
metabolism varies from species to species. In bacteria and some marine invertebrates, purines are degraded via
uric acid, allantoin, allantoic acid, and urea, and then to ammonia and carbon dioxide. With the phylogenetic
evolution, the chain of reactions necessary to convert purines to their final metabolic products has become
progressively truncated through successive loss of urease, allantoicase, allantoinase, and urate oxidase. Most
mammals are known to contain urate oxidase and thus excrete allantoin as the end product of purine
metabolism, whereas man, arthropoid apes, and some genera of New World monkeys remain the only
mammals which lack this enzyme, and as a consequence excrete uric acid as the end product of purine
metabolism. Uric acid is also the end product of purine metabolism in birds and some reptiles. The uricase has
often been classified as a copper-dependent enzyme, but in bacteria, yeast, fungi and bovine liver is free of
copper. Transitory intermediates have been reported to occur during the uricase reaction. The proposed
mechanism assumes an oxidation by O2 during the course of the enzyme-catalysed reaction and the evolution
of CO2 during the decay of the intermediate. The chemical mechanism of the urate oxidase reaction has not
been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPRspectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual
presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (hog liver,
porcine liver, Bacillus Fastidiosus, Candida Utilitis), was performed in the presence of DEPMPO (5diethoxyphosphoryl-5-methyl-pyrroline-N-oxide) as spin trap. DEPMPO was added to reaction mixture and a
radical adduct was observed in all cases. The generation of an urate radical, using Fe(II) and H2O2, in the
presence of DEPMPO, gave the same radical adduct. Therefore, for the first time, the presence of a radical
intermediate in the uricase reaction was experimentally proved.
107
P16
Cognitive abilities in a series of patients with Hypoxanthine guanine phosphoribosyltransferase
deficiency
A. Verdu1, R.J. Torres2, I. De Antonio2, J.G. Puig2
1
Hospital Virgen de La Salud, TOLEDO, Spain
2
OBJECTIVE
To estimate cognitive abilities in a series of 12 children with deficiency of hypoxanthine guanine
phsophoribosyltranseferase
METHODS
Estimates of some aspects of cognition by the use of 1. Standardized IQ test (Kaufman Brief Intelligence
Test,K-BIT), 2. Standardized Test of Non Verbal Intelligence based in abstrac/visual reasoning (TONI-2), 3. A
questionnaire designed to systematize caregiver observations. Age range form 7 years 2 months to 17 years 6
months. Each subject tested in 3 sessions of 30-40 minutes.
RESULTS
The mean estimated IQ (K-BIT) was 53.4 (range 43-78). The mean IQ as measured by TONI-2 was 83.7 (range
72-110). Questionnaire:-Good range of self-awareness, sociability, alertness, orientation, emotional reactions
and memory for past events,
-Mild to moderate impairment for short term memory, -Moderate
impairment for attention/concentration.
CONCLUSIONS
1. Most of these subjects show a mild to moderate cognitive impairment. There seems to be a typical pattern of
weakness and strenghts (verbal reasoning more affected than visual/abstract reasoning). 2. Mental retardation is
not invariable as some subjects show a low average intelligence. 3. All subjects showed deficient
attention/concentration and self-abusive tendencies that interfered with the testing process. 4. The social and
emotional behaviour of patients with Lesch-Nyhan disease suggests that cognitive abilities may be better than
showed by standardized tests.
108
P17
The motor features of Lesch-Nyhan disease
J.E. Visser1, S.G. Reich2, J.C. Harris2, G. Barabas3, G.E. Eddey3, H.A. Jinnah2
1
UMC St Radboud, NIJMEGEN, The Netherlands
2
3
Matheny School and Hospital, PEAPACK, NJ, United States of America
Introduction.
Deficiency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) causes Lesch-Nyhan disease (LND),
characterized by hyperuricemia and a neurobehavioral phenotype including self-injurious behavior, cognitive
dysfunction, and motor disability. Although the genetic, metabolic and behavioral aspects of LND have been
addressed in several studies, the nature of the motor disorder has received less attention.
Many reports describe LND as choreoathetosis with spasticity. However, other reports describe dystonia with
hypotonia. These marked differences in the published literature might arise from phenotypic variability,
temporal variability due to disease progression, or varied use of descriptive terminology. To distinguish among
these possibilities, we conducted a prospective evaluation of the motor disorder in our patients.
Methods.
The patient population consisted of 19 cases ranging from 6-40 years of age. The diagnosis was based on
HPRT enzyme activity <1% with typical hyperuricemia, self-injurious behavior, and motor disability. Variant
cases with higher levels of residual HPRT and those without self-injury were excluded. A detailed neurological
examination was conducted by a specialist in motor disorders and videotaped for review by a second examiner.
Descriptive terminology was applied according to established guidelines, with a 4-point severity scale
(0=absent, 1=mild, 2=moderate, 3=severe).
Results.
Dystonia was the most common and most severe motor feature in all 19 cases (average score, 3.0±0.0). Chorea
was observed in 13 cases and it was generally not severe (0.8±0.7). Ballismus was observed in 7 cases, again
not severe (0.4±0.6). Hypotonia was evident in 16 cases. Spasticity occurred in only 5 cases and was generally
mild (03±0.6). In most of these cases, spasticity of the legs or one arm was combined with hypotonia
elsewhere. Mild rigidity occurred in 2 cases. True cerebellar ataxia was not observed.
Conclusions.
The motor syndrome of LND is best described as an action dyastonia superimposed upon hypotonia. Although
other extrapyramidal signs such as chorea and ballismus may occur, these are less frequent and less severe than
dystonia. True spasticity is not common; when it occurs it is often asymmetric or limited to the legs in a pattern
suggestive of myelopathy. This motor syndrome is consistent with dysfunction of the basal ganglia.
109
P20a
Lesch Nyhan Disease, essentials of diagnosis and management - a European perspective
G.T. McCarthy1, H.A. Simmonds2, L.D. Fairbanks2
1
Chailey Heritage Clinical Services, NR LEWES, EAST SUSSEX, United Kingdom
2
The purposes of this presentation are to describe the essential diagnostic features and investigations of this very
rare condition; to share methods of management, which have been found to be effective; to promote the need
for support between families; and to outline research in progress; finally to promote the benefits of pooling data
across Europe via the internet.
WHAT IS LESCH NYHAN DISEASE? A description of LND will introduce the poster followed by
presentation of the condition over time DIAGNOSIS the essential diagnostic investigations will be explained
and problems which can mask the diagnosis
GENETIC INFORMATION
This includes X linkage, carrier detection using DNA probes, pre-natal diagnosis. genetic counselling
MANAGEMENT OF THIS COMPLEX CONDITION
Motor Disorder
Speech and Communication
Management of self-injurious behaviour - psychological management including parental understanding of
stress management; intellectual development Restraints
Medical Problems associated– swallowing, hiatus hernia, nutrition, seizures, apnoeic attacks; dietary
management.
Medical Treatment
Allopurinol medication and the need for regular metabolic and renal monitoring will be explained.
RECENT RESEARCH ADVANCES: i) imaging techniques, ii) investigation of neuronal development in
HPRT deficiency, iii) studies of self-injurious behaviour in young children with LNS
EUROPEAN PERSPECTIVE
Princess Margaret Nucleotide Metabolism Centre
Why is a collaborative approach essential?
FAMILY SUPPORT NETWORKS: - sharing information across Europe i). The Purine Metabolic Patients
Association (PUMPA) in the United Kingdom; ii) Baschirotti Institute Italy, Paola Cargiolli; iii) LNS Francais.
110
P20b
Nucleotide degradation products in cerebrospinal fluid (CSF) in inherited and acquired pathologies
H.A. Simmonds1, L.D. Fairbanks1, J.A. Harris2, J.A. Duley1
1
2
CSF nucleotide degradation products have been reported in neurologiclal disorders, meningitis, renal failure
and hypoxia. The present study, using RPLC, compares concentrations in patients genetically deficient in
adenylosuccinase (ADSL), purine nucleoside phosphorylase (PNP), Lesch-Nyhan Disease (LND:
hypoxanthinephosphoriboxyltransferase deficiency HPRT) and its variants (the majority on allopurinol),
with neurological controls and in tuberculous meningitis.
The results a) confirm grossly elevated concentrations of SAICAR 314µmol/l (control mean <0.01) and
SAdo 401µmol/l (control 0.9) in ADSL, but normal hypoxanthine and xanthine; b) demonstrate the absence
of purine bases in PNP, inosine replacing hypoxanthine in concentrations comparable to hypopcanthine in
LND, but concentrations of guanosine are 3 fold that of xanthine in untreated LND; c) confirm hypoxanthine
alone is grossly elevated in LND not on allopurinol. Although hypoxanthine and xanthine are elevated by
allopurinol, concentrations are not different between classic LND and partial variants and oxipurinol is
present in all on allopurinol; d) show grossly elevated uric acid and its precursors (xanthine, hypoxanthine
and inosine) in tuberculous meningitis.
Although CSF purines in these genetic disorders are elevated with the defect, as in plasma, concentrations of
uric acid are uniformally low compared with plasma. (except PNP - virtually absent in both). This finding,
together with the presence of oxipurinol in allopurinol-trested LND patients, indicates a limited ability of
plasma components to cross the normal blood-brain barrier and confirms the absence of functional xanthine
dehydrogenase in human brain. The elevated hypoxanthine relative to xanthine in untreated LND illustrates
the normal recycling of hypoxanthine via HPRT to ATP in human brain. The higher CSF guanosine in PNP
contrasts with the inosine/guanosine ratio in plasma and suggests the turnover of GTP is high. However, the
normal xanthine in LND argues against this. The finding of detectable amounts of SAICAR as well as SAdo
only in LND, accords with some increased activity of the synthetic route in the absence of hypoxanthine
salvage via HPRT. The elevated uric acid plus precursor purines in tuberculous meningitis confirms severe
damage to the normal blood-brain barrier.
111
P21
Genotype and clinical features of adenine phosphoribosyltransferase (APRT) deficiency in French
families
I. Ceballos-Picot, S. Chevaliez, H. Lassal, D.M. Daudon, P. Perignon
University of Paris, PARIS, France
The purpose of our study was to characterize the clinical, diagnostic, and pronostic features of complete
adenine phosphoribosyltransferase (APRT) deficiency in french patients, as well as to determine their
genotype. Twenty one individuals with complete APRT deficiency (type I), (diagnosed by enzymatic APRT
activity determination in erythrocytes and analysis of urinary calculi or crystals by infrared spectroscopy) were
identified in 18 families from 1990 to 2003. There were 13 females and 8 males, and the median age at
diagnosis was 28 years (range 1 to 72 years). 60% of cases were adults, suggesting that total APRT deficiency
is not essentially a disease of children as was once thought. However the time from onset to correct diagnosis
was usually much longer in adults. Eighteen patients were index cases and three asymptomatic adult patients
were diagnosed during screening of first-degree relatives. This screening is important because an effective
treatment with allopurinol is available if the condition is diagnosed early and the prognosis depends on renal
function remaining at the time of diagnosis. The eighteen index cases had symptomatic disease. 2,8dihydroxyadenine (DHA) urolithiasis led to mild and moderate renal insufficiency for 15 patients; 3 patients
had advanced renal failure. The analysis of genomic alteration performed on ten patients, by polymerase chain
reaction-based mutation analysis, revealed for six patients a T insertion at the exon4/intron4 junction
(1833insT) resulting in the lack of exon 4 in the APRT mRNA or a A/T substitution at this junction (1833+1AT. The other mutations are located in exon 2 (295C-G), exon 3 (1422G-A and 1444delCT) and exon 5
(2079insA; 2087T-C; 2176delTTC). These results showed a mutational hot spot at the intron 4 splice donor site
(probably resulting in the lack of exon 4 in the APRT mRNA) in French patients which was also detected in a
variety of Caucasian patients throughout Europe.
Supported by EC project BMH4-CT98-3079
112
P22
Adenylosuccinate lyase deficiency: study of physiopathologic mechanism(s)
V. Race, S. Marie, G. Van den Berghe, M.F. Vincent
Adenylosuccinate lyase (ADSL) is an enzyme catalysing two distinct reactions in purine nucleotide synthesis.
ADSL deficiency, first described in 1984, is characterised by the accumulation, in cerebrospinal fluid (CSF)
and urine, of succinylaminoimidazolecarboxamide (SAICA) riboside and succinyladenosine (S-ado), the
dephosphorylated derivatives of the two substrates of the enzyme, SAICA-ribotide and adenylosuccinate
(SAMP). The symptoms of the disease are exclusively neurologic and the affected children display variable but
mostly profound psychomotor retardation, often accompanied by epilepsy and autism.
This work aims at unraveling the physiopathologic mechanism(s) leading to the selectively neurological
symptoms. We investigated two major hypotheses: (i) a deficiency in the terminal products of the de novo
pathway and (ii) a toxic effect of the succinylpurines (SAICAriboside and S-ado) which accumulate in huge
amounts in CSF.
To test the first hypothesis, we measured intracellular nucleotide concentrations in skin fibroblasts from 7
severely and 1 mildly retarded patients. Since these fibroblasts still exhibit a residual ADSL activity, we
rendered them more dependent on de novo synthesis by abolishing the purine salvage pathway. The
intracellular concentrations of adenylic, guanylic and uridylic nucleotides were measured by HPLC: no
significant differences could be detected as compared to control cells. An exception was an accumulation of
SAMP which was only detected in the fibroblasts from the patient with a near complete loss of activity toward
SAMP, who is paradoxically mildly retarded.
We next considered a possible neurotoxic effect of the succinylpurines. This effect was evaluated on the
viability of rat embryonic cortical neurones using the MTT test. Neither SAICAriboside nor S-ado affected cell
viability. The possibility of an interaction between the succinylpurines and the ionotropic glutamate NMDA
receptor was excluded by the facts that they did not affect (i) glutamate toxicity (ii) binding of MK801 on the
NMDA receptor (iii) calcium movements inside the cell. Until now, our results show that an explanation
regarding the cause of the neurological manifestations in ADSL deficiency remains elusive.
113
P23
Adenylosuccinate lyase deficiency - first UK case
A.M. Marinaki1, M. Champion1, M.A. Kurian1, H.A. Simmonds1, S. Marie2, M.F. Vincent2, G. Van den
Berghe2, J.A. Duley3, L.D. Fairbanks1
1
2
3
Adenylosuccinate lyase (ASDL) catalyses two steps in purine synthesis - the 8th step in the de novo pathway:
conversion of SAICAR (succinylaminoimidazole-carboxamide ribotide) to AICAR (aminoimidazolecarboxamide ribotide), and conversion of S-AMP (adenylosuccinate) to AMP (adenylate) in the purine
nucleotide cycle. SAICAriboside (SAICAr) and S-Ado(succinyladenosine) accumulation occurs when this
enzyme is deficient. Clinical presentations vary from mild psychomotor retardation to epilepsy with
convulsions starting within the newborn period. The lower the ratio of S-Ado/SAICAr in body fluids the more
severe the clinical presentation.
The female patient presented on day 14 with a progressive neonatal encephalopathy and seizures (myclonus
and generalised tonic clonic events). Neurological examination revealed marked axial and peripheral
hypotonia, MRI showed widespread white matter changes. She died at 4 weeks of age.
Analysis of urine by HPLC showed SAICAr and S-Ado present in large quantities (S-Ado 0.925mmol/l :
SAICAr 1.325mmol/l (ratio 0.7), correlating with the severe clinical picture in the patient. Further investigation
detected these compounds in the plasma (34µmol/l/27µmol/l) and CSF(921µmol/l/477µmol/l) respectively.
Fibroblast cultures were grown, and the molecular analysis undertaken. cDNA analysis showed an 9G>C
transition resulting in a A3P amino acid substitution. Sequencing of genomic DNA showed the patient was
heterozygous for this mutation. The patient is thus a compound heterozygote with this novel 9G>C mutation on
one allele and a second, still unknown, mutation on the other. It is possible that mRNA expressed from the
second allele is unstable.
This is the first case of ADSL to be reported in the UK. In a previous study 10,000 urines, from a selected
population of UK children with undiagnosed neurological deficits, were screened for SAICAr and S-Ado and
no cases were detected. Thus, ADSL deficiency is very rare in the UK.
114
P24
Elevated erythrocyte CDP-choline levels associated with b-thalassaemia in patients with transfusion
independent anaemia
A.D.J. Laurence1, M. Layton2, J.A. Duley3, H.A. Simmonds3
University College Hospital London, LONDON, United Kingdom
2
3
1
Haemolytic anaemia with basophilic stippling is characteristic of pyrimidine 5’-nucleotodase (UMPH1)
deficiency (1). The selective accumulation of CDP-choline in high concentrations in the erythrocytes of a
patient with haemolytic anaemia and a normal UMPH1 activity was first reported by Paglia et al (1). A defect
in CDP-choline phosphotransferase which catalyses the last stage of lecithin biosynthesis was suggested. The
selective accumulation of CDP-choline is thought to occur in the erythroblast as CDP-choline
phosphotransferase has been shown to be inactive in the mature erythrocyte. The accumulation of CDPethanolamine as well as CDP-choline in a small cohort of patients with normal UMPH1 activity and either
unexplained haemolytic anaemia, or secondary to chronic renal failure, led us to postulate the existence of a
defect in both phosphotransferases. Correction of the haematological profile following successful renal
transplantation in one renal failure patient implied possible heterozygosity for this defect (2)
Here we report a series of ten patients with transfusion independent b-thalassaemia; eight heterozygote and two
heterozygote with Hb E, which also show elevated levels of both CDP-choline (86.4uM +/- 48µM) and CDPethanolamine (34.6µM +/- 34.5µM). This compares with levels of less than 3µM in healthy controls. In
contrast to the patients previously reported with raised CDP-choline/ethanolamine associated with no other
cause of their anaemia, the patients in this report all had CDP-choline levels in excess of their CDPethanolamine (p=0.002).
In summary elevated CDP-choline in patients with no defined cause for their haemolytic anaemia. has been
suggested as a possible indicator of CDP-choline phosphotransferase deficiency Here we associate it with
transfusion independent b-thalassaemia.
(1) Paglia DE, Valentine WN, Nakatani M, Rauth B (1983) Proc Natl Acad Sci 80;3081-3085.
(2) Laurence A, Duley JA, Simmonds HA (1998) Adv Exp Med Biol 155-159
115
P25
Nucleotides can afford protection in cirrhosis induced by thioacetamide
S. Pawa
Jamia Hamdard University, NEW DELHI, India
The present work was intended to study nucleotides as anti-hepatotoxic agent against thioacetamide (TAA)induced cirrhosis in Wistar rats. The liver pathology of the Wistar rats treated chronically with TAA closely
mimics that of human disease Cirrhosis was induced by oral intake of TAA in drinking water (300 mg/l) for
four months. Nucleotides pretreatment significantly decreased lipid peroxidation, which is an important index
of oxidative stress. Thioacetamide caused decrease in the activities of peroxide-metabolizing enzymes
(glutathione peroxidase, and catalase), and a noteworthy increase in the activity of xanthine oxidase (XO). XO
catalyzes the hydroxylation of a wide variety of purines, pyrimidines, and nitrogenous compounds and
generates reactive oxygen species as by-product. Pretreatment with nucleotides resulted in attenuation of
biochemical parameters associated with TAA-induced cirrhosis. Appreciable regeneration of the injured liver
was also seen in nucleotide-pretreated group. The mechanism by which these nucleotides influence tissue repair
is unclear, however, it can be hypothesized that it may manifest its effect via xanthine oxidase pathway.
Increase in the activity of XO by TAA results in significant depletion in purines and pyrimidines, thereby
aggravating tissue damage. Supplementation with nucleotides replenishes the loss caused by TAA, thereby
acting as potential hepatoprotectant.
116
P26
Serum Folate Level in Mozambique's Children
E. Marinello1, R. Leoncini1, R. Guerranti1, G. Cinci1, A. Tabucchi1, F. Carlucci1, D. Vannoni1, A.B. Agostinho2
1
2
National Institute of Saude, MAPUTO, Mozambique
Folate and vitamin B12 deficiencies are two unequivocal nutritional anemias in man. Folate is essential for
normal development of red blood cells and is essential, as coenzyme, in nucleotide metabolism, DNA and RNA
synthesis. It is involved in protein metabolism as well as tissue growth and cell function. It has been used in the
prevention and treatment of folic acid anemia. Measurements of folate levels constitutes a direct and reliable
means of determining the existence of folate deficiency. Vitamin B12 is essential for normal folic acid
metabolism; it helps the formation of red blood cells and the maintenance of central nervous system. The
deficiency or incapacity of absorption caused the disease known as pernicious anemia.
We have made a study on seric Folate/Vitamin B12 content in children frequenting schools of Maputo
(Mozambique). The children were healthy and subjected to complete blood count and many different tests
(glycemia, cholesterol, Ca, Mg, total protein, albumin, Fe); also weight and height were registered. The subjects
were 173 (age ranging between 6 and 16 years) subdivided in 80 males and 93 females. No analytical
parameter showed statistical difference related to sex for this reason we have studied the children as an unique
group.
The values that we have found for serum folate and vitamin B12 are respectively: 6.32pg/ml ± 3.47 and
782.70ng/ml ± 573.07.
All the data have passed the Kolmogorov-Smirnov test for normal distribution.
The analysis of correlation between various analyte has shown an interesting relationship between folate and
platelet content. The equation describing this relationship is: [folate]= 0.01573[platelet]+3.574 with p<0.0001
(extremely significant). Also MVC and HCT show an extremely significant positive correlation with folate
content.
These data are not underlined in previous papers in literature and could reflect a stimulating and specific effect
of folate on cell proliferation and could be an interesting approach to study the nucleotide metabolism in these
subjects, and specifically in their megakaryocytes. The same study could be replicated in Italian scholar
population.
