TABLE OF CONTENTS

TABLE OF CONTENTS
PHYSICS, BIOPHYSICS, AND RISK ESTIMATION
Technical Characteristics of the Columbia University Single-Ion
Microbeam .....................................................................................................................1
Gerhard Randers-Pehrson, Charles R. Geard, Gary W. Johnson, and
David J. Brenner
Electrostatic Lens Design for the Columbia Microbeam ...............................................5
Alexander D. Dymnikov, David J. Brenner, Gary Johnson, and
Gerhard Randers-Pehrson
The Low-Energy Neutron Facility .................................................................................10
Stephen A. Marino, Dusan Srdoc, and Gary W. Johnson
Risks from Less than 10 Millisievert: What Do We Really Know?
How Can We Learn More? ........................................................................................15
David J. Brenner
The Risk of Fatal Cancer from Pediatric Computed Tomography ............................19
David J. Brenner, Carl D. Elliston, Eric J. Hall, and Walter E. Berdon
(Department of Radiology, Columbia University)
A Polymer, Random Walk Model for the Size-Distribution of
Large DNA Fragments After High-LET Radiation .................................................21
David J. Brenner, with Artem Ponomarev and Rainer Sachs (both from
the Univeristy of California/Berkeley), and Lynn Hlatky, (Harvard
Medical School)
MICROBEAM: CELLULAR STUDIES
Induction of a Bystander Mutagenic Effect of Alpha Particles on
Mammalian Cells.........................................................................................................23
Hongning Zhou, Gerhard Randers-Pehrson, and Tom K. Hei
Intra- and Inter-Cellular Reponses Following Cell-Site-Specific
Microbeam Irradiation ...............................................................................................26
Charles R. Geard, Gerhard Randers-Pehrson, Stephen A. Marino,
Gloria Jenkins-Baker, Tom K. Hei, Eric J. Hall, and David J.
Brenner
i
Single Alpha-Particle Traversals and Tumor Promoters.............................................28
Richard C. Miller, Satin Sawant, Gerhard Randers-Pehrson, Stephen
A. Marino, Charles R. Geard, Eric J. Hall, and David J. Brenner
Bystander Effect of Radiation on Oncogenic Transformation....................................30
Satin G. Sawant, Gerhard Randers-Pehrson, Charles R. Geard, and
Eric J. Hall
Role of Oxyradicals in DNA Damage Induced by Cytoplasmic
Irradiation in Mammalian Cells ................................................................................32
An Xu, Gerhard Randers-Pehrson, and Tom K. Hei
CELLULAR STUDIES
RBE and Microdosimetry of Low-Energy X Rays .......................................................35
Stephen A. Marino, Dusan Srdoc, Satin Sawant, Charles R. Geard, and
David J. Brenner, in collaboration with Zugen Fu (SUNY/
Stonybrook)
Establishment of an Alpha-Particle-Induced Estrogen-Dependent
Breast Cancer Model...................................................................................................40
Gloria M. Calaf and Tom K. Hei
Genotoxicity Versus Carcinogenicity: Implications From Fiber
Toxicity Studies............................................................................................................42
Tom K. Hei, An Xu, Darren Louie, and Yong-Liang Zhao
Induction of Reactive Oxygen Species by Crocidolite Asbestos in
Mammalian Cells.........................................................................................................46
An Xu and Tom K. Hei
Focal Adhesion Motility Revealed in Stationary Fibroblasts ......................................49
Lubomir B. Smilenov with Alexei Mikhailov (Massachusetts General
Hospital), and Robert J. Pelham, Jr., Eugene E. Marcantonia, and
Gregg G. Gundersen (Departments of Pathology and Anatomy and
Cell Biology, Columbia University)
Transformation of Human Bronchial Epithelial Cells by the
Tobacco-Specific N-Nitrosamine, NNK.....................................................................52
Hongning Zhou and Tom K. Hei
Protein Expression in Tumorigenic Human Breast Epithelial Cells
Transformed by Alpha Particles................................................................................54
Gloria M. Calaf and Tom K. Hei
ii
Microsatellite Instability in Tumorigenic Human Bronchial
Epithelial Cells Induced by Alpha Particles and Fe-56 Ions ...................................57
Chang Q. Piao and Tom K. Hei
Malignant Transformation of Human Bronchial Epithelial Cells
by Arsenite ...................................................................................................................60
Chang Q. Piao and Tom K. Hei
Radon, Arsenic, and Mutagenesis ..................................................................................62
Su X. Liu and Tom K. Hei
CYTOGENETIC STUDIES
Chromosomal Aberrations in Tumorigenic Human Bronchial
Epithelial Cells Transformed by Chrysolite Asbestos .............................................65
Masao Suzuki, Chang-Qing Piao, and Tom K. Hei
Cytogenetic Effects of Heavy-Ion Beams in Normal Human
Bronchial Epithelial Cells ...........................................................................................67
Masao Suzuki, Chang-Qing Piao, and Tom K. Hei
MOLECULAR STUDIES: CELL-CYCLE CHECKPOINTS
Physical Interactions Among Human Checkpoint Control Proteins
HHUS1p, HRAD1p, and HRAD9p, and Implications for the
Regulation of Cell-Cycle Progression ........................................................................70
Haiying Hang and Howard B. Lieberman
Two-Hybrid Interactions Between the Human HRAD9p
Checkpoint Control Protein and the Tumor Suppressor p53.................................72
Sarah J. Rauth, Wei Zheng, and Howard B. Lieberman
Defective G2 Checkpoint by Inactivation of 14-3-3σ
σ Gene
Influences Telomere Function ....................................................................................73
Sonu Dhar, Jain Kaung (University of Texas), Jeremy A. Squire
(University of Toronto), Charles R. Geard, Raymund J. Wellinger
(Universite de Sherbrooke), and Tej K. Pandita
iii
MOLECULAR STUDIES: DAMAGE RESPONSIVENESS
Ionizing Radiation Activates ATM Kinase Throughout the Cell
Cycle .............................................................................................................................76
Tej K. Pandita, Howard B. Lieberman, Dae-Sik Lim (St. Jude’s
Children’s Research Hospital), Sonu Dhar, Wei Zhen, Yoichi Taya
(National Cancer Center Research Institute, Japan), and Michael B.
Kastan (St. Jude’s Children’s Research Hospital)
Ataxia Telangiectasia: Chronic Activation of Damage-Responsive
Functions is Reduced by Alpha-Lipoic Acid.............................................................80
Magtouf Gatei (Queensland Institute of Medical Research), Dganit
Shkedy (Tel Aviv University), Kum Kum Khanna (QIMR), Tamar
Uziel (TAU), Yosef Shiloh (TAU), Tej K. Pandita, Martin F. Lavin
(QIMR), and Galit Rotman (TAU)
Activation of Abl Tyrosine Kinase by Ionizing Radiation Requires
ATM But Not DNA-PK...............................................................................................82
Sanjeev Shangary and Tamara Lataxes (both from the University of
Pittsburgh Medical Center), Tej K. Pandita, Guillermo E. Taccioli
(Boston University), and R. Baskaran (UPMC)
Atm Inactivation Results in Aberrant Telomere Clustering During
Meiotic Prophase1 .......................................................................................................84
Tej K. Pandita, Christoph H. Westphal (Harvard Medical School),
Melanie Anger (University of Kaiserslautern), Satin G. Sawant,
Charles R. Geard, Raj K. Pandita (Albert Einstein College of
Medicine), and Harry Scherthan (Univ. of Kaiserslautern)
Influence of ATM Function on Telomere Chromatin Structure.................................88
Lubomir Smilenov, Sonu Dhar, and Tej K. Pandita
Expression of the Catalytic Subunit of Telomerase in Developing
Brain Neurons: Evidence for a Cell-Survival-Promoting
Function........................................................................................................................94
Weiming Fu, Michael Killen, and Carsten Culmsee (all from Sanders
Brown Research Center on Aging, University of Kentucky), Sonu
Dhar, Tej K. Pandita, and Mark P. Mattson (SBRCA)
iv
The Human Homologue of Schizosaccharomyces pombe Rad9
protein, HRAD9p, Interacts with Bcl-2/Bcl-xL and Promotes
Apoptosis ......................................................................................................................95
Howard B. Lieberman, Haiying Hang, Kevin Hopkins, and Wei Zheng,
in collaboration with Kiyoshi Komatsu (University of South Florida
College of Medicine), Toshiyuki Miyashita (National Children’s
Research Center, Japan), Sandy Cuddeback (USFCOM), Massao
Yamada (NCRC), and Hong-Gang Wang (USFCOM)
MOLECULAR STUDIES ORIENTED TOWARDS CANCER
Identification of THG1 as a Potential Suppressor of Testicular
Tumorigenesis..............................................................................................................97
Yuxin Yin
Molecular Mechanism of Radiation-Induced Transformation of
Human Bronchial Epithelial Cells by High-LET Radiation .................................100
Yong L. Zhao, Chang Q. Piao, and Tom K. Hei
Expression of Transforming Genes and Allelic Imbalance in
Human Breast Epithelial Cells Induced by High-LET Radiation........................103
Debasish Roy, Gloria Calaf, and Tom K. Hei
Aberrant Hypermethylation of the 14-3-3σ
σ Gene is Associated
with Gene Silencing in Breast Cancer .....................................................................106
Anne T. Ferguson, Ella Evron, Christopher Umbricht (all from Johns
Hopkins Oncology Center), Tej K. Pandita, Heiko Hermeking
(JHOC), Jeffrey Marks (Duke University Medical Center), Andrew
Futreal, Martha R. Stampfer (both from Berkeley National
Laboratories), and Saraswati Sukumar (JHOC)
STUDIES RELATED TO RADIATION THERAPY
A More Robust Biologically Based Ranking Criterion for
Treatment Plans.........................................................................................................109
David J. Brenner and Rainer K. Sachs (University of California/
Berkeley)
Tumor Heterogeneity and its Effect on Parameters Estimated
Using the Linear-Quadratic Model..........................................................................111
David J. Brenner and Eric J. Hall
v
THE RADIOLOGICAL RESEARCH ACCELERATOR FACILITY
The Radiological Research Accelerator Facility: An NIHSupported Resource Center......................................................................................115
Director: David J. Brenner, Ph.D., D.Sc., Manager: Stephen A.
Marino, M.S.
THE RADIATION SAFETY OFFICE
Radiation Safety Office Staff ........................................................................................123
The Radiation Safety Office..........................................................................................124
Director: Salmen Loksen, M.S., CHP, DABR
ACTIVITIES AND PUBLICATIONS
Professional Activities....................................................................................................139
The Columbia Colloquium and Laboratory Seminars...............................................144
Publications ....................................................................................................................145
vi
Technical Characteristics of the Columbia University Single-Ion
Microbeam
Gerhard Randers-Pehrson, Charles Geard, Gary Johnson, and David Brenner
Introduction
Microprobes for the study of radiation damage to biological systems have been
known since the 1950s (1) but were not used extensively until a recent resurgence of
interest (2,3,4), due in part to recent developments in computer-based microscopy
systems that allow rapid location and accurate positioning of target cells.
A single-ion microbeam facility comprises a number of elements arranged to
deliver reliably counted numbers of ions to a chosen biological target. The elements are:
1) a source of ions of the appropriate energy, 2) a means of limiting the location of the
ions to an area less than the area of the target, 3) a means of locating and moving the
biological targets to the beam position, 4) a means of detecting each ion as it traverses the
target, and 5) a means of shutting off the beam after the arrival of the chosen number of
ions. The characteristics of each of these elements determine the type of experiment that
can be performed at the facility.
Van de Graaff Accelerator
The source of ions for our microbeam is a model D1, 4.2-MV Van de Graaff
accelerator. This machine, which was originally the injector for the Cosmotron at
Brookhaven National Laboratory, was converted to a dedicated radiobiology facility in
1966 and later moved to Irvington New York where it presently operates. The terminal is
fitted with a duo-plasmatron ion source, which can produce beams of the isotopes of
hydrogen and helium. Most of the work to date on the microbeam has been performed
with alpha particles of 6 MeV corresponding to an LET of approximately 90 keV/µm at
the center of the cells. Experiments could be performed with stopping alpha particles or
with hydrogen ions with a lowest LET of 30 keV/µm limited by transparency of the
collimator system.
Collimator
The area of the beam of ions can be limited either by collimation or by focusing.
For the present system we chose to use collimation because of the simplicity of set up and
operation compared to focused systems. The collimator consists of a pair of apertures
laser-drilled in 12.5-µm thick stainless steel foils separated by 300 µm (Lenox Laser,
Phoenix, Maryland). The limiting aperture is a 5-µm diameter hole in the first foil. The
second aperture, which is 6 µm in diameter acts as an anti-scatter element. The relative
alignment of the two apertures is fixed during manufacture as a three-layer sandwich with
the spacer in between. Monte Carlo modeling of this geometry and comparison to the
energy spectrum of transmitted particles indicates that about 91% of them are within the
unscattered core of the beam, having a diameter of 5 µm. Approximately 7% of the beam
is contained in a halo around the core resulting from particles that scatter in the edge of
1
the first aperture and then pass through the anti-scatter aperture. The halo has a diameter
of about 8 µm at the cell irradiation position. The remaining 2% of the particles are
scattered by both apertures and appear at larger distances from the target position. The
beam characteristics are appropriate for the originally intended targets for the microbeam,
namely the nuclei of mammalian cells in culture.
Imaging and control program
The most important factor in determining the throughput of a microprobe system
for irradiating cells is the ability of the microscopic video analysis system to recognize
the targets and move them into position. A program written in Visual Basic under the
Windows NT operating system controls the video analysis system and microscope stage
motion. Cells are grown attached to a thin polypropylene foil treated with Cell-Tak.
Polypropylene was chosen because it is non-fluorescent. The stock cell suspension is
diluted so that a chosen number of cells will be contained in a 2-µl drop of medium. The
cells are stained by exposure to a 50-nM solution the vital DNA stain Hoechst 33342 for
30 minutes prior to analysis. This low stain concentration requires the use of a channelplate image intensified CCD camera (GenSysII and CCD-72, Dage-MTI, Michigan City,
Indiana) to obtain a high contrast image. A narrow band epi-fluorescence cube (XF-06,
Omega Optical, Brattleboro, Vermont) selects the 366-nm line from a mercury arc lamp
for the observation. The video signal is captured by a Matrox Genesis image processing
board using the Matrox Imaging Library (Matrox Electronic Systems, Dorval, Canada)
Each image is a 10-frame average that has been smoothed with a mean intensity filter and
corrected for non-uniformity of illumination. A threshold in intensity is set to separate
the cells from the background. For normal nuclear irradiation, the centroid of each cell is
found relative to the position of the exit aperture (located by laser light shining through
the collimator system). Each cell is then positioned over the exit aperture by the stepping
motors driving the microscope stage (Daedal, Inc., Harrison City, Pennsylvania). The
computer maintains a list of centroids of cells that have been irradiated to prevent an
accidental second irradiation. The combined precision of the video analysis and stage
positioning is estimated to be about 2 µm. The total time required to irradiate a single
dish of 2,000 cells is approximately 10 minutes corresponding to a throughput of 12,000
cells/hour.
Ion detection and beam shutter
In order to shut off the beam after delivering a certain number of ions, a reliable
counter must be used. Our main counter, which is used when the ion beam has a
sufficient residual range after passing through the target cells, is a P10 gas filled pulsed
ion counter mounted on the high power objective of the observation microscope.
Because the counter is above the cell culture, it is necessary to aspirate off all but a thin
layer of medium for the duration of the radiation exposure. Humidified air with 5% CO2
is passed over the culture to prevent dehydration through a passage in the counter body.
An entrance window of 2.5-µm thick optically clear mica separates the gas in the counter
from the gas over the cell culture. The path length for the particles is 8 mm. When the
counter is operated at 300 volts, it is working without gas gain and provides a very stable
signal well separated from the noise. The signal from the detector preamp is shaped by a
2
standard NIM amplifier, which feeds an SCA and computer controlled scaler. The gate
period output of the scaler is fed to a high voltage amplifier (Technisches Büro S. Fischer,
Ober-Ramstadt, Germany) connected to electrostatic deflection plates to turn on the beam
until the chosen number of particles has arrived. The rise and fall time of the deflection
voltage is such that 4 times out of 10,000 there will be an extra particle delivered to the
sample. There is a background signal of about 7 counts per day due to alpha particle
activity in the glass elements of the microscope objective or in the body of the counter.
A second counter is available for studies with stopping particles or particles with a
small residual range. It is a Schottky barrier detector constructed from a 2-µm thick
silicon wafer. It provides a signal with excellent resolution and 100% efficiency but
suffers from light sensitivity and radiation damage, so it will only be used for experiments
where the gas counter is inappropriate.
Alternative irradiation protocols
In addition to the straightforward standard protocol where we deliver a counted
number of ions to the center of each cell nucleus present in the dish (5,6) we are
developing and using several other irradiation protocols. The first of these new protocols
was developed to irradiate the cytoplasm of each cell. The image analysis system found
the long axis of each cell and then the computer system delivered particles at two target
positions 8 µm away from each end of the cell. In these experiments, the computer
generated exclusion zones around each nucleus to ensure that the target positions from
one nucleus was not accidentally within the nucleus of a nearby cell. Wu et al. (7)
reported mutation induction by cytoplasmic irradiation using this technique.
We are using two variants of the standard protocol to study bystander effects. In
the first case, all of the cells are imaged but the computer randomly irradiates only a
chosen fraction of them. Zhou et al. (8) are using this technique. The second approach is
to have a mixture of cells growing in the irradiation dish, but only a fraction of them are
stained with Hoechst and are therefore visible to the image analysis system. The other
cell might be stained with a dye of another color so they can be distinguished during later
analysis. This is the technique preferred by Geard et al. (9)
Future Developments
It is clear from the present requests for beam time and discussion with users of the
Columbia microbeam facility, that the main interest for future use is to study bystander
effects and to irradiate sub-cellular components. Both of these classes of experiments
require better special resolution and the absence of a beam halo. It is not possible to
obtain a beam with those properties using a collimator system. We are therefore
designing a new microbeam facility that will use a compound electrostatic lens system to
obtain a beam spot of sub-micron precision (10). A prototype of the lens has been
constructed and is undergoing test on the present microbeam station. The prototype is
expected to provide a beam with 2 µm diameter and essentially no halo, while the final
objective is to obtain a beam with a diameter of 0.3 µm.
3
Another limit of the present facility is that it can only be used to provide light ions
with a limited range of LET. We plan to correct this limit by installing a laser driven ion
source that will produce ions with masses up through iron and thereby LET’s as high at
4500 keV/µm. The combination of a large variety of ions and a focusing system will
require new diagnostic techniques to ensure that all the parameters of the system are set to
optimum values. We are designing an electron microscope to image the impact position
of each ion by focusing the secondary electrons produced.
Conclusion
The Columbia University microbeam facility has proved capable of satisfying its
original objective of studying the ability of single alpha particles to produce
transformational and mutational events in mammalian cells irradiated through the
nucleus. New studies of sub-cellular targeting of radiation and bystander effects require
upgrade of the facility to obtain a smaller diameter, halo-free beam.
References
1. Zirkle and W. Bloom Irradiation of Parts of Cells. Science 117, 487-493 (1953).
2. C. R. Geard, D. J. Brenner, G. Randers-Pehrson and S. A. Marino, Single-particle irradiation of
mammalian cells at the Radiological Research Accelerator Facility: induction of chromosomal changes.
Nucl. Inst. And Meth. B54, 411-416 (1991).
3. J. M. Nelson, A. L. Brooks, N. F. Metting, M. A. Khan, R. L. Buschboom, A. Duncan, R. Miick and L.
A. Braby, Clastogenic effects of defined numbers of 3.2 MeV alpha particles on individual CHO-K1
cells. Radiat. Res. 145(5), 568-574 (1996).
4. M. Folkard, B. Vojnovic, K. M., Prise, A. G. Bowey, R. J. Locke, G. Schettino and B. D. Michael, A
charged-particle microbeam: I. Development of an experimental system for targeting cells individually
with counted particles. Int. J. Radiat. Bio. 72, 375-385 (1997).
5. T. K. Hei, L-J Wu, S-X Liu, D. Vannais, C. A. Waldren and G. Randers-Pehrson, Mutagenic effects of
a single and an exact number of α particles in mammalian cells. Proc. Natl. Acad. Sci. USA 94, 37653770 (1997).
6. R. C. Miller, G. Randers-Pehrson, C. R. Geard, E. J. Hall, and D. J. Brenner, The oncogenic potential
of a single alpha particle. Proc. Natl. Acad. Sci. USA 96, 19-22 (1999).
7. L.-J. Wu, G. Randers-Pehrson, A. Xu., C. A. Walden, C. R. Geard, Z.-L. Yu and T. K. Hei, Targeted
cytoplasmic irradiation with alpha particles induces mutations in mammalian cells. Proc. Natl. Acad.
Sci. USA 96, 4959-4964 (1999).
8. H. Zhou, . G. Randers-Pehrson, T. K. Hei, Bystander mutagenic effects of alpha particles in
mammalian cells. Radiat. Res. In press.
9. C. R. Geard, G. Randers-Pehrson, S. A. Marino, G. Jenkins, T. K. Hei, E. J. Hall and D. J. Brenner,
Intra- and Inter-cellular responses following cell-site-specific microbeam irradiation, Radiat. Res. In
press.
10. A. D. Dymnikov, D. J. Brenner, G. Johnson, and G. Randers-Pehrson, Electrostatic lens design for the
Columbia microbeam. Radiat. Res. In press.
4
Electrostatic Lens Design for the Columbia Microbeam
Alexander Dymnikov, David Brenner, Gary Johnson, and Gerhard Randers-Pehrson
Introduction
Microscopy with high-energy ions is a relatively new technique. The first nuclear
microprobe (1,2) with magnetic Russian quadruplet lens focusing (3) was built in 1970
and opened up many new investigative fields. Now an increasing number of laboratories
are applying nuclear microprobes to a very wide range of problems in science and
technology. Microbeams are used in biology and medicine, in microelectronics and
photonics, in arts and archaeology, in geology and planetary science, in environmental
science, in ion lithography, and in material science.
Our objective is to construct a microprobe with electrostatic focusing to study the
response of a biological cell localized irradiation. We are planning to obtain a microbeam
having a diameter of about 0.4 µm. This microbeam will deliver a predetermined number
(e.g. one) of charged particles (such as α-particles or protons), with micrometer accuracy,
through each of a number of cells growing on a dish.
Microprobe focusing lenses
Focusing of ion beams of MeV energy is mostly accomplished by quadrupole
lenses. The great majority of these employ a combination of magnetic quadrupole lenses
(4). Another way to obtain a microbeam is to use solenoids as probe-forming lenses. But
manufactured coils do not have a perfect rotational symmetry. Existing microprobes with
solenoids do not produce a resolution of less than a few microns (5).
Two stages of synthesis of new design
We have divided the synthesis of the new design in two stages. During the first
stage we create the preliminary microprobe design with probe-forming system having the
following geometry. The total length lt = 1.3 m (the distance between the object slit and
the target), the lens length l = 0.26 m (the sum of all lengths of lenses and spaces between
lenses) and the working distance g = 0.1 m (the distance between the last lens and the
target). The purpose of this design with rather small demagnification (- 4.2) is to be a
prototype of the final design with lt =3.7 m. The final design synthesized for a second
stage is operating with the second mode of excitations and has rather big positive
demagnification (~50 – 80).
As the result of our numerical investigation of different field configurations, the
electrostatic Russian quadruplet (RQE) has been chosen for the probe-forming lens in the
preliminary design. We have chosen an electrostatic focusing system because its focusing
strength depends only on the accelerating voltage used to produce the ions. This is
important for us because we intend to add a heavy ion capability to our system.
5
Our RQE consists of four quadrupoles, each of them formed by four cylindrical
rods with the same radius r and semiaperture a, and with length l i . Geometrical and
electrical data are the following: the distance s 0 of the first quadrupole to the object
aperture, the separation s i between the i-th and i+1-th quadrupoles; finally the
polarization is chosen so that in every plane V1 = −V4 and V2 = −V3 .
Maximum operating voltage
A feature of both these designs is the method by which the relative strengths of the
individual quadrupoles are selected. In order to obtain the maximum operating voltage,
we want to operate all the lenses at approximately the same voltage ( V1 ≈ −V2 ). That is,
they will all be operated near the breakdown strength of the system without having one
element be the weak link. We therefore choose the lengths of the electrodes such that the
proper focusing will be obtained with essentially the same voltage on each electrode.
Optimal beam envelope and optimal matching slits
We have performed a series of analytical and numerical calculations to obtain the
best design for our system. Beam focusing is understood as the result of non-linear
motion of a set of particles. As a result of this motion, we have a beam spot on the target.
The set has a volume, the phase volume, or emittance. For a given brightness, the phase
volume is proportional to the beam current and vice versa. The beam has an envelope
surface and all beam particles are located inside this surface, i.e., inside this beam
envelope. For the same phase volume (or beam current) the shape of the beam envelope
can be different. We consider that the beam envelope is optimal if the spot size on the
target has a minimum value for a given emittance. The beam of a given emittance is
defined by a set of two matching slits: objective and aperture slits. For a given emittance
em, the shape of the beam envelope is the function of the half-width (or radius) r1 of the
objective slit and of the distance l12 between two slits. The size r2 of the second (aperture)
slit is determined by the expression: r2 = em*l12/r1. The parameters r1opt, r2opt and l12opt
determine the optimal beam envelope or the optimal matching slits.
Optimal probe-forming system
The probe-forming system consists of two systems: the matching slit system and
the focusing system. Usually the focusing system has two field parameters (two
excitations) and several parameters of its geometry. For this case, from two conditions of
stigmatism we find the first approximation of two excitations as a function of the
geometry. For each geometry we can find the optimal matching slits. The geometry,
which gives the smallest spot size, is the optimal geometry. For this geometry and for the
optimal matching slits we find the optimal excitations giving the minimum spot size. The
optimal probe-forming system comprises the optimal excitations, optimal matching slits
and optimal geometry. For each emittance we find the parameters of the optimal probeforming system. We consider the non-linear motion of the beam accurate to terms of third
order for systems with rotational or quadrupole symmetry and to terms of second order
for systems with dipole symmetry.
6
Matrix approach
The essential feature of our optimization is a matrix approach for non-linear beam
motion. In this approach we obtain and use analytical expressions for the matrizant (or
transfer matrix) and for the envelope matrix of the third order. This matrix technique is
known as the matrizant method (6). We use this technique for solving the equations of
motion, the non-linear differential equations in 4-dimensional phase space, for example,
for the field with rotational or quadrupole symmetry accurate to terms of third order.
These equations are replaced by two vector linear equations (for the x- and y- planes) in
the 12-dimensional phase moment space or by one equation in the 24-dimensional phase
moment space. Writing the non-linear equations in a linearized form allows us to
construct the solution using a 12×12 (or 24×24) matrizant.
We consider the evolution of the phase moment vector, which contains the
elements of the phase moments of first and third order. The envelope matrix is taken as
the matrix of the second moments of the distribution of this vector over the totality of the
phase coordinates. We consider the case of a small density beam; then the beam self-field
as well as particle collisions can be neglected, and the distribution function satisfies the
Liouville’s equation. The integration is done over the object and aperture slits. We find
the analytical form of the 12×12 (or 24×24) initial envelope matrix as a function of em, r1
and l12. This matrix is normalized by equating the first diagonal element to r122. Thus the
average radius of the beam is determined by the first diagonal element of the envelope
matrix, which is a function of the position along the axis.
Two figures of merit
To perform an optimal synthesis we use two different figures of merit. The first is
the average radius of the beam. For a given geometry of the focusing system we compute
the first approximation of lens excitations, κ i , which provide the stigmatic property of
the system. Then we find the optimal r1 and l12 that give the smallest value of the average
radius in the Gaussian image plane for each emittance. The minimum spot is not located
at the Gaussian plane but we can move this spot to the Gaussian plane by changing the
excitations and finding the optimal ones. In the set of n particles we select two particles:
the reference (or axial) particle and the particle which is the most distant from the
reference particle. At the end of the optimization we take the second figure of merit, the
distance ρ between these two particles, and determine the optimal lens excitations (the
second approximation) that give the minimum ρ, using 1,000 particles with randomized
positions and divergences. This gives us the possibility to obtain the minimum spot
without a tail.
Influence of the energy spread
We have investigated the influence of the energy spread ∆E/E (the energy
resolution of the accelerator) on the optimal spot for the RQE. To keep the increase in the
average radius of the optimal spot less than 10%, we need to have ∆E/E less than 0.0001
for em =1 µm×mrad, 0.0002 for em = 3 µm×mrad and 0.0004 for em = 10 µm×mrad. The
same results are obtained for the requirements on the stability of the power supply.
7
Aberration due to misalignment
An increase of the beam spot size can also be caused by a lateral displacement of
the slit system with respect to the RQE longitudinal axis. Our calculations show that to
limit the increase in the average radius of the optimal spot to less than 10%, we need to
have the tolerance for this displacement of less than 0.1 mm.
Construction and fabrication
One of the main features of the RQE design being used is that part of the
alignment of the electrodes is accomplished by using four 0.01 m diameter ceramic
(macor) rods 0.3 m long for the entire set of four quadrupoles. The rods are centerless
ground to a tolerance of 6 µm for the diameter and 12 µm for camber (straightness). This
design essentially eliminates misalignment of the quadrupole axes, which would induce
parasitic aberrations. Evaporating a thin layer of gold onto the entire cylindrical surface in
bands creates the 16 positive and negative electrodes. The insulating sections between
bands is the original ceramic surface with a relief machined at the end of each section.
Rotational misalignment of chosen construction
For the chosen construction we can consider the possible small rotation of the
entire set of quadrupoles. Our calculations show that for the increase in the average radius
of the optimal spot to be less than 10% we need to have a tolerance for this axial rotation
less than 1.2 mrad over the whole length of the lens.
Main parameters of the prototype lens system
As a result of our optimization we have obtained the following optimal
parameters: r=a=5mm, l1 = l 4 = 3 cm, l 2 = l 3 = 6.5 cm, s 0 = 94 cm, s1 = s 3 = 2 cm, s2 = 3
cm, V1 ≈ −V2 ≈ 15kV (for 3 MeV protons).
For the condition of satisfying all requirements for the energy spread and
misalignments with optimal r1opt, r2opt and l12opt for every emittance we obtain the
minimum spot size ρ. We have found the following values of ρ, r1opt, r2opt in µm and l12opt
in mm for three emittances.
For em = 1 µm×mrad: ρ = 0.508, r1opt = 1.859, r2opt = 5.133, l12opt = 9.54.
For em = 3 µm×mrad: ρ = 1.18, r1opt = 4.47, r2opt = 10.215, l12opt = 15.1.
For em = 10µm×mrad: ρ = 2.88, r1opt = 10.40, r2opt = 24.83, l12opt = 27.4.
References
1. J. A. Cookson, J. W. McMillan and T. B. Pierce, The nuclear microprobe as an analytical tool. J.
Radioanal. Chem. 48, 337-357 (1979).
2. J. A. Cookson and F. D. Pilling, The use of focused ion beams for analysis. Thin Solid Films. 19, 381385 (1973).
3. A. D. Dymnikov and S. Ya. Yavor, Four quadrupole lenses as an analog of an axially symmetric
system. Sov. Phys. Tech. Phys. 8, 639-643 (1964).
4. G. J. F. Legge, A history of ion microbeams. Nucl. Instr. and Meth. B 130, 9-19 (1997).
8
5.
6.
A. Stephan, J. Meijer, M. Hofert, H. H. Bukow and C. Rolfs., A superconducting solenoid as probe
forming lens for microprobe applications. Nucl. Instr. and Meth. B 89, 420-423 (1994).
A. D. Dymnikov and R. Hellborg, Matrix theory of the motion of a charged particle beam in curvilinear
space-time. I. General Theory. Nucl. Instr. and Meth. A 330, 323-342 (1993).
9
The Low-Energy Neutron Facility
Stephen Marino, Dusan Srdoc, and Gary Johnson
Development has continued on a facility to produce a series of low-energy neutron
spectra that have maximum neutron energies from 40 to 110 keV, minimal dose
contributions from higher energy neutrons and γ rays, and dose rates usable for irradiation
of cultured cells. The facility is based on the 7Li(p,n)7Be reaction because of its large cross
section and uses a rotating target design to minimize the heating of the lithium, which
melts at 180°C.
The target fixture, shown in Fig. 1, has been greatly modified from the preliminary
design, as was described in last year’s Annual Report, and is the result of a number of
simplifications and improvements. The original Teflon rotating water seals proved to be
inadequate - they began leaking heavily after only a moderate amount of use. New double
Viton seals have been installed and have shown no signs of leaking, even after many days
of use.
A new evaporator has been constructed which is able to evaporate lithium layers at
least 40-keV thick (Fig. 2). The design is based on that of a smaller evaporator that has
been used successfully in the past, but has a limited lithium capacity. The evaporation boat
is a stainless steel tube with a 3-mm diameter hole near one end to expel the lithium. This
end of the tube has a copper plug, which is screwed to a copper tube that surrounds the
boat and acts as one electrode for heating it. The other end of the tube is flattened and
connected to a copper rod, which acts as the other electrode. A ceramic insulator is
epoxied to both electrodes to form the internal vacuum seal of the assembly. The brass
vacuum housing is water-cooled and has been designed to position the hole in the
evaporation tube as close as possible to the target surface in order to maximize the
efficiency of lithium deposition.
The thickness of the lithium layers evaporated onto the rotating target surface are
measured by observing the width of the Li(p,γ) resonance at 440 keV with a NaI detector.
The first target made was approximately 5.5 keV thick at a proton energy of 1.9 MeV. A
measurement of the Li(p,n) threshold (1.880 MeV) was made using a BF3 proportional
counter surrounded by moderating material. This measurement was used to calibrate the
bending magnet and was the basis for all the measurements that followed.
Two spherical proton recoil counters were used to measure the neutron spectra for
this target. One counter is 40 mm in diameter, has a .030” thick stainless steel wall, and
was filled with 1 atmosphere of hydrogen. The other counter (LND model 20790) is 12.7
mm in diameter and measurements were made with 1 and 2 atmospheres of hydrogen. In
all cases, the chambers were pumped to a few microns pressure, filled with high-purity
hydrogen passed through an oxygen-absorbing filter, and the chambers were evacuated and
filled three times successively. A series of calibration spectra were obtained for each
chamber size and pressure combination with neutrons produced at 0° using the thin lithium
10
target in order to obtain the relationship of channel number to neutron energy and to derive
response functions for the unfolding process. The response functions used were obtained
by fitting two straight lines to each of these proton recoil spectra. One line was fit to the
linear portion of the main spectrum, and the second at the terminal or “edge” portion.
From the slopes of these fits, proton recoil spectra at any energy could be derived.
Proton recoil spectra measurements were then made at 100° for 86, 56 and 40 keV
neutron spectra at distances such that the detectors subtended the same angle as the 3-mm
diameter cell samples would at 2.5 cm. Additional measurements were made with a 1-cm
long uranium gamma-ray filter in place for the 100 keV neutrons.
The proton spectra from the counters were unfolded to obtain the neutron spectra
based on a method developed by Gold (1). This is an iterative method based on successive
approximations that seeks to find an appropriate solution to the problem M + E = RN,
where M is the true proton recoil spectrum, E is the error added by the measurement, R is
the response matrix of the detector, and N is the neutron spectrum, which is to be
determined. The solution is such that M – RN ≤ E and all elements of N are non-negative.
The unfolded spectra for the three neutron energies measured without the gammaray filter are shown in Fig. 3. These are normalized to the proton beam charge so that the
relative areas under the curves are the relative neutron fluxes per unit beam current. The
numbers next to the curves are the fluence-weighted mean neutron energies. All three
spectra have a peak around the mean energy of the direct neutrons (32, 54, and 95 keV by
calculation). Neutrons above the peak are due to the scattering of neutrons with higher
energies produced at more forward angles. The portion of the spectrum below the peak is
produced by multiple scattering of neutrons produced at forward angles and by the
scattering of neutrons at angles greater than 100°.
Measurements of microdosimetric spectra were obtained with a 1” diameter tissueequivalent (TE) Rossi counter for 86, 56 and 40 keV neutrons at 100 with and without γray filters. Propane-based TE gas was flowed through the chamber at a pressure that
simulated a tissue diameter of 1 µm. The counter was positioned 25 cm from the target in
order to subtend the same angle as the cell samples. A measurement was also made of the
spectrum for the 478 keV gamma rays from the Li(p,p`γ) reaction using 1.86 MeV protons,
below the threshold of the Li(p,n) reaction.
Plots of yd(y) for the three neutron energies are shown in Fig. 4. As can be seen
from these plots, the frequency of events above 10 keV/µm decreases markedly as the
neutron energy decreases. This is due to the decrease in neutron yield relative to the
gamma-ray yield of the target as the incident proton energy is decreased. The proton peak
shifts to lower y values as the neutron energy decreases because the primary neutrons
produce proton recoils with energies at or below the Bragg peak. These protons do not
have sufficient energy to have the maximum possible energy loss of 147 keV/µm (proton
“edge”) in the 1 µm site size. Proton recoils with y values near this edge are from higher
energy neutrons scattered from forward angles. The proton peak extends down into the
gamma-ray region below 10 keV/µm, especially for the 40 keV neutrons, because of the
11
low proton recoil energies. The values of yD obtained are 16.3, 20.7 and 31.7 keV/µm for
the 40, 56 and 86 keV neutrons respectively. Insertion of the uranium gamma-ray filter
results in a relative increase in the proton peak (yD = 35.1 keV/µm) for 86 keV neutrons
since the gamma-ray attenuation is larger than the loss due to neutron scattering.
It may be possible to reduce the scattering of higher energy neutrons by further
modifying the target assembly. The vacuum housing cover has already been thinned over
most of its surface, however it may be possible to further thin a section of the downstream
vacuum cover in line with the proton beam. The outer centimeter of the target disc can be
thinned to half its present thickness. Most of the higher energy neutrons pass through these
areas, sometimes at fairly large angles that increase their pathlengths and the probability of
scattering.
After approximately 1.4 coulombs of beam use (equivalent of 4 hours at 100 µA), a
second measurement of the target thickness using the Li(p,γ) resonance was made. The
maximum target yield was the same as for the first measurement and the target thickness
was 3% larger than the initial measurement, indicating no loss of lithium but possibly some
oxidation of the target over the six-week period between the measurements. During the
various spectroscopy measurements, a beam spot 5 x 5 mm and currents up to 15 µA had
been used with the target rotating at only 900 rpm, half the design speed.
A lithium target approximately 14 keV thick at 1.9 MeV was evaporated for
dosimetry measurements. Graded γ-ray filters 7.9 mm in diameter consisting of 6-10 mm
of depleted uranium, 0.5 mm of Sn, 0.5 mm of Cu and 1 mm of Al were placed to shield
the dosimeter. Total dose measurements were made using a TE ionization chamber used
with gas multiplication and having a spherical cavity 6.4 mm in diameter that was centered
in the cell sample position. The γ-ray dose rate was measured with the same chamber using
a proton energy of 1.865 MeV, just below the threshold for neutron production, and scaled
by the relative cross-section of the Li(p,p’γ) reaction. Results of these measurements for a
filter with 8 mm U are given in Table I.
Fig. 1. Photograph of the
rotating target assembly as
viewed from slightly
upstream. The box in the
lower left is the control unit
for the infrared themometry
system. A fiber optic cable
connects the unit to the
target vacuum chamber,
center right. The drive belt
for rotating the target can be
seen in the center of the
picture.
12
Fig. 2. The lithium
evaporator. The upper
assembly is the vacuum
housing that connects to the
target vacuum chamber. The
copper tube, which is one
electrode, has the boat
attached to it on the left. A
copper rod, used as the
second electrode, attaches to
the other end of the boat.
4
Fig. 3. Results of
neutron spectroscopy.
Relative Neutron Flux
40 keV
86 keV
56 keV
3
2
1
0
0
20
40
60
80
100
120
140
160
180
Neutron Energy (keV)
0.5
0.4
86 keV n
1 µ m site size
y d(y)
Fig.4. Microdosi-metric
spectra for 1 µm site size
using a walled spherical
TE proportional counter
without γ-ray filters.
56 keV n
0.3
40 keV n
0.2
40 keV n
0.1
56 keV n
86 keV n
0.0
0.1
1
10
y (keV/ µm)
13
100
1000
Table I. Results of dosimetry using a U/Sn/Cu/Al γ-ray filter
Mean neutron
energy (keV)
40
56
86
γ-ray dose rate at
100 µA (mGy/hr)
7.4
7.6
8.1
Total dose rate at
100 µA (mGy/hr)
66
107
186
Reference
1. Gold, R. An Iterative Unfolding Method for Response Matrices. Argonne National
Laboratory Report ANL-6984, 1964.
14
Risks From Less Than 10 Millisievert: What Do We Really Know?
How Can We Learn More?
David J. Brenner
When assessing data on the carcinogenic effects of low doses of radiation, it is
always possible to find data sets which contain one or more data points that are lower than
controls, and thus can be interpreted as showing that low doses of radiation have zero or
even negative carcinogenic potential. Here it is argued that, in drawing biophysical or
health-policy conclusions, to focus only on these data sets to the exclusion of others, is not
prudent.
The basic issue is that, even if a linear relationship between risk and dose did apply
at low doses, the low-dose risks are sufficiently small, particularly for protracted exposures,
that one would expect fluctuations in the data; in other words the occasional low-dose study
would be expected to yield the occasional data-point estimate that is lower than the control
(zero dose) value. Thus the fact that some low-dose studies do yield an occasional data point
estimate that is lower than controls is not a convincing argument against low-dose linearity.
Of course if many or most studies yielded zero or negative risks at low doses, this would be
a different matter, but it is argued here that this is not the case.
Consequently, when using radiation carcinogenesis data to assess dose-effect
relationships, particularly at low doses where the risks are small, it is important to evaluate
all credible relevant data.
As an example, in a recent editorial (1), Harald Rossi discussed three data sets from
the literature relating to low-dose radiation carcinogenesis – one for lymphoma in mice, one
for leukemia in A-bomb survivors, and one for lung cancer after protracted medical
exposures. In each case there is at least one point that is lower than the controls (though in
most cases the decrease is not statistically significant). From these data, Rossi concluded, at
least in these cases, that a “rather confident” answer can be given regarding the carcinogenic
risks of low doses (< 10 mSv or 1 rad) of low-LET radiation such as x or γ rays.
The first data set that Rossi (1) discussed is for radiation-induced lymphomas in
male BC3F1 mice (2). This system is not ideal because the control incidence is so high
(57%), making it difficult to quantify the effects of a low-dose of radiation above this
background. It would therefore be prudent also to consider data from other mouse strains in
which the control incidence is lower – and which would have a greater chance of picking up
the effects of low radiation doses. As an example, Figure 1 shows data for radiation-induced
thymic lymphomas in male RFM mice (3), where the control incidence is 7%; here there is a
steady increase in risk with dose.
Turning to low-dose radiation risks in man, Rossi’s second example (1) was for
radiation-induced leukemia in A-bomb survivors. Figure 2 shows updated (1950-1990) data
15
(4) both for leukemia and for solid cancers (the latter constituting about 90% of the cancer
excess). For the solid cancers, no point estimates of the risk are lower than that of the
controls, and again there is a steady increase in risk with dose, at low doses. For the
leukemias, there is one point which is less than the controls, though not significantly so; the
raw data in the dose window for that point are 59 observed leukemias, with 62 expected
based on the controls. It would be hard to make any confident statements based on those
numbers.
Finally, Professor Rossi (1) discussed lung-cancer mortality in populations exposed
to protracted medical exposures. The suggestion that protracted exposure to small doses of
low-LET radiation results in decreased lung-cancer risks is well taken, and agrees well with
animal data (5), as shown in Figure 3. However, the suggestion that protraction eliminates
the risk of radiation-induced lung cancer at low doses does not appear justified. Figure 4
shows the data from the Canadian TB fluoroscopy cohort (6); none of the data points are
significantly lower than the controls. Figure 4 also contrasts these fractionated exposure data
with the corresponding A-bomb survivor data. The similarities between the dose/
fractionation patterns in the human data (Fig. 4) and the animal data (Fig. 3) are quite
striking, and this would argue against the non-significant decrease in risk at low doses in the
protracted exposure human data being anything but statistical fluctuation. Again these
considerations highlight the fact that when risks are small (which is true for protracted lowdose radiation-induced lung cancer), and the observed number of cancers is small (which is
true for the human data in Fig. 4), fluctuations in the data are bound to occur.
To reiterate the point: epidemiological studies of low-dose radiation risks are
necessarily quite limited in power - the risks are small and the numbers of excess cancers are
small. In such situations there will sometimes be cases where the point estimates of the risks
are zero or negative, but to focus exclusively on these situations to the exclusion of others
where this is not the case will necessarily result in a distorted picture. For example, although
Rossi (1) discusses radiation-induced lung cancer for protracted medical exposure (6), a full
analysis would also include the corresponding breast-cancer data (7) from the same study;
these data are shown in Figure 5, where a monotonic increase in breast-cancer risk with dose
is apparent at low doses.
It is not argued here that there are no low-dose thresholds for radiation induced
malignancy -- there may well be. Radiation-induced sarcomas are likely candidates here,
where the target cells are typically dormant, and need large doses to produce sufficient tissue
damage to stimulate cellular proliferation. Rather, it is argued that radiation risks at low
doses are small and hard to quantify, and so the data will necessarily contain statistical
fluctuations. Many data sets show a monotonic increase of risk with dose at low doses; some
contain one or more point estimates of radiation risks that are less than controls. It is unwise
to focus only on the latter in drawing mechanistic conclusions, and particularly imprudent in
a radiation protection context.
The conclusion then, is that even studies of very large groups of individuals exposed
to doses below 10 mSv have extremely limited power and are prone to statistical
16
fluctuations which can obscure the true situation. Should we give up? I would suggest that
there are two viable approaches to assess the risks at such low doses.
The first viable approach is, of course, to understand mechanisms. Professor Rossi
alludes, for example, to the “microdosimetric” argument, essentially that at low doses, as
one lowers the dose, one is simply proportionately reducing the number of cells damaged,
rather than changing the nature of the cellular damage. Combined with the monoclonal
origin of most cancers, this is an a-priori argument for linearity at low doses. Of course
there are many steps in that argument that need to be tested - but they are, in principle, quite
testable.
A second viable approach is to focus more on systems where one would expect the
low-dose risks to be larger, and thus better quantifiable. As an example, the younger the
exposed individual, the larger the proportion of proliferating cells and the larger the
expected risk; thus the continued study of malignancies in individuals exposed either in
utero or in early childhood to very low doses of radiation, seems a fruitful direction. Another
direction is to study populations which might be expected to have a genetically-based
increased sensitivity to radiation. Thus further studies of low dose radiation risks in, for
example, ataxia telangiectasia heterozygotes may prove fruitful.
References
1. Rossi HH, Radiat Protec Dosim; 1999; 83:277-80.
2. Covelli V, Di Majo V, Coppola M, and Rebessi S, Radiat Res 1989; 119:553-561.
3. Ullrich RL and Storer JB, Radiat Res 1979; 80:303-316.
4. Pierce DA, Shimizu Y, Preston DL, Vaeth M, and Mabuchi K, Radiat Res 1996; 146:1-27.
5. Ullrich RL, Jernigan MC, Satterfield LC, and Bowles ND, , Radiat Res 1987; 111:179-184.
6. Howe GR, Radiat Res 1995; 142:295-304.
7. Howe GR and McLaughlin J, Radiat Res 1996; 145:694-707.
% Incidence (age adjusted)
30
Thymic lymphoma in male RFM mice
25
20
15
10
5
0
0
1
2
Dose (Gy)
Fig. 1: Age-adjusted incidence of thymic
lymphoma in male RFMF/Un mice (3).
17
3
Excess absolute risk per person
0.5
Excess relative risk
Solid Cancer
0.4
0.3
0.2
0.1
0.0
0.0
0.2
0.4
0.6
0.8
0.015
Leukemia
0.010
0.005
0.000
1.0
0.00
Weighted colon equivalent dose (Sv)
0.20
0.40
0.60
0.80
1.00
Weighted marrow equivalent dose (Sv)
40
Lung cancer in BALB/c mice
Lung cancer
4
Acute
30
Relative Risk
% Incidence (age adjusted)
Fig. 2: (a) Excess relative risk of mortality (1950-1990) from solid cancers in A-bomb survivors (4);
only points below 1 Sv are shown. (b) Excess absolute risk of mortality (1950-1990) from leukemia
in A-bomb survivors (4); only points below 1 Sv are shown.
20
Protracted
3
Acute
2
10
Protracted
1
0
0.0
0.5
1.0
1.5
2.0
0
1
Dose (Gy)
Equivalent dose (Sv)
Fig. 3: Age-adjusted incidence of lung
adenocarcinoma in female BALB/c mice following
very low dose rate or acute ?-ray exposure (5).
2.5
Relative Risk
2
Fig. 4: Lung cancer mortality in Canadian TB cohort
exposed to protracted multiple fluoroscopies, compared to
A-bomb survivors exposed to a single acute exposure (6).
Only points below 3 Sv are shown.
Protracted medical exposure
2.0
breast
1.5
lung
1.0
0
1
2
Equivalent dose (Sv)
Fig. 5: Lung cancer vs. breast cancer mortality in Canadian TB cohort exposed to rotracted
multiple fluoroscopies (6, 7). Only points below 3 Sv are shown.
18
The Risk of Fatal Cancer from Pediatric-Computed Tomography
David Brenner, Carl D. Elliston, Eric Hall, and Walter E. Berdon (Dept. of Radiology,
Columbia University)
The use of computed tomography (CT) has increased dramatically in the past two
decades, fueled in part by the development of helical CT. For example, the estimated
annual number of CT examinations in the U.S. rose about sevenfold from 2.8 million in
1981 to 20 million in 1995. By their nature, CT examinations contribute disproportionately
to the collective diagnostic radiation dose to the population; for example, it has been
estimated that, in the UK, about 4% of diagnostic radiological procedures are currently CT
examinations, but their contribution to the collective dose from diagnostic radiology is
about 40%.
It was estimated in 1989 that about 4% of CT examinations (currently
corresponding to about 1 million per year in the US) were performed on children under the
age of 15; it is, however, likely that the proportion of childhood CT examinations is
currently increasing (indeed an average value of 6% was estimated in 1993). The increased
frequency of pediatric CT is largely due to the advent of fast helical CT, reducing the need
for sedation, making more types of CT examinations more practical in younger, sicker, or
less cooperative children, as well as allowing newer pediatric CT applications such as
dynamic studies of pulmonary physiology, 3D airway imaging, or diagnosing appendicitis.
Whilst pediatrics represent a comparatively small, though increasing, fraction of the
overall number of CT examinations, we show here that the combination of higher effective
radiation doses to children for a given examination and, more importantly, the much larger
lifetime risks per unit dose which apply to children (see Fig. 1), result in lifetime cancer
risks attributable to the radiation exposure which are significantly higher in children than in
adults.
Organ doses as a function of age-at-diagnosis were estimated for common CT
examinations, and attributable lifetime cancer mortality risks (per unit dose) for different
organ sites applied. The larger doses and increased lifetime risks in pediatric CT produce a
sharp increase in risk, relative to adult CT (see Fig. 2). Estimated lifetime cancer mortality
risks attributable to the radiation exposure from a single abdominal CT examination in a
newborn are 1 in 430, and 1 in 1,100 for a newborn head CT - an order of magnitude
higher than for adults. In the US, about 600,000 abdominal and head CT examinations per
year are currently given to children under the age of 15, and about 500 of these individuals
are estimated ultimately to die from a cancer attributable to the radiation from their
examination. Although the risk-benefit balance for pediatric CT is generally strongly tilted
towards benefit, in light of the risks and of the increasing frequency of pediatric CT, it is
desirable and practical to reduce the dose from pediatric CT examinations.
19
16
Total
Other
Digestive
Breast
Leukemia
Lung
Females
14
Risk per Unit Dose (Percent per Sv)
Risk Per Unit Dose (Percent per Sv)
16
12
10
8
6
4
2
0
0
10
20
30
40
50
60
70
Total
Other
Digestive
Leukemia
Lung
Males
14
12
10
8
6
4
2
0
0
80
10
20
30
40
50
60
70
80
Age at Acute Exposure
Age at Acute Exposure
Fig. 1 . Breakdown, by sex and cancer type, of lifetime attributable cancer mortality risks as a function of age
at a single acute exposure, as estimated by the National Academy of Sciences BEIR V Committee.
Lifetime Attributable Risk (Percent)
0.30
0.25
0.20
0.15
0.10
Abdominal
Head
0.05
0.00
0
10
20
30
40
50
60
70
80
Age at CT Examination (Years)
Fig. 2. Lifetime attributable cancer mortality risk, as a function of age at examination, for a single typical CT
examination of the head and of the abdomen.
20
A Polymer, Random-Walk Model for the Size-Distribution of
Large DNA Fragments after High-LET Radiation
David Brenner, with Artem Ponomarev and Rainer Sachs (both from University of
California, Berkeley), and Lynn Hlatky (Harvard Medical School)
DSBs (DNA double strand breaks) produced by densely-ionizing radiations are not
located randomly in the genome - recent data indicate DSB clustering along chromosomes.
Stochastic radiation-induced DSB clustering at large scales, from >100 Mbp down to
<0.01Mbp, have been modeled using Monte Carlo computer simulations.
A random walk, coarse-grained polymer model for chromatin has been combined
with a simple radiation track-structure model in software called DNAbreak. The chromatin
model neglects molecular details but systematically incorporates the increase in average
spatial separation between two DNA loci as the number of basepairs between the loci
increases. The approach generalizes the random-breakage model, whose broken-stick
fragment-size distribution is applicable to low LET radiations.
The technique allows biophysically based extrapolations of high-dose DNA
fragment-size data to relevant doses sufficiently low that one-track action dominates. Doseresponse relations for DNA fragment-size distributions, which are linear at low doses, were
found to be non-linear when there is a significant probability of overlapping among DSB
clusters from different tracks along one chromosome, an effect important for large
fragments and high doses.
It was also found that fragment-size distributions are very similar whether or not
chromatin undergoes significant rearrangement by Brownian motion between hits by
different tracks. Fragment-size distributions obtained using DNAbreak match current
experimental data on large fragments about as well as distributions previously used in a
less mechanistic approach (1).
Reference
1. Sachs RK, Brenner DJ, Hahnfeldt PJ, Hlatky LR, A formalism for analysing largescale clustering of radiation-induced breaks along chromosomes. Int. J. Radiat.
Biol. 74:185-206 (1998).
21
MICROBEAM: CELLULAR STUDIES
Induction of a Bystander Mutagenic Effect of Alpha Particles on
Mammalian Cells
Hongning Zhou, Gerhard Randers-Pehrson, and Tom Hei
Epidemiological studies of uranium mine workers and experimental animal studies
suggest a positive correlation between exposure to alpha particles emitted from radon and its
progeny and the development of lung cancer (1,2). The mechanism(s) by which alpha
particles cause lung cancer has not been elucidated, although a variety of genetic lesions,
including chromosomal damage, gene mutations, induction of micronuclei, and sister
chromatid exchanges (SCE) have been associated with the DNA-damaging effects of alpha
particles (3,4).
For over a century since the discovery of X rays, it has always been accepted that the
deleterious effects of ionizing radiation such as mutation and carcinogenesis are due mainly to
direct damage to DNA. However, recent circumstantial evidence suggests that extranuclear or
extracellular targets may also be important in mediating the genotoxic effect of irradiation
(3,4,5). It was found, for example, that very low doses of alpha particles induced clastogenic
responses (principally SCE) in both CHO and human fibroblast cultures at levels significantly
higher than expected, based on microdosimetric calculation of the number of cells estimated
to have been traversed by a particle. The additional responding cells which received no
irradiation were "bystanders" of either directly hit cells or resulted from agents released from
the irradiated medium (3,5). Subsequent studies suggested that reactive oxygen species (ROS)
might contribute to the induction of SCE among the bystander cells (6). While circumstantial
evidence in support of a bystander effect appears to be consistent, direct proofs of such
extranuclear/extracellular effects are not available.
Using a precision charged-particle microbeam, our laboratory showed recently, and for
the first time, that irradiation of cellular cytoplasm with either a single or an exact number of
alpha particles resulted in gene mutation in the nucleus while inflicting minimal toxicity and
that free radicals mediate the process (7). The results with the well-established free radical
scavenger, DMSO, and the thiol-depleting drug buthionine S-R- sulfoximine (BSO) provide
further support of the idea that ROS modulates the mutagenic response of cytoplasmic
irradiation. Using a precision charged-particle microbeam, 5 to 20% of randomly selected AL
cells was irradiated with 20 alpha particles. As shown in Figure 1, the actual mutant yield
obtained, when 5 to 20% of cells were irradiated with 20 alpha particles each, was
significantly higher than the expected yield assuming there were no bystander modulation
effects (p<0.05). The results suggest that non-irradiated cells acquire the mutation phenotype
indirectly. In other words, irradiated cells may induce a bystander mutagenic response in
neighboring cells not directly traversed by alpha particles. Furthermore, analysis by multiplex
PCR shows that the types of mutants induced are significantly different from those of
spontaneous origin. the majority of spontaneous CD59- mutants (31 of 47, or 66%) had
retained all of the markers analyzed. In contrast, about 82% of mutants induced with
bystander mutagenesis of 20 alpha particles traversals through 20% of the cells each had lost
at least one additional marker which included 28% complex mutations (p<0.01). Pre23
treatment of cells with the radical scavenger, DMSO had no effect on the mutagenic
incidence. In contrast, cells pretreated with a 40-µM dose of lindane, a gap junction inhibitor,
significantly decreased the mutant yield (Figure 2). The doses of DMSO and lindane used in
these experiments were non-toxic and non-mutagenic. Our studies provide direct evidence
that irradiated cells may induce bystander mutagenic response in neighboring cells not
directly traversed by alpha particles, and that signal transduction pathways other than
oxidative stress play a critical role in mediating the bystander phenomenon.
CD59- Mutants per 105 Survivors
250
200
150
100
50
0
Control
20 α, 5%
20 α, 10%
20 α, 20%
Figure 1. Mutant fraction obtained from populations of AL cells in which 0, 5, 10 or 20 % of
whose nuclei were traversed by 20 alpha particles. Data were pooled from 3 to 8 independent
experiments. Error bars represent ± SEM.
24
CD59- Mutants Per 105 Survivors
250
1. Control
2. 20α, 20%
3. 20α, 20%,
40µM Lindane
4. 40µM Lindane
200
150
100
50
0
1
2
3
4
Figure 2. Effects of gap junction inhibitor lindane (40 µM, 2 hours before and 3 days after
irradiation) on mutant yields in AL cells, 20% of which had been irradiated with 20 alpha
particles through their nuclei. Data were pooled from 3 independent experiments. Error bars
represent ± SEM.
References
1. Samet et al, J. Natl. Cancer Inst. 81: 745-757, 1989.
2. Lubin et al, Natl. Cancer Inst., 89: 49-57, 1997.
3. Nagasawa et al, Cancer Res. 52: 6394-6396, 1992.
4. Deshpande et al, Radiat. Res. 145: 260-267, 1996.
5. Mothersill et al, Radiat. Res. 149: 256-262, 1998.
6. Narayanan et al, Cancer Res. 57: 3963- 3971, 1997.
7. Wu et al, Proc. Natl. Acad. Sci. USA. 96: 4959-4964, 1999.
25
Intra- and Inter-Cellular Responses Following Cell-Site-Specific
Microbeam Irradiation
Charles Geard, Gerhard Randers-Pehrson, Stephen Marino, Gloria Jenkins-Baker,
Tom Hei, Eric Hall, and David Brenner
A charged-particle microbeam has, in a definitive manner, the capacity to place
defined numbers of radiation tracks in a controlled spatio-temporal framework, both
within and between individual cells. At the Columbia microbeam facility we have
developed protocols to place exact numbers of charged particles through nuclear
centroids of cells; at defined distances off the nuclear centroid; at defined positions in the
cytoplasm relative to the nucleus; at defined positions in the cellular milieu (deliberately
missing cells), and through defined fractions of cells in a population. Vital dye staining
protocols have also been developed to allow the targeting of sub-cellular entities, or of
known cells in mixed cell populations (i.e., hit versus non-hit or bystander cells). Cells
can also be imaged off line using conventional transmission microscopy and their
positional coordinates recorded before moving the entire stage and cell dish, with submicrometer precision, to kinematic mounts on the on-line microbeam microscope. In this
way cellular staining and reflected fluorescence may be avoided if desired, with little
impact on irradiation speed.
The accuracy of the current Columbia microbeam system is such that more than
90% of particles are within 3.5 µm of a designated coordinate which, together with
cellular throughputs up to 15,000 cells per hour (depending on the application), has
allowed for definitive assessments of exact single-particle responses for mutation and
oncogenic transformation; this obviates the uncertainties of Poisson-distributed particle
numbers from broad beam or isotopic sources.
The basic paradigm that the directly damaged cell nucleus is the pre-eminent
responder to radiation has been brought into question with findings of cell responses to
cytoplasmic irradiation only. In addition, microbeam irradiation of known small fractions
(e.g. 10 or 20% of cells) in a population has produced responses in non-hit or
“bystander” cells. Co-culturing known hit and non-hit cells has allowed evaluations of
responses in cells that are otherwise handled identically. A fluence-dependent bystander
effect has been definitively demonstrated for reduced cell growth, for induced delay in
cell cycle progression, for the induction of micronuclei, for the differential expression of
p53 and p21, for mutation, and probably for oncogenic transformation.
What have we found so far?
1. One trans-nuclear alpha particle can produce a micronucleus -- further increases are
fluence dependent.
2. The nucleus is non-uniformly sensitive to alpha-particle damage.
3. One trans-nuclear alpha particle can initiate cell-cycle delay -- further delay is fluence
dependent.
26
4. One trans-nuclear alpha particle can initiate a p53 response -- dependent on cell site
(cytoplasm/nucleus), time, and fluence.
5. Cytoplasmic alpha-particle irradiation can initiate a p53 response.
6. A bystander effect has been clearly demonstrated: non-hit cells show hit-cell-fluencedependent cell-cycle delay, slowed growth, enhanced micronuclei, and enhanced p53/
p21 response.
27
Single Alpha-Particle Traversals and Tumor Promoters
Richard Miller, Satin Sawant, Gerhard Randers-Pehrson, Steve Marino, Charles Geard,
Eric Hall, and David Brenner
In most homes, radon gas is present in such low concentrations that relevant
bronchial cells are very rarely traversed by more than one alpha particle. However, radon
cancer risk estimates are derived to a significant degree on extrapolation of
epidemiological data from uranium miners whose bronchial cells were frequently
exposed to multiple alpha-particle traversals. Recently, we reported results from a series
of experiments in which oncogenic transformation was assessed after predefined exact
numbers of alpha particles - delivered through the single-particle microbeam -- traversed
cell nuclei (1). Using positive controls to ensure that the dosimetry and biological
controls were comparable, the measured oncogenicity from exactly one alpha particle was
significantly lower than for a Poisson-distributed mean of one alpha particle, implying
that cells traversed by multiple alpha particles contribute most of the cancer risk.
Therefore, extrapolation from high-level radon risks could overestimate low-level
(involving only single alpha particles) radon risks.
Of course several caveats are required before such a conclusion could be applied
to an epidemiological situation:
1. Our published studies so far refer only to cells that have not been damaged by
tobacco, and it is likely to be the case that most (85-95%) of the lung-cancer deaths
that can be attributed to radon are actually the result of a synergistic interaction
between alpha-particle damage and tobacco damage. So a direct interpretation of our
results would be that the radon risk estimates for non smokers exposed to low levels
of radon may be somewhat overestimated.
2. Our studies are in cells that are physically quite flat (in the direction of the alpha
particle beam), so the path length of alpha particles through these cells is probably
smaller than that through target cells in the lung.
3. Our studies are in an in-vitro rodent system, and thus potentially not directly
applicable to the appropriate human bronchial cells which are at risk. On the other
hand we are looking only at relative effects (e.g. the effects of 1 alpha particle
compared to 2 alpha particles), not absolute effects, so there is no a-priori reason why
this in-vitro rodent system would produce misleading results regarding these relative
effects.
It is important to recognize that homeowners are exposed to many potential
carcinogens and promoters of tumorigenesis. Carcinogenesis is a multistep event that, in
most cancer models, begins with exposure to a carcinogen during the initiation stage,
followed by the promotion stage where tumor promoters are believed to have an impact
on the expression of the initiated event (2). It has been known for some years that the
tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly increases the
frequency of x ray- and neutron-induced oncogenic transformation (3). To explore the
situation for a single alpha particle traversal through a cell nucleus, C3H10T½ mouse
28
cells were exposed in vitro to single alpha-particle traversals through cell nuclei-achieved with the single-particle microbeam -- followed by treatment with TPA. The
frequency of oncogenic transformation induced by the combination of a single alpha
particle and a tumor promoter was compared to cells exposed to single alpha-particles
without TPA.
When compared to cells exposed to alpha particles alone, a synergistic increase in
oncogenic transformation frequencies occurred with cells treated to the combination of
single alpha particles and TPA. We compared these results with measurements of the
TPA enhanced transformation frequency of x rays - at an x-ray dose that produced the
same transformation frequency (without TPA) as a single alpha particle (without TPA).
Our preliminary results yield no significant difference in the TPA-induced enhancement
between single alpha particles and x rays, suggesting that, in this case at least, RBE’s of
relevance to the radon problem are not very sensitive to changes in the tumor promoter
environment
References
1.
R. C. Miller, G. Randers-Pehrson, C. R. Geard, E. J. Hall, D. J. Brenner. The oncogenic transforming
potential of the passage of single alpha particles through mammalian cell nuclei. Proc Natl Acad Sci
USA 96, 19-22 (1999).
2.
E. J. Hall, T. K. Hei. Modulating factors in the expression of radiation-induced oncogenic
transformation. Environ Health Perspect. 88,149-155 (1990).
3.
A. Han, M. M. Elkind. Enhanced transformation of mouse 10T½ cells by 12-O-tetradecanoylphorbol13-acetate following exposure to X-rays or to fission-spectrum neutrons. Cancer Res. 42, 477-483
(1982).
29
Bystander Effect of Radiation on Oncogenic Transformation
Satin Sawant, Gerhard Randers-Perhson, Charles Geard, and Eric Hall
Purpose
Several studies have shown evidence for the bystander effect of radiation using
endpoints such as the accumulation of p53 protein, frequencies of sister chromatid
exchange, and micronuclei formation. Utilizing the charged-particle microbeam as a means
of localized energy delivery system, this study investigates the bystander effect of radiation
on oncogenic transformation of mouse C3H10T½ cells.
Materials and Methods
Mouse C3H10T½ fibroblast cells were grown in complete Eagle's basal medium.
For the microbeam exposure, 1000-1200 exponentially growing cells were plated into
specially prepared microbeam dishes coated with Cell-tak, an adhesive protein. Alpha
particles accelerated by a 4-MV Van de Graaff accelerator to energy of 5.3 MeV (stopping
power 90 keV/µm) were used for the microbeam irradiations. For the broad-beam
exposures, exponentially growing C3H10T½ cells were plated at a density of 200,000 cells
per dish onto thin-bottomed (6-µm Mylar) 35-mm-diameter stainless-steel dishes. To
ensure compatibility with the microbeam experiments, same-energy α particles (5.3 MeV)
were used for the broad-beam experiments. Immediately after irradiations, the cells were
trypsinized from the irradiation containers and replated into 100-mm-diameter cell-culture
dishes. Cells were incubated for 7 weeks with fresh culture medium changes every 2
weeks, before being fixed and stained to identify morphologically transformed types II and
III foci. In parallel studies, dishes were plated with about 30 viable clonogens that had been
subject to exactly the same conditions and incubated for 2 weeks. The resultant colonies
were then stained to determine plating efficiencies and surviving fractions of control and
irradiated cells.
Results
The transformation incidence per surviving cell for a) experiments where all cells
were irradiated with eight α particles through the nucleus is 0.99 × 10-4, and b) for parallel
experiments where none of the cells were exposed to radiation is 31.06 × 10-4 (Table 1).
This brings the expected transformation incidence per surviving cell in a population where
only 10% of the total cells are exposed to eight α-particles to 3.99 × 10-4. However, the
observed transformation frequency of 15.15 × 10-4 is an order of magnitude higher than the
expected incidence of transformation per surviving cell. In sharp contrast to the above
finding, mixing experiments performed using irradiated and unirradiated cells, by
microbeam as well as broad-beam exposure, failed to show transformation frequency
greater than the expected (data not shown).
Conclusions
Targeting α particles, using a microbeam, to 10% cells within a population
produced more transformed foci than expected. This study provides direct evidence for the
30
presence of bystander effect of radiation. In addition, the failure to obtain higher
transformation frequency in mixing experiments indicated that the cells in close proximity
to the irradiated cells are influenced only.
Table 1. Clonogenic survival rates, numbers of viable cells exposed in transformation
studies, number of transformed clones produced, and transformation frequencies for
microbeam irradiations
Percent of
cells
irradiated
Exact no. of
α particles
Clonogenic
surviving
fraction (PE)
Number of viable
cells exposed × 104
Number of
transformants
produced
TF/104 surviving
cells
0
0
(0.46)
1.01
1
0.99
100
8
0.19
0.32
10
31.06
10
8
0.79
1.85
28
15.15
31
Role of Oxyradicals in DNA Damage Induced by Cytoplasmic
Irradiation in Mammalian Cells
An Xu, Gerhard Randers-Pehrson, and Tom K. Hei
Radon and its alpha-emitting progeny, which are ubiquitous in indoor
environments, have been established as a human carcinogen (1). The EPA has estimated
that approximate 20,000 cases of bronchogenic carcinoma occurring in U. S. each year
may be related to them (2). To develop a better risk assessment of residential radon
exposure, it is essential to understand the genotoxic effects of low dose exposure. We
have previously shown in experiments with radical scavenger and inhibitor of
intracellular glutathione that the mutagenicity of cytoplasmic irradiation depends on the
generation of reactive oxygen species (ROS) (3). However, the exact mechanistic role for
ROS in the process of mutations and carcinogenesis has not been elucidated. DNA
damage induced by ROS is important in mutagenesis and carciongenesis (4). The
modified base 8-OHdG, one of the most abundant oxidized DNA bases, is considered as
the most sensitive biomarker of DNA damage due to ROS, especially hydroxyl radical,
attacking at C8 of guanine (5). Competent cells usually successfully repair such damage;
but if unrepaired, the presence of 8-OHdG in DNA templates may cause the miscoded
incorporation of nucleotides in the replicated strand, which may contribute to the
development of mutations. Here, we report the results of studies undertaken to investigate
the induction of 8-OHdG by cytoplasmic irradiation in mammalian cells.