117
P27
Deoxyuridine accumulation in urine in thymidine phosphorylase deficiency (MNGIE)
A.M. Marinaki1, J.A. Duley2, E.A. Carrey1, S.R. Hammans3, L.D. Fairbanks1
1
2
3
Southampton General Hospital, SOUTHAMPTON, United Kingdom
INTRODUCTION. The autosomal recessive condition mitochondrial neurogastrointestinal encephalomyopathy
(MNGIE) is caused by a deficiency of thymidine phosphorylase (TP) and is characterised by mtDNA multiple
deletions and depletion. TP catalyses the catabolic conversion of thymidine to thymine. Thymidine accumulates
in the urine of TP deficient patients and is a diagnostic marker for the disorder. We report the presence of the
unusual nucleoside deoxyuridine in the urine of a patient with MNGIE due to TP deficiency.
RESULTS. A random urine specimen from a previously described female MNGIE patient now 38 years old
(Science 1999, 283: 689-92) was fractionated by anion exchange LC prior to reversed phase HPLC. An
unknown peak co-eluting with uric acid, visible after fractionation or incubation with uricase, was identified as
deoxyuridine (150 µmol/mmol creat, normally not detected), by retention time, shared spectral characteristics
to an authentic deoxyuridine standard and co-elution of the peak with an added standard. Thymidine
(125 µmol/mmol creat, normally not detected), uracil (77 µmol/mmol creat, control range 1.4-15.4 µmol/mmol
creat), and thymine (48 µmol/mmol creat, normally not detected) were also elevated. Interestingly, uric acid
production on a creatinine basis was grossly raised (0.8 µmmol/mmol creat, normal range 0.3+/- 0.05), but may
be a consequence of cachexia since the patient studied was severely underweight.
DISCUSSION. Our results confirm that thymidine is a useful marker in screening for TP deficiency. However,
raised uracil and thymine are also found in dihydropyrimidine dehydrogenase and dihydropyrimidinase
deficiencies. The presence of deoxyuridine in urine of TP deficient patients thus provides a second unique
metabolic marker for MNGIE.
We propose a mechanism of increased intracellular thymidine pools leading to the synthesis and accumulation
of the nucleotide TMP. Thymidylate synthetase is inhibited by raised levels of TMP, resulting in the
accumulation of dUMP. We suggest that dUMP then has two metabolic fates: degradation by nucleotidase to
deoxyuridine and excretion in the urine; or phosphorylation to dUTP. Incorporation of dUTP into mtDNA
would be consistent with the pattern of mtDNA deletion and depletion characteristic of TP deficiency.
118
P28
The Genetic Basis of the Interaction Between Pyrimidine 5' Nucleotidase deficiency and
Hemoglobin E
E. Escuredo1, J.A. Duley2, D.C. Rees3, A.M. Marinaki1
1
2
3
King's College Hospital, LONDON, United Kingdom
INTRODUCTION. Hemoglobin E is a hemoglobin variant, occurring with a polymorphic prevalence
throughout Southeast Asia and Northeast India. We have previously described a family in which an interaction
between a biochemical deficiency of pyrimidine 5’ nucleotidase (P5’N-1) deficiency and HbE resulted in an
unexpectedly severe anemia with features of hemolysis and dyserythropoiesis. In this study we explored the
genetic basis of the severe clinical phenotype.
RESULTS. A novel P5’N-1 splice site mutation (IVS8+1-2 delGT) was found in the family by genomic
sequencing of the P5’N-1 gene, and is predicted to result in skipping of exon 8 and premature termination of
the protein. A homozygous IVS8+1-2 delGT genotype correlated with a severe phenotype in the propositus
when a homozygous HbE genotype was co-inherited. However, the phenotype of HbE trait appeared to be
unchanged by the co-inheritance of a single P5’N-1 mutant allele. The ratio of P5’N-1 activity to that of
deoxypyrimidine nucleotidase (P5’N-2) in HbE heterozygous and homozygous patients with a normal P5’N-1
genotype, were decreased (P5’N-1/P5’N-2 0.24-0.55, controls >0.7).
Pyrimidine metabolism was also studied in the erythrocytes of 6 patients with HbE/beta-thalassemia. The
compound heterozygous genotype, HbE/beta-thalassemia is an important cause of significant thalassemia,
resulting in a variable, often severe condition. The variation in clinical phenotype is largely unexplained and it
is possible hemoglobin instability may be exacerbated by acquired or genetic deficiency of P5'N-1. Inhibition
of P5’N-1 relative to P5’N-2 was variable (range 0.39-0.97). Although there was a trend towards lower enzyme
ratios with decreasing Hb and an increasing percentage of HbF, this was not significant.
CONCLUSION. The severe phenotype seen in the propositus in this study consequent on the two genetic
defects raises the possibility that acquired or inherited variation in P5'N-1 activity may modulate the severity of
HbE and other beta-thalassemia syndromes.
119
P29
Purine nucleoside phosphorylase deficiency: Is there a mitochondrial pathology?
A.M. Marinaki1, J.A. Duley2, A.R. Gennery3, L.D. Fairbanks1
1
2
3
Newcastle Upon Tyne NHS Trust, NEWCASTLE, United Kingdom
INTRODUCTION. Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive disorder of
purine metabolism. PNP-deficient children present early with a range of neuromuscular problems including
spastic diplegia, tetraparesis, ataxia, hypertonia or hypotonia, tremor, behavioural problems, varying degrees of
mental retardation, and are typically diagnosed as cerebral palsy. Neurological symptoms often precede
immunodeficiency due to severe T-cell depletion and dysfunction. B-cell dysfunction may lead to
autoimmunity. Biochemically, the disorder is characterised by low plasma uric acid and gross purine
overproduction in the form of (deoxy-)nucleosides in the urine. Red cell nucleotide abnormalities include
accumulation of dGTP with low GTP, and raised NAD.
FAMILY STUDY: The first child (male) presented with an autoimmune haemolytic anaemia and was
diagnosed as PNP deficient at 2 years. He died post-BMT of gut graft-versus-host disease. The propositus
(female) was the second child in the family. A prenatal diagnosis of an affected child was made at 12 weeks,
but the mother elected to continue with the pregnancy as a suitable bone marrow donor had been identified. The
child was transplanted at 2 months and received immunosuppressive therapy (CyA and high dose methylprednisolone). Neurologically, the child appears well and developmentally normal, active with eye contact and
smiling. She is mildly floppy but without clear evidence of neuromuscular disorder as at 8 months age.
However, at 3 months post-BMT she developed a large pericardial effusion and hypertrophic obstructive
cardiomyopathy (HOCM), very rare in a child this age, was diagnosed. This has been accompanied by
pneumonitis, refluxing and aspiration difficulties which will require fundoplication.
MOLECULAR STUDIES. Sequencing of PNP cDNA showed a homozygous 383A>G transition,
heterozygous in the parents, resulting in a Asp128 to Gly substitution. This mutation has been previously
reported in PNP deficiency
CONCLUSION. Studies of a PNP deficient mouse model suggest that the accumulation of dGTP in
mitochondria results in abnormal mtDNA repair. This raises the intriguing possibility that the cardiomyopathy
in the propositis may have a mitochondrial pathogenesis linked to PNP deficiency through an imbalance in
nucleotide pools.
120
P30a
Urinary methylxanthine and autistic disorder: absence of previously reported correlation
C. Salerno
Although the exact prevalence of biochemical defects in autism is unknown, inherited disorders on purine
biosynthesis and interconversion, such as adenylosuccinate lyase deficiency and 5'-nucleotidase
superactivity, have been often associated with autistic symptoms, focusing the attention on the metabolic fate
of endogenous and exogenous purines in patients with behaviour abnormalities. Great expectations in
patient's families arose from recent reports [1, 2] that 7-methylxanthine levels in urine samples are greatly
decreased in a significant fraction (47%) of autistic children, while urinary xanthine levels are increased.
Consequently, it was proposed that determination of methylxanthine and xanthine in the urine has a
diagnostic value for autism and that methylxanthine could be used to treat individuals exhibiting symptoms
of autistic disorder. We developed a HPLC method for separation of urinary uric acid, xanthine,
hypoxanthine, and most of the methylxantine metabolites (caffeine, theophylline, theobromine, 1methylxanthine, 1-methylurate, 3-methylxanthine, 3-methylurate, 7-methylxanthine, 7-methylurate). Urinary
levels of methyxanthines were strictly dependent on the diet. Theobromine, 7-methylxanthine, 7methylurate, and 2-methylxanthine were identified in the urine upon administration of chocolate.
Comparison of data obtained with 59 autistic patients and 64 age- and sex-matched normal volunteers did
not reveal any difference in the levels of xanthine and methylxanthine derivatives in the two groups. In light
of these findings, it appears that abnormalities in methylxanthine and xanthine excretion cannot be used to
discriminate autistic disorders and represent distinctly uncommon symptoms in autism.
[1] Zhang J, Rivers G, Peyser J, Zack B, Horvath K, Rushe J (2000) Analysis of compounds in the urine of
autistic children with HPLC and mass spectrometry. World Congress of Pediatric Gastroenterology, Boston
[2] Rusche JR, Zhang J (2002) Methylxanthines in the diagnosis and treatment of autistic disorder. US Patent
Application 20020019407 A1
121
P30b
Novel mutations and a 'hot-spot' in Purine Nucleoside Phosphorylase deficient patients
E.G. Grunebaum, J. Zhang, C.M. Roifman
Hospital for Sick Children, TORONTO, Canada
The lack of purine nucleoside phosphorylase (PNP) enzyme activity in human and in mice results in thymus
abnormalities and T lymphocyte dysfunction. Patients with PNP deficiency typically suffer from recurrent
infections, neurologic impairment and autoimmune phenomena. Bone marrow transplantation is available for
some of these patients and ongoing research in gene therapy may provide an alternative approach, particularly
if the transformed cells have a survival advantage over the patient’s naïve cells.
The laboratory findings of lymphopenia, reduced serum uric acid and abnormal PNP enzyme activity assist in
the diagnosis of PNP-deficient patients, which is further confirmed by genetic analysis. Since the identification
of the gene coding for the PNP protein, several mutations have been found. Here we report 3 novel mutations in
3 unrelated patients recently diagnosed at the Canadian Center for Primary Immunodeficiency Diseases as
suffering from PNP deficiency. These include a G348A, C700T and C769G, resulting in putative Ala117Thr,
His257Asp and Arg234Stop, respectively. Two of these patients also had a C to T transition at codon 172,
which leads to a premature stop at amino acid 57. Combined with mutations previously described in the
literature, the C172T change appears to be particularly common among patients with PNP deficiency. It was
found in unrelated families originating from Asia, North-America and Europe thereby suggesting that this is a
specific “hot spot” in the PNP gene. The C172T abolishes the only Taq1 restriction site in exon 2, enabling
rapid diagnosis of this mutation.
The identification of mutations in the PNP gene may allow better genetic counseling as well as early
intrauterine or even pre-implantation confirmation of PNP-deficiency. Restriction site analysis for the more
frequent mutations enables quicker and easier diagnosis.
122
P31
The frequency and diversity of the haplotypes near adenine phosphoribosyltransferase (APRT) locus are
different between patients with APRT deficiency and normal controls
A. Taniguchi
The susceptible genes for common diseases are supposed to be common origin among different families
(common disease common variant hypothesis). Based on this hypothesis, the haplotype near a susceptible gene
among patients should be different from normal people, suggesting a genome-wide association studies utilizing
haplotypes of SNP are powerful method to search susceptible genes for common diseases. The purpose of this
study is to examine the potential of association studies utilizing haplotypes for detection of common variant
genes.
The incidence of the adenine phosphoribosyltransferase (APRT) deficiency is very high in Japanese. The
carrier frequency of the most frequent mutant allele (M136T), designated as APRT*J, was estimated to be
0.73%, which showed a founder effect for APRT*J. We genotyped 80 Japanese cases with APRT deficiency
including 42 cases with APRT*J/APRT*J and 93 cases with controls (APRT*1/APRT*1), for 23 SNP markers
that spanned about 2000 kb. An expectation-maximization algorithm estimated 21 haplotypes (frequency > 2)
from these data. Haplotype block defined by D’ (the standard coefficient of linkage disequilibrium), revealed
40 to 50 kb in APRT*1/APRT*1. The length of haplotype block of APRT*J/APRT*J were much longer than
that of APRT*1/APRT*1, showing APRT*J originated from a common ancestors. The haplotype frequencies
of APRT*J/APRT*J or patients with APRT deficiency including APRT*J/APRT*J are extremely different
from APRT*1/APRT*1. The result of this study showed the genome-wide association studies utilizing
haplotypes are valuable as a means to detect disease susceptibility genes for common diseases.
123
P32
Pyrosequencing of Thiopurine S-methyltransferase (TPMT) alleles in a Swedish general population and
in IBD-patients
S. Haglund1, M. Lindqvist2, S. Almer2, C. Peterson2, J. Taipalensuu1
1
County Hospital Jonköping, JONKÖPING, Sweden
2
Linköping University, LINKÖPING, Sweden
BACKGROUND: The thiopurine drugs; 6-mercaptopurine, 6-thioguanine and azathioprine, are used in the
treatment of leukaemia, inflammatory bowel disease (IBD) and to prevent organ transplant rejection. About 3040% of IBD-patients fails to benefit from this treatment.
One important factor that may explain interindividual differences in therapeutic efficacy and development of
adverse reactions is genetic variations in the enzymes metabolising these drugs. The therapeutic efficiency and
presence of adverse reactions partly depend on the levels of thiopurine S-methyltransferase enzymatic activity.
The TPMT enzymatic activity has been associated with genetic polymorphism at the gene locus for TPMT on
chromosome 6.
AIMS: The aims of this study were to 1) develop pyrosequencing as a tool to genotype TPMT for nine single
nucleotide polymorphism’s (SNPs) known to reduce TPMT activity, 2) examine a Swedish general population
(n = 800) for the most frequently reported TPMT alleles (TPMT*3A, TPMT*3B, TPMT*3C and TPMT*2)
and 3) investigate the relationship between TPMT genotype and phenotype in a group of IBD-patients (n = 24)
and healthy volunteers (n = 6), selected because of low, intermediate or normal TPMT enzymatic activity. In
this latter group all nine SNPs were analysed.
METHOD: We established a PCR and pyrosequencing based method to screen for all known SNPs resulting in
reduced TPMT enzymatic activity, i.e. nine SNPs in all.
RESULTS: Genotypes found in the general population were TPMT*1/*3A (*3A allelic frequency 3,8%),
TPMT*1/*3C (*3C allelic frequency 0,44%), TPMT *1/*3B (*3B allelic frequency 0,13%) and TPMT*1/*2
(*2 allelic frequency 0,06%).
None of the nine individuals judged as normal metabolisers from their enzymatic activity were carrier of any of
the investigated SNPs. Eight individuals with low activity were genotyped as either TPMT*3A/*3A (n=4),
TPMT*3A/*3C (n=2) or TPMT*1/*3A (n=2), whereas 13 individuals with intermediate enzymatic activity
were genotyped as either TPMT*1/*3A (n=12) or TPMT*1/*2 (n=1).
CONCLUSION: Pyrosequencing is a rapid and reliable method for precise and parallel identification of
multiple sequence variants.
Next to wild type, the most frequent alleles in Sweden are TPMT*3A and TPMT*3C.
There was a close correlation between genotype and phenotypic expression, i.e. enzymatic activity.
124
P33a
Polymorphism in the MTHFR gene affects thiopurine methyltransferase activity
A.M. Marinaki1, M. Arenas1, J.A. Duley2, E.A. Shobowale-Bakre1, A. Ansari1, J.D. Sanderson1, L.D.
Fairbanks1
1
2
In this study, we investigate the effect of common genetic polymorphism in the MTHFR
(methylenetetrahydrofolate reductase) gene on erythrocyte activity of TPMT (thiopurine methyltransferase). SAdenosyl-methionine (SAM) is the methyl donor for the reaction catalysed by TPMT, and is converted to Sadenosyl-homocysteine (SAH). The adenosyl moiety of SAH is subsequently cleaved and homocysteine
remethylated to methionine. A common polymorphism in the MTHFR gene, the thermolabile 677C>T variant,
is associated with decreased MTHFR enzyme activity and impaired remethylation of homocysteine to
methionine leading to mild homocysteinaemia. We proposed that the MTHFR 677C>T variant may indirectly
affect TPMT activity.
The frequency of the MTFHR 677TT homozygous genotype was compared between 52 patients with
intermediate TPMT activity (4-8U) and a wild type TPMT genotype, and 55 patients with normal TPMT
activity and genotype (in the range 12-15U). The MTHFR 677TT genotype was significantly under-represented
in the normal activity group (p=0.0296). In order to establish whether MTHFR genotype was associated with
adverse drug reactions to AZA, MTHFR 677TT genotype frequencies in a cohort of 61 IBD patients who had
experienced adverse drug reactions to AZA therapy were compared to 68 patients who had no side effects. The
MTHFR 677TT genotype was not significantly associated with adverse drug reactions to AZA. Although the
MTHFR 677C>T allele and 677TT genotype appeared to be under-represented in a subset of 16 patients with
hepatitic, pancreatic or abdominal pain side effects, the association was not quite significant.
The accumulation of methylated thio-nucleotides in patients has been suggested as a cause of pancreatic and
hepatic side effects. Our results suggest that MTFHR genotype indirectly influences TPMT activity, and imply
that a wild type MTHFR genotype may be necessary for a high methylator phenotype. More patients with
pancreatic or hepatitic side effects should be genotyped for MTHFR to establish the role of folate metabolism
in methylated thionucleotide toxicity.
125
P33b
Allele frequency of inosine triphosphate pyrophosphohydrolase gene polymorphisms in a Japanese
population
A.M. Marinaki1, S. Sumi1, M. Arenas1, L.D. Fairbanks1, S. Harihara2, K. Shimizu3, A. Ueta4, J.A. Duley1
1
2
Tokyo University Japan, TOKYO, Japan
3
Naruto University, NARUTO, Japan
4
Nagoya City University, NAGOYA, Japan
The enzyme inosine triphosphate pyrophosphohydrolase deficiency (ITPase) catalyses the
pyrophosphohydrolysis of inosine triphosphate (ITP) to inosine monophosphate (IMP). ITPase is present in
many human tissues as well as RBCs and in addition to ITP, can also utilise other purine compounds as
substrates. ITPase deficiency is a clinically benign autosomal recessive condition characterised by the
abnormal accumulation of inosine triphosphate (ITP) in erythrocytes. A deficiency of ITPase may predict
adverse reactions to therapy with the thiopurine drug 6-mercaptopurine and its prodrug azathioprine.
We have reported the structure of the ITPA gene and identified five SNPs in 8 families with ITPase
deficiency. One missense mutation (94C>A) and one intron mutation (IV2+21 A>C) affected enzyme
activities. Homozygotes for the 94C>A (Pro32 to Thr) mutation had zero erythrocyte ITPase activity while 9
heterozygotes averaged 22.5% of the control mean. ITPase activity of IV2+21 A>C homozygotes averaged
about 60% of the control mean. Compound heterozygotes 94C>A/IV2+21 A>C were 10% of the control
mean. In addition, three silent polymorphisms (138G>A, 561G>A and 708G>A) were found. In this study,
we examine the frequencies of ITPA polymorphisms in 100 healthy Japanese individuals.
DNA was extracted from blood samples after informed consent had been obtained. The 5 SNPs in ITPA gene
were determined using PCR-RFLP methods.
In the Japanese sample, the allele frequency of the 94C>A mutation was 0.135, twice the frequency in
Caucasians (0.06). The IV2+21 A>C was not found in Japanese, although it occurred with a frequency of
0.130 in Caucasians. Allele frequencies of the 138G>A, 561G>A and 708G>A were 0.57, 0.18 and 0.06 in
the Japanese population, and with the exception of the 138G>A polymorphism similar to allele frequencies
in Caucasians (0.31, 0.12 and 0.05 respectively).
Polymorphism in the TPMT gene are associated with adverse drug reactions to thiopurine drugs. However,
the frequency of TPMT deficiency-associated alleles is reported to be <1% in Japanese populations. It is thus
possible that polymorphism in the ITPA gene may explain adverse drug reactions in Japanese patients treated
with thiopurine drugs.
126
P34
Genetic determinants of the thiopurine methyltransferase intermediate activity phenotype
M. Arenas1, J.A. Duley2, C.M. Lewis1, E.A. Shobowale-Bakre1, E. Escuredo1, A. Ansari1, J.D. Sanderson1,
L.D. Fairbanks1, A.M. Marinaki1
1
2
INTRODUCTION. Polymorphism in the TPMT gene is associated with reduced TPMT activity. Variable
number tandem repeats (VNTR*3 to VNTR*9) in the promoter region of the TPMT gene are also reported to
modulate the TPMT activity phenotype. In this study we investigate concordance between TPMT activity in the
intermediate activity range and open reading frame (ORF) and VNTR genotypes.
METHODS. Patient were selected for non-overlapping TPMT activities within the intermediate activity range
(4 - 8 U, 108 patients) and normal range (12 - 15 U, 53 patients), and were genotyped for VNTR type, and
TPMT*3A, TPMT*3C and TPMT*2 ORF polymorphisms.
RESULTS. 41% of patients from the intermediate TPMT activity group were heterozygous for a TPMT ORF
polymorphism. Marked linkage disequilibrium was noted between VNTR*6b - TPMT*3A (D’=1), VNTR*4b
- TPMT*3C (D’=0.67) and VNTR*6a - TPMT*1 (D’=1) alleles. To investigate the influence of the number of
VNTR repeat units on TPMT activity, the number of Type A, B or total repeat units summed for both alleles in
the ORF heterozygous, wildtype intermediate and normal activity groups and were compared in an analysis of
variance. For Type A and total repeats, significant differences (p<0.05) in the median number of repeats were
found between the heterozygous intermediate activity group and the wild type intermediate or normal activity
groups. There was no significant difference in the median Type A, B or total repeat number between the two
wild type groups. CONCLUSION. Our results suggest there is poor concordance between TPMT ORF
genotype and activity in the carrier range and that VNTRs do not significantly modulate basal TPMT enzyme
activity.