Approximately 200 AL cells were seeded overnight into specially constructed
microbeam dishes in medium containing 1 mM dcAMP to enhance cell spreading. For
cytoplasmic irradiation, dual fluorochrome dyes, Hoeschst 33342 and Nile Red, were
used to stain the nucleus and cytoplasm, respectively, as described previously (3). After
being stained for 30 min, cells were irradiated either in the presence or in the absence of
8% DMSO, and then were added to the medium 10 min before and 3 min after
irradiation. The dose of DMSO used was nontoxic and nonmutagenic under the
conditions used in this study .The irradiated cells were fixed with 5% acid-alcohol at –
20oC. Induction of 8-OHdG in AL cells was quantified by using the monoclonal antibody
1F7, which is specific for 8-OHdGs coupled with immunoperoxidase staining and an
image analysis software as described (6). Briefly, irradiated cells were treated with
normal goat serum in Tris buffer to block nonspecific binding sites, and incubated with
primary antibody 1F7 overnight. Mouse ABC reagent (Burlingame, CA) was added and
the reaction was terminated after 20 min. A Cell Analysis System 200 microscope
(Becton Dickinson, San Jose, CA) was used to measure the relative intensity of nuclear
staining in cells using the Cell Measurement Program software package.
Although a faint background staining was evident in the control cells, there was a
dose-dependent induction of 8-OHdG in AL cells irradiated through cytoplasm (Figure 1).
A two-fold increase in the relative staining intensity of 8-OHdG in AL cells irradiated
with 8 alpha particles was detected. However, there was no more increase in 8-OHdG
32
staining with further increase in the number of particle traversals. DMSO is a wellestablished hydroxyl radical scavenger in mammalian cells. We demonstrated that
concurrent addition of DMSO suppressed the formation of 8-OHdG in irradiated cells
(Figure 2). The level of 8-OHdG induced by 8 alpha particles was reduced to close to
background in the presence of DMSO (p<0.05). These data were consistent with our
previous findings on mutation induction by cytoplasmic irradiation with or without
DMSO. Our results highlight the involvement of ROS, particularly the hydroxyl radical,
in mediating the mutagenicity of cytoplasmic irradiation.
300
Relative staining intensity of 8-OHdG
Relative staining intensity of 8-OHdG
References
1. IARC. Radon. 43: 173, 1988.
2. Environment Protection Agency, 1992.
3. Wu et al., Proc. Natl. Acad. Sci. USA, 96: 4959, 1999.
4. Cerutti et al. Science, 227: 375, 1985.
5. Kasai et al. Carcinogenesis, 7: 1849, 1989.
6. Yarborough et al., Cancer Research 56: 683, 1996.
AL cells
250
200
150
100
50
0 2 4 6 8 10 12 14 16 18
300
250
1. Control
2. Control + 8% DMSO
3. 8α
4. 8α + 8% DMSO
200
150
100
50
1
Number of alpha particles
2
3
4
Figure 2 Effect of DMSO on the induction of 8-OHdG
in AL cells irradiated by 8α with or without 8% DMSO.
Data were pooled from three independent experiments.
Figure 1 Relative staining intensity of 8-OHdG
in irradiated AL cells. Data were pooled from
three independent experiments. Bar: ±SEM
Bar: ±SEM
SESEMSEM
33
CELLULAR STUDIES
RBE and Microdosimetry of Low-Energy X Rays
Stephen Marino, Dusan Srdoc, Satin Sawant, Charles Geard, and David Brenner,
in collaboration with Zugen Fu (SUNY/Stony Brook)
There is a good deal of evidence, both experimental and theoretical, that X rays
below about 50 keV are more biologically effective per unit dose than higher-energy
gamma rays. From a public-health perspective, screening mammograms are typically
given with low doses of 20-25 kVp X rays. Given the increasing emphasis on
mammographic screening for breast cancer, it is of societal importance to provide realistic
risk estimates for breast cancer induction from mammographic X rays, in keeping with a
recent ACS recommendation that "the stated 'risks' from mammography should be further
quantified."
In order to measure the effects of low-energy X rays relative to gamma rays, two
rodent cell lines are being irradiated in the energy range 5-25 keV with monoenergetic X
rays produced by the National Synchrotron Light Source (NSLS) of Brookhaven National
Laboratory (BNL). C3H10T1/2 mouse cells are being observed for oncogenic
transformation, the in vitro analog of carcinogenesis, and Chinese hamster cells are scored
for chromosomal changes.
The NSLS X-ray facility is a 2.5 to 2.8-GeV electron synchrotron with a storage ring
that produces synchrotron radiation by bending the electron beam. We used beam line
X23A2 to make initial microdosimetry measurements but are now using beam line X23A2,
which has a sophisticated calibration system for the position of the crystal used to define
the X-ray energy. In addition, it is essentially free of harmonics (3x, 5x main energy) and
permits use of the entire energy range of interest without lengthy down time to change
crystals, as was the case with the X23A2 beam line. The crystal position is calibrated by
measuring the absorption edge of a nearby element. Since there is a small error (50 to 100
eV) in energy as the crystal is moved away from the calibration energy, we will only
operate at available calibration points. Energies in the range of interest would be 5.465
keV (vanadium K edge), 9.659 keV (zinc K edge), 15.200 keV (lead LII edge) and 20.000
keV (molybdenum K edge). Both beam lines are operated by the National Institute of
Standards and Technology (NIST).
The irradiation and dosimetry fixtures used for this experiment are very similar to
those used for charged-particle irradiations using the RARAF track segment facility (1)
and at the Tandem Van de Graaff facility at BNL (1989-1993). All dosimetry and
irradiations are performed under computer control using a personal computer. A custom
program written in Quick Basic and used for the charged-particle experiments at BNL and
RARAF has been adapted for use here.
Special cell dishes (Fig. 1) have been constructed of Kynar (C2F2), which has a massenergy attenuation coefficient more closely matched to that of the cells than does
polystyrene, normally used as the call growth substrate. The Kynar surface of the dish on
which the cells are plated is 18 mm in diameter and less than 80 µm thick in order to
35
minimize the attenuation of the X rays, especially at lower energies. Cells do not attach
well to the Kynar, so a thin layer of CelTak is spread on the surface, as in the microbeam
experiments. Because the X-ray beam is horizontal, the cell dishes are vertical and the side
of each dish opposite the Kynar surface is sealed with a layer of Mylar 6 µm thick held in
place with a metal ring. The dishes are filled with medium through one of the ports on the
edge of the dish in order to prevent the cells from drying out during the irradiation.
Up to 20 cell dishes can be placed on the irradiation wheel (Fig. 2). A stepping
motor with a right-angle 20:1 gear reducer rotates the dishes through the X-ray beam,
which is collimated to a rectangular area 2 mm high by 20 mm wide. The rotator assembly
is positioned on a table in the radiation safety hutch at the end of the beam line so that the
center of rotation of the rotator system is at the same height as the beam. We have initially
used a motor controller that produces 400 steps/motor revolution (400 steps/dish), but
because the X-ray beam is so narrow (<14 motor steps), a microstepping motor controller
has been purchased to increase the stepping resolution to 2000 steps/revolution. The
stepping rate is determined by the dose to be delivered and the dose rate, as monitored by
an ionization chamber through which the X-ray beam passes before reaching the cells. The
gas used in this ionization chamber (He, N, Ar) is selected to minimize the attenuation of
the X rays at a particular energy while still absorbing enough energy to produce an
acceptable signal. Because the X rays are generated by a storage electron ring, the dose
rate doesn’t fluctuate, but declines slowly with time as the number of electrons in the ring
declines. The storage ring is filled with beam twice a day.
The dose rate is measured using an ionization chamber made of Kynar with a 2.4 mm
gap between electrodes, equivalent to approximately 2.4 µm at unit density. The thickness
of the front wall of the chamber is matched to that of the cell dishes. The diameter of the
collector is 16 mm and the chamber is filled with a special gas mixture that was designed
by Carl Elliston of our laboratory. The composition of this gas (40.9% CH4, 38.2% Ne,
10.9% C3H8, 7.1% CO2, 2.6% N, 0.3% Ar by partial pressure) matches the response of cell
nuclei to X rays within 0.5% over the energy range 4 keV to 40 MeV. During the several
irradiations of cell wheels with 15.2 keV X rays that have been performed so far, the ratio
of the dose rate measured by the ionization chamber relative to the monitor chamber has
changed by no more than 1%, each wheel full of dishes taking approximately 40 minutes.
In conjunction with the cell irradiations, microdosimetric spectra of the X-ray beams
are being measured. Preliminary spectra have been obtained using a 1/8” diameter right
circular cylindrical wall-less chamber in a large tank with a thin Mylar entrance window.
This system was used to measure microdosimetric spectra of charged particles at the
Tandem Van de Graaff at BNL. The helix and center wire are stainless steel. The tank
was filled with propane-based muscle TE gas mixture at a pressure such that the chamber
simulated a 1-µm tissue diameter. The spectrum obtained for 20 keV X rays is presented
in Figure 3. The steel center wire contributes low-energy characteristic X-rays (~ 5 keV)
when struck by higher energy X rays, distorting the spectrum at low values of y (2). To
avoid this problem, a new wall-less detector is being designed that is completely nonmetallic. The center wire will be a carbon fiber 12.7 µm in diameter and the collecting
volume will be defined by a double helix made of A150 TE plastic. There is a significant
36
restriction in space horizontally because beam line X23A3 is within a few inches of the Xray beam in the X23A2 hutch, so the large tank used for the first measurements cannot be
centered on the beam. The new counter will be placed in a TE plastic cylinder 2.54 cm in
diameter so that it can be centered in the X-ray beam.
Figure 1. Cell dishes for low-energy X rays with (right) and without Mylar seal.
37
Figure 2. Cell irradiation wheel with cell dishes in positions 1 and 2.
38
20 keV monoenergetic X rays
0.8
yD(y)
0.6
0.4
0.2
0.0
0.1
1
10
100
y, keV/µm
Fig. 3. Microdosimetric spectrum for 20.0 keV X rays for a simulated diameter of
1 µm obtained using a wall-less counter.
References
1. Colvett, R.D. and Rohrig, N. Charged-particle beams for radiobiology at RARAF. In Annual Report on
Research Project, ERDA Report COO-3243-5, pp. 38-54, National Technical Information Center,
Springfield, VA, 1976.
2. Kliauga, P. and Dvorak, R. Microdosimetric measurements of ionization by monoenergetic photons.
Radiation Research 73:1-20, 1978.
39
Establishment of an Alpha-Particle-Induced
Estrogen-Dependent Breast Cancer Model
Gloria Calaf and Tom Hei
It is well accepted that cancer arises in a multi-step fashion where exposure to
environmental carcinogens is a major etiological factor. The aim of this work was to
establish an experimental breast cancer model in order to understand the mechanism of
neoplastic transformation induced by high-LET radiation in the presence of 17 β estradiol
(E).
Exponentially growing immortalized human breast epithelial MCF-10F cells were
plated 3 days before irradiation at a density of 3x105 cells in 60 mm dishes made of a
specially constructed stainless steel ring with a 6-µm mylar bottom. Cells were irradiated
with graded doses of α particles (150 keVµm) accelerated in the 4-MeV van de Graaff
accelerator at the Columbia University Radiological Research Facility and subsequently
cultured in the presence of 17 β estradiol (E) for periods up to 10 months post irradiation.
MCF-l0F cells irradiated with double doses of 60 cGy α particles in the presence
of (E) showed gradual phenotypic changes including altered morphology, an increase in
cell proliferation relative to the control, telomerase activity, saturation density, a
decreased response to growth factors, anchorage-independent growth, chromosomal
aberrations, and invasive capabilities before becoming tumorigenic in nude mice (Figure
1). In α−particle-irradiated cells cultured in the presence of E, increased BRCA1, BRCA2
and RAD51 expression were detected by immunofluorescence staining and quantified by
confocal microscopy (Figure 3).
Figure 2 shows the invasive characteristics of control and MCF-10F cells after the
various treatments scored at either 12 h or 20 h after plating onto the matrigel basement
membrane. The number of cells that migrated through the membrane was clearly a
function of time in addition to E treatment. Neither the normal breast epithelial cells nor
the immortalized MCF-10F cells showed any significant invasive capability. Addition of
E to the growth medium significantly enhanced the invasive phenotype of MCF-10F cells
in every treatment group examined. It should be noted that the BP1Tras clone, derived
from MCF-10F cells transformed with benzopyrene and then transfected with the c-Haras oncogene, demonstrated the highest invasive behavior and was consistent with its
tumorigenic phenotype.
The expression of several oncoproteins frequently associated with breast cancer
was determined among the various immortalized and transformed MCF-10F cells with or
without estrogen treatment. Quantification of the immunofluorescent imaging of stained
cells showed a significant increase in BRCA1 and BRCA2 in MCF-10F cells irradiated
with α particles and treated with E compared to control cultures (Figure 3, Top). The
staining intensity for BRCA1 among individual cells was fairly uniform and showed a
40
gradual increase in expression between the control MCF-10F cells and their tumorigenic
counterpart (60E/60E). The difference in staining intensity for the tumor suppresor
proteins between the two groups was highly significant (P< 0.05). Irradiated cells that
were transformed but non-tumorigenic (e.g., 60, 60/60 cGy) showed an intermediate
staining intensity. The tumorigenic cell line (60E/60E) showed more intense staining.
Furthermore, addition of E enhanced the expression level of all of the oncoproteins
examined. In general, expression of BRCA2 paralleled that of BRCA1, except that the
expression level was much higher. It should be noted that the expression of BRCA2 in
MCF-7 cells was the highest among the cell lines examined.
Expression of RAD51, which had frequently been shown to be associated with
BRCA1 and BRCA2 was determined in control and transformed MCF-10F cells with or
without E (Figure 3, Bottom). Quantification of the immunofluorescent imaging of
stained cells showed a significant increase in RAD51 in MCF-10F cells irradiated with a
double dose of 60 cGy α particles and concurrently treated with E (60E/60E and 60E/60)
in comparison to control cultures. Similarly, the tumorigenic cell lines BP1Tras and
MCF-7 showed high RAD51 protein expression. Similar to our findings with BRCA1,
addition of E significantly enhanced the expression of RAD51 in all irradiated groups.
These studies showed that high-LET radiation, such as that emitted by radon
progenies, and in the presence of estrogen, induced a cascade of events indicative of cell
transformation and tumorigenicity in human breast epithelial cells.
MCF-10F: Spontaneously immortalized cell line human breast
epithelial cells: Effects of high-LET radiation and estrogen
6OE/6OE α-particle-treated cells.
Figure 1. Schematic diagram illustrating the gradual
morphological alterations in irradiated MCF-10F cells.
Figure 2. Invasive characteristics of control and MCF-10F-treated cells after the
various treatments were scored either 12 or 20 h after plating onto the matrigel
basement membrane.
Figure 3. Relative amounts of BRCA1 and BRCA2 (top) and RAD51 (bottom)
protein expressed by MCF-10F-treated cells. It was determined by
immunofluorescent staining, visualized by using confocal microscopy, and
quantified by a computer program which gives the area and intensity of the
staining.
41
Genotoxicity versus Carcinogenicity: Implications from Fiber
Toxicity Studies
Tom Hei, An Xu, Darren Louie, and Yong-liang Zhao
Although the association between exposure to asbestos fibers and the
development of lung cancer and mesothelioma has been well established in man, the
carcinogenic potential of other natural and man-made fibers/particles are not clear.
Various in vitro genotoxicity studies have been employed to assess their in vivo
carcinogenic potential. A variety of highly quantitative assays ranging from DNA strand
breaks to neoplastic transformation in rodent cells have been used successfully to
compare and contrast the genotoxic potential of various fiber types. These systems vary
in complexity and degree of relevance to the human target-tissues of interest.
Nevertheless, in vitro genotoxic studies are useful in identifying physiochemical
properties of fibers/particles that are likely to affect their in vivo carcinogenic behavior.
Asbestos as a gene and chromosomal mutagen
Earlier attempts at defining the mutagenic potential of asbestos fibers at either the
hprt or oua loci in a variety of mammalian cells have yielded largely negative results (1).
The negative gene mutation data suggest either that asbestos is a non-genotoxic
carcinogen or that mutants induced at these loci are non-viable. Given the strong
evidence that fibers induce chromosomal alterations in mammalian cells, it is likely that
asbestos induces mostly large multilocus deletions that are non-compatible with survival
of the mutants. Using the human-hamster hybrid (AL) cells in which mutations were
scored at a marker gene (CD59) located on human chromosome 11 (11p13) that the AL
cell carries as its only human chromosome, Hei et al showed previously that both
crocidolite and chrysotile fibers were indeed mutagenic and induced mostly deletions
involving millions of basepairs (2,3). In recent years, several other mutagenic assays that
are proficient in detecting either large deletions, homologous recombinations or score
mutants located on non-essential genes have been used successfully to demonstrate the
mutagenic potential of various fiber types (1, for review).
Role of fiber-cell interaction in mediating asbestos genotoxicity
The correlation between fiber dimension and carcinogenic potency suggests the
importance of fiber-cell interactions. The ability of cells to phagocytose asbestos fibers
both in vitro and in vivo has been well documented (4). Fibers less than 5 µ in length are
usually completely phagocytosed whereas those greater than 25 µ are generally not. This
inability to completely engulf long fibers has been termed “frustrated phagocytosis”
which has been associated with increased membrane permeability and increased
oxyradical production. Figure 1 shows the effect of a diminished phagocytic ability on
chrysotile-induced mutagenicity in AL cells. Cytochalasin B at a dose of 1 µg/ml, while
being minimally cytotoxic (surviving fraction ~0.82) and largely non-mutagenic,
reduced to 1/3 the percentage of AL cells containing phagocytosed fibers in cells treated
with a 2 µg/cm2 dose of UICC chrysotile fibers as well as the number of internalized
42
fibers per phagocytic cell (data not shown). Concurrent treatment of fiber-exposed cells
with cytochalasin B significantly reduced fiber-induced CD59− mutant yield. In cells
exposed to a 2 µg/cm2 dose of fiber, concurrent treatment with cytochalasin B reduced
the induced mutant yield to a level similar to that of cytochalasin B treatment alone
(Figure 1). There is evidence that oxyradicals play an essential role in fiber toxicology
(5). Although iron has been shown to be an important source of reactive oxygen species
with the iron-rich crocidolite fibers, not all iron containing minerals, for example iron
oxide, are toxic. Furthermore, the observation that tremolites and erionites which contain
little or no iron are mutagenic in the AL cells suggest that fiber-cell interaction may be an
important pre-requisite in fiber mutagenesis (6).
Figure 1. Induced CD59− mutants in AL cells treated with UICC chrysotiles with
or without concurrent cytochalasin B, which inhibits cellular phagocytosis. Data
are pooled from 3 to 5 experiments. Bars, + SD.
Neoplastic transformation as a genotoxic endpoint
Morphological transformation assays based on rodent cell systems such as C3H
10T1/2, NIH 3T3, and Syrian hamster embryo cells occupy a useful intermediate position
between the bacterial mutagenesis assays, which are quick and inexpensive, and animal
studies, which are cumbersome and inordinately expensive. These assays afford an
opportunity to evaluate both qualitative and quantitative aspects of fiber/particle-induced
oncogenic transformation as well as mechanisms involved in the neoplastic process.
Upon treatment with mineral fibers, transformed cells which loss contact inhibition of
growth form multi-layered growth and criss-crossing cells at the peripheral over a
contact-inhibition background of non-transformed cells. The morphology of the foci can
be correlated with neoplastic potential with type III foci being the most tumorigenic when
injected into syngeneic animals. While asbestos has not been shown to be oncogenic
transforming in C3H 10T1/2 cells (7), it has largely been found to be active in Syrian
hamster embryo cells. In general, morphological transforming potential of mineral
fibers/ particles depends on fiber dimension, treatment time, cell model systems, and that
glass fibers that are long and thin tend to be neoplastic transforming as well in the Syrian
hamster embryo system (8). However, transformed SHE cells are largely nonimmortalized and call into question their relevance to the neoplastic process in human
cells where cellular immortalization is a pre-requisite requirement for their tumorigenic
conversion.
Transformation studies with human epithelial cells
One of the main difficulties in studying mechanisms of asbestos carcinogenesis is
the lack of a suitable human-cell model system whereby the various tumorigenic stages
can be dissected and the molecular changes associated with each stage examined. Up to
the present moment, no primary human-cell model is available for this area of studies
because the frequency for human cell transformation has been estimated to be in the
range of 10-15, an incidence too low to be reproduced in any laboratory setting (9).
43
Treatment of normal human mesothelial cells with amosite asbestos has been shown to
extend the proliferative lifespan of 4 out of 16 independently derived primary cultures
(10). However, these cells eventually all senescence and enter crisis. Using a human
papillomavirus immortalized human bronchial epithelial (BEP2D) cells, Hei et al.
showed recently that a single, 7-day treatment with a 4-µg/cm2 dose of chrysotile induced
neoplastic transformation of these cells in a step-wise fashion at a frequency of ~10-7, as
shown in Figure 2. The immortalization step, therefore, increases the transformation
yield of primary human epithelial cells by more than a million fold. Tumorigenic
BEP2D cells show no mutation in any of the ras oncogenes (11). Results of cell fusion
studies between asbestos-induced tumorigenic and parental BEP2D cells demonstrated
that the tumorigenic phenotypic induced by chrysotile treatment could be completely
suppressed by fusion with non-tumorigenic control cells. These data indicate that nontumorigenic BEP2D cells complement the loss of putative suppressor element among
tumorigenic cells, and suggest that loss of suppressor gene(s) is an important mechanism
of fiber carcinogenesis.
Figure 2. Schematic diagram illustrating the multistep process in the neoplastic
transformation of immortalized human bronchial epithelial cells by chrysotile
fibers.
Genotoxicity data on refractory ceramic fiber
Refractory ceramic fiber is a class of man-made vitreous fibers first produced in the
mid 1950s and used primarily as high-temperature insulation in industry settings. RCF
fibers can be classified into three categories: pure RCF that is a blend of alumina and
silica (RCF-3), kaolin-based (RCF-1), or blends of alumina and silica containing other
metal oxides. Inhalation studies with RCF-1 fibers have been carried out in both rats and
hamsters. Treatment of animals at the maximum tolerable dose of 30 mg/m3 for 6 hr/day
for 24 months resulted in incidence of lung tumors in rats ranging from 3.5 to 13%
whereas no lung tumor was detected in similarly treated hamsters (12). Several in vitro
genotoxic endpoints have been used to examine the DNA damaging potential of RCF-1
fibers in mammalian cells. In general, RCF-1 fibers have been shown to be less
biologically reactive when compared to either crocidolite or chrysotile. While RCF-1
fibers are significantly longer than chrysotiles, they are much larger in diameter as well
(6). As a result, both the surface area and the number of fibers per unit weight of
samples are smaller than either chrysotiles or erionites. These data highlight the
importance of fiber numbers in determining in vitro genotoxicity of particles/fibers.
Summary
In summary, in vitro genotoxic data are useful in identifying physiochemical
properties of fibers/particles that affect their in vivo carcinogenicity. Genotoxicity,
however, does not necessarily equate carcinogenicity in all cases. Carcinogenicity of
44
fibers/particles is a complex interplay of many factors, including dose, fiber
characteristics, fiber-cell interaction, cell and tissue responses to foreign particles, and,
finally, inflammation and progressive neoplastic changes.
References
1.
See Jaurand, IARC 1996 for review.
2.
Hei et al., Cancer Res. 52:6305, 1992.
3.
Xu et al., Cancer Res. 59:5789, 1999.
4.
Miller et al., Environ. Res. 15:139, 1978.
5.
See Kamp et al., Free Rad. Biol. & Med. 12:293, 1992 for review.
6.
Okayasu et al., Brit. J. Cancer 79:1319, 1999.
7.
Hei et al., Brit. J. Cancer 50:717, 1984.
8.
Hesterberg et al., Cancer Res. 46:5795, 1986
9.
Hei et al., Adv. Space Res. 18:137,1996.
10.
Xu et al., Carcinogenesis 20:773,1999.
11.
Hei et al., Environ. Hlth. Persp. 105:1085, 1997.
12.
See Ellouk and Jaurand, Environ. Hlth. Persp. 102: 47, 1994 for review.
45
Induction of Reactive Oxygen Species by Crocidolite Asbestos
in Mammalian Cells
An Xu and Tom Hei
Crocidolite fibers are known to cause cellular damage, leading to asbestosis,
bronchogenic carcinoma, and mesothelioma in humans (1). However, the mechanism(s)
responsible for the toxic and carcinogenic effects of asbestos is not yet clear. In vitro
studies with asbestos and concurrent exposure to radical scavenging enzymes such as
superoxide dismutase (SOD), catalase have indicated a close relationship between the
induction of reactive oxygen species (ROS) by fibers and asbestos-mediated toxicity (24). Previous studies from this laboratory have shown that there is a dose dependent
increase in the formation of 8-OHdG, which is one of the most specific forms of DNA
damage induced by ROS, in AL cells treated with crocidolite fibers (5). The objective of
the present study was to quantify the induction of ROS in asbestos-treated AL cells.
ROS generation in AL cells was detected with 5-(and 6-)-chloromethyl-2’7’dichlorodihydrofluorescein diacetate (CM-H2DCFA), which produced a green
fluorescence when oxidized (6). Cells preincubated with a 1 µM dose of CM-H2DCFA
for 40 min at 37oC were exposed to fibers either in the presence or absence of 0.5%
DMSO. ROS induction was quantified by using an ACAS570 Interactive Laser
Cytometer which was based on an acousto-optically modulated Ar-ion laser turned to 488
nm to excite the fluorescence in the cells. To detect the release of H2O2 from asbestostreated AL cells, Amplex Red and Horseradish peroxidase (HRP) (Molecular Probes Inc.,
Eugene, OR) reaction mixture with fibers with or without concurrent catalase (1000U/ml
final) in 96-microplate were prewarmed for 15 min (7). Exponentially growing cells were
trypsinized and washed with Krebs Ringer phosphate glucose (KRPG) buffer twice. 2.5 x
106/ml cells were added to the microplate and incubated for 4 hr. The fluorescence of
each well was measured by a fluorescence microplate reader using excitation in the range
of 530~560nm. In order to generate a H2O2 standard curve, different dilutions of H2O2 in
KRPG buffer were added to the same volume of reaction mixture in microplates and
fluorescence was determined after 15min when the reactions were stabilized.
Figure 1 shows the dose effect of crocidolite fibers on the induction of ROS in AL
cells. The relative fluorescence intensity in AL cells treated with a 6 µg/cm2 dose of fibers
was more than five-fold that of the control. However, there was no further increase in the
intensity of fluorescence with 9 µg/cm2 fibers. Figure 2 shows 0.5% DMSO dramatically
suppressed the induction of ROS in AL cells treated with a dose of 6 µg/cm2 fibers
(p<0.005), which was consistent with our previous study of the effect of DMSO on the
formation of 8-OHdG. Crocidolite fibers induced a dose-dependent increase in the
release of H2O2 from AL cells. But the concentration of H2O2 was not increased at the
dose of 9 µg/cm2 (final concentration). The presence of catalase in the reaction mixture
significantly reduced the release of H2O2 from the treated cells (p<0.005). These data
46
provide further corroborating evidence that the mutagenic effect of crocidolite fibers on
mammalian cells is mediated through the induction of reactive oxygen species.
References
1. Rom et al,. Am. Rev. Respir. Dis. 143: 408, 1991.
2. Hei et al., Carciongenesis, 16: 1573, 1995.
3. Goodglick et al, Cancer Res. 46: 5558, 1986.
4. Mossman et al., Lab. Investig. 54: 204,1986.
5. Xu et al., Cancer Res. 59: 5922, 1999.
6. Long et el., Environ. Health Perspect. 105: 706, 1997.
7. Monanty et al., J. Immunol. Methods 202: 133, 1997.
47
60
70
A L cells
Relative fluorescence
Relative fluorescence
70
50
40
30
20
10
0
0
2
4
6
8
10
50
Control
Control + 0.5% D M S O
6µg/cm 2 fibers
6µg/cm 2 fibers
+ 0.5% DM S O
40
30
20
10
0
1
2
Concentration of fibers (µg/cm )
Figure 1 Relative fluorescenc intensity for the induction
of ROS in A L cells treated with graded doses of fibers.
Data were pooled from three independent experiments.
60
1.
2.
3.
4.
2
3
4
Figure 2 Relative fluorescence intensity for the induction
of ROS in A L cells treated with fibers with or without concurrent
treatment of DMSO. Data were pooled from three independent
experiments.
0.5
1.
2.
3.
4.
5.
6.
H2O2 (µM)
0.4
0.3
Con
2 µg/cm 2
6 µg/cm 2
9 µg/cm 2
Con + Catalase
6 µg/cm 2 + Catalase
0.2
0.1
0.0
1
2
3
4
5
Figure 3 Release of H 2 O 2 in A L cells treated with graded dose of fibers
with or without catalase at a dose of 1000U/ml.Data were pooled from three
independent experiments.
48
6
Focal Adhesion Motility Revealed In Stationary Fibroblasts
Lubomir B. Smilenov, with Alexei Mikhailov (Massachusetts General Hospital, Department
of Molecular Biology), and Robert J. Pelham, Jr., Eugene E. Marcantonio and Gregg G.
Gundersen (Departments of Pathology and Anatomy and Cell Biology, Columbia University)
Adhesive contacts between cells and the substratum are critical for spreading and
migration of many cells and are mediated by integrin receptors (1). In fibroblasts, these
integrins concentrate in specific regions within the plasma membrane, called focal
adhesions (FA), as where actin stress fibers and associated proteins are anchored (2).
During fibroblast migration, FAs form at the leading edge of the cell, remain fixed as the
cell migrates over them and then detach at the rear (3). The mechanisms that regulate
polarized FA formation and detachment in migrating cells are largely unknown.
To study FAs in living cells, a chimera of GFP, the transmembrane and cytoplasmic
domains of the β1 integrin subunit, and the signal sequence from the α1 integrin subunit is
generated, such that the GFP would be extracellular. Stable cell lines with low levels of
GFP-integrin expression were selected in order to limit the effect of the chimera on integrin
function (4-6). The GFP-integrin labeled all FAs as shown by co-localization with
endogenous integrins and the FA marker vinculin. The GFP-integrin cell lines were similar
to the parental cell line in morphology, adhesion to fibronectin, growth, and spreading on
fibronectin, demonstrating that the chimera had no detectable effect on the cells’ adhesive
properties (data not shown).
In stationary cells, FAs labeled with GFP- integrin showed a surprising amount of
movement (Fig.1). These movements: 1) were linear, 2) usually occurred without change in
FA area or shape, 3) occurred relative to the substratum and cell edge, and 4) involved
distances of greater than one FA length. The motile FAs were distributed throughout the
cell; most moved toward the cell center, but some moved along the cell edge. New FAs
formed near the cell edge as others moved inward. FAs infrequently split in two or
elongated during movement. The rate of movement was 0.12 + 0.08 µm/min (N=128
FAs; 9 cells), similar to the rate of 3T3 cell migration. We defined a motile FA as one that
moved at least one plaque length within one hour. By this criterion, 65 + 27 % (N=692
FAs; 10 cells) of the FAs in individual cells were motile. Variability in FA motility may
reflect differences in the cell cycle, metabolic activity or local substratum conditions
Similar FA movements occurred in stationary cells within a monolayer, at the edge of a
wounded monolayer, and in well-spread cells in sparse cultures, suggesting that FA
movement is independent of cell density and cell-cell interactions.
In contrast, little movement of GFP-integrin labeled FAs was observed in migrating
cells stimulated to migrate by wounding a monolayer or after cell division. Interference
reflection microscopy (IRM) can estimate the distance between the ventral cell surface and
the substratum. Portions of the cell within 15 nm of the substratum, as in FAs, appear as
dark contrast against a gray background in the zero-order IRM image. The patterns of GFP
49
Fig. 1. Moving FAs in GFP-integrin cells and parental cells remain in close contact with the substratum. (A
and B). Stationary GFP-integrin cell imaged with fluorescence and IRM microscopy. (C). Stationary
parental cells imaged with IRM alone. (A). Fluorescence images from a timelapse recording (in minutes)
showing four moving FAs detectable by GFP fluorescence (blue lines are a fiduciary mark). (B). IRM
images of GFP-integrin cell corresponding to the fluorescence images shown in Panel A. FAs are black. (C):
IRM images from a timelapse recording (in minutes) showing three moving FAs in a parental cell. Bars,
Panels A to C, 2.5 µm.
fluorescence and IRM contrast in stationary cells were closely matched, indicating that
GFP-labeled FAs were closely apposed to the substratum. (Fig.1). When FAs moved, they
maintained close apposition to the substratum (dark contrast by IRM ). However, for some
fast moving FAs, we observed a transient diminution of IRM contrast. Hence, though most
moving FAs remained closely apposed to the substratum, for some rapidly moving FAs the
interaction was reduced. Using IRM, similar FA movements were observed in stationary
parental 3T3 cells.
That FAs exhibit nonmotile and motile states coordinated with cell migration,
suggests the existence of a "molecular clutch" to alternate between these states. The
transition between these states must reflect the balance between tension and adhesion. So,
the molecular clutch may regulate either the affinity of the integrin for the ECM or the
tension applied to the FA by the actin cytoskeleton or both. Regulation of integrin affinity
has been noted previously and there is an optimum affinity at which cells are capable of
migrating. However, neither high substrate concentrations of fibronectin nor addition of
Mn2+ blocked FA motility. While these conditions increase binding in cell adhesion
assays, it is unclear whether they increase integrin affinity under our conditions.
50
Regulation of tension by altering actin contraction is known to be mediated by factors such
as Rho and Ca2+ and our results with nocodazole suggest that changes in tension can alter
the velocity of FA movements. Increasing tension without altering FA affinity for the
ECM, may decrease cell traction, and also contribute to FA movements that are involved in
remodeling the ECM, although ECM remodeling is currently thought to occur on a much
longer time scale. Whatever the composition of the molecular clutch, the existence of
moving FAs shows directly that a cell is able to regulate its interactions with the ECM in a
previously unexpected fashion.
References
1. R.O. Hynes, Cell 69, 11 (1992); D. A. Lauffenburger and A. F. Horwitz, Cell 84, 359 (1996).
2. C.S. Izzard and L. R. Lochner, J. Cell Sci. 21, 129 (1976); K. Burridge, K. Fath, T. Kelly, G. Nuckolls, C.
Turner, Ann. Rev. Cell Biol. 4, 487 (1988).
3. C.M. Regen and A. F. Horwitz, J. Cell Biol. 119, 1347 (1992); S.P. Palecek, C. E. Schmidt, D. A.
Lauffenburger, A. F. Horwitz, J.Cell Sci. 109, 941 (1996).
4. S.E. LaFlamme, L. A. Thomas, S. S. Yamada, K. M. Yamada, J. Cell Biol. 126, 1287 (1994).
5. L.B. Smilenov, R. Briesewitz, E. E. Marcantonio, Mol. Biol. Cell 5, 1215 (1994).
6. NIH 3T3 cells were stably transfected with pLen GFP-β and pSVneo as described in 5.
51
Transformation of Human Bronchial Epithelial Cells
by the Tobacco-Specific N-Nitrosamine, NNK
Hongning Zhou and Tom Hei
It has been recognized for more than four decades that tobacco smoking is causally
associated with several types of human cancer such as lung, oral cavity, and esophageal
cancer. Cigarette smoke is a mixture of about 3,800 chemicals containing at least 40 known
human carcinogens (1). Studies have indicated that 4-methylnitrosamine-1-3-pyridyl-1butanone (NNK) is the most carcinogenic among tobacco-specific nitrosamines with
approximately 80-770ng NNK per cigarette, depending on the type of tobacco (2).
Although previous studies have shown that NNK is carcinogenic in mice, rats and hamsters
(3), little information is available regarding the clastogenic effects of tobacco-specific
nitrosamines in mammalian cells. There is recent evidence that NNK can transform
hamster pancreatic duct cells, human immortalized oral keratinocytes in vitro and
adenovirus 12-SV40 immortalized human bronchial epithelial cells (BEAS-2B) in
xenograft system, but human bronchial epithelial cells have not been neoplastically
transformed in vitro by exposure to the tobacco-specific nitrosamine, NNK (4,5,6).
In the present study, we use the human papillomavirus-immortalized bronchial
epithelial cells (BEP2D) to study the various stages of neoplastic transformation induced
by NNK. Cells are routinely cultured in serum-free LHC-8 medium supplied with
epidermal growth factor and other growth supplements as described (7). Exponentially
growing BEP2D cells were treated with NNK at graded doses of 100 and 400 µg/ml for
either 1 day or 7 days. At passage 15, the cells treated with NNK for 7 days were retreated
with the same doses for 7 days; at passage 20, the cells treated with NNK for 1 day were
retreated with the same doses for 1 day. Medium was changed every three days, and cells
were subcultured weekly. Following treatment, cells were assayed for changes in growth
kinetics, saturation density, resistance to serum-induced terminal differentiation, and
anchorage-independent growth. As shown in Figure 1, the survival fraction of BEP2D cells
treated with graded doses of NNK for either 1 day or 7 days was dose dependent. NNKtreated for 1 day showed not much toxicity to the cells, but NNK-treated for 7 days showed
toxicity; the lethal mean dose was about 1000 µg/ml. At passage 23, NNK-treated cells
acquired resistant to serum-induced terminal differentiation phenotype such that the plating
efficiency in serum-containing medium was much higher than that of control (Table 1). At
passage 30, NNK-treated BEP2D cells acquired anchorage-independent growth ability in
soft agarose (0.24%) as compared to 0.03% for control (Table 1). However, there was no
significant different in growth kinetics between NNK-treated and control cells. The
doubling time was between 28 to 34 hrs. These data suggested that NNK-treated cells have
already acquired the transformed phenotype in that they are resistant to serum-induced
terminal differentiation and anchorage-independent growth in soft agarose. Studies are
currently underway to evaluate the tumorigenic potential of these putative transformed
cells in nude mice and the possible mechanisms surrounding this.
52
Survival Fraction
1
1 day
7 days
0.1
0
200
400
600
800
1000
1200
1400
1600
NNK(µg/ml)
Figure 1 Survival fraction of BEP2D cells treated with
graded doses of NNK for either 1 day or 7 days.
Table 1. Resistance to serum-induced terminal differentiation and anchorage-independent
growth in soft agarose of BEP2D cells and its NNK-treated variants.
Group
BEP2D
NNK100-7
NNK400-7
NNK100-1
NNK400-1
Plating Efficiency
LHC-8
4% FBS LHC-8
0.39
0.42
0.33
0.32
0.58
0.02
0.11
0.11
0.07
0.12
Formation of
8% FBS LHC-8
References
1. Hecht et al, Cancer Res. 54 (supple): 1912s-1917s, 1994.
2. Baker et al, Recent Adv. Tob. Sci. 6: 184-224, 1980.
3. Hoffmann et al, Cancer Res. 45: 935-944, 1985.
4. Klein-Szanto et al, Proc. Natl. Acad. Sci. USA. 89: 6693-6697, 1992.
5. Kim et al, Cancer Res. 53: 4811-4816, 1993.
6. Baskaran et al, Carcinogenesis 15: 2461-2466, 1994.
7. Hei et al, Carciongenesis. 15: 431-437, 1994.
53
0.00
0.08
0.07
0.02
0.08
Colony (%)
0.03
0.18
0.24
0.11
0.19
Protein Expression in Tumorigenic Human Breast Epithelial Cells
Transformed by Alpha Particles
Gloria Calaf and Tom Hei
Breast cancer is a complex disease in which numerous genetic aberrations occur. It
is unclear which of these abnormalities are causative of breast tumorigenesis. However, on
the basis of the currently accepted view of breast cancer as a multi-step process, it is
possible that specific abnormalities may be required in the progression from a normal to
an invasive tumor cell. The knowledge of specific genetic changes is critical to an
understanding of the natural history of breast tumors. These changes may involve specific
genetic loci that contribute directly to one or more attributes of transformation, i.e.,
deregulated proliferation and invasion, while other changes confer genetic instability that
increases the possibility of acquiring subsequent, specific lesions relevant to
tumorigenesis. However, there is much remains to be learned in order to understand the
key factors behind the evolution of breast cancer. It is well accepted that the
transformation of a normal cell to one that is malignant can result from mutations in genes
that encode key growth regulatory proteins. Among them c-myc, c-jun and c-fos, c-Ha-ras,
p53, and many others can lead to aberrant cell growth, hyperproliferation, and eventually
cancer.
Since the identification of genes involved in breast cancer are of critical
importance in understanding the progression of this disease, the aim of this work was to
define whether these oncogenes play a functional role in radiation-induced transformation
of human breast epithelial cells. Identification of factors involved in cell proliferation and
transformation has been facilitated by studies using breast cancer cell lines representative
of different tumor phenotypes. In vitro model systems have been extensively used in the
study of radiation-induced transformation. Since there is little or no information available
on the radiation induced breast cancer, an in vitro breast cancer model utilizing epithelial
cells at different stages of the neoplastic process provide a unique opportunity for studying
radiation carcinogenesis. We have recently developed a model in which the spontaneously
immortalized MCF-10F breast epithelial cells were irradiated with high-LET radiation.
These cells have the morphological characteristics of normal breast epithelial cells and do
not exhibit anchorage independence, invasiveness and tumorigenity in nude or SCID mice.
Exponentially growing cells were irradiated with graded doses of 150 keV/µm 4He ions
accelerated in the 4-MeV van de Graaff accelerator at the Columbia University
Radiological Research Accelerator. These high-energy particles have a LET value
comparable to the α particles emitted by radon-daughter products. MCF-10F cells were
irradiated with either a single or double doses of 30, 60 or 100 cGy 4He ions prepared by
subculturing for 10-15 passages and 12-14 weeks between doses. After irradiation, cells
were subsequently cultured in the presence or absence of 10-8 M estradiol 17-β (E).
Only cells irradiated with one or double doses of α particles, either in the presence
of E before or after irradiation, formed agar-positive clones after 25 passages with a
colony-forming efficiency in agar of 1% and had invasive capabilities. Table 1 shows the
54
list of cells utilized in these studies and their morphological phenotypes in relation to
anchorage-independent growth, invasive capabilities, and tumorigenicity. Only MCF-10F
cells irradiated with the double doses of 60 cGy α particles in the presence of E (60E/60E)
induced tumors in the SCID and nude mice.
Table 1. Characteristics of Radon-Irradiated Human Breast Epithelial Cells. The
anchorage-independent and tumorigenic characteristics of various MCF-10F cells
irradiated with either a single or double dose of radon-simulated a particles. Only cells
irradiated with one or a double dose of 60-cGy a particles formed agar-positive clones
after 25 passages with a colony-forming efficiency in agar of 1%.
Cell Line
MCF-10F
MCF-10F+E
MCF-10F
MCF-10F
MCF-10F+E
MCF-10F
MCF10F
MCF10F+E
MCF-10F+E
MCF-10F
MCF-10F
Dose (cGy)
x No. of
Exposures
0
0
60 x 1
60 x 1
60 x 1
60 x 2
60 x 2
60 x 2
60 x 2
60E/60
60E/60E
Passage
+46
+14
+23
+25
+30
+19
+25
+22
+25
+25
+25
Anchorage
Independence Invasion
+
+
+
+
+
+
+
+
+
+
+
+
Tumorigenicity
+
Alterations in the expression of several oncogenes including c-myc, c-jun, c-fos, cHa-ras and the tumor suppressor gene p53 were observed in α−particle-irradiated cells,
and in those cells subsequently cultured in the presence of E, as detected by
immunofluorescence staining and quantified by confocal microscopy.
An increase in c-myc protein expression was detected in all irradiated population
compared with control MCF-10F cells. Such increase was irrespective of E treatment
(Figure 1). However, there was little or no significant difference in c-myc expression
between cells irradiated with either a single 60-cGy dose or with a double dose of α
particles. Figure 2 represents the quantification of c-Ha-ras protein expressions in αirradiated MCF-10F cells with or without pre-and post-treatment with E. The tumorigenic
breast carcinoma MCF-7 cells and the positive control clone BP1Tras showed a similar
expression level of c-Ha-ras oncoproteins, and at a level roughly 4 times that of the
control MCF-10F cells. In contrast, most irradiated cells without E treatment showed low
expression level of c-Ha-ras and there was no significant difference in expression level
between those receiving a single vs a double dose of α particles. However, cells irradiated
with a double dose of α particles followed by E treatment (60E/60E) had a 3-fold higher cHa-ras expression than the non-irradiated cells with or without E treatment. Figure 3
represents the quantification of the immunofluorescent imaging of c-jun protein
55
expressions in α-irradiated MCF-10F cells with or without pre-and post-treatment with E.
It was evident that cells receiving a double dose of α particles had a significantly higher
expression level of c-jun than those cells irradiated only once. Results showed an increase
in mutant p53 oncoproteins in MCF-10F cells irradiated with a double dose of α particles
either in the presence or absence of E in comparison to control MCF-10F (Figure 4). The
tumorigenic 60E/60E cell line showed an expression level which was significantly greater
than the non tumorigenic cell lines (P< 0.05) and at a level 3 fold-higher than the control
MCF-10F cells. Among the two tumorigenic control lines, MCF-7 cells showed an
expression level which was two times greater than that of the control MCF-10F cells,
whereas the clone BP1Tras had a p53 expression level similar to the 60E/60E cells. The
tumor formation induced in the immunologically depressed animals with cells irradiated
with the double dose of 60-cGy α particles in the presence of E (60E/60E-treated cells)
suggest to us that tumorigenicity may be related to the higher expression of the early
oncogenes c-myc, c-jun, the c-fos, c-Ha-ras oncogene and the tumor supressor p53. Other
oncogenes may likely be involved since other irradiated cells did not form tumors but had
high levels of p53 expression. Overall, our data suggest that over-expression of these
oncogenes are important in the process of cell transformation of human breast epithelial
cells. Furthermore, MCF-10F transformation model induced by an environmental agent, as
radon-simulated α particles as well as an endocrine factor, as estrogens, will allows us to
examine the various aspects in the regulation of gene expression and will provide us the
basis for understanding the process of breast carcinogenesis.
No figures available.
56
Microsatellite Instability in Tumorigenic Human Bronchial Epithelial
56
Cells Induced by α Particles and Fe Ions
Chang-Qing Piao and Tom Hei
Lung cancer is considered to be a disease caused by exposure to environmental
carcinogens. High-LET radiation such as α particles emitted by radon progenies is one of
the important etiological factors. The HPV-18 immortalized human bronchial epithelial
cells (BEP2D) have previously been malignantly transformed by either a single low dose of
30 cGy α particles or 60 cGy of 56Fe ions. In addition, tumor cell lines from irradiated
BEP2D cells have been established from nude mice in our laboratory (1). However, further
investigation is needed for a better understanding of the mechanisms involved in malignant
transformation of BEP2D cells induced by low dose of high-LET radiation. It has been
well known that carcinogenesis is a progressive multistage process. Genomic instability
induced by a single low dose of high-LET radiation may contribute to clonal selection with
accumulating genetic changes and ultimately leading to malignant conversion.
Microsatellite repeats have been shown to be useful markers for genetic instability and are
frequently detected in pathology samples of lung cancer at chromosome 2p, 3p, 3q, 9p,
11p,11q, 13q and 18q (2,3,4,5). In this study, microsatellite alterations at genomic markers
of chromosome 3q and 18q, selected based on their alteration in lung cancer, were analyzed
in a total of 11 tumor cell lines, induced by either a single low dose of 30 cGy α particles
or 60 cGy of 56Fe ions together with control BEP2D cells.
For analysis of microsatelite instability, high-molecular-weight DNA from tumor cell
lines and control BEP2D cells were isolated and subjected to PCR amplification using
primer pairs for the various polymorphic markers (Table 1) obtained from Research
Genetics, Inc. (AL). The Gene Phor System (Pharmacia, NJ) was used for analysis of
microsatellite alterations. Sample consisted of 3 µl of PCR products, 2 µl of denaturing
solution heated at 50oC for 10 min. and 2 µl of loading buffer was fractionated by
electrophoresis using GeneGel Clean 15/14 gel (Pharmacia, NJ) which was rehydrated for
3h in 13 ml of supplied gel buffer containing 7 M urea, and run at 200 V for 2 h. at 50oC
(6). The gel was stained using DNA sliver staining kit (Pharmacia, NJ). Alteration of
bands relative to control were analyzed.
Table 2 lists the tumor cell lines analyzed and their microsatellite alterations in a total
of 16 microsatellite markers (11 of them on chromosome 8q, 5 of them on chromosome
3p). Instabilities in loci D18S34, D18S38, D18S474, and D3S1038 were detected in all of
the 11 tumor cell lines examined. Instability in loci D18S877 and D3S1067 were detected
only in all 4 secondary tumor cell lines. Novel bands were revealed in most of the tumors
examined while band expansion was observed in some of the tumors (Figure 1). Neither
deletion nor LOH was found. Furthermore, no difference in banding pattern was found
between the tumor cell lines induced by α particles and 56Fe ions.
57
Although BEP2D cells are immortalized by HPV-18, which disturbs normal p53 and
Rb functions, they are non-tumorigenic even in late passage. The present finding that
alteration of microsatellites persists in tumor cell lines but not in BEP2D cells strongly
supports the notion that additional genetic changes are needed for tumorigenic conversion
of BEP2D cells induced by high-LET radiation.
Table 1. Markers of Microsatellite
Marker
Chromosomal location
D18S-877
18q11.1 - q11.2
D18S-34
18q12.2 - q12.3
D18S-535
18q12.3
D18S-454
18q12.3 - q21.1
D18S-474
18q21.1
D18S-46
18q21.1
D18S-363
18q21.1
DCC
18q21.1 - q21.2
D18S-858
18q21.2
D18S-38
18q21.1 - q21.31
D18S-58
18q22.3 - q23
--------------------------------------------------------------------------------------D3S-1284
3p13 - p14
D3S-1289
3p21.1 - p14.3
D3S-1067
3p21.1 - p14.3
D3S-1038
3p25
D3S-1611
3p21.3
Table 2. Microsatellite Alteration in Tumor Cell Lines
Tumor Cell Lines Induced by α Particles
___________________________________________________
Marker
R30T1 R30T1L2 R30T5 R30T5L2 H1AT H2BT H2BT2
Tumor Cell Lines Induced by 56Fe
_______________________________
Fe60T1 Fe60T2 Fe60T3 Fe60T4
D18S877
+
+
+
+
D18S34
+
+
+
+
+
+
+
+
+
+
+
D18S535
D18S454
D18S474
+
+
+
+
+
+
+
+
+
+
+
D18S46
D18S363
+
+
+
+
+
+
+
+
+
+
+
DCC
D18S858
D18S38
+
+
+
+
+
+
+
+
+
+
+
D18S58
-----------------------------------------------------------------------------------------------------------------------------------------D3S1284
D3S1289
D3S1067
+
+
+
D3S1038
+
+
+
+
+
+
+
+
+
+
+
D3S1611
-
58
No figures available.
References
1. Hei et al. Carcinogenesis, 15: 431, 1994 and Adv. Space Res., 22:1699, 1998.
2. Shridhar et al. Cancer Res., 54:2084, 1994.
3. Fong et al. Cancer Res., 55:28, 1995.
4. Rosell et al. Int. J. Cancer, 74:30, 1997.
5. Takei et al. Cancer Res., 58:3700, 1998.
6. Windle et al. Mutation Res., 267:199, 1992.
59
Malignant Transformation of Human Bronchial Epithelial Cells
by Arsenite
Chang-Qing Piao and Tom Hei
Epidemiological investigations have indicated that exposure to arsenite is associated
with increased risks of human cancer of the skin, respiratory tract, hematopoietic system,
and urinary bladder. It has been documented in tin miners that long-term exposure to
arsenic via inhalation results in lung cancer. However, the mechanism by which arsenite
causes cancer is not well understood, and underlies the need of a human cell model. In
this study, we show, and for the first time, that arsenite can induce malignant
transformation of immortalized human bronchial epithelial cells in a step-wise fashion.
Human papillomavirus (HPV-18) immortalized human bronchial epithelial cells
(BEP2D) have been in culture for more than 180 population doubling and have near
diploid karyotype. They exhibit anchorage-dependent growth and are non-tumorigenic in
nude mice. The cells are routinely cultured in serum-free LHC-8 medium supplemented
with growth factors as described (1). Exponentially growing BEP2D cells were treated
with graded doses of sodium arsenite ranging from 0.5 to 3 µg/ml for 48 h. Arsenite
induced a dose-dependent cytotoxicity in BEP2D cells, as shown in Figure 1, with a mean
lethal dose of ~ 1.7 µg/ml. For the transformation assay, two doses of arsenite (1.5 and 2.0
µg/ml) were selected based on their moderate toxicity in BEP2D cells. BEP2D cells were
treated for 6 courses of 3 days each. After each treatment period, cells were trypsinized and
subcultured for 1 week before the next course. Total time peroid for the treatment was
about two months. After the last treatment, arsenite-treated cells showed a faster growth
rate and a higher saturation density (A155 = 3.5 x 105/cm2, A206 = 4.5 x 105/cm2, BEP2D
= 2.0 x 105/cm2). Arsenite-treated cells as well as BEP2D cells were continuously
cultured for another 4 weeks, and then were tested for resistance to TGF-β1-induced
growth inhibition as well as anchorage-independent growth. The cells were subsequently
inoculated into nude mice for tumorigenic analysis at 8 weeks after treatment.
There is evidence that TGF-β inhibits the growth of most epithelial cells, however,
most cancer cells are resistant to TGF-β mediated inhibition of growth (2). The cells
transformed by arsenite acquired phenotype of resistance to TGF-β1-induced growth
inhibition in a way similar to positive control tumor cell line (TB2B) induced by asbestos
from BEP2D cells (Figure 2). Their plating efficiency in soft agar was about 1%. The
transformed cells (A260) were assayed for tumorgenicity: 5 tumors developed in 8 nude
mice, with size of tumors reaching to more than 1 cm in diameter after 3 months of
injection.
The results of the present study demonstrated that arsenite induces malignant
transformation of immortalized human bronchial epithelial cells after prolonged treatment
of up to two months and provide a unique opportunity to study the pattern of molecular
alterations at the genetic level in a step-wise fashion.
60
No figures available.
References
1. Hei et al. Carciongenesis 15:431, 1994.
2. Reiss, M., Oncology Res. 9:447, 1997.
61
Radon, Arsenic, and Mutagenesis
Su Liu and Tom Hei
Arsenite enhances radon-induced bronchogenic carcinoma among miners by
mechanisms that are not established. In a large epidemiological study of Chinese tin
miners known to be exposed to both radon and arsenic, the apparent risk of radon
exposure was substantially reduced when adjustment was made for arsenic exposure (1).
Recent studies have demonstrated that the human carcinogen arsenic is in fact a strong
dose-dependent mutagen to mammalian cells in vitro, and that it induces mainly large
chromosomal mutations (2). Furthermore co-treatment of AL cells with the oxygenradical-scavenger dimethyl sulfoxide (DMSO) significantly reduces the mutagenicity of
arsenite. Assessment of the carcinogenic and mutagenic effects of two or more
environmental agents in combination has become an important issue, as the risk from
joint exposure may be substantially higher than predicted from the sum of risk of the
individual agents. To elucidate the interaction between the arsenic and radon exposures,
we used the human-hamster hybrid (AL) cells assay to examine the mutagenic potential of
alpha particles, either alone or in combination with sodium arsenite.
To determine cytotoxicity and mutation frequency, exponentially growing AL cells
(4 x 10 ) were plated onto Mylar dishes. After 48-hr incubation, cells were exposed to
arsenite for 24 hr, followed by irradiation with either a 25 or 50-cGy dose of 4He ions
(150 keV/µm) accelerated at the Radiological Research Accelerator Facility of Columbia
University. After irradiation, cultures were washed, trypsinized, and replated for both
survival and mutagenesis assays as described before (3). For mutant analysis,
independently derived CD59- mutants were isolated by cloning and expanded in culture.
Analysis of mutant spectrum was assessed by multiplex PCR as described previously (4).
4
Survival fraction of AL cells treated with graded doses of arsenite with or without
concurrent exposure to a 50-cGy dose of 4He is presented in Figure 1. A dose of 0.5
µg/ml of arsenite resulted in 85% of cells retaining clonogenic potential relative to the
control. In combination with a 50-cGy dose of alpha particles (survival = 0.64±0.04),
arsenite induced a clonogenic survival in AL cells which was consistent with a synergistic
interaction of the two, that is, the resultant survival level from combined exposure fell
outside the statistical range of the calculated values assuming the two agents acted in an
additive manner. This result demonstrated that arsenite increases alpha-particle-induced
cytotoxicity.
Mutation induction at the CD59- locus in AL cells treated with graded doses of
arsenite with or without concurrent irradiation with a 50-cGy dose of 4He is presented in
Figure 2. A 50-cGy dose of alpha particles induced a net mutant yield of 92±17 per 105
survivors. The background CD59- mutant fraction of AL cells used in these studies
averaged 32 per 105 survivors. In combination with arsenite, a synergistic mutant yield
was observed when AL cells were treated with lower doses of arsenite (0.1 and 0.5µg/ml).
62
The measured CD 59- mutation yields in AL cells treated with a combination of alpha
particles and arsenite were significantly higher than the predicted yield, assuming a
simple additivity. This finding suggests that arsenite enhanced alpha-particle-induced
CD59- mutants in a more than additive manner.
The cumulative deletion maps for all CD59- mutants analyzed are shown in Figure
3. Arsenite treatment at a dose of 0.5µg/ml in combination with alpha particles (50-cGy
dose) significantly increased the proportion of multi-locus deletion among AL cells
exposed to both carcinogens as compared to those treated with a single agent.
Our present study showed, for the first time, clear evidence that arsenite and alpha
particles induced both cell killing and mutagenesis in mammalian cells in a more-thanadditive fashion.
No figures available.
References
1. Xuan at al., Health Physics, 64:120, 1993.
2. Hei at al., Proc. Natl. Acad. Sci. USA, 95:8103, 1998.
3. Zhu at al., Radiation Research 145:251, 1996.
4. Hei at al., Proc. Natl. Acad. Sci. USA, 94:3765, 1997.
63
Chromosomal Aberrations in Tumorigenic Human Bronchial Epithelial
Cells Transformed by Crysolite Asbestos
Masao Suzuki, Chang-Qing Piao, and Tom Hei
It is well known that numerical changes of specific chromosomes may play an important
role in the expression of transformed phenotypes. There is evident that the stepwise karyotypic
changes correlate with specific transformed phenotypes in rodent cells (1). Weaver et al. (2) also
demonstrated that the common numerical changes in specific chromosomes occurred to
tumorigenic cell lines, which were transformed by radon-simulated alpha particles based on the
human papillomaviraus immortalized human bronchial epithelial cell line (BEP2D).
In the present study, we examined the numerical and structural changes of chromosomes
in order to clarify the mechanisms of asbestos-induced neoplastic conversion in vitro.
Furthermore, we demonstrate the use of a high-resolution G-banding in karyotype analysis using
a Calyclin-A mediated premature chromosome condensation (G2 PCC) technique.
Figure 1 shows the distribution of chromosomes in normal human
bronchial epithelial (NHBE) cells at passage 4. It was evident that
88% of the cells had a normal modal number of chromosomes (2n
= 46).
Figure 2 shows the distribution of chromosomes in metaphase
spreads (a) and G2 PCCs (b) in the immortalized BEP2D cells.
These results based on both metaphase spreads and G2 PCCs
indicated a similar trend in chromosome distribution, and the
BEP2D cells clearly were aneuploid compared with the parental
NHBE cells.
Karyotype analysis in 9 immortalized BEP2D cells (at passage 50) using G-banding with
G2 PCCs are summarized in Table 1. The results indicated that 78% of ch.#3, 11% of ch.#7,
44% of ch.#10 and 100% of ch.#12 were monosomy and 78% of ch.#5, 11% of ch.#8, 78% of
ch.#14, 22% of ch.#15, 11% of ch.#19 and 22% of ch.#20 were trisomy. A notable result was
monosomy of ch.#3 (78%). The results for monosomy of ch.#10, #12 and trisomy of #5, #8, #14
were consistent with the data for BEP2D by Weaver et al. (2).
65
Tab l e 1. C h rom o s o m e k ary o ty p e s i n G 2 P CC s o f B E P 2 D c el l s (P. 50 )
G 2 P CC
K ary o ty p e
# 2
47 , X Y, + 5, -1 2, + 1 4
# 4
44 , X Y, -3, + 5 , -7 , -10 , -12 , + 14
# 7
45 , X Y, -3, + 5 , -1 0 , -1 2 , + 1 4 , d e l(1 5 )(q te r --- > ? : )
# 9
# 10
45 , X Y, de l ( 1)(p t er ---> ? : ), -3 , + 5, d el (1 0)(p t er - --> ? : ),
-1 0, -1 2, + 1 4
46 , X Y, de l ( 1)(p t er ---> ? : ), -3 , + 5, + 8 , - 1 2
# 14
47 , X Y, -3, -1 2, + 1 4, + 1 9, + 2 0
# 15
# 19
48 , X Y, 2p + , - 3, + 5 , d e l(6 )(q te r ---> ? : ), -1 2,
d el ( 1 3)(q t er ---> ? : ), + 1 4 , + 1 5 , + 2 0
45 , X Y, -3, + 5 , -1 0 , -1 2 , + 1 4
# 24
46 , X Y, de l ( 1)(q t er ---> ? : ), -1 2 , + 1 5
No figures available.
References
1. Watanabe et al., Cancer Res., 50:760-765, 1990.
2. Carcinogenesis, 18:1251-1257
66
Cytogenetic Effects of Heavy-Ion Beams in Normal Human Bronchial
Epithelial Cells
Masao Suzuki, Chang-Qing Piao, and Tom Hei
At a time of manned space exploration, the potential exposure of astronauts or crews of a
spacecraft and/or an aircraft to low-flux galactic cosmic rays (GCR) and the subsequent
biological effects on the crews have become one of the major concerns of space science. It is
well known that high-LET charged particles are more effective in causing biological effects in
vivo and in vitro than low-LET radiations. However, there are few reports available on energyand ion-source-dependent dose-response relationships of biological effects induced by high-LET
charged particles, especially since biological studies using high-energy and charged (HZE)
particles are very limited. In this study, we examined the effects on cell death, mutation
induction, and chromatin break in normal human bronchial epithelial (NHBE) cells by high
energy 56Fe-ion beams.
Primary NHBE cells, which were derived from male donors, were obtained from
Clonetics Corporation (San Diego, CA). NHBE cells at passage 4 were used in this study.
Exponentially growing cells inoculated in T25 flasks (Falcon 3109 ) were irradiated with graded
doses of 56Fe-ion beams (1GeV/n) accelerated by the Alternating Gradient Synchrotron (AGS) at
Brookhaven National Laboratory (BNL). The dose-averaged LET value of the beams was
estimated to be 140 to 150keV/µm at the sample position.
Figure 1 shows the dose-response curve for clonogenic survival. The survival
curve showed little or no shoulder. At the D10 surviving level, the RBE for cell
death was estimated to be ≅ 2.2, compared with the data from 137Cs gamma rays
reported previously in these cells (1).
Figure 2 shows the dose-response curves for both initially measured and residual
chromatin-break induction in prematurely condensed chromosomes detected with
the Calyclin A-mediated premature chromosome condensation (PCC) technique.
Residual breaks were detected after a 24-hour postirradiation incubation. The
results indicated that the induction of initially measured breaks was about 1.7
times higher than that of residual breaks and the percentage of residual breaks
was about 60% at 24 hours after irradiation. The induction of residual breaks in
normal human fibroblast cells caused by C- and Ne-ion beams with similar LET
ranges was reported to be 50% at 110-130keV/µm (2,3). These data show that the
induction of residual breaks by Fe-ion beams is higher than those by C- and Neion beams.This result suggests that heavier ions may cause severer damage at both
the cellular and molecular levels, and that differences in track structure of energy
deposition with different kinds of ion sources play an important role in the
biological effects caused by high energy heavy ions.
67
Fig. 2. Dose-response curves of initially measured and residual
chromatin breaks detected by the PCC technique for NHBE
cells irradiated with 56 Fe-ion beams.
No figures available.
References
1. Suzuki & Hei, Mutat. Res., 349 : 33-41,1996.
2. Suzuki et al., Adv. Space Res., 18:(1/2)127-(1/2)136, 1996.
3. Suzuki et al., Int. J Radiat. Biol., 72:497-503, 1997.
68
MOLECULAR STUDIES:
CELL-CYCLE CHECKPOINTS
Physical Interactions Among Human Checkpoint Control Proteins
HHUS1p, HRAD1p and HRAD9p, and Implications for the
Regulation of Cell Cycle Progression
Haiying Hang and Howard Lieberman
Schizosaccharomyces pombe hus1 promotes radioresistance and hydroxyurea
resistance, as well as S and G2 phase checkpoint control. The human homologue of hus1,
HHUS1, has been isolated. HHUS1p can be co-immunoprecipitated with two other
human checkpoint control proteins, HRAD1p and HRAD9p, indicating that all three are
associated in a complex. Two-hybrid analysis reveals that they all can interact directly in
pairwise combinations. A typical two-hybrid result is illustrated in Figure 1.
Furthermore, additional two-hybrid studies indicate that two or more HHUS1p molecules
can bind to each other and this protein can also bind the N-terminal region of HRAD1p.
In contrast, the C-terminal portion of the checkpoint protein HRAD9p is essential for
interacting with HHUS1p and the C-terminal region of HRAD1p. Since the N-terminal
portion of HRAD9p was previously demonstrated to participate in apoptosis, this protein
likely has at least two functional domains, one that regulates programmed cell death and
the other cell cycle checkpoint control. Truncated versions of HHUS1p are unable to
bind HRAD1p, HRAD9p or another HHUS1p molecule, suggesting that this protein must
be intact to associate with other proteins successfully. HRAD1p-HRAD1p and
HRAD9p-HRAD9p interactions can also be demonstrated by co-immunoprecipitation,
but not by two-hybrid analysis, suggesting that the proteins associate as part of a complex
but do not interact directly. Northern blot analysis indicates that HHUS1 is expressed in
different tissues, but the mRNA is most predominant in testis where high levels of
HRAD1 and HRAD9 message have been detected. These studies suggest that HHUS1p,
HRAD9p and HRAD1p form a complex in human cells, and may function in a meiotic
checkpoint in addition to the cell cycle delays induced by incomplete DNA replication or
DNA damage. However, these proteins may individually join other complexes to mediate
additional cellular processes. For example, HRAD9p may regulate cell cycle checkpoints
through its interactions with HHUS1p and HRAD1p, but may participate in apoptosis
when independently or simultaneously binding Bcl-2 and Bcl-xL, two other protein
interactions observed. Understanding the structure, function, and coordination of these
complexes should provide important insight into mechanisms of cell-cycle checkpoint
control, as well as other cellular responses to damaged DNA.
70
Figure 1. Two-hybrid interaction between LexA-HRAD9p and AD-HRAD1p fusion
proteins. S. cerevisiae EGY48 cells containing pLexA and pB42AD, either devoid of
inserts (denoted by a dash) or bearing the genes indicated, were initially grown on SD
agar medium with glucose, then streaked onto SD agar with X-gal and galactose instead
of glucose. Five independent transformants for each two-plasmid pair combination were
examined.
71
Two-Hybrid Interactions Between the Human HRAD9p Checkpoint
Control Protein and the Tumor Suppressor p53
Sarah Rauth, Wei Zheng, and Howard Lieberman
The human homologue of fission yeast S. pombe rad9 has been isolated, and plays
a role in checkpoint control as well as apoptosis. The encoded protein was found to coimmunoprecipitate with p53, another protein also involved in the regulation of cell-cycle
progression after DNA damage and in the control of programmed cell death. Two-hybrid
analysis was performed to determine whether these proteins only associate as part of a
larger protein complex or perhaps could bind directly. As indicated in Figure 1 by the
levels of beta-galactosidase activity illustrated, LexA-HRAD9p and AD-p53p fusion
proteins demonstrate a strong two-hybrid interaction. Furthermore, preliminary data
using fragments of each gene indicate that the C-terminal two-thirds of HRAD9p,
important for binding human checkpoint control proteins HHUS1 or HRAD1, interacts
with p53. The functional significance of these interactions, as well as studies designed to
localize more precise sequences important for the protein-protein interactions observed
are currently under investigation.
Figure 1. Two-hybrid interaction between LexA-HRAD9p and AD-p53p fusion proteins. S. cerevisiae
EGY48 cells containing pLexA and pB42AD, either devoid of inserts or bearing HRAD9, p53 or SV40
large T as indicated, were initially grown on SD agar medium with glucose, then streaked onto SD agar with
X-gal and galactose instead of glucose. Five to seven independent transformants for each two-plasmid pair
combination were examined by a qualitative agar plate assay. Like transformants gave similar results.
Two independent transformants from each group were selected to quantitate beta-galactosidase activity.
Columns: 1, 2: pB42-p53 + plexA-HRAD9; 3, 4: pB42-p53 + plexA; 5, 6: pB42 + plexA-HRAD9; 7:
pB42-p53 + plexA-SV40 large T, as positive control. Each column represents the average of three trials.
Standard deviations are indicated.
72
Defective G2 Checkpoint by Inactivation of 14-3-3σ Gene
Influences Telomere Function
Sonu Dhar, Jain Kaung (MD Anderson Cancer Center, University of Texas, Houston),
Jeremy A. Squire (Department of Medical Biophysics, University of Toronto, Ontario,
Canada), Charles Geard, Raymund J. Wellinger (Department de Microbiologie et
Infectiologie, Faculte de Medecine, Universite de Sherbrooke, Quebec, Canada), and
Tej K. Pandita
14-3-3 proteins have distinct mammalian isoforms which show a remarkable
evolutionary conservation, extending to lower eukaryotes and plants. These proteins
appear to modulate a large variety of functional proteins and enzymes that are involved in
control of cell cycle, cell death, and mitogenesis. 14-3-3 proteins are thought to function
as adaptor proteins that allow interaction between signaling proteins that do not associate
directly with each other. The association of 14-3-3 with different kinases in cytosol and
membrane may contribute to kinase activation during intracellular signaling. The 14-3-3σ
gene has been implicated in the G2 checkpoint. It was originally identified as an
epithelial-specific marker, HME1, which was downregulated in a few breast cancer cell
lines but not in cancer cell lines derived from other tissue types. Recently, we found that
the expression of 14-3-3σ is lost in 94% of breast tumors. This gene sequesters the
mitotic initiation complex, cdc2-cyclin B1, in the cytoplasm after DNA damage. This
prevents cdc2-cyclin B1 from entering the nucleus where the protein complex could
normally initiate mitosis. In this manner, 14-3-3σ has been implicated in G2 arrest,
thereby allowing the repair of DNA damage. Cell cycle checkpoints influence genomic
stability and are considered to be guardians of genome integrity.
It has been suggested that genomic stability is maintained by telomeres that
protect fusions of chromosome ends. Shortening or loss of telomeres is correlated with
chromosome end associations that could be the cause of genomic instability and gene
amplification. Chromosome end-to-end associations, also called telomeric associations
(TA), seen at metaphase, have been reported in cells derived from tumor tissues,
senescent cells, the Thiberge Weissenbach syndrome, Ataxia telangiectasia individuals,
and following viral infections. These have been linked to genomic instability and
carcinogenicity. Chromosome end-to-end associations involve telomeres, which are
essential for the stability and complete replication of eukaryotic chromosomes. Telomeres
are maintained by telomerase, a reverse transcriptase that adds TTAGGG repeats onto the
3’ ends of vertebrate chromosomes. It has been shown that a dominant negative allele of
human telomeric protein TRF2 induces loss of telomeric G-strand overhangs,
subsequently enhancing end-to-end chromosome fusions.