127
P35
Association between Thymidylate Synthase polymorphism with the microsatellite instability in colorectal
cancer
A. Calascibetta1, L. Rausa1, L. Gullotti1, R. Buettner2, R. Sanguedolce1
1
University of Palermo, PALERMO, Italy
2
University of Bonn, BONN, Germany
5-Fluorouracil (5Fu) is the main drug used for the treatment of colorectal cancer and Thymidylate Synthase
(TS) is its target enzyme. TS gene has regulatory tandemly repeated sequences in its 5’ untranslated region, that
are polymorfic in tumours and depending on ethnic factor; more over this gene has an other polymorphism
located in the 3' untraslated region, characterised by the absence of a 6 bps olygonucleotide. These
polimorfisms have been reported to influence the TS-mRNA expression and the TS levels. Colorectal cancer
often shows an other kind of genomic instability called Microsatellite Instability (MSI), that at present time is
considered one of the most important marker of tumour genomic instability. High colorectal cancer MSI
frequency is associated with a significant better survival and with a lower incidence rate of metastasis. While 5’
and 3' UTR polymorphisms are related with a particular genomic instability, the aim of the study is to try to
find any relationship between the 5’ with the 3' UTR polymorphisms, the MSI, the TSmRNA expression, with
the TS levels and with the prognosis and drug treatment, in 54 paraffin embedded primary colorectal cancer
samples using an ABI PRISM sequencer. Our data show that the genotype 2R / 2R is more frequently
associated with MSI + and lower TS levels. More over we did not find any significant association between the
2R / 3R and 3R / 3R genotypes with MSI + and MSI –, while these genotypes were associated with a higher
TS expression. In the same group of patients we evaluated the presence of polymorphism in the 3' UTR: we
found in only 5 patients the presence of the -6bp/-6bp genotype that it was not related with MSI, the 5' UTR
polymorphism and with the TS levels. As consequence we can hypothesise that patients bearing colorectal
cancer with MSI+, the 2R/2R genotype and with low TS levels could have a better prognosis and they could
not be drug resistant.
128
P36
Polymorphic tandem repeats in the thmidylate synthase gene and TS protein and mRNA levels in
different tissues of colorectal cancer patients
R. Mauritz, I.J. Beumer, K. Smid, C.J. Van Groeningen, G.J. Peters
High expression of thymidylate synthase (TS) has been related to poor response of colorectal cancer patients to
treatment with 5-fluorouracil (5-FU). Recent studies suggest that a tandem repeat polymorphism within the
enhancer region of the TS gene promotor influences TS expression: the triple repeat (3R/3R) genotype might
be associated with higher TS levels than the double repeat (2R/2R) or heterozygous (2R/3R) genotype. Most
studies thus far focussed on the relation between TS genotype and survival of patients treated with 5-FU-based
chemotherapy. Only few studies explored the relation between TS genotype and in vivo TS expression and
although all reports suggest an increase in TS activity with the number of tandem repeats, conflicting data are
reported concerning TS mRNA levels in relation to the TS genotype.
We are currently undertaking a retrospective analysis of the TS polymorphism in normal and malignant tissues
of patients with advanced colorectal cancer. DNA is obtained from either freshly frozen tissue, paraffin slides
or frozen tissue sections. Subsequently the polymorphic status is assessed by PCR amplification. By now, 61
samples from 46 patients were analysed. We found 20 patients with the 2R/3R genotype, 14 with the 3R/3R
and 12 with the 2R/2R variant. Conform previous studies we found a high level of homology between the TS
polymorphic status of normal and malignant tissues from one patient. The TS genotypes will be correlated to
TS enzyme and mRNA expression levels in addition to survival data.
129
P38a
Dihydroorotate dehydrogenase expression analysis in normal and drug-resistant cells and tissues
M.L. Loeffler1, M. Hayek-Ouassini1, K. Steger1, A. Klein2, T. Monses3, W. Knecht2
1
Philipps-University Marburg, MARBURG, Germany
2
Institute Physiological Chemistry, MARBURG, Germany
3
Hautklinik, GIESSEN, Germany
Analysis of the temporal or permanent and spatial expression of proteins is a crucial step towards understanding
gene function and regulation and elucidating physiology and pathophysiology of enzymes in metabolic
pathways. Here, we present our approach and establishment of methods to follow the expression of the fourth
enzyme of pyrimidine biosynthesis, dihydroorotate dehydrogenase (DHODH) protein and mRNA in various
cells and tissues as precedent for further investigation. (1) Polyclonal immunoglobulins raised against DHODH,
as described in Adv. Exp. Med. Biol. 431: 507-513 (1998), were applied for immunochemical quantification of
the enzyme in two lines of mouse B-lymphocytes by Western blotting/ECL. The parental cells were sensitive to
leflunomide (Arava), a potent inhibitor of DHODH, while adapted cells exhibited a 40-fold tolerance to the
drug. The corresponding overexpression of DHODH protein was demonstrated with homogenates of theses
cells. (2) The detection of DHODH mRNA expression in homogenates of tissue and cultered cells from the rat
reproductive tract by means of RT-PCR was achieved. (3) By non-radioactive hybridization technique the
DHODH mRNA distribution was examined in paraffin sections of the appropriate rat tissue and kidney. (4)
Immunocytochemical proof of DHODH protein in sections from the same tissues verified that the pattern of
expressed RNA corresponded to the distribution of the encoded protein. (5) The occurrence of protein
correlated to catalytic enzyme histochemistry of DHODH that was visualized by application of the nitroblue
tetrazolium/formazan technique for dehydrogenase activity in cryostat sections previously (Histochemistry Cell
Biol. 105:119-128 (1996).
Support: Marburg-Giessen DFG Graduiertenkolleg and P.E. Kempkes-Stiftung FB Medizin Marburg.
130
P38b
Phosphorylitic cleavage of deoxyribonucleosides catalysed by human deoxycytidine kinase - a side
reaction or a function. Preliminary Biochemical and NMR Studies
E.V. Usova1, T.V. Maltseva2, S. Eriksson1
1
Swedish Univ. of Agricultural Science, UPPSALA, Sweden
2
Medivir AB, STOCKHOLM, Sweden
Human deoxycytidine kinase (dCK) is a key enzyme in the 5’-phosphorylation of purine and pyrimidine
deoxynucleosides with dCyd as the most efficient substrate. The ability of dCK to degrade
deoxyribonucleosides to free nucleobases and (most likely) deoxyribose-1-phosphate was demonstrated for the
first time by 1H -31P correlation spectroscopy and by 3H-labelling methods. The new reaction was studied
with regard to several factors such as concentration of enzyme and deoxyribonucleosides, temperature and pH
of the buffer. The reaction depended on inorganic phosphate and all natural deoxyribonucleosides could be
cleaved but the specific activity of phosphorylitic reaction compared to the kinase reaction was in most cases
low, about 2-10%. However, with dThd which is very poor kinase substrate the rate of cleavage was several
fold higher than its phosphorylation. The formation of free nucleobases occurred with reduced dCK because the
reaction was highly dependent on the presence of reducing agents such as DTT. Thus, recombinant dCK can
most likely act as a phosphorylase similar to the nucleoside phosphorylase family of enzymes. This catalytic
activity is of importance for the design of in vitro experiments with dCK and it might play a physiological role
in the metabolism of some deoxyribonucleosides.
131
P39
The Effect of Signalling Pathway Modulations on the Activity of the Deoxycytidine Kinase
M. Staub, G. Keszler, Z.C. Csapó, T. Spasokoukotskaja, S. Virga, M. Sasvári-Székely
Semmelweis University, BUDAPEST, Hungary
Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the salvage of three natural
deoxyribonucleosides as well as several therapeutic nucleoside analogues, which in turn can enhance its
enzymatic activity and improve the efficacy of the cytostatic therapy, as shown earlier. The increase of dCK
activity has been suggested as a compensatory mechanism of cells, induced by damage of DNA. The
potentiated dCK activity is able to supply the repair of DNA with dNTPs in quiescent cells. The possible
functions of signalling molecule(s) leading from DNA damage to activation of dCK have been investigated
in this work.
The tyrosine kinase inhibitors, Tyrphostin and Genistein, (between 10 and 300 mM) elevated dCK activity by
200% during 120 min treatment of human lymphocytes. The unspecific phosphatase inhibitor NaF (15 mM),
used as an inhibitor of the small G-protein GTPase activity, can also elevate the dCK activity to 200% of the
untreated activity. Calyculin A, the inhibitor of Protein Phosphatase 2A (PP2A), also increased the dCK
activity in treated cells by the same range. Moreover gamma-irradiation (1-3 Grays) provoking double-strand
DNA breaks, enhanced also significantly dCK activity in lymphocytes. The elevation of dCK activity induced
by all treatments mentioned above, can be released by 10 mM dCyd. On the other hand, hyperosmotic stress
of cells, induced by sorbitol treatment, was found to decrease the activity of dCK.
Conclusion: Several signalling molecules seem to function between DNA damage and increase of dCK activity
i.e. the elevated DNA repair capacity of cells.
132
P40
Unusual phosphate donors for human deoxyrybonucleoside kinases
K. Krawiec1, B. Munch-Petersen2, S. Eriksson3, D. Shugar1, B. Kierdaszuk1
1
University of Warsaw, WARSAW, Poland
2
3
Krzysztof Krawiec 1, Birgitte Munch-Petersen 2, Staffan Eriksson 3, David Shugar 1,4
and Borys Kierdaszuk 1
1 University of Warsaw, Institute of Experimental Physics, Department of Biophysics, 93 Żwirki i
Wigury St., PL-02-089 Warsaw, Poland; 2 University of Roskilde, Department of Life Sciences and Chemistry,
DK-4000 Roskilde, Denmark; 3 Swedish University of Agricultural Sciences, Department of Veterinary
Medical Chemistry, Biomedical Centre, S-75123 Uppsala, Sweden; 4 Institute of Biochemistry and Biophysics,
Polish Academy of Sciences, 5a Pawińskiego St., PL-02-106 Warsaw, Poland
Inorganic tripolyphosphate (PPPi) and pyrophosphate (PPi) were examined as potential phosphate donors for
all four human deoxyrybonucleoside kinases: deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK),
cytosolic thymidine kinase (TK1), and mitochondrial thymidine kinase (TK2); and additionally the
deoxynucleoside kinase (dNK) from Drosophila melanogaster. This is the first report on the substrate activity
of PPPi, which was proved to be a good phosphate donor for dGK, as well as for dCK with dCyd, but not
dAdo, as acceptor substrate, illustrating also dependence of donor properties on acceptor. Products of
phosphorylation were shown to be 5˘-phosphates. In contrast to ATP, the phosphorylation reaction
follows strict Michaelis-Menten kinetics, with Km values of 74 and 92 µM for dCK and dGK, respectively, and
Vmax values 40-50% that for ATP. With the other three enzymes, as well as for dCK with dAdo as acceptor,
no, or only low levels (<1 % of that for ATP) of activity were observed. PPi was inactive (<0.1%) as a
phosphate donor with all enzymes, but was a competitive inhibitor vs ATP, as was PPPi in systems with no or
low donor activity.
Activities of the enzymes towards PPPi perfectly correlates with classification of kinase activities, which were
previously suggested using their activities with analogues of 2'- and 3'- triphosphates of adenosine.
Deoxyribonucleoside kinases which easily accepts all mentioned analogues can also effectively utilize PPPi as
a phosphate donor, while kinases with more restricted specificity towards those analogues, are also restricted
towards PPPi.
The extension of mentioned investigations, including tests with longer polyphosphates and more complete set
of acceptors, are in progress.
133
P41
The multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster can phosphorylate
glycerol
B. Munch-Petersen1, B. Stitt2, C. Zhu1, R. Browne2, J. Piskur3, C. Grubmeyer2
1
2
Temple University, PHILADELPHIA, United States of America
3
Deoxyribonucleoside kinases are found in nearly all organisms except in fungi and a few bacteria. These
enzymes participate in the salvage pathway providing nucleic acids precursors, and they also activate a number
of medically important enzymes. In vertebrates, it seems to be a common characteristica to possess more than
one kinase with overlapping specificities.
Recently, in the fruitfly Drosophila melanogaster, we found that there was only a single deoxyribonucleoside
kinase, Dm-dNK, but in contrast to other kinases, this enzyme is able to phosphorylate all four
deoxynucleosides as well as a broad number of nucleoside analogues. In two other insects, Bombyx mori the
silk butterfly, and Anopheles gambiae, the malaria mosquito, we also found a multisubstrate
deoxyribonucleoside kinase with the capacity of phosphorylating all four deoxyribonucleosides. Currently,
several development projects are in progress using the Dm-dNK for biotechnological production of DNA
reagents and for gene therapy.
We have investigated further the substrate properties of the Dm-dNK and found a surprising new property of
the enzyme. In addition to nucleosides, it is able to phosphorylate glycerol. Data on this new property and
activity towards other polyalcohols and sugars will be presented. Because of this new substrate property, we
suggest to rename the enzyme to: Multifunctional deoxyribonucleoside kinase.
134
P42
Amino acid residues determining the substrate specificity of deoxyribonucleoside kinases
B. Munch-Petersen1, M.P.B. Sandrini2, W. Knecht2, H. Eklund3, J. Piskur2
1
2
3
SLU, UPPSALA, Sweden
The multisubstrate deoxyribonucleoside kinase from insects – dNK (EC 2.7.1.145) is closely structurally
related to the two human mitochondrial deoxyribonucleoside kinases, thymidine kinase 2 (TK2) and
deoxyguanosine kinase (dGK). TK2 and dGK have recently been found to be essential for mitochondrial DNA
integrity and mutated forms of these enzymes cause mitochondrial DNA depletion with lethal consequences.
Increased knowledge about structure-function relationship of deoxyribonucleoside kinases helps to understand
the relation between structure, function and substrate specificity, which is a prerequisite for the development of
novel mutant enzymes for suicide gene therapy. The recently solved 3D structures of the Drosophila
melanogaster dNK and human dGK are very similar. When the dNK and dGK structures were used for
modelling of TK2 and dCK respectively, there were only a few differences in the residues closest to the base.
Substitution of a few of these residues changed the specificity from pyrimidine to purine deoxyribonucleosides.
Lately, we isolated, cloned and characterized the sole deoxyribonucleoside from Anopheles Gambia, which is
also a multisubstrate kinase but in contrast to the drosophila dNK, the mosquito enzyme has essentially the
same specificity towards both purine and pyrimidine deoxyribonucleosides. Since the residues in the binding
pockets for the mosquito and drosophila dNK’s appears to be identical, other more distant residues must also be
involved in determination of the substrate specificity.
135
P43
Mitochondrial deoxyguanosine kinase/thymidine kinase mutations and mitochondrial DNA depletion
syndrome
L. Wang, S. Eriksson
Mitochondrial DNA (mtDNA) synthesis is irrespective of cell cycle phase and therefore a constant supply of
mitochondrial DNA precursors is vital for mtDNA maintenance. Mitochondrial deoxyguanosine kinase (dGK)
and thymidine kinase 2 (TK2) catalyse the initial phosphorylation of deoxynucleosides which is the rate
limiting step in mtDNA precursor synthesis. Deficiency in either dGK or TK2 have been observed in patients
with severe mtDNA diseases, e.g. earlier onset of progressive live failure, lactic acidosis and neurological
abnormalities due to mutations in the dGK gene and reduction of respiratory function and devastating
myopathy due to mutations in TK2 gene. We used site-directed mutagenesis to introduce those point mutations
or base insertion, observed in patients, in the dGK and TK2 genes. The recombinant mutant enzymes were
expressed in E. coli and purified and characterised with regards to substrate specificity and catalytic efficiency.
Thr-77 to Met mutation of TK2 resulted in an enzyme with low catalytic efficiency as compared with wild type
TK2 and also altered substrate specificity. Arg-142 to Lys mutation of dGK resulted in an inactive enzyme.
The 4 bp insertion in exon 7 which introduced a stop codon and C-terminal 22 amino acids truncation of dGK
protein and this C-terminal truncated dGK protein was also inactive. Glu227Lys mutant dGK enzyme showed
very low residual activity. In summary mutations in dGK genes probably result in cells with dGK deficiency,
which will probably starve the mtDNA synthesis with depleted dATP and dGTP pools. TK2 mutations likely
resulted in imbalanced dTTP and dCTP pools in mitochondria. Insufficient supply of dNTPs may well be the
cause of mtDNA depletion. These results implicate that dGK and TK2 play an important role in mtDNA
precursor synthesis and the maintenance of mtDNA integrity.
136
P44
Biochemical and Biological Evaluation of 3-(Carboranylalkyl) thymidines as Substrates for Human
Thymidine Kinases 1 and 2
A.S. Al-Madhoun1, W. Ji2, J. Johnsamuel2, J. Yan2, W. Tjarks2, S. Eriksson3
1
Medical BioSciences, SLU, UPPSALA, Sweden
2
College of Pharmacy, COLUMBUS, OHIO, United States of America
3
A small library consisting of two series of thymidine derivatives containing o-carboranylalkyl groups at the N-3
position was prepared. In both series, alkyl spacers of 2-7 methylene units were placed between the ocarborane cage and the thymidine scaffold. In one series, an additional dihydroxypropyl substituent was
introduced at the second carbon atom of the carborane cage. In the series of N-3 substituted carboranyl
thymidines without additional dihydroxypropyl substituent, three steps were required to obtain the target
compounds in overall yield as high as 75%, while in the series of N-3 substituted carboranyl thymidines with
additional dihydroxypropyl substituent nine to ten steps were necessary with significantly lower overall yield.
All target compounds were good substrates for TK1 while they were, if at all, poor substrates for TK2. There
was only a minor difference in phosphorylation rates between N-3 substituted carboranyl thymidines with
additional dihydroxypropyl substituents with TK1 (range: 13 to 49% relative to thymidine) and their
counterparts lacking this group (range: 11 to 57% relative to thymidine). In order to gain a better
understanding of the phosphorylation by TK1, apparent Km, Vmax and Ki values for these analogues were
determined. Taking together, the tether lengths of two and five methylene groups in both series were found to
be good substrate for TK1 and a hypothesis for this result is proposed. The results of stability studies including
incubation with human and bacterial thymidine phosphorylase and human cytosolic nucleotidases will be also
presented. In vitro studies related to cytotoxicity and uptake will be included.
137
P45
Cloning and biochemical characterisation of thymidine kinase in Ureaplasma urealyticum
C.M.E. Carnrot
Biomedical Center, UPPSALA, Sweden
Ureaplasma urealyticum (U.u), belonging to the mycoplasmas, is a human pathogen colonising the urogenital
tract. Two genes coding for a presumed thymidine kinase (TK) and deoxynucleoside kinase (dAK) have been
identified in the genome of U.u. These enzymes are most likely required for the salvage pathway in DNA
precursor synthesis. Here we present the cloning, expression and biochemical characterisation of U.u TK. The
enzyme is pyrimidine specific and phosphorylates thymidine, with a Km value of 5.4 mM and a Vmax of 3.6
mmol mg-1 min-1. 5’-fluorodeoxyuridine (5FdU), 3’-azidothymidine (AZT) and deoxyuridine are also good
substrates, and the latter appears to have a very high Km value. 5FdU and AZT have Km values of 9.3 and 44.6
mM respectively. Their corresponding Vmax values are 4.7 and 0.6 mmol mg-1 min-1. All nucleoside
triphosphates (NTPs) or dNTPs but not dTTP can act as phosphate donors. ATP is the most efficient phosphate
donor with a specificity constant of 4.5 x 104 M-1s-1. dTTP acts as a feedback inhibitor with a Ki of 0.18 mM.
Also thymidine inhibits TK but noncompetitively, at concentrations >10 mM. The availability of recombinant
TK and knowledge of the properties of this enzyme will facilitate studies of the physiological role of TK in U.u
and future possibilities to use TK as target in the development of new drugs against mycoplasma infections.
138
P46
Hypoxanthine effects on cyclic amp levels in human lymphocytes
Introduction: Adenosine A2 receptor activation stimulates adenylate cyclase (increasing cAMP levels).
Inhibition of adenosine uptake by hypoxanthine could lead to accumulation of extra-cellular adenosine that
binds to adenosine A2A receptors and increases cAMP production. A2A receptors are highly concentrated in
the basal ganglia and may modulate dopaminergic activity. Hypoxanthine excess could be implicated in the
pathogenesis of the neurological symptoms of Lesch-Nyhan syndrome. The aim of this study is to evaluate the
effect of hypoxanthine on receptor-mediated adenosine action in human cells using peripheral blood
lymphocytes.
Methods: To test hypoxanthine effect on A2A receptor activation, cells were first incubated with 5 uM or 25
uM CGS-21680, or without agonist and then different hypoxanthine concentrations (0, 5, 25 or 50 uM) were
added and cells incubated for 60 min at 37oC.
Results: We determined the effect of 5uM (close to mean normal concentration of hypoxanthine in
cerebrospinal fluid) vs. 25 uM (near Lesch-Nyhan concentration 17,5 +- 2,8) extra-cellular concentrations of
hypoxanthine on basal and CGS 21680-stimulated levels of cAMP in PBL cultures. Basal cAMP levels were
significantly increased with the addition of 25 uM and 50 uM hypoxanthine versus 5 uM hypoxanthine. cAMP
levels in PBL stimulated with 5 uM CGS 21680 were significantly increased with the addition of 25 uM and 50
uM hypoxanthine versus 5 uM hypoxanthine. In PBL stimulated with 25 uM CGS 21680, no significant
differences were found between cAMP levels with the addition of hypoxanthine 5 uM, or without
hypoxanthine. However, hypoxanthine 25 uM caused a significant increase in cAMP levels compared with
basal or 5 uM hypoxanthine concentrations.