Recently, we have shown that the ATM gene which is defective in the cancerprone disorder Ataxia telangiectasia, influences chromosome end-to-end associations and
telomere length. ATM dysfunction results in abnormal checkpoint responses in multiple
phases of the cell cycle, including G1, S, and G2. Cells defective in the G1 checkpoint
73
e.g., cells with compromised p53 function (RC-10.3, RKO.p53.13, SW480), have higher
frequencies of cells with chromosome end-to-end associations. However, it is not known
whether cells defective in the G2 checkpoint have normal telomere behavior. The purpose
of the present study was to determine the influence of the 14-3-3σ gene on the telomere
structure because this gene is involved in G2 checkpoint after DNA damage.
Telomeres are terminal complexes of repetitive DNA sequences and proteins at
the end of chromosomes that are associated with the nuclear matrix. They are thought to
be released from the nuclear matrix by the breakdown of nuclear membrane components
at the time cells exit from G2 to M phase. Checkpoints maintain the order and fidelity of
the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and
cancer. The 14-3-3σ gene promotes G2 arrest following DNA damage. The 14-3-3σ
protein is cytoplasmic with perinuclear localization. Here we demonstrate that
-/inactivation of such a gene influences telomere metabolism. 14-3-3σ cells show a
reduction in G-strand overhangs, a loss of telomeres, frequent chromosome end-to-end
associations, and terminal nonreciprocal translocations. A possible basis for these
findings is an aberrant nuclear matrix breakdown during the G2/M transition. This is
supported by the frequency of ionizing-radiation-induced G2-type chromosome
-/aberrations being higher in 14-3-3 σ cells as compared to their parental cells, and 14-3-/3 σ cells having constitutively phosphorylated status of MPM2 epitopes. This suggests
that 14-3-3σ influences the phosphorylation status of MPM2,which in turn leads to
aberrant nuclear matrix breakdown with consequences for normal chromosome telomere
structure and the maintenance of genomic integrity.
74
MOLECULAR STUDIES:
DAMAGE RESPONSIVENESS
Ionizing Radiation Activates ATM Kinase Throughout the Cell Cycle
Tej K. Pandita, Howard Lieberman, Dae-Sik Lim (Department of Hematology/Oncology, St.
Jude Children’s Research Hospital, Memphis, TN), Sonu Dhar, Wei Zheng, Yoichi Taya
(National Cancer Center Research Institute, Tokyo), and Michael B. Kastan (Department
of Hematology/Oncology, St. Jude Children’s Research Hospital)
Ataxia telangiectasia (A-T) is a rare, pleiotropic, autosomal human recessive
disorder characterized by progressive neurological degeneration, growth retardation,
premature aging, oculocutaneous telangiectasias, specific immunodeficiencies, high
sensitivity to ionizing radiation (IR), gonadal atrophy, genomic instability, defective
telomere metabolism, and cancer predisposition. Cells derived from A-T individuals
exhibit a variety of abnormalities in culture, such as a higher requirement for serum
factors, hypersensitivity to ionizing radiation, and cytoskeletal defects. The gene that is
mutated in A-T has been designated ATM (A-T, mutated) and its product shares the PI-3
kinase signature of a growing family of proteins involved in the control of cell-cycle
progression, processing of DNA damage, and maintenance of genomic stability. ATM
appears to be required for initiation of multiple DNA damage-dependent signal
transduction cascades that activate cell-cycle checkpoints (1,2). One of the bestcharacterized mammalian cell-cycle checkpoints involves accumulation/stabilization of
p53 protein and subsequent G1 arrest or apoptosis (1,3). Cells derived from A-T
individuals or Atm null mice are very poor at this induction of p53 following ionizing
irradiation, though induction following UV irradiation appears relatively normal (3-6).
For this reason, it has been suggested that ATM is involved in specific signaling pathways
induced by ionizing radiation exposure (3-6).
Recently, it has been reported that the phosphorylation of p53 on serine-15 is
impaired in A-T cells after IR (7) and that the ATM kinase is capable of phosphorylating
this site in p53 (8-10). Though these results suggest the importance of ATM in the
phosphorylation of p53, it is not known whether this activation of ATM by IR is cell-cycle
dependent. For example, it is conceivable that ATM activation by IR could be dependent
on specific types of DNA lesions introduced during a particular phase of the cycle, such as
DNA replication-dependent events during S phase. However, since ATM signals to p53,
and p53 is involved in a G1 checkpoint after IR, this activation is likely to occur at least
during the G1-phase of the cycle. Ataxia telangiectasia cells also exhibit specific defects
in S phase and G2 checkpoints which are intact in SV-40 transformed cells from non-A-T
patients, and in tumor cell lines with mutant p53 (11). Thus, there are p53-independent
pathways in which ATM participates following IR. These observations raised the
question of whether ATM activation after IR occurs in all phases of the cell cycle.
Cells deficient in ATM function have higher initial chromosomal damage and
greater amounts of residual chromosomal damage in G1 as well as in G2 after treatment
with ionizing radiation, and are sensitive to ionizing radiation induced cell killing in all
phases of the cell cycle (11-13). ATM might therefore play a critical role in all phases of
76
the cell cycle after ionizing-radiation treatment. We set out to determine the cell survival,
ATM kinase activity and, simultaneously, the serine-15-phosphorylation state of p53 in
cell populations enriched in the G1, S or G2/M-phases of the cell cycle. Asynchronous
exponentially growing populations of GM536 lymphoblastoid cells were fractionated by
centrifugal elutriation into populations enriched for different phases of the cell cycle. The
quality of cell cycle enrichment was monitored by flow cytometry for DNA content, and
independent determinations of cell-cycle stage were assessed by premature chromosome
condensation (12). The G1-phase-enriched populations contained greater than 98% of
their cells in that phase. The S-phase and G2/M-enriched populations were about 88% and
70 to 80% pure, respectively.
To determine whether centrifugal elutriation influences the physiology/
reproductive capability of the cells, we compared cells that did or did not undergo this
procedure for viability and survival after ionizing-radiation exposure. We elutriated the
cells and pooled fractions together. The flow analysis of the pooled fractions showed
similar frequencies of G1-, S- and G2/M-phase cells as was found in the exponentially
growing asynchronous population of cells before fractionation. GM536 cells, elutriated or
not elutriated, were treated with different doses of gamma rays. Cell viability as
determined by the trypan blue exclusion test showed no difference in elutriated versus
mock-treated cells.
Cell survival after ionizing-radiation treatment was determined by two independent
assays, i.e., growth curve analysis and limiting dilution analysis (12). GM717 (Ataxia
telangiectasia) lymphoblastoid cells were used as a control. No difference in cell survival
after irradiation was detected between the cells that were elutriated versus not elutriated.
In addition, the normal cells (GM536) were much less sensitive to IR than the GM717
(Ataxia telangiectasia, A-T) cells, with survival characteristics for all cell-cycle-dependent
results given in Table 1. The enhanced sensitivity of G1-phase cells to ionizing radiation
relative to cells in other phases of the cell cycle was consistent with previous studies (12).
Cells at different phases of the cell cycle show differences in radiosensitivity, and
those deficient in ATM function show abnormal checkpoint responses in G1, S and G2.
Recent studies revealed enhanced ATM kinase activity in response to DNA damage (810). We were interested to determine whether ATM protein levels and/or kinase activity
Table 1. Survival characteristics after ionizing radiation.
_______________________________________________________________________
Cell Phase
D0 (Gy)
n
_______________________________________________________________________
GM717 (A-T) (asynchronous population)
0.37
1.00
GM536 (normal) (asynchronous population)
0.93
1.37
GM536 (normal) (G1-phase)
0.69
1.25
GM536 (normal) (S-phase)
1.04
1.42
GM536 (normal) (G2/M-phase)
0.89
1.25
_______________________________________________________________________
77
were enhanced in all phases of the cell cycle following IR treatment. Cell-cycle- phase
enrichment was accomplished by centrifugal elutriation. First, we determined that
centrifugal elutriation had no influence on the induction of ATM protein or on its kinase
activity. These features of the ATM protein were compared in cells that were elutriated
and pooled versus those not subjected to the enrichment procedure. Both cell preparations
(elutriated and nonelutriated) were treated with 5 Gy of gamma rays and incubated for
different times prior to cell lysis for determination of ATM protein levels and kinase
activity. Elutriation did not influence ATM protein levels or its kinase activity after
ionizing-radiation treatment. We then analyzed the G1-, S- and G2/M-phase-enriched cell
populations separately after treatment with 10 Gy of gamma rays. ATM protein levels and
kinase activity were analyzed at different times post-irradiation. ATM protein levels were
identical in all phases of the cell cycle, and did not change after irradiation. However,
when ATM kinase activity was assessed, enhanced activity after IR was found
immediately in all phases (G1, S or G2/M) and this enhanced activity remained constant
for one hour post-irradiation. These results suggest that ATM has a critical role in sensing
ionizing-radiation-induced DNA damage in each of these phases of the cell cycle.
A known target of ionizing-radiation DNA-damage-induced phosphorylation is the
p53 protein. Phosphorylation of p53 at serine-15 in response to ionizing radiation
correlates with both the accumulation of total p53 protein as well as its transactivation of
downstream genes. The cell-cycle-phase dependence of p53 phosphorylation is not known.
Activation of p53 results in a G1 cell-cycle arrest or apoptosis that contributes to
suppression of malignant transformation and the maintenance of genomic integrity (13).
The p53 tumor suppressor protein is a transcription factor that is activated in response to
treatment with a variety of DNA-damaging agents, including ionizing radiation (3). Since
G1 cells are more sensitive to killing by ionizing radiation, while the enhancement of
ATM kinase activity levels post-irradiation is similar among G1-, S-, and G2/M-phase
cells, p53 accumulation and phosphorylation at serine-15 may be differentially altered in
G1 cells.
We investigated p53 accumulation and phosphorylation in vivo following ionizingradiation treatment of cells in different phases of the cell cycle. As for the other endpoints
tested, we found that the enrichment protocol had no effect on the radiation-induced
accumulation of p53. Cells were elutriated and all the fractions pooled, treated with 5 Gy
of ionizing radiation, and p53 levels determined at different time periods. No differences
in the accumulation of p53 were observed between cells elutriated versus those not
elutriated.
To assess p53-serine-15 phosphorylation after treatment with ionizing radiation,
p53 was first immunoprecipitated using a specific monoclonal antibody. The amount of
p53 protein loaded was adjusted to similar amounts per lane of the gel. As a control for the
serine-15 antibody, some cells were treated with the proteosome inhibitor acetyl-Leu-Leunorleucinal (ALLN), resulting in the stabilization of p53 protein levels by inhibiting its
degradation. Under these conditions, Western blot analysis of lysates prepared from
ALLN-treated or irradiated cells demonstrated equivalent amounts of p53 protein.
78
Western blot analysis demonstrated that p53 was phosphorylated on serine-15 in response
to ionizing radiation. p53 serine-15 phosphorylation was not observed in ALLN-treated
cells even after irradiation. No difference was observed in the levels of p53 serine-15
phosphorylation after treatment with ionizing radiation between cells elutriated and pooled
versus those not elutriated.
To determine whether the p53 response to ionizing radiation is cell-cycle-phase
dependent, we examined p53 protein levels in unirradiated or irradiated G1, S, and G2/M
cells and found no cell-cycle phase differences. Similarly, when p53 serine-15
phosphorylation was compared among G1-, S-, and G2/M-phase cells after gamma
irradiation, no significant differences were detected. p53 is involved in a G1 checkpoint
and phosphorylation of p53 can be linked with the G1 checkpoint. It is not clear whether
the phosphorylation of p53 at serine-15 has any role in S- and G2/M-phase cells after
treatment with DNA-damaging agents. Cells deficient in p53 function have normal Sphase as well as G2-phase checkpoints after ionizing-radiation treatment in contrast to the
altered G1 checkpoint, while A-T cells exhibit specific defects in G1- as well as S-phase
and G2-phase checkpoints. These studies suggest that the ATM kinase may have other
functional targets in S phase and G2 phase of the cell cycle. Thus, these results suggest
that activation of the ATM kinase by DNA damaging agents is important for signaling in
all phases of the cell cycle.
References
1. Morgan SE, and Kastan MB. (1997) Cancer Res. 57, 3386-3389; Morgan SE and Kastan MB. (1997).
Adv. Can. Res. 71, 1-25.
2. Shiloh Y. (1995) Eur. J. Hum. Genet. 3, 116-138.
3. Kastan MB, Zhan Q, el-Deiry WS, Carrier F, Jacks T, Walsh WV, Plunkett BS, Vogelstein B, and
Fornace AJ Jr. (1992) Cell 71, 587-597.
4. Khanna KK and Lavin MF. (1993) Oncogene. 8, 3307-3312.
5. Canman CE, Wolff AC, Chen CY, Fornace AJ Jr, and Kastan MB. (1994) Cancer Res. 54, 5054-5058.
6. Xu Y and Baltimore D. (1996) Genes Dev. 10, 2401-2410.
7. Siliciano JD, Canman CE, Taya Y, Sakaguchi K, Appella E and Kastan MB (1997) Genes Dev. 11,
3471-3481.
8. Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, Smorodinsky NI, Prives C, Reiss Y,
Shiloh Y, and Ziv Y. (1998) Science 281, 1674-1677.
9. Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, Appella E, Kastan M.B. and
Siliciano JD. (1998) Science 281, 1677-1679.
10. Khanna KK, Keating KE, Kozlov S, Scott S, Gatei M, Hobson K, Taya Y, Gabrielli B, Chan D, LeesMiller SP and Lavin MF. (1998) Nat. Genet. 20, 398-400.
11. Morgan SE, Lovly C, Pandita TK, Shiloh Y, and Kastan MB. (1997) Mol. Cell. Biol. 17, 2020-2029.
12. Pandita TK and Hittelman WN. (1992) Rad. Res. 130, 94-103.
13. Pandita TK and Hittelman WN. (1992) Rad. Res. 131, 214-223.
14. Hartwell LH and Kastan MB (1994) Science 266, 1821-1828.
79
Ataxia Telangiectasia: Chronic Activation of Damage-Responsive
Functions is Reduced by Alpha-Lipoic Acid
Magtouf Gatei (Queensland Institute of Medical Research, Royal Brisbane Hospital,
Australia), Dganit Shkedy (Department of Human Genetics and Molecular Medicine,
Sackler School of Medicine, Tel Aviv University, Israel), Kum Kum Khanna, (Queensland
Institute of Medical Research), Tamar Uziel and Yosef Shiloh (Dept. Human Genetics and
Molecular Medicine, Tel Aviv University), Tej K. Pandita, Martin F. Lavin (Queensland
Institute of Medical Research), and Galit Rotman (Dept. Human Genetics and Molecular
Medicine, Tel Aviv University)
The human genetic disorder ataxia telangiectasia (A-T) is characterized by
neurodegeneration, immunodeficiency, premature aging, telangiectasis, genomic
instability, cancer predisposition, and extreme sensitivity to ionizing radiation. A
hallmark of A-T cells is hypersensitivity to agents that cause oxidative damage by
generating reactive oxygen species (ROS) including ionizing radiation (IR), various
radiomimetic drugs and H2O2. This hypersensitivity can be accounted for, at least in part,
by failure to repair a significant fraction of DNA double-strand breaks. In addition, these
cells are impaired in their ability to activate radiation-induced signal-transduction
pathways, most notably those that control cell-cycle checkpoints. The gene responsible
for A-T, ATM, seems to play a central role in sensing oxidative damage to DNA and in
the subsequent activation of a signaling network, leading to repair of the damage and
cellular recovery and survival.
Cells derived from A-T patients show spontaneous higher base levels of
chromosome damage (1). This may be the cause for the constitutive activation of certain
cellular functions that have been reported occasionally in A-T cells. Singh and Lavin (2)
described the constitutive presence in the nucleus of A-T cells of a DNA-binding protein
that is present in the cytoplasm of normal cells, but migrates to the nucleus in response to
treatment by agents that generate free radicals. Constitutive activation of NF-κB and
abnormal elevation of interferon-β (IFN-β) and IFN-β-inducible genes in A-T cells were
also reported. The amount of p21 associated with cyclin A/cdk2 and cyclin B/cdc2 was
higher in A-T cells than in controls. Basal levels of Gadd45 protein, another gene
activated by p53, were also elevated, and the phosphorylated form of cdc2 was more
abundant in A-T fibroblasts. Hyperphosphorylation of Rb and constitutive activation of
E2F-1 in A-T cells were also reported.
We recently hypothesized that the chronic activation in ATM-deficient cells of
pathways responsive to agents that generate ROS, could indicate a tenuous state of
oxidative stress in these cells. This prediction is supported by recent studies showing
markers of oxidative stress in organs of Atm-deficient mice and in cell lines derived from
A-T patients. To further support our hypothesis we analyzed A-T cells for basal levels
and modifications of some known proteins which normally respond to genotoxic stress,
and found that the basal levels of p53, the serine 15-phosphorylated form of p53,
80
p21WAF1/CIPI and the phosphorylated form of cdc2 are chronically elevated in these
cells. Treatment of A-T cells with the antioxidant α-lipoic acid significantly reduced the
levels of p53 and p21 proteins, pointing to the involvement of reactive oxygen species in
this chronic activation. These results suggest that the absence of functional ATM might
result in a mild but continuous state of oxidative stress, which could account for several
features of the pleiotropic phenotype of A-T.
References
1. Pandita TK, Pathak S, Geard CR. (1995). Chromosome end associations, telomeres and telomerase
activity in ataxia telangiectasia cells. Cytogenet. Cell Genet . 71, 86-93.
2. Singh SP and Lavin MF. (1991) DNA-binding protein activated by gamma radiation in human cells. Mol.
Cell. Biol., 10, 5279-5285.
81
Activation of Abl Tyrosine Kinase by Ionizing Radiation Requires
ATM But Not DNA-PK
Sanjeev Shangary and Tamara Lataxes (Molecular Genetics and Biochemistry, University
of Pittsburgh Medical Center), Tej Pandita, Guillermo E. Taccioli (Department of
Microbiology, Boston University), and R. Baskaran (Molecular Genetics and Biochemistry,
Univ. of Pittsburgh Medical Center)
The product of the proto-oncogene c-Abl is a non-receptor tyrosine kinase that is
ubiquitously expressed and localized in both the nucleus and cytoplasm. The c-Abl
protein is required for the normal growth and function of the organism because mice that
are nullizygous for Abl die 14 to 15 days after birth for unknown reasons. The c-Abl
protein contains an unusually long C-terminus that is essential for Abl’s function because
mice containing c-Abl with an intact kinase domain but lacking the C-terminus also
exhibited neonatal lethality. In the C-terminus of c-Abl, a binding site for Abl’s nuclear
substrate RNA polymerase II has been identified. In addition, several other functional
domains such as a nuclear localization signal (NLS), a DNA binding domain (DBD), and
an actin binding domain (ABD) have also been identified. Recently, a nuclear export
signal (NES) has been identified in the extreme C-terminus of c-Abl (1).
The tyrosine kinase activity of c-Abl is normally tightly regulated during the cell
cycle. This can be explained by the binding partners of Abl. The kinase domain of Abl
binds to the C-terminus of Rb (retinbolastoma protein). When Rb becomes
hyperphosphorylated by Cdk4/6, Abl loses its association with Rb and gains its tyrosine
kinase activity. Other members of Abl binding partners include Abl interacting proteins
Abi-1, Abi-2, and PAG, whose binding also leads to suppression of Abl kinase activity.
In addition to the cell cycle, the kinase activity of Abl is also regulated by
exogenous stimuli such as ionizing radiation (IR), cis-platin, MMS, mitomycin C,
hydrogen peroxide, and a number of other agents. Interestingly, c-Abl is not activated by
UV treatment. This observation led to the identification of ATM (mutated in the human
disorder ataxia telangiectasia) as an upstream regulator of Abl kinase. Using a yeast twohybrid approach, Shafman et al. (2) have shown that ATM directly interacts with Abl and
that activation of Abl kinase activity by ionizing radiation requires the ATM gene product
(2). Consequently, c-Abl is not responsive to IR in AT cells, which lack functional ATM.
The mechanism of ATM activation of Abl has been elucidated. Following irradiation,
ATM kinase phosphorylates Abl on a specific Serine (S465) residue located in the kinase
domain of Abl, resulting in the activation of its kinase activity. Together, these results
have unequivocally identified ATM as an upstream regulator of Abl kinase in response to
radiation exposure.
In addition to ATM, the DNA-dependent protein kinase (DNA-PK), which is also
a member of a subgroup of the phosphatidyl-3-inositol kinase superfamily can
phosphorylate Abl in vitro resulting in activation of its kinase activity. In response to
82
ionizing-radiation treatment, cells obtained from SCID mice showed reduced activation
of Abl kinase. These observations have led to the identification of DNA-PK as yet
another upstream regulator of Abl kinase.
In response to IR, the DNA-PK can also phosphorylate Abl and activate its kinase
activity. To examine the physiological relevance of these two kinases in IR-induced Abl
phosphorylation and activation, we assayed for Abl, ATM, and DNA-PK activity in ATM
and DNA-PK-deficient cells. Our results show that despite the presence of higher-thannormal levels of DNA-PK kinase activity, c-Abl is not activated by IR in AT cells. On
the other hand, activation of ATM and Abl kinase is observed in cells that are completely
deficient for the catalytic subunit of DNA-PK. Furthermore, activation of Abl by IR
correlates well with activation of ATM kinase activity by IR in G1 and S phase.
Interestingly, cells in the G2/M phase exhibited enhanced Abl activity irrespective of
exposure to IR. Together, these results indicate that ATM may regulate Abl kinase at
every phase of the cell cycle in response to ionizing radiation. Furthermore, activation of
Abl correlates well with activation of ATM in G1, S, and G2/M phases. That is, ATM
regulates Abl kinase activity irrespective of cell-cycle phase.
References
1. Taagepera S, McDonald D, Loeb JE, Whitaker LL, McElroy AK, Wang JY, Hope TJ (1998) Nuclearcytoplasmic shuttling of C-ABL tyrosine kinase. Proc Natl Acad Sci U S A 95:7457-62.
2. Shafman T, Khanna KK, Kedar P, Spring K, Kozlov S, Yen T, Hobson K, Gatei M, Zhang N, Watters D,
Egerton M, Shiloh Y, Kharbanda S, Kufe D, Lavin MF (1997) Interaction between ATM protein and cAbl in response to DNA damage. Nature 387:520-3.
83
Atm Inactivation Results in Aberrant Telomere Clustering During Meiotic
Prophase1
Tej K. Pandita, Christoph H. Westphal (Harvard Medical School, Boston, MA), Melanie Anger
(University of Kaiserslautern, Germany), Satin Sawant, Charles Geard, Raj K. Pandita (Albert
Einstein College of Medicine, Bronx, NY), and Harry Scherthan (Univ. of Kaiserslautern)
Telomeres have been considered as key structures of meiotic chromosomes. Meiosis is a
specialized cell division that ensures the proper segregation of genetic material and formation of
viable haploid gametes. The most critical events of meiosis occur during prophase I, when
homologous chromosomes get aligned (prealign), synapse (pair) and recombine with each other.
During early meiotic prophase, telomeres redistribute and accumulate at a limited sector of the
nuclear membrane to form a chromosomal bouquet. A number of studies suggest that bouquet
formation mediates prealignment of homologues and thereby facilitates synapsis. The only
known telomeric proteins that have been implicated in bouquet formation are the products of
Taz1 of fission yeast (1) and NdjI/TamI of budding yeast (2,3).
We have investigated the effects of inactivation of Atm on telomere clustering during
male mouse meiosis. Our search was prompted by the observation that cells derived from A-T
patients, among other features, show an altered telomere metabolism and structure (4-6).
-/Telomere FISH to spermatocytes I of Atm null mice revealed that premeiotic and leptotene Atm
mice nuclei show a similar telomere distribution and signal number as compared to control.
-/Spermatocytes I of Atm mice, however, showed aberrant synapsis and telomere distribution, in
that undisrupted spermatocyte nuclei frequently displayed clustered telomeres and a large
chromocenter. Synaptonemal complex (SC) immunostaining in combination with telomere FISH
to these nuclei revealed fragmentary, strong SCP3 signals at and around the clustered telomeres,
with the SC protein signals often aberrantly extending between several chromosome ends. Such a
distribution of SC proteins and telomeres was not observed in spermatocytes of normal mice,
where synapsis has been shown to initiate more internally, and is usually delayed at the
heterochromatic proximal ends of the acrocentric mouse chromosomes. In male mouse meiosis, a
bouquet arrangement of chromosome ends resolves soon after the initiation of synapsis (early
zygotene) and renders only a very low percentage of bouquet nuclei readily detectable. The
-/higher frequency of spermatocytes with locally clustered telomeres encountered in Atm testes
preparations suggests that the bouquet arrangement is maintained for a considerably longer
period or is arrested in the absence of functional ATM protein. The prevalence of a bouquet
arrangement could result from pairing partner switches, non-homologous synapsis and/or
illegitimate recombination events which interconnect accumulated telomeres at the cluster site,
thereby preventing their dispersion during zygotene. Since telomere clustering occurs normally at
the leptotene/zygotene transition, an elevated number of spermatocyte I nuclei showing a bouquet
arrangement could also be a consequence of an arrest during leptotene/zygotene stages of meiotic
-/prophase. This timing would be consistent with reports that the spermatogenic arrest in Atm
animals occurs as early as leptotene or zygotene (7).
84
ATM protein has been shown to be associated with chromatin (8), and ATM may be
involved in the control of recombination (7). The abrogation of Atm function results in meiotic
prophase arrest associated with aberrant synapsis and fragmentation of SCs. ATM also shows
some homology to TEL1/MEC1 genes of budding yeast which are involved in telomere
maintenance and meiotic and mitotic cell-cycle checkpoint control. Since Atm and Dmc1deficient mice as well as many recombination mutants of budding yeast fail to form normal SC, it
is possible that an absence of Atm function alters the progression of recombination. Consistent
with this hypothesis, proteins involved in normal recombination processes, like Rad51, DMC1
-/and Atr are mislocalized as early as leptotene in Atm meiocytes (7). Aberrant synapsis and
failure to form normal SC seems to induce apoptosis and fragmentation of chromosomes in Atm
/mouse spermatocytes. Given that telomere dispersion from the cluster site is delayed or
-/prevented in Atm bouquet cells, SC fragmentation could result from the physical stress exerted
by immobile meiotic telomeres on dynamic chromosomes with unrepaired double strand breaks.
Recently, it was shown that a bouquet arrangement transiently forms during wild-type
meiosis of budding yeast (9). The spo11 and rad50S recombination mutants of budding yeast,
which fail to form normal SC (10), form a chromosomal bouquet but fail to resolve this nuclear
organization later at prophase, leading to elevated levels of bouquet nuclei (9). The timing and
occurrence of a bouquet in yeast recombination mutants mirrors that in Atm-deficient
spermatocytes which also fail to resolve the bouquet arrangement. The observations that loss of
+
+
all telomeres in fission yeast TEL1 /rad3 double mutants prevents meiosis (11) and that
telomeres support homologue search (12) strongly suggests that telomeres support homologue
-/alignment. A persisting bouquet arrangement in Atm spermatocytes could contribute to the high
levels of chromosome pairing at a telomere associated chromosome 8 region, despite the widely
-/aberrant synapsis in Atm mice. This interpretation is consistent with the observation that
recombination mutants of yeast form a bouquet (9) and undergo limited levels of homologue
pairing (13,14), which is elevated at telomeric regions (10).
Since ATM influences the organization of telomere chromatin in vegetative cells and
telomeres have been shown to be tethered to the nuclear matrix in somatic cells (5,6), we tested
the interaction of telomeres with the nuclear matrix of spermatocytes from normal and Atm
deficient mice. It was found that 90% of telomere repeats were associated with the nuclear matrix
-/fraction of Atm spermatocytes, whereas only 50% of telomere repeats co-fractionated with the
matrix of control spermatocytes. One interpretation is that the altered persistent interaction of
telomeres with the nuclear matrix could be a cause for the failure of resolution of telomere
-/clustering in spermatocytes of Atm mice.
We considered the possibility that aberrant telomere clustering in Atm null mice may be
due to defective telomerase activity. That is, telomerase might be required for the synthesis of the
correct telomere terminis without which telomere ends might have defective interactions with the
nuclear matrix. We found no differences in the telomerase activity between testes of Atm null
and control mice, suggesting that the aberrant telomere clustering cannot be explained based on
defective telomerase.
85
How ATM influences the interactions of telomeric DNA with the nuclear matrix as well
as telomere clustering is not clear at present. There is growing evidence which suggests that both
the shielding of telomeric ends and their elongation by telomerase is dependent upon telomere
binding proteins. Mammalian telomeres are packaged in telomere-specific chromatin (15).
Telomere length homeostasis in yeast requires the binding of a protein along the telomeric tract
and changes in the telomeric protein complex influence the stability of chromosome ends. Three
mammalian telomere binding proteins namely, TRF1, TRF2, and PIN2 have been identified.
They have DNA binding properties with (TTAGGG)n repeats in vitro irrespective of the presence
of a DNA terminus, properties which are consistent with a presence along the ends of
chromosomes. TRF1 has been implicated in the regulation of telomere length (16) and provides
an architectural role at telomeres. Since it has been shown that protein localization is distorted in
-/leptotene/zygotene Atm spermatocytes (7), it is likely that ATM influences telomeres at the
protein level. In agreement, TRF1 and 588 other genes were found to be similarly expressed in
-/Atm and normal spermatocytes. Hence, it remains to be determined how the Atm gene product
influences meiotic telomeres.
ATM belongs to a growing family of PI-3 protein kinases and shares some homology
with the yeast MEK1 protein which has been shown to be a meiosis-specific kinase required for
proper synapsis and phosphorylation-dependent resolution of sister chromatid cohesion prior to
MI (17). Since a major portion of telomere repeats in Atm null mouse spermatocytes abnormally
associated with the nuclear matrix, it may be speculated that Atm (besides other effects), is
involved in this attachment. The altered interaction of telomere repeats with the meiotic nuclear
matrix may be due to aberrant phosphorylation of as yet unknown components. Future
investigations will have to determine whether ATM is involved in meiosis-dependent
phosphorylation changes of telomeric proteins like Tankyrase, a telomere specific poly(ADPribose) polymerase, which has recently been shown to reduce TRF1 telomere binding activity
through poly-ADP-ribosylation in vitro.
References
1. Cooper J.P., Y. Watanabe and P. Nurse. (1998) Fission yeast Taz1 protein is required for meiotic
telomere clustering and recombination. Nature 392:828-831.
2. Chua, P.R. and G.S., Roeder. 1997. Tam1, a telomere-associated meiotic protein, functions in
chromosome synapsis and crossover interference. Genes Dev. 11: 1786-1800.
3. Conrad, M.N., Dominguez, A.M., and M.E. Dresser. (1997) Ndj1p, a meiotic telomere protein
required for normal chromosome synapsis and segregation in yeast. Science 276: 1252-1255.
4. Pandita, T.K., S. Pathak, and C. Geard. (1995) Chromosome end association, telomeres and
telomerase activity in ataxia telangiectasia cells. Cytogenet. Cell Genet. 71: 86-93.
5. Smilenov, L.B., S.E. Morgan, W. Mellado, S.G. Sawant, M.B. Kastan, and T.K. Pandita. (1997)
Influence of ATM function on telomere metabolism. Oncogene 15: 2659-2665.
6. Smilenov, L.B., S. Dhar, and T.K. Pandita (1999) Altered telomere nuclear matrix interactions and
nucleosomal periodicity in ataxia telangiectasia cells. Mol. Cell. Biol. 19: 6963-6971.
7. Barlow, C., Liyanage, M., Moens, P.B., Tarsounas, M., Nagashima, K., Brown, K., Rottinghaus,
S.P., Jackson, S.P., Tagle, D., Ried, T., and A. Wynshaw-Boris. (1998) Atm deficiency results in
severe meiotic disruption as early as leptonema of prophase. Development 125: 4007-4017
8. Gately, D.P., Hittle, C., Chan, G.K.T., and T.J. Yen. (1998) Characterization of ATM expression,
localization, and associated DNA-dependent protein kinase activity. Mol. Biol. Cell 9:2361-2374.
9. Trelles-Sticken, E., Loidl, J., and H. Scherthan. (1999) Bouquet formation in budding yeast:
Initiation of recombination is not required for meiotic telomere clustering. J. Cell Sci., 112: 651-658.
86
10. Weiner, B.M. and N. Kleckner. (1994) Chromosome pairing via multiple interstitial interactions
before and during meiosis in yeast. Cell 77: 977-991.
11. Naito, T., A. Matsuura, and F. Ishikawa. (1998) Circular chromosome formation in a fission yeast
mutant defective in two ATM homologues. Nat. Genet. 20:203-206
12. Rockmill, B. and G.S. Roeder. (1998) Telomere-mediated chromosome pairing during meiosis in
budding yeast. Genes Dev. 12:2574-86
13. Loidl J., Klein F., and H. Scherthan. (1994) Homologous pairing is reduced but not abolished in
asynaptic mutants in yeast. J. Cell Biol. 125:1191-1200.
14. Nag D., H. Scherthan, B. Rockmill, J. Bhargava, and G.S. Roeder. (1995) Heteroduplex DNA
formation and homolog pairing in yeast meiotic mutants. Genetics 141: 75-86.
15. Smith, S. and T. de Lange. (1997) TRF1, a mammalian telomeric protein. Trends Genet. 13: 21-26.
16. van Steensel, B. and T. de Lange. (1997) Control of telomere length by the human telomeric protein
TRF1. Nature 385: 740-743.
17. Bailis, J.M. and G.S. Roeder. (1998) Synaptonemal complex morphogenesis and sister-chromatid
cohesion require MEK1-dependent phosphorylation of a meiotic chromosomal protein. Genet. Dev.
12:3551-3563.
87
Influence of ATM Function on Telomere Chromatin Structure
Lubimor Smilenov, Sonu Dhar, and Tej Pandita
Ataxia telangiectasia represents one of the ideal models to study mechanisms of genomic
instability and carcinogenesis, as it is an autosomal disorder characterized by progressive
cerebellar degeneration, premature aging, growth retardation, gonadal atrophy,
immunodeficiency, high sensitivity to ionizing radiation, genomic instability, and cancer
predisposition. Cells derived from A-T individuals exhibit a variety of abnormalities in culture
such as a higher requirement for serum factors, hypersensitivity to ionizing radiation, and
cytoskeletal defects. These cells are defective in radiation-damage signal transduction pathways
operating through p53, and its target genes WAF1, cyclin E-Cdk2 and cyclin A-Cdk2 kinases, as
well as the retinoblastoma protein. Primary fibroblasts from humans and mice with a defective
ATM gene grow slowly in culture and appear to undergo premature senescence in culture.
Cells derived from A-T individuals show a prominent chromatin defect at chromosome
ends in the form of chromosome end-to-end associations also known as telomeric associations
(TA) and are seen at G1, G2 and metaphase. Chromosome end associations correlate with
genomic instability and carcinogenicity, and involve telomeres. Telomeres contain both DNA
and protein that together appear to stabilize the ends of eukaryotic DNA. Specifically, they are
composed of characteristic repetitive DNA that protects chromosome ends from exonucleolytic
attack, fusion, and incomplete replication. In yeast, non-telomeric DNA created by enzymatic
cleavage leads to genomic instability and cell-cycle arrest. Yeast telomeres have been shown to
exert a position effect on recombination between internal tracts of telomeric DNA. Human
telomeres are composed of 2 to 30 kb of tandemly arranged telomeric repeats with the sequence
(TTAGGG)n in the strand that runs to the 3' end of the chromosome. Telomeres shorten as a
function of age in cells derived from normal human blood, skin, and colonic mucosa. As a result
of this shortening, it is thought that critical genes at the ends of chromosomes either become
deleted or are activated, thus leading to growth arrest and subsequently to cell death.