Conclusions: Adenosine and dopamine receptors in the basal ganglia are coupled and adenosine is known to
modulate dopaminergic neurotransmission. Thus hypoxanthine, by means of its effect on adenosine uptake and
adenosine receptors, can modulate dopaminergic neurotransmission and dopamine content in the basal ganglia
neurons. This may explain the dopaminergic deficit shown by Lesch-Nyhan patients and in HPRT-deficient
culture cells and could give some clues on the relation between the enzyme deficiency and the dopaminergic
defect.
139
P47
Mechanistic and physiological studies of dUTPases
J. Kovari1, A. Bekesi1, I. Zagyva1, A. Nagy1, O. Barabas2, B.G. Vertessy1
1
Institute of Enzymology, BUDAPEST, Hungary
2
Eötvös Loránd University, BUDAPEST, Hungary
The essential enzyme dUTP pyrophosphatase is responsible for preventive DNA repair via exclusion of uracil.
dUTPase hydrolyses dUTP to dUMP and pyrophosphate, simultaneously reducing dUTP levels and providing
dUMP for dTTP biosynthesis. Lack or inhibition of the enzyme induces thymine-less cell death in all cells and
tissues performing active DNA synthesis such as neoplasia, serving therefore as an important chemotherapeutic
target. Developmental regulation of the Drosophila melanogaster enzyme is suggested to be involved in
thymine-less apoptosis. In addition to sequence motifs conserved amongst dUTPases, the fruit fly enzyme
contains a unique C-terminal Ala-Pro-rich extension. Kinetic, biochemical and biophysical analyses of the fulllength protein and a truncation mutant show that the Ala-Pro-segment is flexible and has no significant role in
catalytic activity. For detailed mechanistic investigations, several point mutants of E. coli- and Drosophila
melanogaster enzymes are being characterised. Formation of the catalytically competent closed enzyme
conformer is induced by the product dUMP and the non-hydrolysable substrate analogues dUDP and dUPNPP
in the Drosophila melanogaster dUTPase, while only by dUPNPP in the E. coli enzyme. Two dUTPase
isoforms were identified in different developmental stages of the fruit fly. Protein levels are drastically reduced
in larvae, arguing for a possible induction of developmental apoptosis. Interestingly, mRNA levels are high
throughout development, indicating translational or post-translational control of dUTPase protein. Fruit fly
dUTPase is the first eukaryotic enzyme characterized in details. Our results reveal significant differences
between prototypes of eu- and prokaryotic dUTPases with respect to conformational flexibility of the active
site, metal ion binding, and oligomerization. These differences are consistent with alterations of the catalytic
mechanism and in hydropathy of subunit interfaces.
Recently, we also succeeded in cloning the gene of nuclear isoform of human dUTPase into an expression
vector, which will allow for detailed characterization of the human enzyme.
140
P48
Crystal structure of a human deoxyribonucleotidase
A. Rinaldo-Matthis
Stockholm University, STOCKHOLM, Sweden
Mammalian 5’ deoxyribonucleotidases (dNT´s) is key proteins in the control of the deoxyribonucleotide pools
sizes, essential for accurate DNA synthesis. They are potential important drug targets and reveals a relationship
to the HAD family, with a phosphoserine phosphatase as the closest neighbor. The mitochondrial 5’
deoxyribonucleotidase (dNT-2) specifically dephosphorylates dUMP and dTMP, protecting mitochondrial
DNA replication from excess dTTP.
We have solved the structure of dNT-2, the first determined structure of a mammalian 5'-nucleotidase. The
structure reveals a relationship to the HAD family, with a phosphoserine phosphatase as the closest neighbor. A
structure-based sequence alignment of dNT-2 with other 5’-nucleotidases also suggests a common origin for
these enzymes.
The structure of dNT-2 has been studied in complex with bound phosphate and beryllium trifluoride plus
thymidine as model for a phosphoenzyme product complex. Based on these structures, determinants for
substrate specificity recognition and the catalytic action of dNT-2 have been outlined.
141
P49
Differential activity of pyrimidine nucleoside kinases in proliferating, resting and adipocyte
differentiated 3T3-L1 cells
S.N. Rylova1, G. Flygh1, F. Albertioni2, S. Eriksson1
1
Swedish Univ. of Argicultural Sciences, UPPSALA, Sweden
2
Karolinska Institute, STOCKHOLM, Sweden
Nucleoside reverse transcriptase inhibitors (NRTI) in combination with protease inhibitors are a highly
effective therapy for HIV infections. Prolonged NRTI treatment of HIV patients is associated with a number
of metabolic complications. The lipodystrophy syndrome (LD), characterized by lipoatrophy (face, limbs,
buttocks), fat accumulation (abdominal, neck) and dysmetabolism (hyperlipidaemia, insulin resistance and
lactic acidosis) presents a serious risk for HIV patients.
One of the proposed mechanisms for LD is NRTI -induced mitochondrial toxicity which results in mtDNA
depletion, disruption of oxidative phosphorylation and possibly induction of apoptosis. The primary cause of
NRTI associated mtDNA depletion is chain termination by NRTI triphosphates. Inhibition of cellular
nucleoside kinases (NK) and altered mt dNTP pools may also be contributing factors.
NRTI induced mitochondrial toxicity is found to be tissue specific and differs in activated and resting cells
due to differential activities of NK’s, converting NRTI analogs to triphosphates. We used 3T3-L1 mouse
preadipocyte cells - a model for white adipose tissue - to measure levels of NK’s, responsible for activation
of the NRTI analogs d4T, AZT, 3TC, some of which are associated with LD. We compared cytosolic (TK1)
and mitochondrial (TK2) thymidine kinases (phosphorylating d4T and AZT), deoxycytidine (dCK) kinase
(phosphorylating 3TC) and thymidylate (TMPK ) kinase (limiting in AZT activation) activities in
proliferating, resting and adipocyte differentiated 3T3-L1 cells and in CEM lymphoblasts.
We found a 30-fold drop in TK1 activity and 3-fold decrease in dCK activities in extracts from resting 3T3L1 compared to proliferating cells. In differentiated 3T3-L1 (9 days after adipocyte induction) there is no
change in TK1 activity, a slight decrease in dCK activity, but a 90% increase in TK2 activity compared to
resting preadipocytes. In contrast, CEM cells have very high activities for TK1 and dCK but below 1% for
TK2. TMPK activity was 7-fold higher in proliferating preadipocytes than in CEM cells. It was 2 fold
lower in resting preadipocytes versus proliferating cells, and slightly higher in differentiated 3T3-L1
adipocytes compared to resting 3T3-L1. These results show that rate limiting NRTI activating enzymes are
differentially expressed during adipocyte differentiation.
142
P50
Cloning of a mitochondrial UMP/CMP kinase from Drosophila melanogaster
A. Karlsson, S. Curbo, M. Amiri, F. Foroogh, M. Johansson
The group of characterized UMP/CMP kinases catalyses the phosphoryl transfer from ATP to CMP, UMP and
dCMP, resulting in the formation of ADP and the corresponding nucleoside diphosphates. The human
UMP/CMP kinase phosphorylates structural analogs of pyrimidine nucleotides that are often administered as
nucleosides in the treatment of cancer and viral infections. We have cloned a UMP/CMP kinase from
Drosophila melanogaster (Dm-UMP/CMP kinase). It has similar enzymatic properties as the human
UMP/CMP kinase and thus uses uridine and cytidine substrates. In contrast to the human UMP/CMP kinase
that is located in the cytosol the Dm-UMP/CMP kinase is located in the mitochondria. So far no human
mitochondrial UMP/CMP kinase has been discovered and it is therefore puzzling to find a mitochondrial
UMP/CMP kinase in the lower species Drosophila melanogaster.
143
P51
Purine and Pyrimidine Salvage in Whole Rat Brain: Utilization of ATP-derived Ribose-1-Phosphate and
5-phosphoribosyl-1-Pyrophosphate Generated in Experiments with Dialyzed Cell-free Extracts
P.L. Ipata, C. Barsotti, M.G. Tozzi
The object of this work stems from our previous studies on the mechanisms responsible of ribose 1-P and 5phosphoribosyl-1-pyrophosphate mediated nucleobase salvage and 5-fluorouracil activation in rat brain
(Mascia, L. et al. (2000) Biochim. Biophys. Acta 1474, 3585-3589; Mascia, L. et al. (1999) Biochim. Biophys.
Acta 1472, 93-98). Here we show that, when ATP at 'physiological concentration' is added to dialyzed extracts
of rat brain, in the presence of natural nucleobases or 5-fluorouracil, adenine-, hypoxanthine-, guanine-, uraciland 5-fluorouracil-ribonucleotides are synthesized. The molecular mechanism of this peculiar nucleotide
synthesis relies on the capacity of rat brain to salvage purine and pyrimidine bases by deriving ribose 1phosphate and 5-phosphoribosyl-1-pyrophosphate from ATP, even in the absence of added pentose or pentose
phosphates. The levels of the two sugar phosphates formed are compatible with those of synthesized
nucleotides. We propose that the ATP-mediated 5-phosphoribosyl-1-pyrophosphate synthesis occurs through
the action of purine nucleoside phosphorylase, phosphopentomutase and 5-phosphoribosyl-1-pyrophosphate
synthetase. Furthering our previous observations on the effect of ATP in the 5-phosphoribosyl-1pyrophosphate-mediated 5-fluorouracil activation in rat liver (Mascia, L. and Ipata, P.L. (2001) Biochem.
Pharmacol. 62, 213-218), we now show that the ratio [5-phosphoribosyl-1-pyrophosphate]/[ATP] plays a major
role in modulating adenine salvage in rat brain. On the basis of our 'in vitro' results, we suggest that massive
ATP degradation, as it occurs in brain during ischemia, might lead to an increase of the intracellular 5phosphoribosyl-1-pyrophosphate and ribose 1-P pools, to be utilized for nucleotide resynthesis during
reperfusion (Barsotti, C. et al. (2002) J. Biol. Chem. 277, 9865-9869). Finally, we also show that the pathway
of ATP degradation in rat brain follows the AMP - IMP - Inosine route or the AMP - Adenosine - Inosine route
at high and low Energy Charge value respectively.
144
P52
Colocalization of Human Cytidine Triphosphate Synthetase 1 with Microtubules
M.J.H. Higgins, L. Graves
Cytidine triphosphate (CTP) has emerged as an important nucleotide regulator of cellular processes such as
differentiation, phospholipid metabolism and DNA replication. The last step in the de novo synthesis of CTP is
catalyzed by CTP synthetase 1 (CTPS1) which amidates UTP to form CTP. Although CTPS1 has been well
characterized in bacteria and yeast, there remains relatively little information about human CTPS1 cellular
biology. In this study, we characterized the cellular localization of human CTPS1 in HEK 293 cells. After
confirming expression of our human CTPS1 construct in HEK 293 cells via immunoblot, we performed
immunostaining of HEK 293 cells using an antibody against the N-terminal Xpress tag attached to human
CTPS1. Immunostaining revealed that the localization of hCTPS was cytoskeletal; however, human CTPS1
did not colocalize with actin as determined by costaining with Alexa488-phalloiden. To investigate whether
the endogenous human CTPS1 also colocalized with the cytoskeleton, we generated an antibody against the Cterminal 14 amino acids and performed immunostaining on untransfected HEK 293 cells. Endogenous human
CTPS1 also localized to the cytoskeleton and, interestingly, colocalized with microtubules. While treatment of
untransfected HEK 293 cells with a specific pharmacological inhibitor of CTPS, CPEC (cyclopentenyl
cytosine), did not greatly affect the localization of human CTPS1, CPEC incubation was associated with an
unexpected amount of microtubule formation in HEK 293 cells. Here we show for the first time that human
CTPS1 localizes to microtubules and treatment with the specific inhibitor of CTPS, the anticancer drug CPEC,
is associated with microtubule formation. Further investigation will include CTPS effects on tubulin
polymerization as well as how tubulin affects CTPS activity. A more complete understanding of where and
how hCTPS1 operates in the cell will not only benefit our basic understanding of pyrimidine metabolism but
also help in efforts to develop more specific anticancer drugs.
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P53
Identification of binuclear zinc coordination for human guanine deaminase by site directed mutagenesis
F.F. Snyder, H. Zhang, F.F. Snyder
Guanine deaminase, which catalyzes the conversion of guanine to xanthine, irreversibly removes the guanine
nucleobase from the cellular pool of guanine containing purines. We have previously cloned human guanine
deaminase (J. Biol. Chem. 1999), characterized 5 cDNA alleles in the mouse (Genome 2002) and identified the
E. coli homolog (J. Bact. 20001). Guanine deaminase shares the 9-residue motif, PGLVDTHIH, believed to be
responsible for coordination of zinc, with other amino and amido-hydrolases. Recent crystallographic studies
of dihydroorotase and phosphotriesterase have identified binuclear metal centers. These include coordination
of the alpha-metal site through the two histidines of the above-mentioned motif, a carboxylated lysine, and an
aspartate. The beta-metal site includes the same lysine and two additional carboxy-terminal side histidine
residues. Exposure of guanine deaminase to the heavy metal atom chelator, 1,10-phenanthroline, results in the
loss of activity. By comparative analysis of the human and E. coli primary guanine deaminase sequence we
identified conserved histidine residues including the lysine residue corresponding to the putative binuclear
metal center. By site directed mutagenesis we have made the following individual modifications to human
guanine deaminase: His82Ala, His84Ala, Lys201Ala, His146Asn, His240Asn, and His279Ala. The specific
activity and affinity with guanine was established for each of these proteins and modification of His 82, 84,
240, and 279 caused greater than 95% loss of specific activity. Substitution of Lys201 resulted in
approximately a 75% decrease in activity. The kinetic findings were also correlated with zinc analysis of the
purified proteins. The wild type protein had approximately 1.7 atoms of zinc per subunit, consistent with the
coordination of two zinc atoms. The histidine substitutions resulted in the partial loss of zinc per subunit. In
contrast replacement of His146, not predicted to be involved in the zinc coordination sites resulted in only a
50% loss of specific activity and no loss of associated zinc. These studies provide evidence for a binuclear zinc
center associated with the catalytic function of human guanine deaminase.
Supported by the Canadian Institute of Health Research
146
P55
GTP Concentrations are Elevated in Erythrocytes of Renal Transplant Recipients when Conventional
Immunosuppression is replaced by the Inosine Monophosphate Dehydrogenase Inhibitor Mycophenolic
Acid Mofetil (MMF)
E.A. Carrey, D.J.A. Goldsmith, S.M. Edbury, A.M. Marinaki, H.A. Simmonds
MMF is an effective immunosuppressant developed for use in organ transplantation. The active metabolite,
mycophenolic acid, is a potent inhibitor of inosine monophosphate-dehydrogenase (IMPDH). IMPDH
catalyses the first step in the synthesis of GMP from IMP derived via either the purine synthetic or salvage
pathways. Elevated GTP concentrations were first noted in erythrocytes of immunodeficient children treated
with another IMPDH inhibitor, Ribavirin (1); IMPDH activity was elevated in such patients compared with
controls (2670pmol/h cf 8.5pmol/h per mg protein). A similar elevation of GTP was reported recently in
erythrocytes of heart transplant recipients given MMF (2). In the present report we measured nucleotides by
HPLC in renal failure patients’ erythrocytes before and after successful transplantation and noted a mean
drop in GTP followed by an abrupt elevation in those on MMF. Since GTP concentrations are elevated in
renal failure and fall after successful transplantation the latter was attributed initially to possible rejection,
but subsequently related to the change from conventional triple therapy to MMF. GTP concentrations in 24
transplanted patients on triple therapy were in the normal range (mean 40µmol/l) but elevated 3 fold in 24
patients on MMF (mean 128µmol/l). Such elevation of erythrocyte GTP by IMPDH inhibitors is curious
since normal human erythrocytes lack functional IMPDH, as confirmed in vitro where radiolabelled
hypoxanthine in intact erythrocytes is found only in IMP. Such an effect in erythrocytes by an
immunosuppressant which demonstrably inhibits lymphocyte IMPDH in vitro, inducing severe GTP
depletion (3), indicates the long-term efficacy of MMF therapy in renal transplant recipients requires careful
monitoring. Studies in progress comparing graft survival in these two patient cohorts have not yet revealed
any differences.
1.Montero C et al. Demonstration of induction of erythrocyte IMP-dehydrogenase activity in ribavirin treated
patients using an HPLC-linked method. Clin Chim Acta 1995; 238:169-178.
2. Wiegel G et al. Effect of mycophenolate mofetil therapy on inosine monophosphate dehydrogenase
induction in red blood cells of heart transplant recipients. Clin Pharmacol Ther 2001;69: 137-144
3. Qiu Yet al. Mycophenolic acid-induced GTP depletion also affects ATP and pyrimidine synthesis in
mitogen-stimulated primary human T-lymphocytes. Transplantation 2000; 69: 890-897
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P56
Cyclase and Phosphodiesterase Activity on Pre-T Lymphoid Human Cells, treated with Dimethyl
Sulfoxide (DMSO)
F. Di Pietrantonio, E. Di Matteo, M. Di Nicola, O. Trubiani, R. Primio, E. Serra, G. Spoto
University ‘G. D'Annunzio’, CHIETI, Italy
Cellular death can take two different forms respectively called necrosis and apoptosis (PCD). These two forms
differ from each other due to morphological and biochemical characteristics and biological incidence and
meaning. Almost all cells can experience PCD independently from the synthesis of new proteins or from the
phase of cellular cycle in which they are. In the immune system, PCD is a mechanism that is widely used to
select the clones that are functionally helpful for T and B lymphocytes during differentiation process. DMSO
typically promotes cellular differentiation due to its ability to arrest cellular growth at the G0-G1 phase.
Nevertheless, in other experimental conditions, for example in cellular cultures of pre-t lymphoid human cells
(RPMI-8402) DMSO induces apoptosis instead of a terminal differentiation. We studied cyclase and
Phosphodiesterase (PDE) activity on this cellular type during apoptosis, has been demonstrated that DMSO
probably induces apoptosis.
Methods: the utilized cells were the lymphocyte pre-t cells (RPMI-8402) in which apoptosis was induced
with reproducible and standardized DMSO. Cellular line was maintained in culture and the cells were let
grow until reaching the number of 2.5 x 106 /ml. During the logarithmic phase of growth, the cells were
treated with 1.5% of DMSO for a time included between 0 and 72 hours. Subsequently, the samples were
washed and re-suspension in PBS and derived for the analysis in HPLC.
Results: The obtained values, in the analysis of the cyclase and phosphodiesterase activity, are absolutely
specular. Adenylate-cyclase and cAMP-PDE activity has demonstrated values just determinable. Guanylatecyclase and cGMP-PDE activity has demonstrated values that were variable depending on the time of
treatment with DMSO.
Discussion: the data obtained from studying these activities agree with the type of the cell treatment with the
DMSO. In fact, the DMSO seems to activate the cells toward a "forced differentiation" because after 24
hours there is a peak in the synthesis of the chains if the gene TCR, which then decreases in the 48th hour.
The dynamics of the transcription gene was different. Its translocation from the cytoplasm to the nucleus
constantly increases from 0 until 72 hour.
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P57
Identification of the 5'-nucleotidase activity altered in neurological syndromes
M.G. Tozzi1, R. Pesi1, M. Camici1, F. Crementieri1, V. Micheli2, G. Jacomelli2, L. Notarantonio2
1
2
Nucleoside monophosphate phosphohydrolases comprise a family of enzymes that dephosphorylate nucleotides
both in intracellular and extracellular compartments. Members of this family exhibit different sequence,
location, substrate specificity and regulation. Besides the ectosolic 5’-nucleotidase, several cytosolic (cN-I, cNII, pN-I, dNT-I) and one mitochondrial enzymes (dNT-II) have been described (1-5). Nevertheless, several
researchers refer to as 5’-nucleotidase any AMP-dephosphorylating activity, lacking a more accurate
identification. An increase of cytosolic 5’-nucleotidase activity has been associated with neurological disorders
(6,7). The identification of the specific enzyme involved in these pathologies would be fundamental for the
comprehension of the linkage between the alteration of enzyme activity and alteration of brain functions. We
present here a relatively simple protocol which enabled us to measure the specific activity of three different
nucleotidases in primary cultures of fibroblasts of patients and controls. Patients have been chosen on the base
of their symptoms including autistic features, speech retardation and impaired urate/creatinine ratio in urine.
Human skin fibroblasts were obtained after informed consent, and were grown at 37°C in RPMI medium
supplemented with 10% foetal calf serum. We measured the activity of cN-I as the rate of radiolabelled AMP
hydrolysis, cN-II as the rate of phosphotransfer from IMP to radiolabelled inosine and dNTI and II as the rate
of radiolabelled dUMP hydrolysis.
Our preliminary results indicate that the described neurological symptoms, in at least one case, are associated
with an increase of cN-I activity. Cultured fibroblasts from patients are utilised for further metabolic studies.
Combined data of metabolic aspects and enzyme activity in erythrocytes and fibroblasts will help identifying
the metabolic pattern characteristic of these neurological syndromes.
1.Sala-Newby G.B. et al J. Biol. Chem. 274, 17789 (1999).
2.Allegrini S. et al J. Biol. Chem. 276, 33526 (2001).
3.Amici A. et al. Blood 96, 1596 (2000).
4.Ramazzo C. et al J. Biol. Chem 275, 5409 (2000).
5.Ramazzo C. et al PNAS 97, 8239 (2000).
6.Page T. et al PNAS 94, 11601 (1997).
7.Pesi R. et al NeuroReport 11, 1827 (2000).