Alternatively, silent senescence genes could become activated by removal of heterochromatic
regions. Recovery of proper telomere length by the activation of telomerase prolongs the lifespan
of a cell. Shortening of telomeres or telomere loss in a variety of cancers and immortalized cell
lines has been found to be the reason for the chromosome end associations that could be the
cause of genomic instability and gene amplification.
There is growing evidence suggesting that both the shielding of telomeric ends and their
elongation by telomerase is dependent upon telomere binding proteins. Mammalian telomeres
are packaged in telomere-specific chromatin. Human and mouse cell lines have their telomeric
tracts attached to the nuclear matrix, which is a proteinaceous subnuclear fraction. Telomere
length homeostasis in yeast requires the binding of a protein along the telomeric tract and
changes in the telomeric protein complex influence the stability of chromosome ends. In
mammals, a nuclear matrix binding site occurs at least once in every kb of the telomere tract.
These studies suggested that mammalian telomeres have frequent multiple interactions with the
nuclear matrix. Whether the ATM gene or downstream effectors influence the interaction of
telomeres with the nuclear matrix is not yet known. Since a chromatin defect in A-T cells is
88
pronounced at telomeres, we compared the interactions of telomeres with the nuclear matrix
among cells with normal and inactivated ATM function before and after treatment with ionizing
radiation.
What factors influence chromosome end association? One possible factor is loss or
shortening of telomeres; another is altered chromatin structure. In our previous studies, we
reported that the frequency of cells with chromosome end associations is higher in G1 phase
than in G2 phase followed by metaphase, and for each phase of the cell cycle, the frequency of
cells with end associations was significantly higher in A-T than in normal cells. It is probable
that the end associations seen at mitosis reflect a continuation of interphase chromosome
behavior, perhaps indicating interactions or linkages between chromosome ends and the nuclear
matrix. Since the telomeric signals are seen at the chromosome end association sites, it is
possible that in the absence of ATM function, the chromosome end associations are the
consequences of the failure of the nuclear matrix holding the telomeres together. The telomeric
signals at the chromosome end association sites in A-T cells suggest that chromosome end
associations could be the primary event that subsequently lead to the loss of telomeres.
Telomeric signals at the chromosome end association sites and changes in the frequency of cells
with chromosome end associations through the cell cycle raise the possibility that A-T cells have
an altered nuclear matrix, leading to defective interactions between telomeric DNA and the
nuclear matrix.
Telomeres are important components of chromosomes, as they have been implicated in
several cellular functions involved in aging and cancer development. They have been shown
cytologically as well as biochemically to be tethered to the nuclear matrix. The nuclear matrix is
a proteinaceous scaffold in the interphase nucleus, which is isolated by removing most of the
nuclear DNA and RNA, along with histones and loosely bound proteins. We determined whether
inactivation of ATM influences the interactions of telomeres with the nuclear matrix.
Telomere-nuclear matrix association
To characterize the nature of telomere anchorage to the nuclear matrix of different cell
types, plateau phase cells were processed by the LIS procedure and the resulting nuclear matrix
halos were cleaved with StyI. The nuclear matrix halos are the insoluble nonchromatin
scaffolding of the interphase nuclei. The nuclear remnant and associated DNA were isolated by
centrifugation and suspended in MWB; for genomic blotting analysis, equal volumes
representing DNA from the identical numbers of halos were fractionated side by side on 1.5%
agarose gels. The amount of telomeric sequence in each sample was determined by storage
phosphorimage analysis. The normal fibroblasts have about 52 to 60% of the telomeric DNA
associated with the nuclear matrix attached (P) fraction and 40 to 48% in the soluble (S) (free)
fraction (Table 1). Summation of the P and S values is equal to total telomeric DNA (T),
suggesting that no telomeric DNA was lost during the extraction procedure. In A-T cells
(GM2052), more than 92% of the telomeric DNA is attached to the nuclear matrix, whereas in
control cells (AG6234) about 52% is attached (Table 1). In another A-T cell strain (GM5823)
more than 95% of the telomeric DNA is attached to the nuclear matrix where as in control cells
(AG1522) about 60% is attached (Table 1). The ratio between the S and P fractions of telomeric
DNA is about 1:19 to 1:15.5 in A-T cells, compared to 1:1.5 to 1:1.2 in normal cells
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Table 1. Percentages of P and S fractions of telomeric DNA.
________________________________________________________________________
P
S
Ratio,
Cell strain
______________
____________
mean P/ mean S
Mean
Mean
(%)
(%)
A-T fibroblasts
GM5823
GM2052
95
92
5
8
19 : 1
11.5 : 1
Normal fibroblasts
AG1522
60
40
1.5 : 1
C21F
59
41
1.4 : 1
AG6234
52
48
1.1 : 1
________________________________________________________________________
Mean values of P and S were obtained from four experiments done separately.
(Table 1). These results suggest that the major portion of telomeres in A-T cells is associated
with the nuclear matrix.
To determine whether altered interactions of telomeres with the nuclear matrix are due to
ATM function, we examined isogenic cells with and without normal ATM function. We found
RKO cells expressing dominant-negative fragments have 87% of telomeric DNA in the P
fraction and 13% in the S fraction, whereas parental RKO cells have 71% in the P fraction and
29% in the S fraction (Table 2). When ratios between means of P and S were determined (Table
2), and compared between RKO and RKOFB2F7 (with expression of dominant-negative
fragment) cells, it was found that RKOFB2F7 cells have 2.73 fold higher ratio than RKO cells,
suggesting that inactivation of ATM influences the telomere associations with the nuclear
matrix. However, the difference in P values of RKO cells with and without expression of
dominant negative ATM fragment was lower than the differences in P values between primary
cells derived from A-T and normal individuals. The possible reason for this could be that RKO
cells are derived from tumors and thus may have other factors that could partly rescue the ATM
phenotype. Further, we determined whether wild type ATM could correct the altered interactions
between telomeres and the nuclear matrix by examining A-T (AT22IJE-T) cells with and
without expression of the wild-type ATM protein. Differences in the values of P and S were
distinct between the parental cell line (ATT221JE-T) and the derivative cells with the wild type
ATM gene (AT221JE-TpEBS7-YZ5) (Table 2). AT221JE-TpEBS7-YZ5 cells with a wild-type
ATM gene have lower amounts of P fraction as compared to P fraction of AT221JE-TpEBS7
cells that contain an empty vector. These results reveal that the expression of the wild type ATM
gene in A-T cells restored the normal telomere nuclear-matrix interactions, as is evident by the
decrease in the amount of the P fraction (Table 2).
90
Table 2. Percent of attached (P) and soluble (S) fractions of telomeric DNA.
Cell strain
RKO
RKOpBABEpuor
RKOFB2F7
P
_____________
Mean
(%)
S
____________
Mean
(%)
Ratio,
mean P/mean S
71
72
87
29
28
13
2.45 : 1
2.57 : 1
6.69 : 1
AT22IJE-T
83
17
4.88 : 1
AT22IJE-T pEBS7
84
16
5.25 : 1
AT221JE-Tp
67
33
2.03 : 1
EBS7-YZ5
________________________________________________________________________
Mean values of P and S are obtained from three experiments done separately. P stands for the
fraction of telomeric DNA attached to nuclear matrix and S stands for telomeric DNA in soluble
fraction. Note that RKOFB2F7 have a higher percentage of P compared to its parental RKO
cells. In contrast, AT221JE-TpEBS7-YZ5 have a lower percentage of P compared to its parental
AT221JE-T cells. Differences in P values between RKO and RKOFB2F7, and between
AT221JE-T and AT221JE-TpEBS7-YZ5, are significant.
Influence of ionizing radiation on telomere-nuclear matrix associations
Since gamma irradiation triggers telomere associations in A-T cells, it was important to
determine the effects of ionizing radiation on the interactions of telomeres with the nuclear
matrix. Plateau-phase cells were treated with a dose of 5 Gy of ionizing radiation, and
proportion of S and P fractions were determined. No change in the ratio of S versus P fractions
of telomeric DNA was seen immediately after treatment with ionizing radiation in either the
control or the A-T cells. However, an increase in telomeric DNA in the S fraction was seen in AT cells 1 h after treatment, whereas no such change was found in normal cells. The ratio of S
versus P fractions of telomeric DNA changed from 1:19 at 0 min to 1:5 at 60 min postirradiation. Since we did not see any change in this ratio of telomeric DNA in normal cells 1 h
postirradiation, we wished to determine whether there are any changes immediately after
irradiation. Therefore, we examined the S and P fractions of telomeric DNA in normal cells at 0,
15, 30 and 60 minute postirradiation and found no differences. These observations suggest that
the interactions of telomeric DNA in normal cells are not influenced by exposure to 5 Gy of
gamma rays.
Telomere nuclear matrix interactions are altered in germ cells
Telomeres are attached to the nuclear matrix of somatic cells. ATM function influences
the interactions of telomere with the nuclear matrix, however, it is not known whether
inactivation of ATM influence such alteration in germ cells. Since such interactions may
influence meiotic telomere mobility, we set out to characterize nuclear matrix/telomere
91
interactions in spermatocytes derived from Atm null and control mice. To characterize the nature
of telomere anchorage in spermatocytes obtained from Atm null and control mice,
leptotene/zygotene cells were collected by elutriation and processed according to the LIS
procedure. The resulting halos were cleaved with StyI and probed with telomere TTAGGG
repeats. It was found that normal mice had about 50% of the telomeric DNA repeats associated
with the nuclear matrix (P) with 50% in the soluble fraction (S). In contrast to normal mice,
spermatocytes of Atm null mice had more than 89% of the telomeric DNA repeats associated
with the nuclear matrix and only 11% in the S fraction. The ratio between the fractions of
soluble versus nuclear matrix attached telomeric DNA is about 1:8 in spermatocytes of Atm null
mice as compared to 1:1 in normal mice. These results suggest that the major portion of telomere
repeats in Atm null spermatocytes remain associated with the nuclear matrix, and that ATM
influences the telomere nuclear matrix interaction in both the somatic as well as germ cells.
Telomere Repeat Binding Factors
To determine whether the abnormalities in telomere nuclear matrix interactions seen in
A-T cells are correlated with alterations in telomere binding factors, we first analyzed the
expression of telomere repeat binding factors (TRF1 and TRF2) in A-T fibroblasts. We used the
RT-PCR approach to determine the expression of TRF1 and TRF2 and found comparable levels
of expression of TRF1 and TRF2 in A-T and normal control cells. Although the expression of
TRF1 and TRF2 were identical between A-T and control cells, it is possible that mutations in
these genes could lead to altered interactions of telomeres with the nuclear matrix. Therefore, we
carried mutational analysis of TRF1 and TRF2 genes in A-T and control cells. Analysis of TRF1
and TRF2 cDNA in A-T cells by cold SSCP protocol detected no mutations.
To test whether TRF1 and TRF2 were localized correctly in the A-T cells, we performed
the immunostaining of the cells and found that both proteins were localized in the nuclei of both
A-T and control cells. These observations suggest that alterations in the structure or expression
of TRF1 and TRF2 are not the cause for altered interactions of the telomeres with the nuclear
matrix in A-T cells.
In an attempt to identify gene products that might be involved with the altered
interactions of telomeres with the nuclear matrix in A-T cells, we used the ATLAS cDNA
microarray to analyze the expression of genes. The expression profiles of primary fibroblasts of
A-T and normal control were compared using polyA+ RNA for synthesizing 32P labeled cDNA,
subsequently hybridized separately to array membranes. No significant differences in the
expression of the 588 genes on the array were found between A-T and normal control cells.
How ATM influences the interactions of telomeric DNA with the nuclear matrix is not
clear at present. There is growing evidence which suggests that both the shielding of telomeric
ends and their elongation by telomerase is dependent upon telomere binding proteins.
Mammalian telomeres are packaged in telomere-specific chromatin. Our study shows that
telomeres in somatic as well as germ cells are associated with the nuclear matrix. However, we
found a significant difference in the ratio of the P versus S fractions of telomeric DNA between
A-T and normal control cells. This difference could be attributed to alterations in the interactions
between telomeric DNA and the nuclear matrix. The present results are consistent with the
92
hypothesis that the telomere nucleoprotein structure or nuclear matrix structure is different in AT cells. The fact that telomere binding to the matrix is greater in A-T cells and is specifically
influenced by irradiation shows that changes in telomere-matrix association could be involved in
the chromosome-destabilizing function of the ATM gene. The role of ATM function in telomere
nuclear matrix interactions is further strengthened by the fact that cells expressing dominantnegative fragment of the ATM gene have altered telomere nuclear matrix interactions. The
altered telomere nuclear matrix interactions seen in A-T cells were reversed by expression of the
wild type ATM gene. Further, a major portion of telomere repeats in Atm null spermatocytes are
also abnormally associated with the nuclear matrix. An influence of the ATM gene product on
the interactions of telomeres with the nuclear matrix might be an important modulator of cellular
processes influencing cellular senescence and cellular transformation.
Our studies demonstrate that the altered telomere nuclear matrix interactions seen in A-T
cells could be because of the absence of normal ATM protein. Genes involved in signal
transduction could influence chromatin structure and that may explain the basis of the cell-cycle
checkpoint defect in A-T cells and the prevalence of chromosome damage. Since a chromatin
defect in A-T cells is pronounced at telomeres, their interactions with the nuclear matrix may
influence chromatin structure and thus the function of neighboring genes.
93
Expression of the Catalytic Subunit of Telomerase in Developing
Brain Neurons: Evidence for a Cell-Survival-Promoting Function
Weiming Fu, Michael Killen, and Carsten Culmsee (all from Sanders Brown Research
Center on Aging, University of Kentucky, Lexington), Sonu Dhar, Tej K. Pandita, and
Mark P. Mattson (SBRCA)
Telomerase, a specialized reverse transcriptase linked to cell immortalization and
cancer, has been thought not to be expressed in postmitotic cells. We now report that
telomerase activity, and its essential catalytic subunit telomerase reverse transcriptase
(TERT) are expressed in neurons in the brains of rodents during embryonic and early
postnatal development. Telomerase activity was detected using PCR-based TRAPELISA assay in brain tissues from embryonic (E18) and early postnatal (P1 and P7) rats,
and was markedly decreased in adult brains. Immunoblot analysis showed that TERT was
expressed at relatively high levels in embryonic and early postnatal hippocampus and
cerebral cortex with no detectable TERT being present in adult brains. TERT
immunoreactivity was present in neurons throughout the developing brain, with
particularly high levels being present in hippocampal pyramidal neurons and many
cortical neurons. By comparison, TERT immunoreactivity was very weak or absent in
cells in the adult brain.
In order to explore roles for telomerase in neurons, we employed cell cultures
from the hippocampus and neocortex of embryonic day 18 rats and mice which are
highly enriched in differentiated postmitotic neurons. TERT immunoreactivity was
present at high levels in neurons in these cultures, where it was localized in both
cytoplasmic and nuclear compartments in the cell body, TERT immunoreactivity was also
present in neurites. Levels of TERT immunoreactivity appeared to be much greater in
neurons than in astrocytes, consistent with a primarily neuronal localization in the
developing hippocampus in vivo. The vast majority of telomerase activity measured by
the TRAP assay in vivo and in culture likely reflects a neuronal rather than a glial source
because very few glial cells are present in the hippocampus and cortex of E18 embryos,
whereas high numbers of glial cells are present in these brain regions in the adult.
Suppression of TERT expression in embryonic hippocampal and cortical neurons
in culture increases their vulnerability to apoptosis and excitotoxicity, and overexpression
of TERT in neural cells suppresses apoptosis induced by trophic factor withdrawal.
TERT exerts its anti-apoptotic action at an early stage of the cell-death process prior to
mitochondrial dysfunction and caspase activation. TERT may serve a cell-survivalpromoting function during development of the nervous system, and is a potential target
for therapeutic intervention in neurodegenerative conditions involving apoptosis and
excitotoxicity.
94
The Human Homologue of Schizosaccharomyces pombe Rad9
Protein, HRAD9p, Interacts with Bcl-2/ Bcl-xL and Promotes
Apoptosis
Howard Lieberman, Haiying Hang, Kevin Hopkins, and Wei Zheng, in collaboration with
Kiyoshi Komatsu (University of South Florida College of Medicine, Tampa), Toshiyuki
Miyashita (National Children’s Research Center, Tokyo, Japan), Sandy Cuddeback (Univ.
of South Florida College of Medicine), Massao Yamada (National Children’s Research
Center, Tokyo), and Hong-Gang Wang (Univ. of South Florida College of Medicine)
DNA damage induces apoptosis through a Bcl-2 suppressible signaling pathway,
but the mechanism is unknown. Sequence comparisons have identified up to four
evolutionarily conserved domains within Bcl-2 family proteins, called Bcl-2 homology
(BH) domains: BH1, BH2, BH3 and BH4. The conserved BH3 domain in particular is
found within many of the pro-apoptotic Bcl-2 family members and is critical for binding
anti-apoptotic proteins as well as for pro-apoptotic activity. The human cell-cycle
checkpoint control protein HRAD9p was found to contain a BH3-like region near its Nterminal (Fig. 1). Furthermore, HRAD9p was capable of interacting with the antiapoptotic proteins Bcl-2 and Bcl-xL but not with the pro-apoptotic proteins Bax or Bad, as
demonstrated by both yeast two-hybrid and co-immunoprecipitation studies. When
overexpressed in mammalian cells, HRAD9p induced apoptosis that can be blocked by
Bcl-2 or Bcl-xL overexpression. These results indicate a novel function for HRAD9 in
regulating apoptosis, in addition to its previously described checkpoint control and other
radioresistance-promoting activities.
Figure 1. Alignment of amino acid residues in the BH3 homology regions of Bcl-2 protein family members
and HRAD9p. Comparison of the BH3-like regions in HRAD9p and Bcl-2 family members (human Rad9,
accession number U53174; human Bax, L22474; human Bak, U23765; mouse Bid, U75506; human Bik,
U34584; human Hrk, U76376; human Bad, AF021792; human Bim, AF032457; C. elegans Egl-1,
AF057309). Amino acids that match the BH3 domain consensus (PS01259) are darkly shaded.
95
MOLECULAR STUDIES
ORIENTED TOWARDS CANCER
Identification of THG1 as a Potential Suppressor of Testicular
Tumorigenesis
Yuxin Yin
Testicular carcinomas are the most common cancers in young men, but the
mechanisms of the initiation of testicular tumors are largely unknown. This group of
tumors has never been found to contain mutations of the p53 tumor suppressor gene,
although p53 is the most commonly mutated gene in human cancers and p53-deficient mice
spontaneously develop tumors, including testicular teratocarcinoma, at a very high
incidence. Therefore, there must be genetic alterations of some other important genes in
the p53 pathway, which may contribute to development of testicular tumorigenesis. One of
our current projects is set to explore the mechanisms involving testicular tumorigenesis and
to search for clues as to how the p53 pathway is dysfunctional in this group of tumors. We
have previously developed a p53 inducible system in which the p53ER fusion protein,
human p53 protein fused to the hormone binding domain of the human estrogen receptor, is
transcriptionally activated in the presence of estrodial. This p53 inducible system,
expressed in a p53-deficient human cancer cell line, H1299, named Hp53ER, allowed us to
demonstrate the importance of p53 as a transcriptional regulator in cell-cycle arrest and
apoptosis. We have been utilizing this system to isolate p53-regulated genes by PCR select
cDNA subtraction, in which two cDNA libraries were made from cells with and without
functional p53 induced by estrodial for subtraction. We have recently identified a human
testis-specific gene with a homeobox domain, designated THG1 for testis homeobox gene
1, which encodes a homeobox protein of 174 amino acids. THG1 transcripts are only
detected in human and mouse testes and THG1 protein is expressed in germ cells.
Importantly, there is no expression of the THG1 transcripts in human testicular tumor cell
lines containing wild-type p53. We have generated a polyclonal antibody against human
THG1 for Western analysis of THG1 expression. The THG1 protein migrates to the
position of around 28 kDa in 15% acrylamide gels. The level of THG1 protein is
significantly increased in Hp53ER cells in the presence of 17b-estrodial that presumably
activates p53 transactivity (data not shown), suggesting that THG1 is upregulated by p53.
We have carried out a series of experiments to determine whether THG1 is important in
testicular tumorigenesis. We found that THG1 regulates the transactivity of p53, and
overexpression of THG1 inhibits the growth of some tumor cellls.
It is well known that MDM-2 is regulated by p53 transcriptionally, and MDM-2
influences p53 transactivity. We were also interested in examining if THG1 has a similar
influence on p53. To determine whether THG1 effects p53 transactivity, we introduced a
mammalian vector expressing human THG1 into Tera-2 cells that contain wild-type p53.
Stable cell lines containing THG1 or an empty neo vector were isolated by neomycin
selection and confirmed by Western blotting for THG1 using the specific anti-THG1
antibody. Transactivity of p53 was determined by luciferase reporters driven by the
promoters of p21, Bax and MDM2, which are known p53-target genes. Tera-2 cells with
or without THG1 were transfected with reporters and then exposed to UV light to induce
97
p53 transactivity. Figure 1 shows that luciferase activity of the p21 reporter is low in the
Tera2/neo cell line after UV irradiation. In Tera2/THG1 cell line, Tera-2 cells containing
THG1, the luciferase activity of the p21 promoter after induction of p53 by UV is increased
more than four fold. However, luciferase activity of the Bax promoter is reduced 3 to 4
fold in Tera2/THG1 cells after UV irradiation. These results suggest that p53 transactivity
is differentially regulated by THG1. THG1 may positively influence induction of p21 by
p53 but negatively influence transcriptional regulation of Bax by p53 when Tera-2 cells
were exposed to UV irradiation.
To explore the mechanism involving THG1 in p53 transactivity, we used two types
of MDM-2 luciferase reporters, indicated as MDM2 (known as pCOSX1-GL2) and
MDM2-p100 (known as pBP100-GL2), which contain different promoter sequences
upstream of the luciferase gene. The MDM2 reporter contains a genomic sequence
flanking three exons of the MDM-2 gene, in which a p53-responsive element is located
near exon 2. MDM2p100, on the other hand, contains only a100 bp promoter sequence
which includes the p53 responsive element. As shown in Figure 1, following transient
transfection, the luciferase activity of the MDM-2 reporter was decreased in Tera-2 cells
expressing THG1, compared with that in Tera-2 without THG1, suggesting that THG1
inhibits transactivation of the MDM-2 promoter. The luciferase activity of the MDM2P100, however, was not changed in the presence of THG1. These data indicate that there
might be additional sequences required beyond the p53 consensus sites for THG1 to
influence p53 transcriptional activity, and that THG1 may regulate p53 transactivity
through binding these promoter sequences.
Since THG1 is not expressed in Tera-2 cells, we examined whether ectopic
expression of THG1 had any effect on cell growth. We tested this possibility by
performing a colony formation assay in which Tera-2 cells were transfected with a CMVdriven THG1 vector, pcDNA3/THG1, or with pcDNA3 as control. These cells were
selected by G418 for resistant colonies. As shown in Figure 2, the number of colonies
formed following G1 transfection is significantly lower than that in the control group
transfected with an empty vector (left panel). We performed similar experiments with
other tumor cell lines. Next, we examined HT 29 and DLD1 cells, two human colon
cancer cell lines containing mutant p53. We also transfected wild-type p53 into these cells
for the colony formation assay. We found that THG1 inhibits colony formation as
efficiently as p53 in HT29 cells (middle panel), indicating that THG1 imposes its inhibitory
effect on tumor cells without the requirement of wild-type p53. Furthermore, the
combination of p53 and THG1 results in even greater inhibition of colony formation of
these cells (Fig. 2, middle panel). Similar effect of THG1 expression on DLD1 was also
observed (right panel). These data suggest that expression of THG1 inhibits cell growth in
these tumor cells, and that THG1 plays a cooperative role in p53-mediated cell growth
control.
Another ongoing project in the lab is to study the gene expression pattern in
tumorigenic human bronchial cells using genechip technology. We have finished cDNA
synthesis and we are preparing for biotin-labeling of cRNA samples.
98
Figure 1. Regulation of the promoters of p53-target genes by p53 and THG1. Tera2/THG1 cell line is a
stable clone containing pcDNA3/THG1 plasmid. Tera2/neo cell line contains the pcDNA empty vector only
as a control. These Tera-2 cells with or without THG1 were transiently transfected with the luciferase
reporters for the promoters of the indicated genes as well as with b-galactosidase for normalization. The
transfected cells were exposed to UV light (20J/m2) and incubated for two hours before harvesting for
luciferase assay. Luciferase was performed using a chemiluminscent reporter gene assay system (TROPIX).
The procedures were followed according to the manufacturer's protocol for the combined detection of
luciferase and β-galactasidase. The data presented are the average of three independent experiments of
duplicate cultures.
Figure 2. Inhibition of colony formation of tumor cells by THG1. Exponentially growing tumor cells
were plated at equal number (3x105 cells/100 mm dish) and transfected with either pcDNA3 as a control,
pcDNA/THG1 indicated as THG1, pCMVp53 indicated as p53, or a combination of the two vectors as
indicated. G418 selection was performed two days after transfection. Colony formation was determined as
more than 100 cells/colony. The number of stained colonies in each group was counted for statistical
analysis. The results were presented as the mean +/- standard deviation of three independent experiments in
which there were five dishes of cells per group. The results in panels were from: Tera-2 (left panel); HT29
(middle panel); DLD1 (right panel).
99
Molecular Mechanism of Radiation-Induced Transformation of
Human Bronchial Epithelial Cells by High-LET Radiation
Yong Zhao, Chang Piao, and Tom Hei
Carcinogenesis is a multi-stage process with accumulating genetic and epigenetic
alterations during progression (1). Analyses of gene mutation or expression in a series of
tumor cell lines induced by carcinogens is often the first step towards gaining an insight
into the genetic alterations of a particular cancer. Among all known human carcinogens,
radon is an important etiological factor for human lung cancer. However the molecular
mechanism of radon carcinogenesis is not clear. The well-established tumorigenic human
bronchial epithelial cell lines provide a useful model to study the mechanism involved in
human bronchial carciongenesis induced by radon α particles (2). These BEP2D cells
which are immortalized by human papillomavirus become tumorigenic and produce
progressively growing subcutaneous tumors upon inoculation into nude mice after
exposure to α particles. However, BEP2D cells are anchorage dependent and nontumorigenic even in late passage. The data suggest that abnormal p53 and Rb functions
are not sufficient criteria for tumorigenic development and additional genetic changes are
needed. In order to elucidate the genetic events involved in the transformation of BEP2D
cells induced by α particles, we simultaneously analyzed the expression of 588 cellular
genes, which represent about 3% of total transcripts (e.g. 20,000 genes) expressed in a
single cell using a cDNA expression array.
For screening the profile of gene expression using cDNA expression array
(Clontech, Palo Alto, CA.), total RNA from both tumorigenic and control BEP2D cells
were isolated using Trizol Reagent (Gibco) followed by DNaseI treatment to remove any
contaminating DNA from RNA samples. PolyA+ RNA was then isolated and from which
[α-32P]dATP-labeled cDNA probes were generated by Reverse Transcription. The
purified cDNA probes were then hybridized to the array nylon membranes and the
hybridization patterns were analyzed by autoradiography and quantified by
phosphorimaging . As shown in Figure 1, the Clontech cDNA array contains six
quadrants which covers different categories of genes, including oncogenes, tumor
suppressor genes, intracellular signal transduction modulators, DNA synthesis and repair
genes, transcription factors, and receptors and growth factors genes. Each cDNA of the
588 preset genes were spotted in duplicate on the membranes. The hybridization signals
at the bottom of the genes were various housekeeping genes used as positive controls.
The differences of gene expression between two cell lines are illustrated in Table 1. A
total of 11 genes were found to be differentially expressed. Meanwhile, we used Northern
blotting method to screen the expression level of mRNA between the two cell lines. The
differential expression of two of these 11 genes (Fra1, Ets-like gene) could not be
confirmed by Northern blotting. Among these differentially expressed genes, DCC
(deleted in colon cancer), p21cip1, hsp27, and cytokeratin14 were being intensively
studied using different cell lines including early passage cells from one week postirradiation (lane 2), late-passage cells prior to their injection into nude mice (lane 3), two
100
primary tumor, two second tumor and one tertiary tumor cell lines (lanes 4-8). The results
from Northern blot analysis are shown in Figure 2. The expression of p21cip1 in early
passage cells was ~2-fold higher than control BEP2D cells. Among late passage and
tumor cells, the average expression levels of p21cip1 were ~4-fold lower than that of the
control BEP2D cells (lanes 3-8). The p21cip1 is a cyclin-dependent kinase inhibitor,
which can effectively inhibit cdk2, cdk4 and cdk6 kinase, and is capable of inducing cell
cycle arrest in G1 when overexpressed. Downregualtion of p21cip1 has been shown to
increase kinase activity and promote cell cycle progression and cellular proliferation (3),
which suggest that p21cip1 may play an important role in malignant progression induced
by radiation. DCC expression had no significant difference between early passage and
control BEP2D cells, but lowered by ~2 fold in late passage and tumor cells compared
with control. DCC is a candidate tumor suppressor gene that is postulated to function as a
trans-membrane receptor. Frequent allelic and loss of its expression has been seen not
only in colorectal cancer, but also in a number of other malignancies (4). Some of the
strongest evidence supporting a role for DCC as a tumor suppressor gene has been
obtained from the studies showing suppression of tumorigenecity in nude mice by
expression of full-length, but not truncated, DCC in nitrosomethylurea- (NMU)
transformed keratinocytes lacking DCC expression (5). When DCC cDNA was
transfected into tumor cells, apoptosis and G2/M cell cycle arrest are induced by the
activation of caspase-3 and inhibition of cdk1, suggesting a possible mechanism by which
DCC suppresses tumorigenesis (6). Our findings support these results and suggest that
loss of DCC tumor suppressor expression may be involved in the tumorigenic progression
in α-particle-treated BEP2D cells. Both p21cip1 and DCC genes were found to be downregulated in late passage and tumor cells, which means that by successive passaging and
clonal selection, the cells with lower expression of p21cip1 and DCC predominantly
grow in late passage and progressively form tumor when inoculation into nude mice.
This suggests that p21cip1 and DCC are two important genes involved in carcinogenic
process induced by α particles. Expression of both the hsp27 and cytokeratin14 was not
significantly different among early and late passage cells compared with control cells.
However, in tumor cells (lanes 4-8) there was ~5-fold decrease in expression as compared
to control cells. These findings suggest that hsp27 and cytokeratin14 may serve as
diagnosis markers of tumorigenicity of human bronchial epithelial cells.
Table 1. List of differentially expressed genes between tumorigenic and control BEP2D cells
Gene
cDNA Array
Northern Blot
DCC tumor suppressor
DNA-dependent protein kinase
Heat shock protein 27
Cytokeratin 14
Integrin β-4
Alpha-catenin
p21cip1
Glutathione-S-Transferase
cdc-25B (M-phase inducer)
Ets-like gen
Fra1
↓
↓
↓
↓
↓
↓
↓
↑
↑
↑
↑
↓ 2 fold
↓ 2.3 fold
↓ 5 fold
↓ 5 fold
↓ 2 fold
↓ 2 fold
↓ 4 fold
↑ 2 fold
↑ 3 fold
No change
No change
101
Figure 1. Differential gene expression in a representative tumorigenic BEP2D cells
induced by a single 60-cGy dose of alpha particles (T5L2P, right hand panel) relative to
control BEP2D cells (left-hand panel). The labeled cDNAs were hybridized to the array
overnight at 65oC. The image was captured on X-ray film after exposure for 2 days at
80oC.
Figure 2. Northern blot analysis showing the relative levels of p21cip1, DCC, HSP27 and
Cytokeratin14 in control BEP2D cells (lane 1), irradiated BEP2D cells one week postirradiation (lane 2), irradiated cells just before inoculation into nude mice (lane 3) and 5
representative tumor cell lines (lanes 4 through 8). The mRNA blot (p21cip1 and DCC)
and total RNA blot (HSP27 and cytokeratin14) were separately hybridized to 32P-labelled
human probe. The relative abundance of RNA per lane was judged to be similar based on
the β-actin level.
No figures available.
REFERENCES
1. Fearon , et al. Cell 61:759-767, 1990.
2. Hei, et al. Carcinogenesis 15(3):431-437, 1994.
3. Crow, et al. Cell Growth Differ. 9(8):619-627, 1998.
4. Cho et al, Curr Opin. Genet. Dev. 5(1):72-78, 1995.
5. Klingelhutz, et al. Int. J. Cancer 53:382-387, 1993.
6. Chen et al., Oncogene 18(17):2747-2754, 1999.
102
Expression of Transforming Genes and Allelic Imbalance in Human
Breast Epithelial Cells Induced by High-LET Radiation
Debasish Roy, Gloria Calaf, and Tom Hei
Breast cancer is the most common female cancer and is showing an alarming yearly
increase. The mean 5-year survival rate of patients is about 60%, although less aggressive forms
should be distinguished from those that rapidly metastasize. It varies greatly according to the age
at onset, the clinical features, the histological characteristics, and the genetic context. These
differences derive from a complex interplay involving exogenous factors (socioeconomic
situation, diet, breast irradiation, oral contraception, geography), as well as endogenous factors
(hormonal imbalance, mastopathies, family history of breast cancer).
There is evidence that a complex and heterogeneous set of genetic alterations are
involved in the etiology of breast cancer. It is believed that breast cancer, like most other cancers,
has its origin in one cell, which, after going through a number of different mutational events,
becomes malignant. Later selective events lead to the development of clones with different
growth characteristics. Although relatively few genes have been found to be mutated in breast
cancer, there are a large numbers of chromosome arms with as yet unidentified genes are known
to be affected. These aberrations include both amplification of oncogenes (MYC, ERBB2, and
CCND1, etc.) and mutations or deletions of tumor suppressor genes ( TP53 and CDH1, etc.)
through microsatellite instability (MI) or loss of heterozygosity (LOH) at chromosomes 1, 3p, 6q,
7q, 8p, 9p, 10q, 11, 13q, 16q, 17, 18q, 22q, and X. The emergence of MI may involve defects in
DNA replication or mismatch repair (MMR) mechanisms, whereas LOH may correspond to
losses or inactivation of TSGs.
Until recently, there have been very few human cell models available for the study of
radiation carcinogenesis. For an accurate risk assessment of human exposure to ionizing
radiation, mainly the high-LET radon alpha particle, and to have a better understanding of the
molecular mechanisms for radiation-induced human breast carcinogenesis, an in vitro model was
established in our laboratory, consisting of spontaneously immortalized, non-tumorigenic human
breast epithelial (MCF-10F) cells. Using this model, we herein report the genetic changes that
were identified during the course of the malignant transformation process induced by alpha
particles.
In pursuit of studying the molecular mechanisms, we examined the incidence of genomic
instability in transformed, non-tumorigenic MCF-10F cells irradiated with 60/60 cGy doses of
alpha-particles using two different approaches. We examined MI/LOH of genes located on
chromosome 6 and 17 that are known to be affected frequently in breast cancer, and identified
differentially expressed oncogenes/tumor suppressor genes using cDNA expression array. The
tumorigenic MCF-10F-BP1Tras (Clone 20) and MCF-7 were used as positive controls for the
MI/LOH study. Eight microsatellite markers were used from each chromosome with a map
position near known tumor suppressor genes, oncogenes or other cancer-related genes which
involved cell-cycle regulation, DNA replication, DNA repair or signal transduction protein
genes.