149
P58
Exposure to Human Blood Decreases Swine Endothelial ECTO-5'-Nucleotidase Activity
Z. Khalpey1, K. Kalsi1, A.H. Yuen1, Z. Kochan2, J. Karbowska2, E.M. Slominska2, M. Forni3, M. Maccherini4,
M.L. Bacci3, P. Batten1, M. Lavitrano5, M.H. Yacoub1, R.T. Smolenski1
1
2
3
University of Bologna, BOLOGNA, Italy
4
5
University of Milan, MILAN, Italy
BACKGROUND: Ecto-5'-nucleotidase (E5'N or CD73) is an extracellular endothelial enzyme involved in the
final step of purine nucleotide breakdown and the formation of anti-inflammatory, antiaggretory and
immunosuppressive adenosine. Understanding of the regulation of expression of this enzyme may provide an
insight into overcoming acute vascular rejection. We evaluated whether confrontation of pig endothelial cells
with human blood induces changes in the activity of E5'N or the other enzymes of adenosine metabolism in pig
heart homogenates and endothelial cells.
METHODS: Pig hearts (wild type and transgenic for human decay accelerating factor-hDAF) were perfused
with fresh human blood for 4 h using a constant flow perfusion system. Pig aortic endothelial cells were used to
study the effect of human plasma on E5'N activity in vitro. Cells were exposed to human plasma for up to 3h.
Enzyme activity was measured in heart or cell homogenates with an HPLC based procedure.
RESULTS: Activity of E5'N was 6.60±0.33 nmol/min/mg protein in wild type pig hearts and 8.54±2.10
nmol/min/mg protein in transgenic pigs overexpressing hDAF. Ex vivo perfusion of pig heart with fresh human
blood for 4 h resulted in a decrease in E5'N activity to 62% and 61% of initial in wild type and transgenic pig
hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. Initial pig aortic endothelial
activity of E5'N was 9.10±1.40, 9.62±1.56 and 9.15±1.87 nmol/min/mg protein in control, heat inactivated and
active complement plasma groups. Activity decreased to 71% and 50% of initial after 3 h exposure to heat
inactivated and active complement human plasma respectively while it remained constant at 9.62±0.88
nmol/min/mg protein (107%) in controls incubated without human plasma.
CONCLUSION: pig heart activity of CD73 decreases following exposure to human blood which may affect
adenosine production and exacerbate hyperacute and acute vascular rejection. Complement may play a
significant role in the observed effect but its exact clarification requires further studies.
150
P59
Liver Transplant: Adenosine Metabolism and Apoptosis
E. Marinello1, F. Carlucci1, A. Tabucchi1, D. Neri2, G. Gerunda2, R. Merenda2, F. Rosi1, F. Floccari1
1
2
Inst. Chirurgia Gen., PADOVA, Italy
Apoptosis has been shown to play a major role in development and in the pathogenesis of numerous diseases, it
has been observed in hepatocytes and sinusoidal endothelial cell following ischemia-reperfusion injury and it
has been postulated as a contributing factor in ischemia-reperfusion graft dysfunction following liver transplant.
To elucidate the mechanism of ischemia-mediated liver alterations, changes of purine nucleotides, adenosine
catabolizing enzyme activities, glutathione antioxidant system (GSH and GSSG), caspase-3 activity and DNA
fragmentation were examined in hepatic biopsy specimens from six Landrace pig which underwent
experimental liver transplantation. Biopsies were obtained before explantation procedure (t1), after cold
ischemia period (t2), and 30’ from reperfusion on recipient (t3).
High energy phosphates (HEP) concentrations and GSH/GSSG ratio significantly decreased after ischemia,
adenosine and inosine monophosphate (IMP) increased; at 30’ from reperfusion, HEP levels inverted the trend
evidenced at t2. Regarding the activity of adenosine catabolizing enzymes, adenosine deaminase (ADA) and
soluble AMP-ase (e-Ns) were grossly reduced both at t2 and t3, no variations were evident for purine
nucleoside phosphorylase (PNP) and IMP-ase (cN-II). On apoptosis we evidenced a progressive increase of
caspase-3 activity during transplant time-course. In three cases, such increase, was associated with a sustained
DNA fragmentation.
Data on HEP evidenced their active degradation to adenosine during ischemic time, a further degradation to
inosine is however slowed down as confirmed from the reduced ADA activity. This behaviour togheder with
the reduction of e-Ns activity is probably related to the preservation of AMP and adenosine, precursors of HEP
via adenosine kinase.
The present study is aimed at clarifying the role of ischemic insult in a liver transplantat procedure and the
contribution of hepatocyte necrosis and/or apoptosis to this process. Our results suggest that, during liver
transplant, the complex mixture of insults results in a cell injury which can lead to apoptotic features.
151
P60
The immunological and kinetical characteristic of AMP-deaminase from normal and Hepatocellular
carcinoma (HCC) human liver
M. Szydlowska, Z. Ledziski, M. Krzyanowski, K. Kaletha
AMP-deaminase (AMP-aminohydrolase, EC 3.5.4.6) is a highly regulated enzyme, catalyzing irreversible
deamination of adenylic acid (5’-AMP), located at a branchpoint of adenylate nucleotides catabolism. It
plays an important role in stabilisation of adenylate energy charge (AEC) and regulation of purine
nucleotides pool in the cell.
The immunologic, kinetic and regulatory properties of AMP-deaminase from human liver neoplasm hepatocellular carcinoma (HCC) purified by double phosphocellulose chromatography were investigated and
compared with these obtained for the enzyme from unaffected tissue.
Specific activity of the enzyme present in the cancer tissue was significantly higher from that found in the
normal tissue. AMP-deaminase extracted from the cancer was less specific towards substrate analogues, and
in contrast to the enzyme extracted from the normal liver hardly reacted on pH and adenylate energy charge
changes tested. At physiological pH 7.0, in the absence and in the presence of important regulatory ligands,
AMP-deaminase from two sources studied manifested similar sigmoid-shaped substrate saturation kinetics,
with half saturation constant parameter (S0.5) being lower for the enzyme isolated from the tumor tissue. In
contrast to AMP-deaminase isolated from the normal human liver, where presence of a large protein
fragment corresponding to the full size of the native enzyme subunit was also detected (a result confirmed by
Western blot analysis), only smaller, proteolytically degraded fragments of AMP-deaminase isolated from
the cancer liver were detected in result of SDS-PAG electrophoresis. Obtained results indicate that intensive
degradation processes affecting tumor tissue can be responsible for differences found.
152
P61a
Biochemical and molecular genetic correlations in adenylosuccinate lyase deficiency
C. Salerno, C. Crif
Adenylosuccinate lyase (ADSL) deficiency is an autosomal recessive inborn error of purine synthesis
characterised by diminished levels of enzyme activity and accumulation of the dephosphorylated substrate
derivatives, SAICA riboside and S-Ado. Molecular analysis of the affected families reveals the existence of
about 30 different mutations in the gene, mostly in compound heterozygous form. It is widely accepted that
impairment of enzyme activity is due either to remarkable susceptibility to thermal stress or to changes in the
active site structure that leads to an anomalous handling of the substrates. However, the significance of
protein destabilization is unclear in the case of P100A/D422Y compound heterozygosis, because the enzyme
shows a Tm value that is decreased by only 8°C (to 51°C, from 59° C of control samples), while the kinetic
constants are very similar to those of the wild-type protein. Analysis of the enzyme structure suggests that
distorsion of amino acid chain by P100A substitution may expose Cys-98 and Cys-99 to oxidising agents.
Indeed, we found that the defective enzyme is strongly inhibited by 4-hydroxy-2-nonenal, the major product
of membrane peroxidation, which is believed to cause some of the tissue damage that occur in vivo under
conditions of oxidative stress. Enzyme is inactivated by a mixed-type cooperative mechanism that is partially
reversed in the presence of dithiotreitol. Amino acid substitution is located in domain I of the protein, in the
proximity of His-86 which behaves as general acid in the catalysis. Correct geometry of this region should be
essential to preserve enzyme function as it has been suggested by a comparative study of the structure of the
hyperthermophilic bacterium P. aerophilum. Indeed, the latter enzyme appears to be reinforced in this point
by a disulfide bridge that fixes Cys-87 (that corresponds to residue 101 in the human sequence) to the helix
3-helix 4 pair. As far as D422Y substitution is concerned, the mutation involves a cluster of helices in
domain III of the protein, on the outskirts of the tetrameric enzyme and far away from the active site, which
may be exposed to a noxious environmental stimulus as well.
153
P61b
AMPD1 C34T Mutation Selectively affects AMP-deaminase activity in the Heart
K. Kalsi, A.H. Yuen, P.H. Johnson, E.J. Birks, M.H. Yacoub, R.T. Smolenski
Objectives: An improved prognosis in heart failure was described in subjects with the C34T (Glu12Stop)
nonsense mutation in the AMP-deaminase 1 (AMPD1) gene. This gene is predominantly expressed in skeletal
muscle and possession of this mutation leads to decreased activity of AMPD and the accumulation of
cytoprotective adenosine in skeletal muscle. However, little is known about the metabolic changes within the
heart, in this study we evaluated the effect of the C34T mutation on cardiac enzymes involved in adenosine
metabolism.
Methods: Screening for AMPD1 genotype and measurement of enzyme activities was performed on 27 patients
with heart failure (HF): 16 requiring implantation of left ventricular assist device (LVAD) and 11 undergoing
elective transplantation. The presence of the C34T mutation was assayed by single-stranded conformational
polymorphism (SSCP) and restriction-fragment length polymorphism (RFLP) on extracted DNA. Cardiac
specimens were homogenized and assayed for: AMP-deaminase (AMPD), ecto-5’-nucleotidase (E5’N), purine
nucleoside phosphorylase (PNP), adenosine deaminase (ADA) and adenosine kinase (AK). Enzyme activities
were analyzed by measuring the conversion of substrates into product by HPLC. Results are expressed as mean
activities ± SEM.
Results: AMPD activity in heterozygous (C/T) individuals was 45% of that found in normal wildtype (C/C)
individuals (0.59±0.02* vs 1.06±0.09 µmol/min/g wet weight, *p= 0.003). There were no significant
differences in activities of any other enzymes when C/T was compared to C/C individuals, 3.96±0.41 vs
4.60±0.42 for E5’N, 1.03±0.21 vs 0.98±0.07 for PNP, 0.36±0.02 vs 0.41±0.04 for ADA, 0.018±0.002 vs
0.016±0.002 for AK.
Conclusions: We have shown for the first time a correlation between AMPD1 mutation and attenuated AMPD
activity in the heart therefore promoting cardioprotection in a localised manner. These changes did not affect
any other enzyme involved in adenosine/nucleotide metabolism.
154
P62
Association of improved cardiac function in donors with C34T mutation of the AMP Deaminase 1 gene
A.H. Yuen, M.H. Yacoub, E.J. Birks, K. Kalsi, P.H. Johnson, R.T. Smolenski
Background: Possession of the C34T mutation in AMP deaminase (AMPD1) gene has been shown to be
associated with attenuation of the progression of heart failure and improved survival in ischemic heart disease.
The aim of this study was to compare the frequency of the mutation in the heart donors, patients with heart
failure and healthy controls, to evaluate if the nonsense mutation in AMPD 1 can protect against myocardial
dysfunction in this critical condition.
Methods: The presence of the C34T mutation was assayed by single-stranded conformational polymorphism
(SSCP) and restriction-fragment-length polymorphism (RFLP) analysis on heart biopsies. Left ventricular
biopsies were collected from patients with HF due to dilated cardiomyopathy (DCM) (n=27) undergoing either
heart transplantation (Tx) or LVAD implantation. Donors were assessed by transoesophageal echocardiography
at the time of harvesting. Those with an ejection fraction (EF) > 40% (associated with good haemodynamics)
were used for Tx (n=22) and a right ventricular biopsy taken at the time of Tx. Donors with the EF <40%
(associated with poor haemodynamics) were considered unsuitable for Tx (unused donors, n=10). A small
piece from the left apex was taken for analysis. Blood samples from 207 healthy volunteers were also
genotyped.
Results: There was no difference in the frequency of the mutation between the patients with HF (14.8%) and
healthy volunteers (13.5%). However, the frequency of the mutation in the healthy donor hearts (31.8%) was
much higher when compared to the patients with HF and the healthy volunteers, and was significantly higher
than the failing donor hearts (5%) (p<0.025).
Conclusion: The presence of the C34T mutation in the AMPD1 gene protect against the development of donor
heart dysfunction. This suggests that attenuation of cardiac dysfunction in critically ill condition may be the
mechanism of improved survival observed in the other clinical conditions.
155
P63
Purine Metabolism in Pigs and Humans and its Implications for Xenotransplantation
R.T. Smolenski1, Z. Khalpey1, H. Dziewit2, E.M. Slominska2, A.H. Yuen1, M. Forni3, M.L. Bacci3, K. Kalsi1, Z.
Kochan2, J. Karbowska2, M. Maccherini4, M. Lavitrano5, M.H. Yacoub1
1
2
3
University of Bologna, BOLOGNA, Italy
4
5
Immune system is major focus of research into xenotransplantation, while metabolic compatibility of animal
organs with humans which could also affect organ survival has been studied little so far. We compared
concentrations of nucleotide substrates and activities of enzymes of nucleotide metabolism in pig and human
blood, heart and kidney. Furthermore, we studied changes in cardiac metabolite concentration following 4h of
ex vivo perfusion with human blood. Donor human heart and kidney specimens and blood of healthy subjects
were used as a reference. Enzyme activities and metabolite concentrations were measured using HPLC with
UV or mass detection. The most prominent difference was in ecto-5'-nucleotidase (E5'N) activity: in hearts:
1.68±0.30 nmol/min/mg wet wt in human vs. 0.46±0.02 in pigs, in kidney 0.74±0.06 nmol/min/mg wet wt in
humans vs. 0.11±0.01 in pigs and in endothelial cells: 39.1±5.8 nmol/min/mg prot in human vs. 9.1±1.4 in pig
cells. Baseline concentrations of ATP, GTP, UTP and CTP were similar in human and pig hearts. However,
specific decrease in GTP concentration has been observed from an initial of 1.35±0.12 nmol/mg dry wt to
0.68±0.06 following perfusion with human blood. There were profound differences in blood purine and
pyrimidine concentrations which for: 1) hypoxanthine, 2) inosine, 3) adenine, 4) uracil, 5) uridine and 6) uric
acid were: 1) 17.7±1.5 and 0.95±0.08 µM 2) 6.45±1.42 and 0.10±0.10 µM, 3) 3.95±0.14 and 0.07±0.02 µM, 4)
17.6±0.9 and 0.35±0.09 µM, 5) 1.41±0.13 and 2.80±0.30 µM, 6) 15.0±1.9 and 290.0±16.0 µM in pig and
human blood respectively. Conclusion: Low activity of E5'N activity in pigs may lead to retention of proinflammatory and pro-aggregatory nucleotides and attenuation of formation of anti-inflammatory and antiaggreggatory adenosine which counteract those effects following pig to human xenotransplantation. Human
blood concentration of nucleotide precursors may not be adequate to maintain guanine nucleotide pool.
Overexpression of E5'N and supply of substrates for GTP synthesis or allopurinol treatment may prolong pig to
human xenograft survival.
156
P64
Overexpression of Human ECTO 5' Nucleotidase in Pig Endothelial Cells and its Implication for
Adenosine Production and Xenotransplantation
F. Osborne1, C. Lawson1, K. Kalsi1, M. Lavitrano2, M.H. Yacoub1, M.L. Rose1, R.T. Smolenski1
Imperial College, HAREFIELD, United Kingdom
2
1
Ecto 5’ nucleotidase (E5’N) or CD73 is an endothelial surface enzyme involved in extracellular nucleotide
breakdown leading to the formation of anti-inflammatory and immunosuppressive adenosine. Expression of
E5’N in pig cells has been found to be one order of magnitude lower than in human endothelium. This is
particularly important for xenotransplantation where metabolism of extracellular nucleotides into adenosine
plays a crucial role in interaction of endothelial cells with inflammatory cells and platelets. The aim of this
study was to establish the feasibility of over-expression of human E5’N in pig endothelial cells and see if this
affects rate of adenosine production in intact cells from exogenous AMP and ATP. The pig intestinal
endothelial cell line (PIEC) was transfected using Effectene with human endothelial cDNA cloned into
pCDNA3.1 bearing the neomycin resistance element and cell clones with the highest activity were selected
using serial dilutions. Cells used were >95% human CD73 positive by flow cytometry. E5’N activity in cell
lysates and rate of degradation of AMP in intact cells were measured by HPLC. Transfected and nontransfected PIEC were compared to human umbilical vein EC (HUVEC). PIEC enzyme activity in lysed
untransfected cells was 9.10±1.4 compared to 109.6±10.5 in transfected compared to 39.1±5.8 nmol/min/mg
prot in HUVEC. Rate of adenosine production in intact cells from exogenous AMP was 0.099 µM/min/mg
protein in non-transfected cells and 47.2 µM/min/mg protein in transfected cells. Expression of human E5’N in
pigs cells caused three-fold increase in the rate of adenosine formation from extracellular ATP. In conclusion,
human E5’N could be functionally expressed on the surface of pig endothelial cells. Increased production of
adenosine not only from AMP but also from ATP demonstrates that E5’N activity is the rate limiting step and
suggests that E5’N over-expression in transgenic pigs would be a successful strategy for xenotransplantation.
157
P65
Lidoflazine combined with nucleotide precursors increases ATP content and Adenosine production in
cardiomyocytes
K. Kalsi, R.T. Smolenski, M.H. Yacoub
Objectives: Lidoflazine (LIDO) acts not only as a calcium antagonist but also has nucleoside transport inhibitor
(NTI) properties and therefore increases adenosine (ADO) concentration. Stimulation of the purine salvage
pathway with nucleotide precursors increases ATP and ADO content. However the combined effect of LIDO
with precursors has not been demonstrated. Our objectives were to investigate the effects of LIDO alone or
combined with Adenine (Ade), Ade + Inorganic phosphate (Pi), Ade + ribose (A/R) or A/R + Pi on ADO
production and ATP content in myocytes.
Methods: Rat cardiomyocytes were isolated using collagenase perfusion technique. Cells were then incubated
for up to 8hr as follows: (1) Control; (2) 10 microM LIDO; (3) LIDO + 100 microM Ade; (4) LIDO + Ade + 5
mM Pi; (5) LIDO + Ade + 2.5 mM Ribose and (6) LIDO + A/R +Pi. 5 microM EHNA (inhibitor of ADO
deaminase) was added to all incubations. After 8hrs, extracts of myocyte suspensions were analysed by HPLC.
Results are expressed as mean values ± SEM, (n=4-6).
Results: ATP content was decreased after 8hr of incubation in (2) to 21.4±1.6 nmol/mg prot. from 22.1±0.9 in
(1), ATP was enhanced to 24.1±1.6, 29.9±1.9, 32.5±2.5* and 35.2±2.8* respectively in (3), (4), (5) and (6)
(*p<0.05). ADO was increased in (2) to 1.6±0.2, but was further increased in (3) to 2.8±0.8*, in (4) to
2.1±0.3*, in (5) to 2.8±0.8* and in (6) to 3.1±0.6* vs (1) 1.4±0.1 (*p<0.05).
Conclusion: We have shown that LIDO alone decreases ATP concentration in isolated myocytes together with
an increase in ADO production. The presence of a combined application of nucleotide precursors (A/R or either
A/R + Pi) and LIDO stimulates adenine nucleotide resynthesis resulting in an augmentation and protection of
the ATP pool and increased ADO production.
158
P66
Investigation of substrate recognition of Drosophila melanogaster nucleoside kinase by site directed
mutagenesis
S.N. Solaroli1, M. Bjerke2, M. Johansson2, A. Karlsson2
1
Huddinge University Hospital, HUDDINGE, Sweden
2
Karolinska Institute, HUDDINGE, Sweden
Expression of herpes simplex virus thymidine kinase (HSV-TK) in tumor cells and subsequent systemic
chemotherapy with the nucleoside analog ganciclovir is the prototype for combined gene/chemotherapy of
malignant tumors. In search for an alternative suicide gene to HSV-TK, we have cloned and characterized a
deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK). The enzyme efficiently phosphorylates
several anti-viral and anti-cancer nucleoside analogs with a higher catalytic rate then other known
deoxyribonucleoside kinases. Dm-dNK has broad substrate specificity but does not phosphorylate ganciclovir
or other guanosine nucleoside analogs. We have performed site directed mutagenesis on residues that, based on
structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of
purine substrates. In particular two mutants, M88R and V84A+M88R, showed activity using ganciclovir as
substrate. Different truncated forms of the enzyme carrying these mutations will be presented as well as data on
the expression of these mutated enzymes in cell culture system.
159
P67
Evaluation of ADA Gene Expression and Transduction Efficiency in ADA/SCID Patients Underwent
Gene Therapy
E. Marinello1, F. Carlucci1, A. Tabucchi1, A. Aiuti2, F. Rosi1, F. Floccari1, R. Pagani1
1
2
San Raffaele H. Scient. Inst., MILAN, Italy
The ADA-deficient subjects have a type of SCID caused by a gene-defect in the enzyme adenosine deaminase
(ADA), that is critical for normal immune function. ADA-deficient SCID has long been considered a model
disease for gene therapy trials because it's caused by a defect in a single gene.
Gene transfer into hematopoietic stem cell (HSC) for adenosine deaminase (ADA)-deficient SCID has been so
far limited by the low levels of engrafted transduced HSC. An improved protocol for gene transfer into
autologous bone marrow (BM) CD34+ cells, combined with non-myeloablative conditioning was designed. To
explore the transfection efficiency for different retroviral vector and fully evaluate the efficacy of gene therapy,
the measurement of ADA activity in transduced cells is essential. A suitable capillary electophoresis (CE)
method was developed for such purpose.
The enzyme activity was evaluated by the quantitation of substrate disappearance and reaction product
formation separated by CE. The conditions were as follow: electrolyte 20 mmol/L sodium-borate (pH 10.00),
uncoated capillary 42 cm x 75 mm i.d., voltage 12 kV, analysis at 254 nm. The method was linear in the range
2-500 mmol/L (r >0.99). The intra- and inter-assay variability showed a CV <4%. Enzyme activities were
linear with respect to sample amounts in the incubation mixture and respect to incubation time (both r >0.99).