103
The results from the PCR-SSCP (Polymerase Chain Reaction - Single Strand
Conformational Polymorphism) analysis indicated that during the early events of radiation
carcinogenesis there was a frequent allelic imbalance either by MI or LOH (Table -1) occuring at
some specific loci of these chromosomes. These findings were consistent with results obtained
using cDNA expression array (Fig.1), and supported the notion that even at the early stages of
post-radiation treatment, there were some specific inherent genomic alterations within the cell.
These observations supported the concepts that alterations in the mismatch repair (MMR)
process and a simultaneous triggering of the oncogenic potential by radiation treatment can lead
to the generation of some altered signals which may be responsible for the neoplastic
transformation of the cell.
Table 1. Frequent allelic imbalance in chromosome 6 and 17.
MARKERS
D6S220
D6S264
D6S281
D6S355
MAP
POSITION
6q25.2-q27
6q25.2-q27
6q27
6q23.3-q25.2
D6S87
6q23
ESR
6q24-q27
IGF2R
6q25-q27
UTRN
17q11.2-q12
D17S513
17p13
D17S520
D17S579
17p13
17q12-q21
D17S849
17p13.3
THRA1
TP53
10F 60/60α
α
6q24
D17S250
D17S1322
MCF10F
17q21
17q11.2-q12
17p13.1
= Complete Deletion
= LOH
= MI
= No Change (remain the same as control MCF-10F)
104
10FBP1Tras
MCF-7
Fig. 1. Differential expression of eight oncogenes/tumor suppressor genes. C =
Control MCF-10F, E = MCF-10F treated with 60/60 cGy alpha particles.
No figure available.
105
Aberrant Hypermethylation of the 14-3-3σ Gene is Associated with
Gene Silencing in Breast Cancer
Anne T. Ferguson, Ella Evron, and Christopher Umbricht (all from Johns Hopkins Oncology
Center, Baltimore, MD), Tej K. Pandita, Heiko Hermeking (JHOC), Jeffrey Marks and
Andrew Futreal (Duke University Medical Center, Durham, NC), Martha R. Stampfer
(Berkeley National Laboratories, CA), and Saraswati Sukumar (JHOC)
While many studies have identified critical genetic and epigenetic changes that
mark the transformation of cells in tissues such as colon, pancreas, and lung, similar
studies in breast cancer have met with limited success. Here, we report the identification of
a gene, 14-3-3σ, whose expression is lost in 94% (45/48) of breast tumors. 14-3-3σ was
originally identified as an epithelial-specific marker, HME1, which was downregulated in a
few breast cancer cell lines but not in cancer cell lines derived from other tissue types.
Later studies showed that 14-3-3σ protein (also called stratifin) was abundant in
differentiated squamous epithelial cells, but decreased by 95% in SV40-transformed
epithelial cells and in primary bladder tumors.
We investigated the molecular mechanism underlying the low expression of 14-33σ in breast cancers. We find that genetic alterations such as loss of heterozygosity and
intragenic mutations are not major contributing mechanisms for lack of 14-3-3σ
expression, because loss of heterozygosity at 14-3-3σ were rare (1/20 informative cases)
and no mutations were detected (0/34). Instead, we show that hypermethylation of the
CpG-rich region in the 14-3-3σ gene is associated with its transcriptional silencing in the
majority of breast tumors. We verified this finding by Northern blot analysis. Remarkably,
14-3-3σ mRNA was undetectable in 45 of 48 primary breast carcinomas. On the other
hand, hypermethylation of CpG islands in the 14-3-3σ gene was detected in 91% (75/82) of
breast tumors and was associated with lack of gene expression. Treatment of breast cancer
cell lines that do not express 14-3-3σ with the DNA methyltransferase inhibitor, 5-aza-2’deoxycytidine (5-aza-dC), leads to partial demethylation of the CpG island and reexpression of 14-3-3σ mRNA. Thus, hypermethylation appears to be the principal
mechanism for silencing of σ gene expression.
Hypermethylated DNA is known to interact with at least one methyl-CpG-binding
protein, MeCP2, that forms a transcriptionally repressive complex with the histone
deacetylase and the transcriptional co-repressor, SIN3A. In fact, we found that treatment
of 14-3-3σ-negative cells with the histone deacetylase inhibitor, trichostatin A, also leads
to reactivation of the 14-3-3σ gene. Together, these results suggest that methylationmediated chromatin condensation is responsible for suppressing 14-3-3σ transcription in
breast cancer cells. Hypermethylation and loss of 14-3-3σ expression are the most
consistent molecular alterations in breast cancer identified so far. Consequently, 14-3-3σ
gene methylation may serve as a novel diagnostic marker and target for therapeutic
strategies.
106
Recent studies have shed light on the function of 14-3-3σ. It was identified as a
p53-inducible gene that is responsive to DNA damaging agents. 14-3-3σ apparently
sequesters the mitotic initiation complex, cdc2-cyclin B1, in the cytoplasm after DNA
damage. This prevents cdc2-cyclin B1 from entering the nucleus where the protein
complex would normally initiate mitosis. In this manner, 14-3-3σ induces G2 arrest, and
allows the repair of damaged DNA. Of note, we find that breast cancer cells that do not
express 14-3-3σ accumulate significantly more G2 type chromosomal aberrations than
cells that express 14-3-3σ. These results suggest that 14-3-3σ participates in a G2
checkpoint control in breast cells. We propose that loss of 14-3-3σ gene expression plays a
significant role in breast cancer, as it may facilitate the accumulation of genetic damage
conducive to malignant transformation.
In summary, 14-3-3σ-CpG island methylation is an epigenetic change that is largely
responsible for silencing of the gene and occurs in a majority of breast cancers. Loss of 143-3σ may play a role in the increased sensitivity of breast cancers to radiation therapy.
Further evaluation of 14-3-3σ gene methylation in tissue samples such as nipple aspirate
cells, fine needle biopsies, microdissected premalignant lesions like DCIS, and
pathologically negative sentinel lymph nodes may provide the foundation for its
development as a novel marker for early detection.
107
A More Robust Biologically Based Ranking Criterion for Treatment
Plans
David J. Brenner and Rainer K. Sachs (University of California, Berkeley)
In discussing the ranking of treatment plans based on estimated tumor control
(TCP) and normal tissue complication (NTCP) probabilities, it has often been pointed
out that the usual figure-of-merit score
S = TCP (1 - NTCP ),
(1)
which has been adopted by several authors (1-3), has some undesirable properties. For
example, changing the absolute value of the NTCP in two competing treatments plans
by the same proportion can sometimes change the relative ranking of the plans (4). This
is because, if Eq. 1 is used as the score for ranking plans, for a given value of TCP, a
small fractional change in NTCP (i.e., dNTCP/NTCP) is reflected in the fractional
change in the score as
dS
d NTCP  NTCP 

.
=−
S
NTCP  1 − NTCP 
(2)
The problem here is that the term in brackets in Eq. 2 implies that, when the absolute
value of NTCP is large, the score will be very sensitive to changes in NTCP, but when
the absolute value of NTCP is small, the score will be relatively insensitive to changes
in NTCP. Other suggested figures of merit, such as a ratio of biologically effective doses
(e.g., BEDtumor / BEDlate-responding tissue), suggested by Ling and Chui (5), or more complex
function of these BED's as suggested by Dale and Sinclair (6), are also prone to such
problems.
This situation can easily be remedied by using, for example, a figure-of-merit
score
R = TCP / NTCP.
(3)
In this case, a fractional change in the figure-of-merit score used for ranking treatment
plans depends only on fractional changes in TCP and/or NTCP, as
d R dTCP d NTCP
=
−
,
R
TCP
NTCP
i.e., independent of the absolute values of TCP and NTCP, as one would wish.
109
(4)
An extension of Eq. 3, which would allow for different relative weightings of
TCP and NTCP would be
q
R' = TCP / NTCP ,
(5)
where use of the weighting factor q (> 0) allows the physician's perspective on the
relative importance of NTCP and TCP to be quantified (3). As in Eq. 4, fractional
changes in R' depend only on fractional changes in TCP and NTCP.
(In fact, use of a reciprocal ranking score, 1/R or 1/R' - which would, of course,
be minimized rather than maximized - would have the same advantages in terms of
ranking as R or R', but might be more stable. This is because NTCP is generally small,
and small fluctuations in NTCP when it is in the denominator might cause unreasonably
large fluctuations in the ranking score itself).
In conclusion, a treatment-plan ranking score defined using Eq. 3 or Eq. 5 (or
their reciprocals) does not have the undesirable ranking properties which sometimes
appear when using the standard ranking score defined in Eq. 1. Thus R from Eq. 3, or
the more general R' from Eq. 5, or their reciprocals, are likely to represent more robust
ranking criteria than S from Eq. 1.
References
1. Leibel SA, Kutcher GJ, Harrison LB, et al. Improved dose distributions for 3D conformal boost
treatments in carcinoma of the nasopharynx. Int J Radiat Oncol Biol Phys 1991;20:823-833
2. Jain NL, Kahn MG, Drzymala RE, et al. Objective evaluation of 3-D radiation treatment plans: a
decision-analytic tool incorporating treatment preferences of radiation oncologists. Int J Radiat Oncol
Biol Phys 1993;26:321-333.
3. Amols HI, Zaider M, Hayes MK, et al. Physician/patient-driven risk assignment in radiation
oncology: reality or fancy? Int J Radiat Oncol Biol Phys 1997;38:455-461.
4. Langer M, Morrill SS, Lane R. A test of the claim that plan rankings are determined by relative
complication and tumor-control probabilities. Int J Radiat Oncol Biol Phys 1998;41:451-457.
5. Ling CC, Chui CS. Stereotactic treatment of brain tumors with radioactive implants or external
photon beams: radiobiophysical aspects. Radiother Oncol 1993;26:11-18.
6. Dale RG, Sinclair JA. A proposed figure of merit for the assessment of unscheduled treatment
interruptions. Br J Radiol 1994;67:1001-1007.
110
Tumor Heterogeneity and its Effect on Parameters Estimated
Using the Linear-Quadratic Model
David J. Brenner and Eric J. Hall
The standard linear-quadratic (LQ) approach (1-3) is now routinely used for
analyzing clinical data and, based on its use, typical α/β values are found of around 9-12
Gy for most tumors (1,4), and of around 2-5 Gy for late-responding normal tissue (1,4).
Recently, using this standard LQ approach, an α/β value for prostate tumor of 1.5 Gy
was found (5), in the range of those for most late-responding tissues. If, as we have
suggested (5), typical α/β values (i.e., the sensitivity to changes in fractionation) for
prostate cancers are indeed comparable to those from the surrounding late-responding
tissue, the consequences for prostate cancer radiotherapy would be far reaching -- favoring
small numbers of large fractions, or high dose rate brachytherapy.
The standard LQ model assumes homogeneity, i.e., that all the tumors in the clinical
data set have the same values of α and β. Of course this is not true, and it has been pointed
out many times that estimates of the α parameter increases markedly when heterogeneity is
accounted for. It is of interest, then, to see if our conclusions regarding α/β remain the
same when inter-tumor heterogeneity is taken into account.
We have confirmed that fully taking into account heterogeneity in α and in β does
indeed result in larger values of α and of β, but essentially unchanged values of α/β. This
was done by analyzing the same prostate tumor control data that we originally used (5)
with the standard LQ model, but with an extended model in which both α and β are
selected from independent Gaussian distributions (free parameters [α0, σα] and [β0, σβ]).
The prostate data were fitted to this fully heterogeneous LQ model by sampling many times
(10,000) from each Gaussian distribution (excluding negative values) at each iteration of
the fitting procedure.
The results for the estimated mean values of α/β are shown in Table 1 (second row).
Though the estimated mean values of α and of β were both considerably larger than from
the standard LQ model (they now refer to averages over a whole population, rather than
just those tumors that dominate the dose-cure relationship), the estimated value of the ratio
α/β changed very little (2.1 vs. 1.5 Gy) from our original estimate made (5) with the
standard LQ model.
This conclusion was not unexpected: Bentzen et al. (6) pointed out that fully taking
into account heterogeneity in the LQ model should result in an increased estimated α value,
but the best estimate of α/β should be essentially unchanged. This conclusion was
generalized by Dubray and Thames (7), who showed that, while estimates of individual
radiobiological parameters can be highly sensitive to heterogeneity effects, ratios of
parameters are far less sensitive.
111
Table 1. á/â estimates for prostate cancer, a “typical” tumor, and a “typical” late-responding tissue, as
estimated with the standard LQ model (row 1), an extension to the LQ model incorporating independent Gaussian
distributions for á and â (row 2), and a model incorporating the same (correlated) Gaussian distribution for á and
â (row 3).
Prostate*
Tumor control
(skin cancer)†
Late-responding normal
tissue (skin)‡
Standard LQ model
1.5 Gy
8.5
4.3 Gy
Fully heterogeneous LQ model:
independent Gaussian
distributions for α and β
2.1 Gy
9.5
3.9 Gy
Partially heterogeneous LQ
model: same (correlated)
Gaussian distribution for α and β
4.5 Gy
16.4
4.9 Gy
*Data for freedom from biochemical failure from Stock et al. (8) and Hanks et al. (9).
†Data for tumor control for human skin cancer (11), as analyzed by Thames et al. (4).
‡Data for telangiectasia (score ≥ 2 at 5 years) from Turesson and Thames (12), analyses including an
overall time correction.
It should be noted that, in the heterogeneous LQ model outlined above, α and β are
independent of each other and are governed by independent distributions. There are
heterogeneous models in the literature in which α and β can vary but not independently, so,
for example, a tumor with a high α would also have a high β. However, a strong
correlation between α and β is biologically rather implausible, because α and β reflect very
different types of cell killing mechanisms - α relates to cell killing from small-scale
deletions or insertions at the nanometer level, whereas β relates to exchange-type
chromosome aberration formation at the micrometer level, dominated by large-scale
chromosomal geometry (10).
If α and β were correlated, as Dubray and Thames (7) point out, heterogeneity could
indeed affect estimates of the α/β ratio. To investigate if this is so, we fit such a “partial”
heterogeneity model, in which α and β are correlated, to the same prostate cancer data set
(5) and indeed obtained an increased value of α/β (4.5 vs. 1.5 Gy). However, to compare
the α/β value for prostate cancer derived from this model with those for late sequelae or
those for other tumors, these latter also need to have α/β values estimated with this model.
This we have done for a couple of the classic data sets, one for tumor control and one for
late sequelae, from which “typical” α/β values were originally derived. What can be seen
from the results in Table 1 is that an LQ model that fully accounts for heterogeneity
produces essentially unchanged α/β values from the standard model; by contrast using a
partial heterogeneity model in which α and β are correlated, all estimated values of α/β
increase -- for prostate, for other “typical” tumors, and for late sequelae.
112
So with or without heterogeneity corrections, and even if α and β are correlated, α/β
for prostate is comparable to that for late-responding normal tissues, and is much smaller
than those for most other tumors.
Taking into account heterogeneity is certainly interesting although, as we have
shown, it can have its pitfalls, but the standard LQ model is often more informative in that
it focuses in on those radioresistant tumors that control the dose-cure relationship, which is
why, in standard LQ model analyses, the number of relevant clonogens is often very small.
Beyond models, however, is the fundamental biology. As Duschesne and Peters (13)
and ourselves (5) have pointed out, the central issue is consistency with what we know
biologically: Prostatic tumors typically contain very low proportions of proliferating cells,
and response to changes in fractionation tracks with cellular proliferative status, and so it is
not surprising that prostate tumors appear to respond to changes in fractionation like lateresponding normal tissues.
References
1. Fowler JF. The linear-quadratic formula and progress in fractionated radiotherapy. Br J Radiol 1989;
62:679-94.
2. Thames HD. An 'incomplete-repair' model for survival after fractionated and continuous irradiations. Int J
Radiat Biol 1985; 47:319-39.
3. Dale RG. The application of the linear-quadratic dose-effect equation to fractionated and protracted
radiotherapy. Br J Radiol 1985; 58:515-28.
4. Thames HD, Bentzen SM, Turesson I, et al. W. Fractionation parameters for human tissues and tumors. Int
J Radiat Biol 1989; 56:701-10.
5. Brenner DJ, Hall EJ. Fractionation and protraction for radiotherapy of prostate carcinoma. Int J Radiat
Oncol Biol Phys 1999; 43:1095-101.
6. Bentzen SM, Overgaard J, Thames HD, et al. Clinical radiobiology of malignant melanoma. Radiother
Oncol 1989; 16:169-82.
7. Dubray BM, Thames HD. The clinical significance of ratios of radiobiological parameters. Int J Radiat
Oncol Biol Phys 1996; 35:1099-111.
8. Stock RG, Stone NN, Tabert A, et al. A dose-response study for I-125 prostate implants. Int J Radiat Oncol
Biol Phys 1998; 41:101-8.
9. Hanks GE, Schultheiss TE, Hanlon AL, et al. Optimization of conformal radiation treatment of prostate
cancer: report of a dose escalation study. Int J Radiat Oncol Biol Phys 1997; 37:543-50.
10. Sachs RK, Chen AM, Brenner DJ. Review: proximity effects in the production of chromosome aberrations
by ionizing radiation. Int J Radiat Biol 1997; 71:1-19.
11. Trott KR, Maciejewski B, Preuss-Bayer G, Skolyszewski J. Dose-response curve and split-dose recovery in
human skin cancer. Radiother Oncol 1984; 2:123-9.
12. Turesson I, Thames HD. Repair capacity and kinetics of human skin during fractionated radiotherapy:
erythema, desquamation, and telangiectasia after 3 and 5 year's follow-up. Radiother Oncol 1989; 15:16988.
13. Duchesne GM, Peters LJ. What is the alpha/beta ratio for prostate cancer? Rationale for hypofractionated
high-dose-rate brachytherapy. Int J Radiat Oncol Biol Phys 1999; 44:747-8.
113
THE RADIOLOGICAL RESEARCH
ACCELERATOR FACILITY
THE RADIOLOGICAL RESEARCH ACCELERATOR FACILITY
An NIH-Supported Resource Center
WWW.RARAF.ORG
Director: David J. Brenner, Ph.D., D.Sc.
Manager: Stephen A. Marino, M.S.
Chief Physicist: Gerhard Randers-Pehrson, Ph.D.
Funding
During this year, we were delighted that NIH funding for continued development of
our single-particle microbeam facility was renewed for a further five years.
Research Using RARAF
Table I lists the experiments performed at RARAF during the period May 1, 1998
through April 30, 1999 and the number of days each was run in this period. Twelve
different experiments were run during this 12-month period, about the same as the last two
years.
Six experiments were undertaken by members of the CRR, supported by grants
from the National Institutes of Health (NIH) and the Department of Energy (DOE), and six
by outside users, supported by various grants and awards from NIH, DOE, and NASA.
Brief descriptions of these experiments are given here:
Studies of the mutagenesis of human-hamster hybrid (AL) cells by charged particles
(Exp. 43) resumed this year. Tom Hei, Hongning Zhou and Su-Xian Liu of the CRR
irradiated cells with 4He particles having an LET of 150 keV/µm using the track segment
facility. Cells were treated with graded doses of NNK, a tobacco-specific nitrosamine, or
arsenite and given a single particle dose of 0.25 Gy. The mutation rate at the S1 locus of
human chromosome 11, a single copy of which is present cells, is then observed.
Charles Geard of the CRR has continued studies using the RARAF single-particle
microbeam facility to irradiate cell nuclei with specific numbers of 90 keV/µm 4He ions to
observe micronucleus production, cell growth, and progression through the cell cycle in
normal human fibroblasts (Exp. 71). The cells are irradiated through the nucleus, through
the cytoplasm, or through the surrounding medium. In other experiments, only a fraction
of the cell nuclei are irradiated (1 in 2 to 1 in 80) and the unirradiated cells are observed for
a “bystander” effect, i.e. a response greater than can be accounted for by only the cells
which have been irradiated. Cell densities have been varied from intimate contact between
cells to large separations. Brian Ponnaiya is developing a protocol in which small numbers
of these cells, ultimately a single cell, can be observed for gene expression.
Investigations involving the oncogenic neoplastic transformation of mouse C3H
10T½ cells (Exp. 73) were continued by Richard Miller and Satin Sawant of the CRR.
Cells were irradiated individually through the nucleus or the cytoplasm, or a fraction of the
cells were irradiated through the nucleus. In the latter case, when 10% of the cells were
irradiated, with 8 helium ions, there was a significant increase in transformation rate
compared to what would be predicted if there were no “bystander” effect.
115
The frequency and types of mutations induced at the S1 locus of human-hamster
hybrid (AL) cells by an exact number of 4He ion traversals (Exp. 76) continue to be
investigated by Tom Hei, Hongning Zhou and An Xu of the CRR. Twenty helium ions
given to 20% of the cells resulted in a mutation rate 3-fold higher than expected assuming
no “bystander” effect. The presence of DMSO had no effect, however lindane, which
inhibits cell-to-cell communication, significantly reduced the mutation yield. In other
experiments, only the cytoplasm is irradiated and the cells are examined to determine the
mechanism by which mutations have been observed even though no particles passed
through the cell nuclei. In this case, the addition of DMSO reduced the mutation rate,
implying that radicals play a role in the process.
Lucien Wielopolski and colleagues from Brookhaven National Laboratory
continued the characterization of an accelerator-based boron neutron capture therapy
(BNCT) system (Exp. 81). A moderator/reflector assembly consisting mostly of iron and
Teflon is used to moderate neutrons produced by the Li(p,n) reaction. Neutrons with
energies from a few eV to 100 keV are captured by the boron, which emits an energetic
alpha particle, providing the therapeutic advantage. Ideally the spectrum at the entrance to
where the patient’s head will be positioned should have few thermal neutrons, which will
mostly be absorbed before reaching a tumor, or neutrons with energies above 100 keV,
which won’t become thermalized and contribute unwanted dose to healthy tissue. The γ
-ray dose from the competing Li(p,p`γ) reaction and from neutron capture in hydrogenous
material must also be kept at a minimum since this is also unnecessary dose to the healthy
tissue. Neutron yields and spectrometry measurements are compared with Monte Carlo
calculations used to model the system.
Noelle Metting of Pacific Northwest National Laboratory (PNNL) in Washington
State continued to investigate early responses to DNA damage (Exp. 80). HeLa S3 cells
were irradiated through the nucleus by 4He ions with an LET of 90 keV/µm using the
microbeam facility. The DNA of cells irradiated and then incubated was probed by
enzymatic addition of labeled dNTPs to the 3'-OH ends.
William Morgan of the University of California at San Francisco (UCSF) and Frank
Petrini, of the University of Wisconsin at Madison, in collaboration with Charles Geard of
the CRR, are using the microbeam facility to investigate normal human fibrobalsts derived
from people with Nijmejen breakage syndrome (Exp. 84). These cells are deficient in a
component of the repair process. The cells are observed for intra-nuclear localization of
repair proteins following site-specific irradiation.
Tom Hei and Gloria Calaf of the CRR continued experiments using the track
segment facility to develop a model for neoplastic transformation in immortalized human
breast epithelial (MCF-10F) cells similar to that used for human bronchial epithelial cells
(Exp. 85). Cells from transformed colonies resulting from one or two 0.6 Gy doses of 150
keV/µm 4He ions are observed for altered morphology, increased growth rate, anchorageindependent growth, and invasive capabilities before being implanted into nude mice to
assay for tumor formation.
JaeSub Hong and William Craig of the Columbia University Astrophysics
Laboratory continued their investigation of materials to shield gamma-ray detectors used in
high-altitude balloon flights from neutrons (Exp. 88). Neutron and gamma-ray fluxes and
116
spectra are being measured for initially monoenergetic neutrons in the energy range from
0.2 to 2 MeV after they have passed through various potential shielding configurations.
This research will be the doctoral thesis for Mr. Hong.
A portable neutron spectrometry system to cover the energy range from 20 keV to
500 MeV for use on the space shuttle and the manned mission to Mars is being developed
by a group at the Applied Physics Laboratory of Johns Hopkins University. Calibration of
this system (Exp. 89) is being performed by Richard Maurer, David Roth, Raul Fainchtein
and others in their group. The low-energy portion of the neutron spectra are measured
using He proportional counters and the higher energy section is measured using a 5-mm
thick lithium-drifted silicon detector. Essentially monoenergetic neutrons in the energy
range from 0.5 to 18.5 MeV have been provided using the T(p,n), D,d,n) and T(d,n)
reactions. The neutron doses delivered to the detectors have been measured with a tissueequivalent ionization chamber and converted to fluence using standard fluence-to-dose
conversion factors so that the efficiency of the detectors as a function of energy can also be
determined.
David Boothman of Case Western Reserve University, in collaboration with
Charles Geard of the CRR, is examining the expression of radiation-induced proteins
associated with apoptosis in human mammary epithelial cells (Exp. 90). Cells with and
without a p53 construct are irradiated using the single-particle microbeam. Cells
undergoing apoptosis after irradiation are examined to determine protein expression that
may be associated with this process.
Transformation of primary human lung epithelial cells by 4He ions (Exp. 91) is
being investigated by Tom Hei and Hongning Zhou of the CRR. Explants of cells are
grown into cultures and irradiated with 150 keV/µm 4He ions using the track segment
facility. Because of the low probability of producing a transformed cell, large numbers of
cells must be irradiated for each experiment.
Accelerator Utilization and Operation
Accelerator usage is summarized in Table II. Use of the accelerator for radiobiology
and associated dosimetry was very similar to the average for 1992-98. Over 90% of the
accelerator use for radiobiology and 75% of the accelerator use for experiments was for
microbeam irradiations. These experiments require considerable beam time to obtain
sufficient biological data, especially for low probability events such as transformation and
mutation.
Utilization of the accelerator by radiological physics and chemistry increased somewhat
over last year and was slightly higher than the average for the past 6 years. Two of the
projects (Exps 88 and 89) should continue through at least the next year. Long-term
physics experiments can require large amounts of beam time and can often be run on
relatively short notice if the experimenters do not have a long travel time.
Time spent on radiation safety system inspections was reduced slightly by not
inspecting those systems that are rarely, if ever, used, such as the 137Cs source that is used
only for chamber calibrations or the 50 kV X-ray source. Any target stations that have not
been used for a while are also not inspected. Of course, any facility will be inspected
before it is put back into use.
117
Table I. Experiments Run at RARAF May 1, 1998 - April 30, 1999
Exp.
No.
43
71
73
76
80
81
84
85
88
89
90
91
Institution
Exp.
Type
T. K. Hei,
H. N. Zhou,
S. X. Liu
C. R. Geard,
B. Ponnaiya
CRR
Bio
CRR
Bio
R. C. Miller,
S. Sawant
T. K. Hei,
H. N. Zhou,
A. Xu
N. F. Metting
L. Wielopolski,
et al.
W. Morgan,
J. Petrini
T. K. Hei,
G. Calaf
W. Craig,
J. Hong
R. H. Mauer,
et al.
D. Boothman
CRR
Bio
CRR
Bio
PNNL
BNL
Bio
Phys
UCSF, Univ.
of Wisconsin
CRR
Bio
Experimenter
T. K. Hei,
H. N. Zhou
Columbia
Univ.
Johns
Hopkins Univ.
Case Western
Reserve Univ.
CRR
Bio
Phys
Title of Experiment
Cellular and Molecular studies on the
mutagenesis of charged particles using
human-hamster hybrid (AL) cells
Chromosome aberration and micronucleus
production in human cells lines by specific
numbers of α particles
Neoplastic transformation of C3H 10T½
cells by specific numbers of α particles
Mutation at the S1 locus of human-hamster
hybrid (AL) cells by specific numbers of α
particles
Early responses to DNA damage
Neutron spectroscopy for moderator
assembly for BNCT using Li(p,n) reaction
Genomic instability using specific numbers
of α particles
Neoplastic transformation of human breast
epithelial cells by high-LET radiation
Development of neutron shields for highaltitude gamma-ray detectors
No.
Days
Run
2.5
16.2
19.8
21.2
1.0
6.0
2.0
2.0
6.2
Phys
Calibration of a portable real-time
neutron spectrometry system
2.5
Bio
Expression of radiation-induced proteins
associated with apoptosis
Neoplastic transformation of primary human
lung epithelial cells by high-LET radiation
1.0
Bio
Accelerator reliability was about normal this year. Maintenance and repair time
was slightly above the recent average, and less than half that for 1994-95. No major
repairs to the accelerator were performed, although there was a modification to the
charging control system, which is described in the next section.
Development of Facilities
Development of the microbeam and low-energy neutron facilities are described here
briefly. More detailed descriptions of the development of these facilities are given
elsewhere in this report.
The single–particle microbeam has a number of developments and modifications
that are nearing completion:
118
0.5
Table II.
Accelerator Use, May 1997 - April 1998
Percent Usage of Available Days
•
•
•
Radiobiology and
associated dosimetry
27%
Radiological physics
and chemistry
6%
On-line facility
development and testing
23%
Off-line facility
development
33%
Safety system
2%
Accelerator-related
repairs / maintenance.
9%
A quadrupole quadruplet lens to focus the particle beam to ~2µm diameter has been
designed, constructed, and successfully tested for high voltage capability
A high voltage power supply has been purchased for the quadruplet and is being
modified to turn off if there is a sudden voltage change (break-down)
The voice-coil positioner for cell dishes has been refined and a control circuit designed
The low-energy neutron facility produces neutron spectra with dose-mean energies
of 86, 56, and 40 keV. It is based on the Li(p,n) reaction and requires a rotating target to
avoid melting the lithium at high beam currents. The target system is fully functional:
• The water and vacuum seals do not leak, even at twice the design motor speed
• A beam current of 60 µA for several hours did not reduce the thickness of the lithium
• Neutron spectra show only a moderate amount of scattered higher-energy neutrons
• Neutron dose rates are adequate and the percentage γ-ray dose is acceptable
• Multiple small cell samples can be irradiated simultaneously at the same dose rate
We have partially installed the new voltage control system that was purchased for
the Van de Graaff last year. This system is designed to regulate the terminal voltage to ±1
keV whereas the previous system, installed about 1970, can only regulate to ±3-5 keV.
The new generating voltmeter (GVM) and corona head have been mounted on the
accelerator tank and the signal cables have been run. While the new corona head could be
mounted on a spare port, allowing us to keep the old one in place, the new GVM had to
replace the old one, so we could not maintain two parallel systems and switch between
them. The new GVM has been in use for 2 months but the corona head has not as yet been
tried. The control electronics for the new system do not provide some of the features the
119
old system did, so modifications will be made to the circuitry to obtain a digital readout of
the terminal voltage and the position of the corona head.
Personnel
The Director of RARAF is Dr. David Brenner. The Van de Graaff accelerator is
operated by Mr. Stephen Marino and Dr. Gerhard Randers-Pehrson, with the assistance of
Dr. Haijun Song, a post-doctoral fellow. Staffing at RARAF has increased during the past
year to the point that there are no longer offices available and the biology labs have become
somewhat crowded.
Dr. Dusan Srdoc, who had been collaborating on measurements of microdosimetric
and neutron spectra, left RARAF in March 1999.
Dr. Alexander Dymnikov, an expert on ion beam transport, joined the RARAF staff
in February, 1999. He is doing detailed calculations on the design of the electrostatic
quadrupole lens systems which are being developed to increase the microbeam resolution
initially to 2 µm and eventually to ~0.5 µm.
Mr. Stig Palm from the University of Goteborg, Sweden visited from August
through October 1999 to do irradiations with the single-particle microbeam. His
experiments were related to the study of radioimmunotherapy cancer treatment using
antibodies labeled with 211At, the subject of his recent doctoral thesis.
Mr. Francois Lueg-Althoff, an undergraduate student from the University of
Aachen in Jülich, Germany, arrived in October for a nine-month visit to do his
Praxissemester and Diplomarbeit (practical semester and undergraduate thesis). He has
been assisting the RARAF staff, particularly with microbeam irradiations. As his thesis
project, he will irradiate track-etchant plastic using the single-particle microbeam to
determine the radial distribution of alpha particles at the location of the cells.
Biologists from the Center for Radiological Research not supported by the RARAF
grant spend various amounts of time at the facility in order to perform experiments:
Dr. Charles Geard spends a large part of most working days at RARAF. In addition
to his own research, he is collaborating with several outside users on experiments using the
single-particle microbeam facility.
Dr. Richard Miller worked at RARAF approximately 3-4 days per week until
January 1999, when he took a position with the Radiological Society of North America
(RSNA). He has returned several times to perform or assist in microbeam experiments.
Dr. Satin Sawant, has taken over Richard Miller’s work on transformation using the
C3H10T1/2 cell line. He spends essentially all his time at RARAF, primarily doing
experiments utilizing the microbeam facility.
Dr. Brian Ponnaiya, a post-doctoral fellow, arrived in April 1999. He works at
RARAF full-time, performing microbeam experiments. He has equipped the cell
laboratory for molecular characterization of radiation damage.
There is one full-time biology technician, Ms. Gloria Jenkins. Two other
technicians, Ms. Mei Wang and Ms. Sonu Dhar, are at RARAF part of the time.
Microbeam Meeting
We organized the 4th International Workshop: Microbeam Probes of Cellular
Radiation Response, held in Killiney Bay, Dublin, July 17-18. Roughly 75 scientists (about
120
equal numbers of physicists and biologists) attended the workshop, the fourth in a biannual series. Extended abstracts from the meeting are in press in the Radiation Research
journal and are available on the RARAF website (www.raraf.org).
RECENT PUBLICATIONS OF WORK PERFORMED AT RARAF (1998-1999)
1. Calaf, G.M. and Hei, T.K. Establishment of a radiation and estrogen-induced breast
cancer model. Carcinogenesis 21 (in press, 2000)
2. Calaf, G.M. and Hei, T.K. Establishment of a radiation and estrogen-induced breast
cancer model. Carcinogenesis, in press, 2000
3. Dymnikov, A.D., Brenner, D.J., Johnson, G. and Randers-Pehrson, G. Theoretical
study of short electrostatic lens for the Columbia ion microprobe. Rev. Sci. Instr. (In
Press, 2000)
4. Dymnikov, A.D., Brenner, D.J., Johnson, G.W. and Randers-Pehrson, G. Electrostatic
lens design for the Columbia microbeam. Radiation Research, in press, February 2000.
5. Geard, C.R., Randers-Pehrson, G., Marino, S.A., Jenkins-Baker, G., Hei, T.K., Hall,
E.J. and Brenner, D.J. Intra- and inter-cellular responses following cell-site specific
microbeam irradiation. Radiation Research, in press, February 2000.
6. Hei, T. K., Roy, D., Piao, C.Q., Calaf, G. and Hall, E. J. Genomic instability in human
epithelial cells transformed by high LET radiation. Radiat. Res. 153 (in press, 1999)
7. Mauer, R.H., Roth, D.R., Fainchtein, R., Goldsten, J.O. and Kinnison, J.D. Portable
real time neutron spectrometry II, to be published in the proceedings of the
International Space Station Conference, Albuquerque, NM, January 30-February 3,
2000.
8. Miller, R.C., Martin, S.G., Geard, C.R., Marino, S.A., Randers-Pehrson, G., Brenner,
D.J. and Hall, E.J. High LET-induced Oncogenic Transformation. In Risk Evaluation
of Cosmic-ray Exposure in Long-term Manned Space Mission (F. Fujitaka, et. al., Eds.)
pp. 121-126, Kondasha Scientific Ltd., Tokyo, Japan, 1999.
9. Miller, R.C., Martin, S.G., Hanson, W.R., Marino, S.A. and Hall, E.J. Effect of track
structure and radioprotectors on the induction of oncogenic transformation in murine
fibroblasts by heavy ions. Adv. Space Res. 22: 1719-1723 (1998).