In healthy subjects ADA showed a value of 20.23±9.27, in patients at diagnosis it was 0.08±0.06 nmol/h/mg of
protein. The method permitted a complete screening of gene therapy protocols evidencing the restoration of
intracellular ADA activity in PBL, in various lymphocyte populations and the increase of ADA activity in RBC
from undetectable levels to 20-30% of healthy controls, that was accomplished to the reduction of toxic
deoxynucleotides.
This technique is a valuable, fast and unexpensive screening tool in the evaluation of ADA activity, being
useful to quickly assess the expression of ADA in hematopoietic cells of SCID patients and represents an
important tool for the follow-up of patients treated with enzyme replacement therapy or in clinical gene transfer
protocols.
160
P68
Metabolism of Adenosine in Human Colorectal Tumor
E. Marinello1, D. Vannoni1, A. Bernini2, F. Carlucci1, M.C. Di Pietro1, F. Floccari1, R. Leoncini1, F. Rosi1, A.
Santoro1, A. Tabucchi1, G. Tanzini3
1
2
Inst. Chirurgia Gen., SIENA, Italy
3
Institute of Clin. Chirurgica Gen., SIENA, Italy
The adenosine is produced from AMP by the action of 5’nucleotidase (5’-NT) and it is converted either to
inosine or back into AMP via the reactions catalysed by adenosine deaminase (ADA) and adenosine kinase
(AK) respectively. The steady-state concentration of adenosine is maintained by the activities of these three
enzymes. The presence of adenosine is associated with inhibition of the immune response, coronary
vasodilatation, neurotransmission, hormone stimulation; the cytoprotective functions of adenosine include
stimulation of angiogenesis and inhibition of inflammatory reactions. Many observations suggest that
adenosine may have similar function in cancer.
The aim of this work is to analyse the activities of the three enzymes involved in adenosine metabolism, in
fragments of intestinal mucosa of patients affected by colorectal cancer.
We analysed 20 patients 11 males and 9 females aged between 46 and 80 years. 8 of them were affected by
rectal cancer, 6 by sigma cancer, 4 by right colon cancer and 2 by left colon cancer. 2 of them were T2; 14 were
T3 and 4 were T4; 10 presented lymphonoid metastasis and 3 also pulmonary and liver metastasis. The grade
was so distributed:2 patients were G1, 11 G2 and 6 patient G3. The activities were assayed in fragments of
neoplastic and normal-appearing mucosa, surrounding the tumour in a region close (less than 3 cm) and distant
(at least 10 cm) to the tumour, which were removed during the surgical resection and immediately frozen in
liquid N2.
The results show that the three activities are markedly higher in tumour in comparison to close or distant
normal mucosa (p<0.01 for ADA and AK; p<0.05 for 5’-NT).
Results suggest that the activities of purine metabolizing enzymes increase to cope with accelerated purine
metabolism in cancerous tissues. The contemporary increased of ADA and 5’-NT activities might be a
physiological attempt of the cancer cells to provide more substrates needed by cancer cells to accelerate the
salvage pathway activity.
161
P70
New evidences for regulation of deoxycytidine kinase activity by reversible phosphorylation
C. Smal, S. Cardoen, M. Veiga-da Cunha, S. Marie, G. Van den Berghe, F. Bontemps, L. Bertrand, E. Van den
Neste, A. Ferrant, V. Race
Deoxycytidine kinase (dCK) is a key enzyme in the deoxynucleoside salvage pathway. Besides its natural
substrates, dCK activates various nucleoside analogues used against viral infections, leukaemia and solid
tumours. Knowledge of its regulation can be expected to allow optimization of the activation of these
analogues, and hence to improve their clinical efficacy. In recent years, various genotoxic agents, including
2-chloro-2’-deoxyadenosine, aphidicholin and UV light, have been shown to enhance dCK activity in
leukaemic cells without changing the level of dCK protein (Sasvari-Szekely et al, Biochemical Pharmacol
56, 1175-9, 1998; Van Den Neste et al, Biochemical Pharmacol 65, 573-80, 2003). These observations led to
the hypothesis that dCK activity can be regulated by post-translational modification, most probably by
reversible protein phosphorylation (Csapo et al, Biochemical Pharmacol 61, 191-7, 2001). In accordance
with this hypothesis, we observed that dCK can be 65 % inactivated in a crude cell extract from EHEB cells,
a continuous B-cell line, by treatment with protein phosphatase 2A, responsible for dephosphorylation of
serine and threonine residues. Inactivation was prevented by okadaic acid. These results corroborate that
dCK activity is regulated by reversible phosphorylation and indicate moreover that activation of the enzyme
involves phosphorylation of serine or threonine residues. Accordingly, dCK activity was not affected by
protein tyrosine phosphatase treatment. To analyse its phosphorylation in more details, dCK was expressed
in human embryonic kidney (HEK)-293 cells as a His-tag fusion protein and purified by affinity
chromatography on cobalt-agarose resin. After SDS-page electrophoresis and immunoblotting with anti-His
antibody, dCK appeared as a doublet, possibly due to two different phosphorylation levels resulting in a
mobility-shifted signal. To verify this hypothesis, purified expressed dCK was treated with λ phosphatase.
This resulted in a profound decrease of dCK activity, accompanied by an increase of its electrophoretic
mobility. Our results provide additional arguments in favour of a regulation of dCK activity by reversible
phosphorylation.
162
P71
Cyclopentenyl cytosine sensitises SK-N-BE(2)c neuroblastoma cells to cladribine
J. Bierau1, A.B.P. Van Kuilenburg1, A.H. Van Gennip2, R. Leen1, L. Zoetekouw1, H. Caron1
1
2
Cyclopentenyl cytosine (CPEC) is a drug targeted at CTP synthetase and possesses anti-tumour activity against
neuroblastoma in vitro. In neuroblastoma, incubation with CPEC causes depletion of the intracellular
(deoxy)cytidine nucleotide pools, and retardation in the S-phase of the cell cycle. The effects caused by CPEC
in neuroblastoma make the combination of CPEC and S-phase active deoxynucleoside analogues attractive for
chemotherapy.
Cladribine (CdA) is an analogue of deoxyadenosine and is an effective drug in the treatment of haematological
malignancies such as hairy cell leukaemia and acute myeloid leukaemia. However, no anti-tumour activity of
CdA against solid tumours has been observed in clinical trials. CdA is an inhibitor of DNA synthesis as well as
DNA repair and a synergistic effect was obtained when this drug was combined with DNA damaging agents.
Although CdA is a purine analogue, the first and rate-limiting step in its activation is catalysed by
deoxycytidine kinase (dCK). The depletion of dCTP achieved via inhibition of CTP synthetase by CPEC may
thus lead to an enhanced uptake and anabolism of CdA.
In this study, it is demonstrated that CPEC sensitised the CdA-resistant human neuroblastoma cell line SK-NBE(2)c (ED50 >> 1.5 µM) towards CdA. Pre-treatment with 100 or 250 nM CPEC sensitised SK-N-BE(2)c
cells to cladribine the 96-hr ED50 values being 419 ± 125 nM and 70 ± 30 nM, respectively. Preincubation
with 100 nM CPEC for 24 hr increased the amount of intracellular CdA-nucleotides 1.2 to 4.7-fold, CdAMP
being the major metabolite to accumulate. DNA synthesis was inhibited by 45 % by 100 nM CPEC and > 95 %
by the combination of CPEC and CdA. CdA itself did not inhibit DNA synthesis. Our results demonstrate that
the cytotoxic effects of CdA can be enhanced by inhibition of CTP synthetase and suggest that CAMP may be
metabolite causing the increased cytotoxicity observed.
163
P72
Gemcitabine and cyclopentenyl cytosine: a promising combination for the treatment of neuroblastoma
J. Bierau1, A.B.P. Van Kuilenburg1, A.H. Van Gennip2, R. Leen1, R. Meinsma1, H. Caron1
1
2
Neuroblastoma is the most common solid malignancy of childhood. Despite intensive chemotherapeutic
regimens, the survival rate of children suffering from metastasized neuroblastoma remains very poor. Poor
prognosis is often associated with amplification of the MYCN-oncogene. We have observed that 2',2'difluorodeoxycytidine (Gemcitabine, dFdC) has potent anti-tumor activity against neuroblastoma in vitro. dFdC
is a pro-drug that is activated by phosphorylation to its nucleotides, of which deoxycytidine kinase (dCK)
catalyzes the first and rate-limiting step. dFdC was tested on a panel of human neuroblastoma cell lines
consisting of MYCN-amplified and MYCN-single copy cell lines. In both types of cell lines, low ED50 values
(nM range) were observed. Despite the fact that the specific deoxycytidine kinase (dCK) activity was 60%
higher in MYCN-amplified cell lines than in MYCN-single copy cell lines, this did not correlate with the ED50
values observed. However, while treatment with dFdC induced cell death in MYCN-amplified cell lines,
MYCN-single copy cell lines underwent neuronal differentiation. Shep21N cells, in which MYCN-expression
is controlled by tetracyclin sensitive promotor, underwent cell death after incubation with dFdC when MYCN
was expressed, but no cytotoxicity was observed when MYCN was not expressed. This may be caused by the
fact that the dCK activity was higher in MYCN expressing Shep21N cells than in Shep 21 cells that did not
express MYCN. Pre-incubation with the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC) significantly
lowered the ED50 values of 13 out of 15 cell lines. In SK-N-BE(2)c cells that had been incubated with 100 nM
CPEC for 1-4 days, the anabolism of dFdC was increased to 6-44 times as compared to untreated control cells,
as measured by metabolic labeling experiments. This was paralleled by an significant increase in the expression
of dCK-mRNA, dCK proteins and increase of dCK activity. We feel that the combination of dFdC and CPEC
may hold promise for the treatment of high-risk neuroblastoma
164
P73
Modulation of cytarabine resistance in childhood acute myeloid leukemia
I. Hubeek1, G.J. Peters1, A.J.F. Broekhuizen1, Ch.M. Zwaan1, U. Creutzig2, G.J.L. Kaspers1
1
2
AML-BFM Study Group, MÜNSTER, Germany
Cytarabine is an important drug in the treatment of childhood acute leukemia. Resistance to this drug is
therefore a clinically relevant problem. To modulate cytarabine resistance we combined cytarabine with 6
potential resistance modifiers: cladribine and gemcitabine (nucleoside analogs), decitabine (hypomethylating
agent), aphidicolin (inhibitor of DNA polymerases alpha, delta and epsilon), and flavopiridol and UCN01
(inhibitors of cyclin-dependent kinases).
The single agents and their combinations were studied in the leukemic cell line HL60 and ex vivo in blast cells
isolated from 10 children with de novo AML using the MTT assay. In the HL60 cell line cytarabine was
combined with the different modulators at the ratios of their respective IC50s. In the patient samples cytarabine
was tested in 6 different concentrations, the modulators were tested at a low concentration (± 10% cell kill as a
single agent) and a higher concentration (± 30% cell kill as a single agent). Drug interactions were investigated
by computer-assisted analysis using the Calcusyn software (median drug effect method, Chou & Talalay), by
calculation of the combination index ((CI); additivity ± 1; synergistism <1; antagonistism >1).
In the HL60 cell line we observed moderate synergism between cytarabine and aphidicolin (CI=0.81),
cladribine (CI= 0.76), flavopiridol (CI=0.77) and gemcitabine (CI=0.82). The interaction between cytarabine
and decitabine was synergistic (CI=0.3). In contrast, co-incubation of cytarabine with UCN01 resulted in
antagonism (CI=1.47) In the AML patient samples co-incubation of cytarabine with aphidicolin, in both the
low and the high concentration, showed strong synergism (median CI= 0.28 and 0.18 respectively, (n=9)). The
combinations of cytarabine/cladribine (median CI= 0.82 and 0.51 (n=10)) and cytarabine/gemcitabine (median
CI= 0.5 (=10)) were all synergistic. Nearly additive and moderately synergistic interactions were observed
between cytarabine/flavopiridol (median CI=0.93 (n=10) and 0.82 (n=8)) and cytarabine/UCN01 (median CI=
0.82 and 0.74 (n=9)). However, the combination of cytarabine with decitabine was antagonistic (median CI>10
(n=6) and CI=5.92 (n=3)). In conclusion, additive or synergistic interactions of cytarabine with aphidicolin,
cladribine, gemcitabine, flavopiridol and UCN01 were observed in this ex vivo model testing pediatric AML
samples, supporting the future evaluation of these combinations in clinical trials with AML patients.
165
P74
Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine
in leukemia and solid tumor cell lines
A.M. Bergman1, C.M. Kuiper1, P. Noordhuis1, K. Smid1, D.A. Voorn1, E.M. Comijn1, G.J. Peters1, F. Myhren2,
M.L. Sandvold2, H.R. Hendriks3
1
2
Clavis Pharma, PORSGRUNN, Norway
3
EORTC-NDDO, AMSTERDAM, The Netherlands
Ara-C is a deoxycytidine (dCyd) analog with activity in leukemia, which requires phosphorylation by
deoxycytidine kinase (dCK) to its active phosphate ara-CTP, but can be deaminated by deoxycytidine deaminase
(dCDA). In addition to dCK deficiency, altered membrane transport of deoxyribonucleosides over the cell
membrane is a mechanism of drug resistance. In order to facilitate ara-C uptake and prolong retention in the cell,
lipophillic pro-drugs were synthesized. Fatty acid groups with a varying acyl chain length and number of double
bonds were esterified at the 5´ position on the sugar moiety of ara-C. The compounds were tested in two pairs of
ara-C resistant cells (the murine leukemia L5 and L4A6 and the rat leukemia BCLO and Bara-C) and two pairs
of cell lines with a resistance to gemcitabine, a deoxycytidine analog with a comparable mechanism of action as
ara-C (the human ovarian cancer A2780 and AG6000 and murine colon C26-A and C26-G). L4A6, Bara-C and
AG6000 have varying degrees of decreased dCK activity, while the mechanism for C26-G is not yet clear. In the
parent cell lines ara-C was more active, but in the various resistant variants several of the analogs were more
active, while the degree of cross-resistance varied. In AG6000 with a total dCK deficiency, all compounds were
inactive. Structure activity relation analysis showed that ara-C derivatives with shorter acyl chains and more
double bonds were more active in the parental and drug resistant cells. Further mechanistic studies were
performed with the elaidic acid derivative of ara-C (CP-4055). CP-4055 inhibited deamination of dCyd partly
and induced DNA synthesis inhibition effectively in C26-A and C26-G cells, but the retention of inhibition was
much longer for CP-4055 than for ara-C. In contrast to ara-C, CP-4055 inhibited RNA synthesis for 60% after
drug exposure. In conclusion, CP-4055 seems to be a promising prodrug, whose effects were different and
longer lasting than for the parent drug.
166
P75
Pyrimidine deoxynucleoside salvage in Ureaplasma urealyticum: nucleoside analogues acted as potent
inhibitors of mycoplasma growth
R. Wehelie1, S. Eriksson2, G. Bölske3, L. Wang2
1
SLU, UPPSALA, Sweden
2
3
National Veterinary Institute, UPPSALA, Sweden
Ureaplasma urealyticum (U. urealyticum) and Mycoplasma pneumoniae(M. pneumoniae) are pathogens of
both genital and respiratory tract. Radiolabeled nucleosides and nucleoside analogues were used to study the
biosynthesis, interconversion, and degradation of pyrimidine nucleosides/nucleotides in U. urealyticum.
[methyl- 3H]-deoxythymidine (3H-dThd) and [5- 3H]-deoxycytidine (3H-dCyd) were metabolised to monoand diphosphates, and no triphosphates were detectable. 3H-dCyd was also converted to deoxyuridine
monophosphate due to the deamination by cytidine deaminase. Subsequently incoperation of these radiolabeled
precursors into DNA were also detected. Small amount of monophosphate was detected from cell cultures
grown in the presence of either [6- 3H]-5-Fluorodeoxyuridine (5-FdUrd) or [6- 3H]-5-Fluorouracil (5-FUra)
indicating the uptake and salvage of these nucleoside analogues, but there was no incoperation of these
precursors into the DNA. The levels of deoxynucleoside kinases and nucleoside monophosphate kinases in U.
urealyticum were determined and we found that deoxynucleoside kinases especially thymidine kinase activity
was upregulated upon the addition of either dUrd or dTHU in the growth medium while nucleoside
monophosphate kinases such as thymidylate kinase activity were not affacted. A number of pyrimidine and
purine nucleoside analogues were found to inhibit the growth of U. urealyticum and M. pneumoniae and
Among those tested analogues fluoropyrimidines were the most potent inhibitors. 5-FdUrd, 5-FdCyd and 5FUra significantly inhibited U. urealyticum growth with IC50 of 0.8, 3 and 40 mM at 104 cfu/ml
respectively, and 5-FdUrd inhibited M. pneumoniae growth with IC50 of 6.2 µM. The inhibitory effect of
these analogues could be reversed by the addition of either dThd, dUrd or dTHU, suggesting that the
mechanism of inhibition was due to the inhibition of thymidylate biosynthesis.
167
P76
Synergistic effects of BCNU and Didox combination chemotherapy in 9L glioma cells
Z. Horvath1, W. Bauer1, Th. Hoechtl1, Ph.S. Saiko1, M. Fritzer-Szekeres1, T. Tihan2, T. Szekeres1
1
2
Johns Hopkins Hospital, BALTIMORE, United States of America
A significant number of malignant gliomas develop resistance to systemic and local 1,3-bis (2-chloroethyl)-1nitrosourea, (BCNU) therapy, a first-choice drug in adjuvant chemotherapy. Increases in DNA repair and
synthesis are implicated as the main causes of BCNU resistance. Compounds inhibiting DNA synthesis and
repair are expected to act synergistically with BCNU and improve the efficacy of BCNU-therapy. Inhibitors of
the enzyme ribonucleotide reductase might be used as additional agents. Ribonucleotide reductase (EC1.17.4.1;
RR) catalyzes the rate-limiting step in DNA synthesis and plays a critical role in maintaining substrates for
DNA repair. Ist increased rate of expression in cancer cells makes RR a critical target in cancer chemotherapy.
Didox (3,4-dihydroxybenzohydroxamic acid) is a potent inhibitor of RR, and has been proven to induce
apoptosis in vitro and in vivo.
We studied the effects of Didox on 9L glioma cells in combination with BCNU. We analyzed intracellular
deoxynucleosidetriphosphate (dNTP) pools and found that Didox depleted significantly the intracellular dNTP
concentrations. After incubation with 15 and 30 µM Didox, the intracellular concentrations of dGTP fell to 34
and 16,7% of control values, respectively. The dCTP and dTTP pools also showed significant decrease (66,9
and 61,3% of control for dCTP and 84,9 and 67,4% of control for dTTP, respectively). The intracellular dATP
pools have experienced an increase of 186,3 and 154,8%. Experiments using cytotoxicity, growth inhibition
and clonogenicity assays showed significant synergism of Didox and BCNU. In chrystal violet assay, Didox as
a single agent yielded an IC50 value of 400µM, whereas BCNU reached an IC50 value of 500µM. After
combined treatment using equimolar concentrations of the two drugs, a combination index<1 was reached in
concentrations between 50 and 500µM, demonstrating synergistic effects. In growth inhibition and colony
formation assays with concentrations ranging from 20 to 100µM, the synergism between Didox and BCNU
was also confirmed. We have also identified increased gene expressions in a number of DNA-repair related
enzymes using large-scale cDNA-arrays after BCNU incubation. These effects could partially be circumvented
after Didox treatment.
These results introduce the combination of Didox and BCNU as an additional option for the treatment of
gliomas.
168
P77
Amidox, an inhibitor of ribonucleotide reductase, potentiates the action of Ara-C in HL-60 human
promyelocytic leukemia cells
W. Bauer, Z. Horvath, Th. Hoechtl, D. Karl, Ph.S. Saiko, M. Fritzer-Szekeres, T. Szekeres
Ribonucleotidereductase (RR) catalyzes the rate limiting step in the de-novo synthesis of
deoxyribonucleotidetriphosphates (dNTPs). Since its activity was shown to be correlated to the proliferation
rate in tumor cells, it is considered to be an excellent target for cancer chemotherapy. Amidox (3,4dihydroxybenzamidoxime), a polyhydroxy-substituted benzoic acid derivative, inhibits RR by interfering with
the iron binding subunit of the enzyme.
In the present study we investigated the antineoplastic effects of Amidox alone and in combination with
Arabinosylcytosine (Ara-C) in HL-60 human promyelocytic leukemia cells.
In growth inhibition experiments with logarithmically growing HL-60 cells Amidox yielded an IC50 of 30µM.
The colony formation of HL-60 cells was inhibited at an IC50 of 20µM as determined by a soft agar assay. We
also investigated in a possible reduction of intracellular dNTPs after incubation with Amidox as could be
expected by inhibition of RR. Exposure of the cells to 75 and 100µM Amidox for 24 hours was shown to
significantly decrease intracellular dCTP, dGTP and dTTP pools, whereas dATP concentration increased, as
determined by HPLC.
Combination of an RR inhibitor like Amidox with Ara-C could lead to synergistic antineoplastic effects by
various mechnisms.The promotion of phosphorylation of Ara-C to Ara-CTP, the active compound, by
deoxycytidinekinase, which underlies feedback inhibition by dCTP, by decreased dCTP levels could be one of
them.
In our experiments the combination of Amidox with Ara-C yielded additive cytotoxic effects in growth
inhibition assays and additive inhibition of colony formation in soft agar assays. We could show that - after
preincubating the cells with 75 and 100µM Amidox for 24 hours and subsequent exposure to Ara-C for 4 hours
- intracellular Ara-CTP levels increased by 576% and 1143%, respectively.
In conclusion, Amidox was shown to have potent antineoplastic effects in HL-60 human promyelocytic
leukemia cells in concentrations that can be achieved in vivo. It increases the formation of Ara-CTP but yields
additive effects with Ara-C concerning growth inhibition and colony formation. Nevertheless the compound
might be further investigated for the treatment of leukemia.
169
P78
Mechanism of action of trifluorothymidine (TFT) in combination with a thymidine phosphorylase
inhibitor (TPI)
O.H. Temmink1, O.H. Temmink1, M. De Bruin1, E.M. Comijn1, M. Fukushima2, G.J. Peters1
1
2
Trifluorothymidine (TFT) is a thymidine analog which is activated by thymidine kinase (TK) to TFT-MP
which may inhibit thymidylate synthase (TS), involved in thymidine nucleotide synthesis. TFT-TP can be
incorporated into DNA, possibly resulting in DNA damage and cell death. TFT can be broken down to
trifluoromethyluracil (TFMU) by thymidine phosphorylase (TP), which can be inhibited by TP inhibitor (TPI)
to enhance the bioavailability of TFT in vivo (the combination TAS-102). We studied the effect of TPI on TFT
sensitivity, in situ TS inhibition, TFT metabolism and its incorporation into DNA. 5-Fluorouracil (5-FU)
resistant H630 cell lines were cross-resistant to TFT (72 h exposure), in contrast to antifolate resistant WiDr
cells, although all resistant variants had increased TS levels. However, at short term exposure (4 h) the 5FUresistant H630 cells and antifolate resistant WiDr cells were both cross-resistant to TFT. TPI did not enhance
TFT sensitivity in these and Colo320TP1 (TP transfected) cells. The concentration of TFT to get 50%
inhibition of TS in both intact H630 and WiDr cross-resistant cells was 40- to 300-fold higher compared to the
parental cells. In situ TS inhibition was very rapid in FM3A/0 mammary cancer cells (>90% after 4h), but also
recovered rapidly (back to 75% after 20 h removal of TFT) when compared to 5FU. The addition of TPI to the
medium did not increase TS inhibition by TFT. In the colon cancer cell lines WiDr, H630, Colo320 (TP
deficient) and the TP transfected Colo320TP1 TFT incorporation into DNA was about 1.09, 0.77, 1.46 and
0.96 pmol/hr/106 cells, resp., and the accumulation of TFT phosphate derivatives was 1.39, 1.37, 2.73 and 1.29,
resp. TPI increased the accumulation of TFT phosphate derivatives in Colo320TP1 cells to 2.42 pmol/hr/106
cells (p<0.05). In the presence of TPI TFMU formation was completely inhibited in all cell lines. The
formation of TFMU by Colo320TP1 cells was also inhibited by almost 50% at equimolar thymidine
concentrations. It can be concluded that cancer cells take up and activate TFT very rapidly, possibly due to
favourable enzyme properties. TPI increased TFT phosphorylation only at very high TP levels.
170
P79
The effect of different fluoropyrimidines with or without a thymidine phosphorylase inhibitor (TPI) on
the expression of platelet derived endothelial cell growth factor / thymidine phosphorylase (PDECGF/TP)
K. Smid1, M. De Bruin1, T. Van Capel1, K. Hoekman1, M. Fukushima2, H.M. Pinedo1, G.J. Peters1
1
2
TP is an enzyme catalyzing the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1phosphate. TP is similar to PD-ECGF, which plays a role in angiogenesis and has been shown to be
upregulated in several solid tumors. Fluoropyrimidines such as 5-fluorouracil (5FU), 5′-deoxy-5fluorouridine (5′DFUR) and trifluorothymidine (TFT), are substrates for PD-ECGF/TP. These are either
activated (5FU and 5′DFUR) or inactivated (TFT) by TP. We therefore studied whether treatment with
5′DFUR and TFT with or without a specific TP inhibitor (TPI) would affect PD-ECGF/TP expression and
function. Three human colon cancer cell lines, WiDR, HT29 and Lovo were treated for 72 h with 5′DFUR or
TFT at their IC50 concentration with or without 10 µM of TPI or with TPI alone. PD-ECGF/TP expression
was analyzed at the mRNA level with competitive template reverse transcriptase polymerase chain reaction
(CT-RT-PCR), the protein expression was measured using western blotting and the activity with 14Cthymidine as a substrate. In Lovo cells TFT plus TPI treatment increased TP protein expression 1.7-fold, but
there was no profound effect of other treatments, TP-mRNA levels in cells treated with 5′DFUR and TFT
showed a 5- and 1.4-fold increase, respectively. In WiDR cells 5′DFUR plus TPI treatment reduced TP
protein expression 2-fold; TFT and TFT plus TPI increased TP protein 2- and 3-fold, respectively. TPI and
5′DFUR treatment reduced TP-mRNA 1.6- and 5-fold, respectively, which was only accompanied by a
lower TP protein expression in 5′DFUR treated cells. In HT29 cells TP protein and TP-mRNA expression of
the 5′DFUR and 5′DFUR plus TPI treated cells were lower, 3.6- and 2.75-fold, respectively. In all cell lines
5′DFUR and TFT treatment did not affect TP activity, however, treatment with TPI (alone or in
combination) completely abolished TP activity in all cell-free extracts. In conclusion: different effects on TP
were observed at equitoxic drug concentrations, indicating that substrate and endproduct regulation of TP is
drug and cell line dependent, and may affect the effects of drugs.
171
P80
Combination studies of trifluorothymidine (TFT) with antifolates and the DNA synthesis inhibitors
irinotecan, oxaliplatin and gemcitabine
O.H. Temmink1, M.F.M. Hoogeland1, M. Fukushima2 G.J. Peters1
1
2
Trifluorothymidine (TFT) is a fluorinated pyrimidine analog which can be activated by thymidine kinase
(TK) to TFT-MP which may inhibit thymidylate synthase (TS), the only de novo enzyme involved in
thymidine nucleotide synthesis (dTTP). TFT-TP can also be incorporated into DNA, possibly resulting in
DNA damage and cell death. TS converts deoxyuridine monophosphate (dUMP) to deoxythymidine
monophosphate (dTMP) and its inhibition leads to dTTP depletion in the cell causing dUTP to be
misincorporated into DNA.The antifolates AG337, ZD1694 and GW1843 are direct TS inhibitors. We
studied the effect of antifolates, irinotecan (as the topoisomerase inhibitor SN-38), oxaliplatin and
gemcitabine on TFT sensitivity by using cytotoxicity assays. A panel of 5 colon cancer cell lines were used
for the experiments. Multiple drug effect analysis was performed using specialized computer software based
on the Chou and Talalay method (Calcusyn, Biosoft 1996). Effects were expressed as mutually nonexclusive
case combination index (CI) in which a CI<1 indicates synergism and CI>1 antagonism. Three experimental
procedures were used to test the interaction of the drugs: either one of the drugs was kept at a constant
concentration (~IC25) or two drugs were added in a 1:1 IC50 equimolar ratio. The combination of TFT with
irinotecan showed additive to antagonistic effects in most cell lines (0.9<CI<1.7) and was procedure
independent. The combination of TFT with oxaliplatin showed synergism in most cell lines (0.5<CI<1) and
for each procedure. The combination of TFT with gemcitabine was dependent on the cell line and procedure
used (0.7<CI<1.4). In the case of the antifolates we focused on two-sided TS inhibition, evaluated by the in
situ TS inhibition assay. About 20-40% TS inhibitory concentrations of each drug was used, and most
combinations resulted in additive TS inhibition, showing that TS is the major target for their activity. In the
future we will also determine the effect of TFT in combination with the other drugs on DNA damage
induction. In conclusion, TS inhibition may modulate the effect of the other drugs.
172
P81
Combination studies of 5-fluorouracil with UCN-01 or staurosporine
J. Sigmond, E.M. Comijn, G.J. Peters
Cytotoxic effects of 5-fluorouracil (5-FU) have been reported to be RNA related or mediated by and inhibition
of thymidylate synthase (TS). The latter effects are cell cycle dependent, and possibly also related to p53
expression. Therefore we evaluated the interaction between 5-FU and UCN-01, an inhibitor of cell cycle
dependent kinase 2 and protein kinase C (PKC) and another PKC inhibitor, staurosporine. Growth-inhibitory
effects of 5-FU combinations were evaluated by multidrug effect analysis and were related with the role of
induction of apoptosis or cell cycle arrest (with FACS analysis). Variants of the colon cancer cell line Lovo
with wildtype (Lovo B2), mutant p53 (Lovo 175x2) and functionally inactive p53 (Lovo Li) were used.Lovo
B2 and Lovo Li cells showed similar IC50 values for 5-FU, staurosporine and UCN-01, while Lovo 175x2
cells were relatively more resistant. Combination indices for the 5-FU combinations showed synergism in Lovo
B2 and Lovo 175x2 while in Lovo Li moderate antagonism was observed. To evaluate effects on cell cycle
distribution, IC100 concentrations were used. In all Lovo variants, an S phase arrest was induced after
exposure to 5-FU (50 µM). Exposure for 24 hr to staurosporine (0.05 µM) or UCN-01 (0.5 µM) induced
accumulation of cells in the G2/M phase or G0/G1, respectively, shifting to an S-phase arrest after 48 hr.
Simultaneous exposure to 5-FU combinations showed an average cell cycle distribution of both drugs when
used alone. In sequential drug administration, the cell cycle distribution was determined by the first drug. More
induction of apoptosis by IC100 of UCN-01 was found in Lovo 175x2 compared to other Lovo variants, which
required no functional p53. Additive effects in induction of apoptosis were observed in both simultaneous and
sequential 5-FU combinations. Based on induction of apoptosis 5-FU addition prior to the PKC inhibitors
seemed preferable. When staurosporine was used as the second drug, induction of apoptosis in Lovo B2 and
Lovo 175 was 2-fold higher than the sum of both drugs alone. These mechanistic studies offer a good basis for
evaluation of development of combination therapies with 5-FU.
173
P82
5’-nucleotidases levels measured in peripheral blood cells from patients with chronic and acute leukemia
M. Razmara1, S. Eriksson2, F. Albertioni1
1
2
Treatment of leukemia has been improved significantly during the last three decades, mainly based on the
introduction of new nucleoside analogs like cladribine (CdA, 2-chloro-2'-deoxyadenosine) and fludarabine.
These nucleosides must be activated by enzymes such as deoxycytidine kinase (dCK) and deoxyguanosine
kinase (dGK). Key catabolic enzymes responsible for degradation of nucleotides are 5´-nucleotidases. Five
major 5´-nucleotidases have been described, 5´-ectonuclotidase (membrane-bound), cytosolic 5´-nucleotidase I
(cN-I), cytosolic 5´-nucleotidase II (cN-II) and both cytosolic and mitochondria 5´(3´)-deoxyribonucleotidases
(dNT-1, dNT-2). We have investigated the activity levels of cN-I and cN-II two key enzymes in the
dephosphorylation of nucleoside analogs used in chemotherapy. Enzyme assays were performed in cell extracts
from 32 patients with acute myeloid leukemia (AML) and chronic lymphoblastic leukemia (CLL) by using a
selective HPLC assay. We found that the activity of cN-I (using adenosine monophosphate as substrate) was
similar in AML and in CLL. The activity of cN-II (using inosine monophosphate as substrate), was much
higher than cN-I both in AML and in CLL cell extracts. We also investigate the degradation of CdAMP in
same cell extracts. The activity of CdAMP degradation was significantly correlated to the activity of cN-I (R=
0.60). The activity of cN-II did not show a correlation to CdAMP degradation. These results suggest that
CdAMP was most probably dephosphorylated by cN-I. Whether high levels of cN-I independently identify a
poor prognosis group of patients remain to be investigated
174
P83
Cyclic guanosine monophosphate role in human carcinoma pathogenesis
G. Spoto1, M. Di Nicola1, F. Santoleri1, S. Soscia1, M. Fioroni2, C. Rubini2, A. Piatelli1
1
University of 'G. D'Annunzio', CHIETI, Italy
2
University of Ancona, ANCONA, Italy
Cyclic guanosine monophosphate (cGMP) is an essential second messenger for different cellular signals and
is hydrolyzed to guanosine 5’-monophoshate by phosphodiesterases (PDEs), whose activity is regulated by
different inputs originated from some signaling-systems.
The cGMP is important in inhibiting PDE3, stimulating PDE2 and activation of kinase protein.
The aim of this study was a correlation between cyclic guanosine monophosphate and human oral squamous
cell carcinoma progression.
Although the cyclic nucleotide role in carcinoma growth and invasion has not been clearly defined, its
function as second messenger of many signaling molecules suggest that it may be a key factor in neoplastic
progression.
We know that in cancerouses progression there is not only excessive cell proliferation, but also a cell death
decrease, which we know is regulated by cGMP.
The cGMP differences between healthy gingival tissues used as controls, Gingival Squamous Cell
Carcinoma, (OSCC ), with (N+) and without (N-) lymph node metastases were evaluated.
The difference between control (0.32 ± 0.10 µM) and N- (0.53 ± 0.24 µM) and N+ (0.56 ± 0.24 µM) values
showed an important increase of cGMP contemporaneously to an increase of neoplastic tissue trasformation;
this may suggest a probable cGMP involvement in this proliferation mechanism. Moreover, our study
showed an higher PDE activity (5.74 ± 1.40 µM) values in N+ than in control samples (5.59 ± 1.15 µM).
In another study, two colon adenocarcinoma cell lines at different stages of differentiation were analysed. In
cell lines with a better differentiation degree an high cGMP concentration (0.0976 µM) was present.
cGMP and enzyme activity, simultaneously confirm that this second messenger is really influenced by
carcinoma progression.
These data showed an increase of intracellular cGMP concentration in cancer specimens; the organism
reacts increasing PDE activity, to try to stabilize cGMP on basal levels.
Therefore, these results were characterized from an elevated standard deviation, that explain both an altered
hydrolysis PDE mediated and an altered guanilate cyclase activity.
In conclusion we belive that elevated cGMP intracellular concentration in human cancer may be interpreted
as a positive cell because it is knows that cGMP (PDE3A inhibitor) is important in neoplastic proliferation
inhibition.
175
P84
Cyclic nucleotides and neuroblastoma differentiation
A. Giacomello, E. Messina, F. Lupi, L. Barile
An elevation of the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP) induces terminal
differentiation in neuroblastoma cells. The rise of cGMP levels during 1-10 µM retinoic acid and 50 nM
mycophenolic acid treatment suggests the possible involvement of this cyclic nucleotide in morphological
neuroblastoma differentiation. In line with this hypothesis LAN-5 and SHEP human neuroblastoma cells
elaborated a network of neuritic processes during a 6 days treatment with 0.1 to 1 mM 8-p-CPT-cGMP or
dbcGMP, with a neurite outgrowth similar to that obtained using 1mM dbcAMP. In neuroblastoma cyclic
nucleotides hydrolysis is achieved by members of at least five phosphodiesterases (PDEs) families (PDE1 to
PDE5). The effect of the specific inhibitors of PDE1 (MMPX), PDE3 (Ciclostamide, Quazinone, Milrinone,
Zardaverine), PDE4 (Rolipram, Etazolate, Zardaverine), PDE5 (Zaprinast, MY-5445, Dipyridamole) on
neuroblastoma growth and differentiation have been studied. LAN5 and SHEP human neuroblastoma cells
were treated for 3 to 6 days with each specific inhibitor at concentrations ranging from 0 to 10 x IC50 µM.
Spectrophotometric quantification of cell proliferation and viability (cell proliferation reagent WST-1)
showed a decreased cell proliferation only in response to Zaprinast and MY-5445. After 6 days of incubation
of both cell lines in the presence of these two PDE5 inhibitors an increase in neurite outgrowth was
observed. Preliminary experiments showed that at least the inhibitory effect of zaprinast on cell growth was
more marked than the inhibition of PDE5 activity and that at the 10 x IC50 drug concentrations of specific
PDE inhibitors intracellular cGMP levels obtained with Zaprinast or MY-5445 were comparable to those
obtained with dipyridamole, a PDE5 inhibitor without significant effects on neuroblastoma growth and
differentiation. These results suggest that Zaprinast and MY-5445 could act by interfering with other
molecular events than direct cGMP-PDE inhibition. In conclusion we have demonstrated an involvement of
cGMP in neuroblastoma differentiationa and a significant antiproliferative and differentiating action of
Zaprinast and MY-5445. These observations suggest that these PDE5 inhibitors could have a potential role in
the management of neuroblastoma patients.
176
P85a
Differentiation-inducing Activity of Purine and Pyrimidine Derivatives on Myelogenous Leukemia Cells:
The most suitable analog depends on the leukemia type
Y. Honma
Saitama Cancer Center Research Institute, SAITAMA, Japan
The patient outcomes following the treatment of acute myelogenous leukemia (AML) have improved with
progress in intensive chemotherapy. However, the therapy is limited by the unresponsiveness of certain
leukemias and severe adverse effects. The current results of treatment for AML clearly call for
improvement. Differentiation therapy has been successful as a treatment for acute promyelocytic leukemia
(APL), a subgroup of AML, but it dose not clinically apply to other hematopoietic malignancies. The
further development of differentiation therapy for leukemia requires new and potent inducers of
differentiation.
Many purine and pyrimidine derivatives can induce the differentiation of human and murine leukemia
cells. The sensitivity to the derivatives varies for each leukemia cell line. Therefore, the differentiationinducing effect of various purine and pyrimidine analogs on several human myelogenous leukemia cells was
examined and reviewed the association of differentiation-inducing activity of purine and pyrimidine
derivatives with leukemia cell types.
Monocytoid leukemia cells were more sensitive to deoxyadenosine analogs than were erythroid or myeloid
leukemia cells. APL cells were less sensitive to purine and pyrimidine analogs than other AML cells at
inhibiting cell growth and inducing differentiation. 1-β-D-Arabinofuranosylcytosine (araC) is effective to
induce differentiation of many AML cells, but not of APL cells. However, a cytidine deaminase-resistant
analog of araC (DMDC) induced differentiation of APL cells, with dose-response curves similar to those of
other AML cells. Among naturally occurring nucleobases, adenine effectively enhanced the differentiation
of myeloid leukemia HL-60 cells, while other nucleobases, adenosine or deoxyadenosine alone did not.
Since N6-modification potentiated the ability of adenine analogs to induce differentiation, we examined the
effects of various N6-modified derivatives on differentiation. All of the derivatives except zeatin were very
potent at inducing differentiation, and isopentenyladenine had the greatest effect. Novel synthetic analogs
of purines and pyrimidines were also examined to induce differentiation of AML cells, and several
compounds might be useful for differentiation therapy of AML. However, the antileukemic analog that is
the most suitable for the treatment of AML may depend on the leukemia subtype, since the sensitivity of
leukemia cells to these analogs varied greatly among leukemia subtypes.
177
P85b
6-mercaptopurine (6-MP) metabolism in man and its fingerprints in erythrocytes (RBC)
S. Gutsche, A. Kotalczyk, H. Iven
Introduction: The metabolism of 6-MP in nucleated cells is well known. Catabolism by xanthinoxidase
leads to thiouric acid, anabolism via HGPRT and purine interconversion to cytotoxic thioguanine-nucleotides
(TGN). Thiopurine S-methyltransferase (TPMT) – an enzyme with a genetic polymorphism – methylates 6MP and most of the metabolites on various steps of the 6-MP pathway. While the metabolism to 6-TGN can
only take place in nucleated cells, 6-TGN is determined in RBC which are also used to phenotype TPMT
activity. This raises the question whether the results obtained in RBC are always represen-tative for other
tissues?
Materials and methods: We phenotyped TPMT-activity and measured 6-MP metabolites in RBC of more
than 250 paediatric ALL patients treated according to the German BFM-2000 protocol. For each patient up
to 13 samples were collected covering the whole treatment period of 2 years. Measurements were performed
using an enzymatic assay for the TPMT activity and HPLC for the metabolites 6-TGN, 6-methyl-MP (6MMP), and 6-methyl-TG (6-MTG).
Results: After oral administration of 6-MP, 6-MMP accumulates in RBC and follows any change in dosing
rapidly while 6-TGN (and 6-MTG) accumulate more slowly. In the individual patient steady state 6-MMP
concentrations and 6-TGN +6-MTG concentrations correlate to the 6-MP dose. However, while the
regression line of 6-TGN+6-MTG versus 6-MP dose goes through the origin, 6-MMP hits the abscissa
approximately at a 6-MP dose of 20mg/m². In the majority of patients TPMT activity was low at diagnosis
increasing dramatically during the next 20 weeks of treatment. There was no correlation between TPMT
activity and the dependent metabolites or leukocyte count.
Conclusions: Erythrocytes are a mirror of the 6-MP metabolism. They reflect 1) the patients compliance, 2)
a saturable first pass metabolism probably useful for a better individualisation of therapy, 3) the effective
formation of cytotoxic 6-TGN´s as a basis for drug monitoring and 4) a certain increase of the TPMT
activity, dependent on transfusion of erythrocytes, enzyme induction and/or a renewal of the erythrocyte
pool. Especially TPMT activity in RBC´s and its predictive value for the therapeutic efficacy should be a
point of further discussion.
Department of Pharmacology, *Center for Pediatrics , Medical University of Luebeck, Ratzeburger Allee
160. D-23538 Luebeck, FRG .
178
P86a
ATP-site directed potential inhibitors of nucleoside triphosphatases (NTPase)/helicases and polymerases
of hepatitis C and other selected Flaviviridae viruses
T. Kulikowski1, M. Bretner1, S. Schalinski2, M. Lang2, H. Schmitz2, P. Borowski2, S. Behrens3
1
Polish Academy of Science, WARSAW, Poland
2
Bernhard Nocht Institute for Tropic. Med, HAMBURG, Germany
3
Justus Liebig University, GIESSEN, Germany
5`O-(4-fluorosulfonylbenzoyl)-esters of ribavirin (FSBR), adenosine (FSBA), guanosine (FSBG) and inosine
(FSBI) were obtained by acylation of the 5’-OH of adenosine, guanosine, inosine, and ribavirin with 4fluorosulphonylbenzoyl chloride (FSBCl) in HMPA. The above derivatives were tested as inhibitors of
NTPase/helicase and polymerase activities of selected Flaviviridae viruses, such as the hepatitis C virus
(HCV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and dengue virus (DENV). When the
helicase activity of viral NTPase/helicases was tested under standard conditions, only moderate inhibition
was obtained (IC ≥ 120µM) and in the case of FSBG even an activation was seen. The preincubation of the
enzymes with the FSB-derivatives induced or amplified inhibitory effect, approximately 10– to 100–fold.
Comparative studies revealed that the extent of inhibition of the unwinding activity is enzyme specific.
When the polymerase activity of HCV was investigated as a function of increasing concentrations of the
FSB-derivatives, all the compounds inhibited the enzyme with the following order of efficiency:
FSBA<FSBG=FSBR<FSBI. Using various NTPs and polymerases of WNV and HCV, we demonstrated
that, similarly to the NTPase/helicase, the inhibition of the polymerase activity by 5’-O-FSB derivatives
depends strongly both on the enzyme and substrate used. In contrast to the NTPase/helicase the
preincubation of the polymerase did not influence the inhibition.
179
P86b
Pre-treatment TPMT phenotyping/genotyping : an excellent guide for initial thiopurine drug dosing
E.A. Shobowale-Bakre1, A. Ansari1, M. Arenas1, A.M. Marinaki1, L.D. Fairbanks1, J.A. Duley2, J.D.
Sanderson1
1
2
Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme. Deficiency of the enzyme is
predictive of thiopurine drugs (i.e. azathioprine, mercaptopurine and thioguanine) intolerance. In Caucasian and
African populations, 10-14% are carriers while 0.3% of the individuals are totally TPMT deficient. Two-thirds
of these carriers have a high risk of drug induced side-effects on a full dose and therapy is typically withdrawn
due to bone marrow suppression. In some clinics, these patients are either switched onto more toxic drugs or
given surgical intervention.
To test the hypothesis that carriers should tolerate half the normal dose, we identified 28 TPMT carriers who
had pre-therapy TPMT phenotyping done in our laboratory mainly from gastroenterology and dermatology
clinics. Two patients had autoimmune hepatitis, 6 skin and or oral ulceration and 3 eczema. Each patient was
initially given azathioprine (aza) at a dose of 0.5 mg/kg which was then increased to 1mg/kg as required for
steroid-sparing according to departmental guidelines. Clinical notes were reviewed after one year of treatment
for adverse effects and clinical response (defined as withdrawal of steroids: complete, partial or non-response).
TPMT phenotype results were confirmed in all patients by genotyping for the three most common TPMT
allelic variants: TPMT *3A (G460A-Ala154Thr and A719G-Tyr240Cys + G460A-Ala154Thr), TPMT*3C
(A719G-Tyr240Cys) and TPMT*2 (G238C-Ala80Pro).
Six of the 28 patients experienced side-effects requiring drug withdrawal (2 pancreatitis, 4 nausea/malaise). The
4 patients that experienced nausea were switched to low dose 6-mercaptopurine and no further side effects were
observed over the treatment period. 24 of the 26 carriers who remained on drug therapy responded favourably.
In conclusion, TPMT heterozygotes showed an excellent clinical response (86%) to thiopurine therapy at
reduced thiopurine dose. TPMT testing is thus a useful tool for guiding thiopurine drug dosing, thereby
avoiding more toxic drug therapy and complicated surgical intervention.
180
P87
A time saving and rapid HPLC-method for the determination of 6-thioguanine and 6-thioguanine
(-nucleotides)
A. Kotalczyk, S. Gutsche, H. Iven
Introduction: After oral administration of 6-mercaptopurine (6-MP) or its prodrug azathiopurine (AZA), 6MP enters the nucleotide pool as thioinosine-monophosphate and is further metabolised to thioguaninenucleotides. On the other hand 6-thioguanine (6-TG) is directly metabolised to thioguanine- nucleotides. The
latter may be incorporated into DNA and RNA and is considered responsible for therapeutic outcome and/or
toxicity. We therefore monitor 6-TG concentrations routinely in RBC of children treated for ALL and in
patients treated with AZA because of autoimmune diseases using the method of Erdmann et al. (Biomed
Chromatogr 4: 47-51;1990) with slight modifications. This method takes advantage of the fact, that 6-TG
can be transformed into a highly fluorescent derivative by oxidation with potassium permanganate. This pre
column derivatisation requires multiple steps and is tedious and time consuming. Therefore we developed
and improved an HPLC- method for 6-TG (- and its mono-, di-, and trinucleotides) using H202 and UVirradiation (Beam Boost) for post column oxidation of 6-TG.
Methods: We used a Waters Nova Pack C-18, 39x 150 mm column. The eluent consisted of 0,02m
phosphate buffer pH 7.9, acetonitrile (85/15; v/v) containing 700mg/ l tetra-n-butylammonium-hydrogen
sulphate. Flow rate was 0.5 ml/min with no recirculation of eluent. Post column: 0.3% H202 at a flow rate of
10 µl/min was added to the eluent before it entered the photochemical reaction-unit (UV:254 nm, coil-length
5 m). Fluorescence was monitored at Ex 315nm, Em 390nm. The sensitivity of the system for an aqueous 6TG solution is 10 pg absolute. The system is very sensitive to any change of the pH of the eluent.
Pre-treatment: RBC were haemolysed in 5 volumes of aqua dest. and kept at 95° C for 15 min. After
centrifugation the clear supernatant was used directly for analysis.
Results: A modification of column temperature did not improve the chromatograms. The nucleotides
decomposed when the column temperature was more then 40° C. We used this system to study the time and
concentration-dependent uptake of 6-TG into erythrocytes in vitro. In accordance with the result of Kong et
al. (Biochem Pharmacol 24: 807-813;1975) we found a rapid uptake and metabolism of 6-TG to 6thioguanosine- mono-, di-, and predominantly -tri-phosphate. Uptake and metabolism was almost complete
after 90 min of incubation. The metabolism was near saturation at a concentration of 3-5 µg 6-TG/100 µl
erythrocytes. Beyond this concentration, in addition to the 6-TG nucleotides the free base also accumulated
in the erythrocytes. In contrast to 6-TG, 6-MP was only detected as thioinosinic acid (6-MPmonophosphate).Within 24 hours only 1% of the 6-TG in RBC was metabolised to 6-methyl-TG by
thiopurine S-methyltransferase (TPMT). This also held true for 6-MP.
Department of Pharmacology, *Center for Pediatrics, Medical University of Luebeck, Ratzeburger Allee
160, D-23538 Luebeck, FRG.
181
P88
Real time RT-PCR methodology for quantification of TPMT gene expression
M. Lindqvist, S. Almer, C. Peterson, P. Söderkvist
University of Linköping, LINKÖPING, Sweden
Thiopurine methyltransferase (TPMT) catalyses the methylation of thiopurines such as mercaptopurine,
thioguanine and azathioprine, which are used to treat acute lymphoblastic leukemia, autoimmune disorders and
inflammatory bowel diseases. Levels of TPMT enzymatic activity are controlled by a genetic polymorphism,
an important factor responsible for individual differences in thiopurine toxicity and therapeutic efficacy. Ninety
percentages of Caucasians are homozygous for two active alleles, 10 % are heterozygous for one inactive allele
and 0.3 % are homozygous for two inactive alleles. The active TPMT gene is located on chromosome band
6p22.3 in humans and more than 10 variant TPMT alleles have been described. A TPMT processed
pseudogene, with a DNA sequence 96 % identical to that of the open reading frame of TPMT cDNA is located
on chromosome band 18q21.1. The aim of this study was to design and develop a real time PCR method, using
Taqman technology, for quantification of TPMT mRNA in leukocytes and to use this method for correlation
studies between gene expression and enzyme activity. The PCR product was sequenced and thereby confirmed
to be the TPMT cDNA. The housekeeping gene Cyclophilin (CYC) was used as internal control and standard
curves were created both for TPMT and CYC using RNA from a MOLT-4 cell line. Quantitative analysis of
TPMT mRNA was performed in comparison to CYC mRNA. TPMT enzyme activity was determined in
lysates from red blood cells (RBC) using a radioactive assay with tritiated S-adenosyl methionine as a methyl
donor. The TPMT mRNA expression was measured in leukocytes from 39 individuals. In the group presumed
to be homozygous for two active alleles (TPMT value 9.40-19.6 U/ml RBC, n=29), the correlation coefficient
between the mRNA expression and the enzyme activity was statistical significant (p<0.001). No significant
correlation was found in the group of individuals with TPMT activity lower than 9 U/ml pRBC (TPMT values
0.3-8.50, n=10). The real time PCR method will be a valuable tool in future studies of TPMT enzyme activity
regulation. Hopefully, this will contribute to better prediction of the effects of thiopurines in individual patients.
182
P89
Quantitative detection of deoxycytidine kinase by real time light cycler PCR in comparison with
conventional competitive template RT-PCR
J. Sigmond, J.R. Kroep, W. Loves, G.J. Peters
Dept. Medical Oncology, VU University Medical Center, AMSTERDAM, The Netherlands
Deoxycytidine kinase (dCK) is essential for the phosphorylation of several deoxyribonucleoside analogues that
are widely used as chemotherapeutic agents. Deoxyribonucleoside analogues such as cytosine arabinoside
(Ara-C) or gemcitabine (2’2’-difluorodeoxycytidine) are used in leukemias and solid tumors, respectively. For
gemcitabine it has been shown that pretreatment dCK levels can predict the response to gemcitabine in vivo.
For evaluation of dCK mRNA expression, conventional Competitive Template Reverse transcriptase
Polymerase Chain Reaction assays were performed (CT-RT-PCR). However, real time PCR enables more
rapid and sensitive detection of mRNA expression using either SYBR Green or fluorescent probes using a
Light Cycler (LC). We developed and optimized a sensitive real time PCR assay for dCK with SYBR green
detection. For this purpose we used cDNA from human xenografts previously used to establish a relation
between dCK activity and gemcitabine sensitivity (Kroep et al, Mol. Canc. Ther. 1:371-6, 2002). A significant
correlation between LC-PCR and CT-RT-PCR (Pearson: r=0.956; p<0.0001) was found. In addition, we
observed a good correlation (Pearson: r=0.972; p=0.003) between LC-PCR and enzyme activity. Furthermore,
gemcitabine sensitivity was correlated (Pearson: r=0.695; p=0.048) with dCK mRNA measured with LC-PCR.
The assay was applicable to determine dCK mRNA levels in needle biopsy specimens of various human
tumors. Our data demonstrate that Light cycler is a rapid and reliable method for the detection and quantitation
of dCK mRNA levels which can be used in the clinic to predict chemotherapeutic agent sensitivity.
183
P90
Immunocytochemical detection of deoxycytidine kinase
I. Hubeek1, G.J. Peters1, A.J.F. Broekhuizen1, I. Talianidis2, E.R. Van Wering3, U. Creutzig4, G. Giaccone1, P.
Van der Valk1, G.J.L. Kaspers1
1
2
Institute of Molecular Biology, HERAKLION, Greece
3
DCOG, THE HAGUE, The Netherlands
4
AML-BFM Study Group, MÜNSTER, Germany
Deoxycytidine kinase (dCK) is responsible for the activation of several clinically important anticancer agents.
These nucleoside analogs are widely used for the treatment of malignancies and are dependent on
phosphorylation by dCK. To determine the expression level of dCK protein in several different malignancies
we have set up an immunocytochemical method, using a rabbit-anti human dCK antibody and applied this
method in 97 samples. Immunocytochemical staining was performed on cryopreserved cytospins of 31
childhood acute myeloid leukemia (AML), 29 childhood acute lymphoblastic leukemia (ALL), 10
retinoblastoma and 12 pediatric brain tumor samples. Furthermore, the leukemic cell lines CCRF-CEM, U937
and HL60 were used as positive controls. Negative controls were performed by omitting the first antibody.
dCK protein expression was determined by scoring the intensity of the staining: negative (0), low (1+),
intermediate (2+), high (3+), or very high (4+), by two independent investigators. In the leukemic cell lines
U937 and CCRF-CEM and in the different tumor types a predominantly cytoplasmic staining was observed. In
4 out of 31 AML samples and in 2 out of 29 ALL samples we also observed staining in the nucleus. The AML
and retinoblastoma samples showed an intermediate to (very) high expression of dCK. The expression level of
dCK in ALL samples ranged from low to (very) high. In contrast, the majority of brain tumor samples
expressed low to intermediate levels, with 2 samples expressing no dCK. In the NCSLC tissue samples the
expression of dCK ranged from low to very high. All NCSLC samples expressed the dCK protein. The
interobserver variability between two observers showed agreement in 72/82 (88%) cases (kappa = 0.83). On
Western blotting the antibody detected dCK appropriately at 30 kD, without additional bands. In conclusion,
immunocytochemistry is an effective method to determine the expression level of dCK in patient samples. In
the vast majority of samples dCK was located exclusively in the cytoplasm. It was possible to discriminate
between patient samples with a low, intermediate and (very) high expression level of dCK. There were clear
differences in dCK expression observed between tumor types.
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P91
Determination of the deoxycytidine kinase activity in cell homogenates with a non-radiochemical assay
using reversed-phase HPLC. Identification of a novel metabolite of 2-Chlorodeoxyadenosine
J. Bierau1, A.B.P. Van Kuilenburg1, R. Leen1, A.H. Van Gennip2, H. Caron1
1
2
Deoxycytidine kinase (dCK) is a deoxynucleoside kinase with a broad substrate specificity. The natural
substrates of dCK are dCyd, dAdo and dGuo [1]. However, dCK also phosphorylates therapeutically important
deoxynucleoside analogues, such as 1-ß-D-arabinofuranosyl cytosine (cytarabine), 2',2'-difluorodeoxyctytidine
(gemcitabine). and 2-Chlorodeoxyadenosine (cladribine, CdA). In fact, it is the rate-limiting enzyme in the
activation of these cytotoxic nucleoside analogues [2]. To date, all procedures to measure dCK activity are
based on the method described by Ives and Durham [3], and rely on thin-layer chromatography or weak ionexchange paper chromatography to separate the radioactive substrate (CdA or dCyd) from the corresponding
nucleoside-5'-monophospate. These procedures proved to be extremely laborious and time consuming. Another
major disadvantage of these traditional analytical methods is that the formation of other metabolites, which
might hamper accurate measurement of dCK activity, may not be detected.
We have developed a non-radioactive procedure to measure the deoxycytidine kinase activity in crude cell free
homogenates. 2-Chloro-deoxyadenosine (CdA) was used as the substrate and was separated from its 5'monophosphate by reversed-phase HPLC. A complete separation of CdA and its metabolites was achieved in
40 minutes. The minimum amount of CdA that could be detected was 1 pmol. The assay was linear with
reaction times up to at least 3 hours. With respect to the protein concentration, the reaction was linear with
protein concentrations up to 760 µg/ml in the assay. An amount of 8 x103 cells was already sufficient to
determine the specific dCK activity in SK-N-BE(2)c cells.
Under the standard reaction conditions described in literature, CdA was not only converted to its 5'monophosphate but also to 2-Chloroadenine and, surprisingly, also to 2-Chlorodeoxyinosine, in MOLT-3 cells.
Therefore, our results demonstrate that CdA is also a substrate for adenosine deaminase. In order to disqualify
artefacts, the cell lines were tested and found to be mycoplasma negative.
References
1. Arner ES and Eriksson S, Pharmacol.Ther. 67: 155-186, 1995.
2. Liliemark JO and Plunkett W, Cancer Res. 46: 1079-1083, 1986.
3. Ives DH and Durham JP, J.Biol.Chem. 245: 2285-2294, 1970.
185
P93
Application of liquid chromatography - mass spectrometry for identification and quantitation of
metabolic alterations in chronic renal failure
E.M. Slominska1, R.T. Smolenski2, M. Lipinski1, H. Dziewit1, N. Leaver2, B. Rutkowski1, J. Swierczynski1,
R.T. Smolenski3
1
2
3
University of Gdansk, GDANSK, Poland
Metabolic abnormalities are the primary consequence of chronic renal failure (CRF) but its full spectrum has
not been fully elucidated. Because of the limitations of standard HPLC with UV detection for analysis complex
samples such as in CRF we aimed to develop liquid chromatographic/mass spectrometric (LC/MS) procedure
for analysis of alterations of blood concentration of nucleotide metabolites in CRF. An MS compatible
reversed-phase chromatographic system has been developed for analysis and identification of nucleosides and
bases in plasma samples. Despite common view that only crude chromatographic separation is necessary while
applying mass detection, we found that adequate chromatographic separation is essential to achieve sufficient
sensitivity and recovery and to simplify the identification of unknown metabolites. In addition, optimization of
conditions of ion source and mobile phase was critical for sensitivity due to profound effect on ionization
efficiency. Full mass range analysis allowed detection of metabolites in micromolar concentration. However,
several fold improvement of the sensitivity was possible in selective ion monitoring mode or even 1000x
sensitivity improvement while operating in MS2 (fragmentation) mode. Analysis of CRF plasma samples in
addition to well established alterations demonstrated less well characterized changes such as accumulation of
adenine, nicotinamide and its metabolites: N-methyl-2-pyridone-5-carboxamide (2PY) and N-methyl-4pyridone-5-carboxamide (4PY). Adenine concentration increased from 71±32nM in healthy subjects to
632±124nM in CRF. 2PY concentration increased from 0.36±0.10µM in healthy subjects to 19.3±6.4µM in
adult patients with CRF while in children with similar degree of CRF 2PY reached the concentration 7.5±2.3
µM; 4PY was 2.10 µM±0.86 and nicotinamide was 0.29 µM±0.07. Adenosine blood concentration was not
was not different from controls in children or adults with CRF. In addition to the effect of age on 2PY
accumulation which was smaller in children with similar degree of CRF both plasma adenine and nicotinamide
metabolite concentrations correlated with renal function impairment, transiently decreased following
hemodialysis and permanently decreased following successful renal transplantation. Conclusion: LC/MS has
unprecedented advantages for studies of metabolic alterations in CRF. It allows identification of new changes
as well as rapid development of analytical techniques for its detailed analysis.
186
P94
Detection of prothrombin and osteopontin in a renal stone found in a hyperuricemic patient using
2D-PAGE and LCMS analysis
K. Kaneko
Teikyo University School of Medicine, TOKYO, Japan
Objectives: The liquid chromatography/mass spectrometry (LCMS) technique has recently been applied on
analysis of various proteins. A renal stone found in a hyperuricemic patient was analyzed with the twodimensional polyacrylamide gel electrophoresis (2D-PAGE) and LCMS.
Methods: A renal stone (size: 7-9 mm) from a patient with hyperuriceamia was examined. After the infrared
spectrophotometry (IR) analysis, the stone was extracted with EDTA and guanidine hydrochloride following
dialysis, concentration, and was applied to 2D-PAGE. Several proteins were isolated, treated with trypsin
digestion, and analyzed with a nano-LC/mass spectrometer (LCMS). The LCMS was equipped with a
nanospray ionization and an ion trapping detection system. A protein search from the obtained MS/MS spectra
was undertaken, and the containing proteins determined.
Results: IR analysis showed the renal stone was found to be mainly composed of calcium oxalate. In 2DPAGE, several proteins were detected in the acid rich area (pI 3-4). After LCMS analysis, osteopontin and
prothrombin were determined to be contained as acidic proteins. These proteins are both related to calcium
binding. It is suggested that Ca-related proteins play an important role in the growing process of the calcium
oxalate stones.
Conclusion: The analytical technique using 2D-PAGE and LCMS was so sensitive that it was possible to detect
small quantities of proteins even in a renal stone. In order to investigate the cause of pathological stone
recurrence, analysis of each stone in detail is thought to be helpful.
187
P95
A non-radioactive sensitive assay to measure 5-fluorouracil incorporation into DNA of solid tumors
P. Noordhuis, U. Holwerda, J.A.M. Van Laar, C.L. Van der Wilt, G.J. Peters
The mechanism of action of 5-fluorouracil (5FU) has been associated with inhibition of thymidylate
synthase and incorporation of 5FU into RNA and DNA. In cell line models this incorporation is usually
measured with radioactive 5FU, but this is not applicable in patients. Previously we described a method for
measurement of 5FU incorporation into RNA, based on a sensitive 5FU assay using gas-chromatography
coupled to mass-spectrometry (GC-MS). This assay was adapted for measurement of 5FU into DNA. For
that purpose we isolated DNA from cells and tissues using standard methods and degraded DNA
enzymatically with DNAse, nuclease, alkaline phosphatase and purified thymidine phosphorylase, to 5FU,
which after derivatization was measured with GC-MS. The assays were validated using the colon cancer cell
line WiDr. In parallel experiments it was found that the incorporation as measured with radiolabelled 5FU
and non-labelled 5FU (with GC-MS), were similar. The murine Colon 26-B tumor was used as an in vivo
model. Incorporation into DNA after treatment with maximal tolerated doses of 5FU or fluorodeoxyuridine
(FUdR) was maximal at 2 hours and was 15.4 and 71.0 fmol/µg DNA respectively. The level of
incorporation remained high for about 3 days after which a decrease was observed to ± 2 fmol/µg DNA after
10 days both for 5-FU and FUdR. In contrast to the RNA incorporation, DNA incorporation did not correlate
with the intra-tumoral 5-FU concentration, neither for 5FU alone or FUdR. This is possibly because FUdR
can also be incorporated into DNA directly in addition to the pathway via 5FU. Consequently FUdR
treatment as compared to 5FU resulted in a higher incorporation into DNA. In conclusion, a sensitive nonradioactive assay for 5FU incorporation into DNA was developed. This method is also applicable for human
tissues.
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Programme and abstract - The Purine and Pyrimidine Society

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