10. Miller, R.C., Sawant, S.G., Randers-Pehrson, G.,. Marino, S.A., Geard, C.R., Hall E.J.
and Brenner, D.J. Single alpha-particle traversals and tumor promoters. Radiation
Research, in press, February 2000.
11. Randers-Pehrson, G., Geard, C.R., Johnson, G.W. and Brenner, D.J. Technical
characteristics of the Columbia University single-ion microbeam. Radiation Research,
in press, February 2000.
12. Zhou, H.N., Randers-Pehrson, G., Waldren, C., Vannais, D., Hall, E.J. and Hei, T.K.
Induction of a bystander mutagenic effect of alpha particles in mammalian cells. Proc.
Natl. Acad. Sci. U.S.A. 97 (in press, 2000).
13. Zhou, H., Randers-Pehrson, G. and Hei, T.K. Studies of bystander mutagenic response
using charged particle microbeam. Radiation Research, in press, February 2000.
121
RADIATION SAFETY OFFICE
RADIATION SAFETY OFFICE STAFF
PROFESSIONAL STAFF
Salmen Loksen, M.S., CHP, DABR, Director, RSO
Ahmad Hatami, M.S., DABMP, CRESO, Assistant Director RSO
Thomas Juchnewicz, M.S., DABR, Assistant Radiation Safety Officer
Ilya Pitimashvili, Ph.D., Radiation Protection Supervisor
TECHNICAL STAFF
Clifford Jarvis, B.S., Chief Technician
Karolin Khalili, B.S., Senior Technician
Kenyel Spaulding, Technician B
Hayeon Kim, M.S., Technician A
Roman Tarasyuk, Technician A
SECRETARIAL STAFF
Yvette Acevedo, Administrative Aide
Diana Morrison, Executive Secretary
Raquel Rodriguez, Clerk A
Milvia Perez, Clerk A
Zugiery DeLeon, Clerk A
CONSULTING STAFF
Jake Kamen, Ph.D., Physicist
Bruce Emmer, M.S., DABMP, DABR, Physicist
123
Radiation Safety Office
Fiscal Year 1998-1999
Introduction
On May 19, 1957, the President of Columbia University distributed a memo entitled
Directive to All University Departments Having a Source of Ionizing Radiation, advising
all parties of the expanded function of the Radiation Safety Committee.
Later, a notice entitled Radiation Safety Guide for Columbia University, dated
February 10, 1959, named Philip M. Lorio as the Health Physics Officer for University
Departments and Laboratories, except the College of Physician & Surgeons, where Dr.
Edgar Watts was the named Health Physics Officer. The Chairman of the Radiation
Safety Committee was Dr. Gioacchino Failla, who initiated the Radiological Research
Laboratory in the Department of Radiology of Columbia-Presbyterian Medical Center
(CPMC).
By agreement between The Presbyterian Hospital in the City of New York (PH) and
Columbia University (CU), the Radiation Safety Office (RSO) was established as an
autonomous unit in 1962 for the purpose of maintaining radiation safety. The Joint
Radiation Safety Committee (JRSC), appointed by the Medical Board of CPMC and the
Vice President for Health Sciences of Columbia University, is charged with the
responsibility of defining and ensuring enforcement of proper safeguards in the use of
sources of ionizing radiation.
Dr. Harald H. Rossi, Director of the Radiological Research Laboratories, was
appointed Chairman of the JRSC. Under his direction, this committee developed a
Radiation Safety Code and Guide, the administration of which is assigned to the
Radiation Safety Officer. Dr. Eric J. Hall, the present Director of the Center for
Radiological Research, now chairs the JRSC.
The present Radiation Safety Office (RSO) came into existence through an agreement
made on February 12, 1991 between New York State Psychiatric Institute (NYSPI), the
College of Physicians and Surgeons of Columbia University (P&S), and The Presbyterian
Hospital in the City of New York (PH). This agreement combined several overlapping
clinical and educational programs, including all programs for ensuring radiation safety.
On December 16, 1996, Mr. Salmen Loksen was appointed Director of the Radiation
Safety Office (RSO). The Radiation Safety Office advises CPMC and NYSPI through the
JRSC, and also participates in the review of research protocols for the Radioactive Drug
Research Committee under the jurisdiction of the U.S. Food and Drug Administration.
124
The Radiation Safety Office is responsible for ensuring compliance with Federal,
State and City regulatory agencies. These regulatory agencies, which mandate rules,
regulations, and guidelines, include:
•
•
•
•
•
United States Food and Drug Administration
United States Nuclear Regulatory Commission
New York State Department of Environmental Conservation
New York State Department of Health
New York City Department of Health Bureau of Radiological Health.
The Radiation Safety Office also ensures compliance with the rules and regulations of
the Radiation Code and Guide of CPMC and the New York State Psychiatric Institute.
The primary services provided by the Radiation Safety Office are:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Providing initial and annual training to personnel involved in handling
radioactive materials or radiation-producing equipment.
Evaluation of education, training and experience of individuals requesting the
purchase of radioactive materials and radiation-producing equipment.
Routine and specialized laboratory inspection.
Calibration of instruments.
Pick-up storage and disposal of radioactive waste.
Emergency response in case of radiation accidents.
Leak testing of sealed radiation sources.
Bioassay (urinalysis for radioactivity).
Consultation for radiation shielding requirements.
Personnel exposure monitoring.
Supervision of contaminated-area cleanup.
Monitoring dental and medical X-ray equipment.
Quality assurance testing of dental X-ray equipment.
Measurement of personnel thyroid uptake.
Receiving, shipping, tracking and testing packages for radioactive
contamination.
Responding to radiation emergencies: spills, personnel contamination and
investigating reports of overexposure.
It is the goal of the Radiation Safety Office at Columbia-Presbyterian Medical Center
to protect employees, patients and the public from exposure to unnecessary ionizing
radiation. Through continuing training, education and consultation the Radiation Safety
Office ensures adherence to all regulatory requirements and guidelines so that exposure
remains as low as reasonably achievable (ALARA).
The Radiation Safety Office is responsible for maintaining licenses allowing the use
125
of radioactive materials. Licenses include the New York City Department of Health
Bureau of Radiological Health Broad Scope Research and Broad Scope Human Use
License and others. In addition, the Radiation Safety Office is responsible for maintaining
permits from the New York City Department of Health Bureau of Radiological Health for
radiation-producing equipment (X-rays) and the New York State Department of
Environmental Conservation for the discharge and disposal of radioactive material to the
environment. These governmental licensing agencies also perform periodic audits and
inspections. The Radiation Safety Office strives to ensure that regulatory violations are
prevented and that any that might occur are expeditiously rectified.
In professional matters involving radiation safety, the RSO reports to the Joint
Radiation Safety Committee, which meets on a quarterly basis. For administrative
purposes, the RSO reports to Dr. Richard Sohn, Associate Dean for Research
Administration and Director of Grants and Contracts.
Radiation Safety Office staff are Columbia University employees. The RSO budget is
funded by New York Presbyterian Hospital, the Columbia University College of
Physicians and Surgeons, and the New York State Psychiatric Institute, via payback
arrangement.
With the full-asset merger between The Presbyterian Hospital in the City of New
York and New York Hospital on December 1, 1997, a single entity known as New York
Presbyterian Hospital was formed with facilities in two major Manhattan locations:
Columbia-Presbyterian Center at West 168th Street in Washington Heights and New
York Weill Cornell Center at East 68th Street on the Upper East Side. The RSO is in the
process of reviewing the Radiation Safety Manuals at both locations to ensure that
uniform policies are established.
Summary of Services
The statistical data detailed below are for the period of the fiscal year, July 1, 1998
through June 30, 1999. Instances of Radiation Safety Office support, activities, incidents
and response, include those from the date of the last Annual Report, December 1998, to
the present, December 1999.
1. Performed routine radiation safety audit/surveys of 351 Columbia University and
New York State Psychiatric Institute research laboratories using radioactive materials.
Results of the audits were communicated to Responsible Investigators and 67
deficiencies were followed up, resulting in the correction of the cited deficiencies.
2. Received and distributed 4,218 packages containing radioisotopes, with a total
activity of 75 Curies, excluding Nuclear Medicine and Radiation Oncology
Shipments. For all shipments the RSO conducts package surveys, ensures correct
distribution to Authorized Users, maintains inventory control and associated records.
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3. Performed 110 thyroid bioassays on radiation workers using isotopes of iodine,
primarily I-125, and occasionally I-123 or I-131.
4. Distributed 1,613 personnel radiation dosimeters on a monthly basis, and 3,384
personnel radiation dosimeters on a quarterly basis, results in 32,892 dosimeters
distributed and collected annually. To maintain dosimetry records the RSO uses a
dedicated computer with direct modem access to the vendor.
5. From January 1999 the RSO began a changeover from standard LiF (TLD) dosimeters
to Luxel optically stimulated luminescence dosimeters. Luxel’s Optically Stimulated
Luminescence (OSL) dosimeter measures radiation exposure due to x-ray, beta, and
gamma radiation through a thin layer of aluminum oxide. After use, the aluminum
oxide is stimulated with a laser light in Landauer’s laboratory causing it to become
luminescent in proportion to the amount of radiation exposure. The luminescence is
measured and a report of exposure result is generated. The Luxel dosimeter has a
sensitivity of 1 mrem for photon compared to the standard LiF sensitivity of 10
mrem. The first department to be changed to the new system was the Department of
Radiology at New York, Columbia-Presbyterian Center. This changeover was
coordinated with the New York Presbyterian Hospital Weill Cornell Center.
Columbia University, New York Presbyterian Hospital and New York State
Psychiatric Institute employees will completely change over to the Luxel optically
stimulated luminescence radiation dosimeter in January 2000. The RSO conducted
two informational-training sessions on the use of and transition to the new Luxel
system.
6. An officer of the RSO participates as an Ad Hoc Member of the Animal Care
Protocol Review Committee, reviewing all procedures using radionuclides in animal
research. 32 protocols involving the use of radioactive materials in animals were
approved.
7. Scheduled and performed 28 routine animal radiation safety surveys in the Institute of
Comparative Medicine in order to ensure the integrity of ongoing experiments and to
protect the Animal Care Staff from unnecessary radiation exposure and radiation
contamination in animal rooms and cages.
8. Provided calibration and maintenance services for 287 portable radiation survey
instruments used throughout the Columbia-Presbyterian Medical Center and the New
York State Psychiatric Institute. The RSO maintains a supply of portable survey
instruments available for loan to Responsible Investigators.
9. Provided support to New York Presbyterian Hospital Departments of Nuclear
Medicine and Radiation Oncology by performing patient and room surveys, posting
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instructions in patient rooms, entering instructions in patient charts, and distributing
personnel radiation monitoring devices. During 1998-1999, radiation safety support
was given for 97 brachytherapy patients and 9 I-131 radiopharmaceutical therapy
patients. Rooms are decontaminated and contaminated patient wastes are removed for
decay in storage and disposal.
10. The RSO operated the Columbia-Presbyterian Medical Center Low-LevelRadioactive-Waste (LLRW) Disposal and the Decay-In-Storage programs. This
program operates from LLRW/Decay-In-Storage facilities maintained by the RSO in
the Columbia University Physicians & Surgeons Building, Russ Berrie Medical
Science Pavilion, Hammer Health Sciences Building and the new New York State
Psychiatric Institute building on Riverside Drive.
11. The RSO maintained and updated the South Carolina Waste Transport Permit, the
Chem-Nuclear Waste Disposal Permit, and prepared waste shipping manifests. A total
of 2,065.5 cubic feet of radioactive wastes were collected from all research users at
CPMC and the New York State Psychiatric Institute. 125 drums, containing 573.8
cubic feet of dry solid LLRW, were shipped to the burial site in South Carolina. 75
drums, containing 340.8 cubic feet of liquid scintillation vial waste, were shipped for
disposal as non-radioactive waste in Florida. Total activity shipped was 816.8
millicuries. An additional 287 drums containing 1,150.9 cubic feet of short half-life
waste was held for decay in storage and cleared for landfill disposal as regular trash.
An additional 4,350 liters of low-level aqueous wastes were disposed of by monitored
sewer disposal.
Presently, due to sharply increased fees and the distinct possibility that the South
Carolina site will shut down, the RSO is actively evaluating other sites for landfill
disposal of LLRW. Criteria being evaluated include not only costs, but also
environmental risk and impact.
The RSO prepared and submitted the annual Low-Level Radioactive Waste Report to
the New York State Energy Research and Development Authority.
12. The RSO maintained a comprehensive program to prevent the release of patient waste
contaminated with radioactivity. 258 bags of 'black bag' waste were removed from
patient rooms and placed in Decay-In-Storage. Low-level radioactive waste monitors,
maintained and checked daily by the RSO on the Milstein and the New York
Presbyterian Hospital loading docks, detected an additional 100 bags of contaminated
'red bag' waste which were removed from the waste stream and placed in Decay-InStorage.
In order to optimize this program the RSO has placed an order for delivery and
installation of a new network-ready monitoring system that will not only alert workers
on the waste area loading docks to contaminated patient waste, but will display the
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alert on a work station in the RSO, and maintain a 24 hour-a-day, 7 day-a-week record
of waste alarm response in order to insure removal of radioactive patient waste from
the regular waste stream.
13. The RSO continued to provide radiation safety support for the Cyclotron Facility and
the associated PET Suite. The RSO continues to advise Cyclotron and PET staff
regarding keeping extremity and whole body exposures ALARA. During 1999 remote
manipulators were installed in a Cyclotron Facility hot cell at the recommendation of
the RSO. Intensive in-services were given to PET Suite technologists, researchers and
medical staff regarding the safe handling of high-energy positron emitting
radiopharmaceuticals. The RSO maintains a liaison with the corporate RSO of PET
Net, Inc., the operator of the Cyclotron under the CPMC license.
14. Throughout 1999 the RSO provided radiation protection engineering consulting
services to the Columbia University Engineering Department and their contractors
involved in the currently underway construction of the new Radioligand Laboratory in
the basement of the Milstein Hospital Building, adjacent to the existing Cyclotron
Facility. The RSO provided calculations regarding and specifications for the
laboratory radioisotope exhaust system, the roof-top filtration and discharge system,
the computerized effluent stack radiation monitoring system, shielding requirements
for hot cells, radioactive gas delivery lines and the pneumatic delivery system,
portable radiation survey meters and alarming area monitors.
15. In association with the Department of Radiology, the RSO maintained a Radiation
Safety Inspection and Audit Program for non-Radiology X-ray equipment at CPMC to
assure compliance with regulatory requirements. The program includes an audit an
evaluation of compliance with Quality Assurance requirements and procedures,
attendance of employees at radiation safety training sessions, and compliance in the
wear and timely return of personnel radiation dosimetry. Prior to the field audit a form
is sent to each non-Radiology X-ray facility requesting a list of individuals
responsible for performing QA/QC functions and an inventory list of all X-ray
equipment and film processors.
16. Performed quarterly inspections and audits of all CPMC clinical facilities using
radioactive materials to ensure compliance with City of New York Radioactive
Materials License conditions and with RCNY Article 175, Radiation Control. These
audits include quarterly inventories of all sealed sources of radioactivity and leak
testing of sources and irradiators as required. Leak test certificates are provided. The
facilities audited include: New York Presbyterian Hospital Nuclear Cardiology, New
York Presbyterian Hospital Neuroanesthesiology, Milstein Hospital Department of
Nuclear Medicine, Milstein Hospital Cyclotron Facility, Milstein Hospital PET Suite,
New York State Psychiatric Institute Brain Scan Department, Allen Pavilion Nuclear
Medicine and Allen Pavilion Nuclear Cardiology.
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In addition, the RSO investigates all major spills, incidents, misadministrations,
anomalous exposures and reports of missing sources, and provides timely notice of
reportable incidents to the City of New York Department of Health Bureau of
Radiological Health.
17. The RSO maintained the City of New York Radioactive Materials Licenses: 75-287801 (Human Use), 92-2878-02 (Teletherapy), 74-2878-03 (Non-Human Use), 58-287804 (Cyclotron Facility) 93-2878-05 (Gamma Knife), and City of New York
Therapeutic Radiation Linac Unit Certified Registration No. 77-0000018 (East 60th
Street) and No. 77-0000019 (168th Street).
18. Additional interactions with the City of New York Department of Health Bureau of
Radiological Health included:
•
Obtained a renewal of the 92-2878-02 (Teletherapy) License for a five-year
period. The renewed License will expire on July 31, 2004.
•
Obtained an amendment to the 75-2878-01 (Human Use) License adding a
Nordian International Gamma Cell 3000 Blood Irradiator containing 56.5
Terabecquerels of Cs-137 for use by Transfusion Services.
•
Obtained written permission to modify the procedure used to assay Oxygen-15
under 75-2878-01 (Human Use) and 58-2878-04 (Cyclotron).
•
Requested an amendment to the 75-2878-01 (Human Use) License increasing the
outpatient Iodine-131 therapy limit from 1.2 gigabecquerels to 8.103
gigabecquerels.
•
Requested amendments to the 75-2878-01 (Human Use), 92-2878-02
(Teletherapy), 93-2878-05 (Gamma Knife) Licenses, and the 77-0000018 and 770000019 Linac Registrations, to add three qualified radiation oncology physicians
approved by the CPMC JRSC as authorized users.
19. As a major function of the maintenance of the City of New York Radioactive
Materials licenses, X-ray registrations and Linac Registrations, the RSO represents
the CPMC and New York State Psychiatric Institute Joint Radiation Safety
Committee during inspections and audits conducted by the City of New York
Department of Health Bureau of Radiological Health. The RSO accompanies the
inspectors, provides access to information and records, participates in the exit
interviews and receives the written report of the City. Inspections performed in 1999
were:
•
September 1, 1999, inspection for compliance with the requirements of 92-287802 (Teletherapy).
130
•
June 17, 1999 through September 20, 1999, inspection for compliance with the
requirements of 74-2878-03 (Non-Human Use).
•
August 27, 1999, inspection for compliance with the requirements of X-ray Permit
No. H98 1005495 72 (Mobile C-arm, leased to Orthopedics).
•
May 11 and 20, 1999, inspection for compliance with the requirements of Linac
Registration No. 77-0000018 (East 60th Street Facility)
•
February 11, 1999 through April 8, 1999, inspection for compliance with the
requirements of 52-2878-04 (Cyclotron) and 74-2878-01 (Human Use).
•
April 7, 1999, inspection for compliance with the requirements of 93-2878-05
(Gamma Knife).
•
February 25, 1999, initial inspection of new Gamma Knife installation in the
Department of Radiation Oncology for compliance with the requirements of 932878-05 (Gamma Knife) and RCNY Article 175.
In all cases either no deficiencies were found or minor deficiencies discovered
were corrected within thirty days of the inspection.
20. As an additional function of the maintenance of the City of New York Department of
Health Bureau of Radiological Health Radioactive Materials licenses, X-ray
registrations and Linac Registrations, the RSO receives BRH Information Notices
from the City of New York Department of Health Bureau of Radiological Health,
which provide guidance for meeting compliance with specific requirements of RCNY
175. During 1999 the RSO received the following BRH Information Notices and
communicated their requirements to the departments concerned:
•
•
•
•
January 3, 1999, BRH-1: Discharge criteria for patients administered radioactive
material.
January 26, 1999, BRH-2: Approved bodies for external radiation oncology
audits.
August 16, 1999, BRH-3: Requirements for linear accelerator facility doors.
August 24, 1999, BRH-4: Requirements for Y2K contingency planning.
21. The RSO maintained the New York State Department of Environmental Conservation
Radiation Control Permit No. 2-6201-00005/00006 required for the discharge to the
atmosphere of exhausts contaminated with radioisotopes from emission points on the
CPMC campus.
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22. Activities with regard to New York State Department of Environmental Conservation
included:
•
Performed a quarterly review of all atmospheric and sewer discharges of
radioisotopes from the CPMC campus as required by our Radiation Control
Permit Conditions and 6 NYCRR 380. The results of this review are
communicated to the New York State Department of Environmental Conservation
and complied into an annual report of discharges.
•
On November 29, 1999, as required by our Permit Conditions, the RSO submitted
to the New York State Department of Environmental Conservation a
comprehensive Permit Modification Request. This comprehensive permit
modification request combines in one unified document the results of two years of
discussion and correspondence with the New York State Department of
Environmental Conservation regarding radioisotope discharges from CPMC
facilities including the Cyclotron, the Nuclear Medicine Department, and
numerous research laboratories throughout the campus.
•
This document, currently under review, places CPMC in compliance with the new
U.S. Nuclear Regulatory Commission constraint limit of 10 mrem per year to the
general public from the discharge of radioactive materials. The permit
modification request characterizes the location, flow-rate and amount of
radioisotopes discharged from 15 emission points on the CPMC campus. It
provides calculations of discharge concentration and the radiation dose to the
general public resulting from CPMC research and clinical operations.
•
On September 13, 1999, obtained approval from the New York State Department
of Environmental Conservation of a Permit Modification to increase the amount
of Carbon-11 discharged to the atmosphere from 7 Curies per year to 17 Curies
per year. This discharge resulted from an increase in the need to produce Carbon11 for radiopharmaceutical research.
23. As a major function of the maintenance of the New York State Department of
Environmental Conservation Radiation Control Permit, the RSO represents the
CPMC Joint Radiation Safety Committee during inspections and audits conducted by
the New York State Department of Health Radiation Control Section. The RSO
accompanies the inspectors, provides access to information and records, participates
in the exit interviews, receives the written report of the inspection, ensures
deficiencies are corrected in a timely manner, and reports to the CPMC JRSC.
Inspections performed in 1999 were:
•
On November 23, 1999, representatives of the New York State Department of
Environmental Conservation Radiation Control Section conducted an
unannounced inspection of CPMC operations under Permit No. 2-6201-
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00005/00006. The inspection concerned the physical facilities and operations
discharging radioisotopes into the atmosphere, the monitoring of those discharges,
and the disposal of solid Low-Level-Radioactive-Waste. No deficiencies were
cited. A future audit of written records is expected.
•
On February 11, 1999, representatives of the New York State Department of
Environmental Conservation Radiation Control Section conducted an
unannounced inspection and audit of CPMC operations under Permit No. 2-620100005/00006. The inspection concerned the physical facilities, operations
discharging radioisotopes into the atmosphere, and the monitoring of those
discharges. The audit involved a review of records required under the Permit and
6 NYCRR 380. On March 31, 1999, the RSO received a letter from the New York
State Department of Environmental Conservation that CPMC was in full
compliance. In the exit interview it was mentioned that the RSO was doing “a
great job.”
24. As an additional function of the maintenance of the Radiation Control Permit, the
RSO implements corrections and requests made by the New York State Department
of Environmental Conservation as a result of their inspections of the operations under
the Radiation Control Permit.
On March 31, 1999, the New York State Department of Environmental Conservation
Radiation Control Section recommended that CPMC incorporate into its Radiation
Safety Program a regular replacement schedule for carbon filters in the Cyclotron
Facility exhaust system in order to minimize discharges of positron emitting isotopes.
This recommendation has been adopted.
25. In order to provide the data necessary for operation under the Radiation Control
Permit and to meet the requirements City of New York RCNY Article 175, the RSO
operates a program for the safe use of airborne radioactivity.
On November 17, 1999, with assistance provided by International Testing &
Balancing Ltd., the RSO performed air flow rate measurements at 9 radioisotope
exhaust stacks and 25 major radio-iodination hoods or positron-emitter hot cells in
use on the CPMC campus. The measured data as well as specifications, sketches and
photographs of the emission points are used for New York State Department of
Environmental Conservation Quarterly Discharge ALARA reviews and Permit
applications and modifications.
Throughout the year, the RSO makes semi-annual measurements of the average face
velocity of approximately 145 fume hoods in which radioisotopes are used or stored.
Researchers whose hoods do not meet safe flow rate standards are directed to have
their hoods repaired.
133
Ventilation was measured in all rooms where radioactive gases or aerosols are used,
and spill gas clearance times are calculated and posted.
26. In order to meet the requirements of RCNY Article 175 and the Conditions of our
City of New York Radioactive Materials Permits the RSO operates an extensive
ALARA Program.
During the 1998-1999 fiscal year 44 ALARA Level 1 and 20 ALARA Level 2
Notification Reports provided by our personnel radiation dosimetry vendor were
investigated and the radiation workers were informed of their exposures. Particular
attention is paid to three occupational groups typically at or exceeding ALARA limits
for whole body, extremity or eyes: workers and researchers in the Cyclotron Facility;
technologists, researchers and physicians in the PET Suite; and physicians in the
Angiography Suite.
No instances of any employees exceeding any RCNY Article 175 exposure limit
occurred at CPMC during 1999. In one instance, on November 9, 1999, a personnel
dosimetry report listed a quarterly deep dose of 7270 mrem for one employee.
However, the dosimetry report listed an “E2" error code indicating that the dosimeter
response did not match the expected response to any known radiation source.
Investigation of the incident by the RSO confirmed that the individual could not have
received the dose indicated by the faulty dosimeter. As of November 22, 1999, the
RSO has requested authorization from Gene Miskin, Director, City of New York
Department of Health Bureau of Radiological Health, to remove this apparently
erroneous result from the employee’s dose history.
27. At the March 26, 1999 meeting of the CPMC Joint Radiation Safety Committee, the
Committee adopted a uniform Pregnancy Policy for Columbia-Presbyterian Medical
Center. This policy is in compliance with recent rulings of the United States Supreme
Court in the area of occupational rights and with the specific requirements of RCNY
Article 175. During 1999 the RSO made an extensive effort to educate and inform
employees of the Medical Center as to the new policy.
During the 1998-1999 fiscal year, 19 employees of the Columbia-Presbyterian
Medical Center completed a declaration of pregnancy form and received health
physics counseling. These individuals were counseled concerning risk factors and
provided with additional monitoring of the fetus for the gestation period. The RSO
continues to closely follow the personnel exposure reports of this group.
28. The RSO continues to maintain a program for emergency response. A system was
established by the RSO with a list of names and beeper numbers, including a group
pager number, and a procedure for Security to contact members of the RSO in an
emergency. Evidence of the effectiveness of this system was demonstrated during the
following emergencies in 1999:
134
•
During the July 7-9, 1999, electrical blackout, Radiation Safety continued to be
effective in providing support to the CPMC community. During this emergency,
radioisotope packages were received and properly stored using dry ice, and were
distributed to Responsible Investigators.
•
On Oct 27, 1999, there was a fire in a Black Building laboratory, which uses
radioactive material. Radiation Safety staff were notified, and responded
immediately. All personnel and equipment used were surveyed for contamination.
•
On Nov 12, 1999, the couch of the Gamma knife became stuck during the QC
procedure. The RSO provided the service company’s engineer with appropriate
personnel dosimetry equipment, and analyzed the dosimetry equipment on an
emergency basis.
•
On Nov 25, 1999, a physician from Radiation Oncology, during removal of
sources after completion of a Low Dose Rate (LDR) brachytherapy procedure,
became aware that one of the Cesium-137 sources was missing. The RSO staff
member accompanying the physician immediately obtained assistance from other
RSO staff members, and they were able to first isolate the area, and then locate the
source where it was lodged in the plumbing and recover the source.
29. The RSO obtained and reviewed a list of all the non-Y2K-compliant radiation
equipment at Columbia University. The RSO contacted some of the companies
manufacturing this equipment, and they made suggestions for dealing with the
problem. The researchers were notified of the recommendations of the manufacturers,
and instructed to test the status of their equipment.
30. 150 hours of radiation safety training were provided to Columbia University, New
York Presbyterian Hospital and New York State Psychiatric Institute personnel. Types
of training included: initial training for new radiation workers, with separate sessions
geared to researchers, hospital and nursing personnel; annual refresher training for all
CPMC staff (including facilities, housekeeping and security personnel) who come in
contact with radiation; in-service training for nurses, physicians and other clinical
personnel.
31. The RSO reviewed applications submitted to the CPMC Radioactive Drug Research
Committee (RDRC) and/or the CPMC Joint Radiation Safety Committee (JRSC) to
administer radioactivity to human test subjects. A total of 33 applications were
reviewed. Of these, 19 were JRSC applications and 14 were RDRC applications. All
were approved, some with modifications.
32. Six new Responsible Investigator applications for non-human use of radioactivity
were reviewed and approved.
135
33. In 1999 the RSO placed the following new facilities into operation:
A Low-Level-Radioactive-Waste Storage and Decay-In-Storage Facility, located in
room SC-17 in the sub-cellar of the Russ Berrie Medical Science Pavilion.
A Low-Level-Radioactive-Waste Storage and Decay-In-Storage Facility located in
room S609B on the service level of the new New York State Psychiatric Institute
building. On March 24, 1999 the RSO certified 5 radioisotope fume hoods and a
dedicated radioisotope exhaust system installed in the new New York State
Psychiatric Institute building as meeting radiation safety flow rate specifications.
34. The RSO participated as part of the Columbia University Health Science Division
(CUHSD) Emergency Management Plan Task Force. The Emergency Management
Plan is necessary in event that any significant occurrence disrupts the normal day-today operation at CUHSD, including University research activity and/or employee
safety. The objective of the plan is to utilize University resources in an effective
manner should interruption of an essential service occur. The plan provides written
policies and procedures to be implemented in event of emergencies including
radiation spills, chemical spills, transit disruption, utility shutdown, etc. A number of
meetings were held in order to formulate policies, and a draft Emergency
Management Plan document was reviewed.
35. The RSO participated as part of the Columbia University Health Science Division
(CUHSD) Institutional Health and Safety Council (IHSC). The Institutional Health
and Safety Council reviewed and approved a revised Laboratory Safety and Chemical
Hygiene Plan and a revised Formaldehyde Exposure Control Program. In addition, the
IHSC has encouraged the utilization of the Web to provide information, education
and training to personnel. The RSO continued development of its Webpage to
improve dissemination of information and communication with Responsible
Investigators and members of the CPMC community
(http://cpmcnet.columbia.edu/dept/radsafety).
36. The RSO continued the dental quality assurance program for Columbia University
dental facilities, to optimize the radiological safety and clinical quality of dental
radiography. The quality assurance program is based on recommendations for quality
assurance that have been promulgated by a number of professional organizations,
including the National Council on Radiation Protection and Measurements (NCRP),
the Bureau of Radiological Health of the FDA, the American College of Radiology
(ACR), and the American Academy of Dental Radiology Quality Assurance
Committee.
37. At the June 23, 1999 meeting of the CPMC Joint Radiation Safety Committee, a
motion was passed that the JRSC is to assume responsibility for both radiation safety
136
and radiation physics for all medical and dental diagnostic radiology machines that
belong to or are otherwise the responsibility of CPMC, irrespective of the location of
the machines. The CPMC RSO is the executive arm of the JRSC and is the body
assigned to effectively implement this motion. The RSO has taken steps to provide
radiation support for all the equipment effected, including equipment located on the
Morningside Campus.
38. On December 15, 1999 RSO officers participated at a joint meeting that included
representatives of New York Presbyterian Hospital-Columbia-Presbyterian Center,
New York Presbyterian Hospital-Weill Cornell Center, and Memorial Sloan-Kettering
Cancer Center, in order to set uniform policy, procedures and criteria for
Radioimmunotherapy Outpatient Release. The multi-institution discussion and
generation of policy documents is helpful in reducing duplication of effort and
ensuring a through review of policy issues.
137
CENTER FOR RADIOLOGICAL RESEARCH
PROFESSIONAL ACITIVITIES
COLLOQUIUM AND SEMINARS
PUBLICATIONS
PROFESSIONAL ACTIVITIES
Dr. David J. Brenner
Chairperson
Columbia University Radiation Safety Committee
Program Committee, Fourth International Workshop: Microbeam Probes of Cellular
Radiation Response, Killiney Bay, Dublin, July 1999
Member
National Council on Radiation Protection and Measurements (NCRP)
Joint Task Force on Vascular Radiation Therapy
NCRP Committee 1-6 on Risk Linearity
American Society of Therapeutic Radiology and Oncology (ASTRO), Refresher Course
Program Committee
Radiation Research Society Program Committee
American Statistical Association Radiation Meeting Program Committee
Dr. Gloria Calaf
Appointed
Adjunct Professor, Univerisity of Tarapaca, Arica, Chile; Department of Biology and
Health (since December 1998)
Teaching
Postdoctoral Degree Course, “Biology of cancer and inflammatory processes,”
University of Tarapaca (November 24-27, 1998)
Dr. Charles R. Geard
Member
American Society of Therapeutic Radiology and Oncology (ASTRO)
Environmental Mutagen Society
Radiation Research Society
Advisory Committee on Radiobiology, Brookhaven National Laboratory
Associate Member, Radiobiology Advisory Team (AMRAT) of the Armed Forces
Radiobiology Research Institute (AFRRI)
Columbia University, Faculty Council (Voting Member)
Editorial Work
Editorial Board, International Journal of Radiation Biology.
Reviewer
International Journal of Radiation Oncology, Biology and Physics
Radiotherapy and Oncology
British Journal of Cancer
Clinical Cancer Research, Mutagenesis
139
Mutation Research
Radiation Research
Ad Hoc Reviewer of Grant Proposals
American Cancer Society
National Institutes of Health
Dr. Eric J. Hall
Member
American Board of Radiology
Radiotherapeutic Written-Test Committee
National Academy of Sciences
American Society of Therapeutic Radiology and Oncology (ASTRO)
Radiation Research Society
International Stereotactic Radiosurgery Society
Member of the Board
American Radium Society
President
Program Committee Chairman
International Association of Radiation Research
President Elect
Columbia University, College of Physicians & Surgeons
Cancer Center, Internal Advisory Committee/Executive Committee
Columbia-Presbyterian Medical Center:
Chairman, Joint Radiation Safety Committee
Chairman, Radioactive Drug Research Committee
National Council on Radiation Protection and Measurements:
Member of Council
Member, Committee 1
Editorial Work
International Journal of Radiation Oncology Biology Physics
International Journal of Brachytherapy
Dr. Haiying Hang
Member
Radiation Research Society
140
Dr. Tom K. Hei
Adjunct Professor
Department of Radiological Health Sciences, Colorado State University, Fort
Collins, Colorado
Department of Ion Beam Bioengineering, Chinese Academy of Sciences, Hefei,
China
Chairman
Ad hoc review panel, Chemical Pathology Study Section, 1998, 1999
Member:
Chemical Pathology Study Section, 1998- present
Ad hoc review panel, Metabolic Pathology Study Section, 1999
Ad hoc review panel, National Science Foundation, 1999
Radiation Research Society
American Association for Cancer Research
Environmental Mutagen Society
Oxygen Society
Student Mentoring
Ph.D. candidate, Department of Ion Beam Bioengineering, Chinese Academy of
Sciences, China
Master degree students, Environmental Heath Sciences, Columbia University
School of Public Health.
New York City high school science students for Intel Science project
Reviewer
British Journal of Cancer
Cancer Research
Carcinogenesis
Occupational and Environmental Medicine
International Journal of Radiation Biology
Radiation Research
Mutagenesis
Environmental Health Perspective
ICRERTT Fellowship- International Union Against Cancer
Research Grant Council- Government of Hong Kong
Vision of Tomorrow Foundation
Editorial Work
Section editor, Advances in Space Sciences
141
Dr. Howard B. Lieberman
Reviewer
Grants
Chairman, NIH Radiation Study Section
Manuscripts
Biotechniques
Gene
International Journal of Radiation Oncology, Biology, and Physics
Nucleic Acids Research
Radiation Research
Member
Advisory Board, Summer Research Program for NYC Secondary School Science
Teachers, Columbia University
American Association for the Advancement of Science
American Society for Microbiology
Environmental Mutagen Society
Genetics Society of America
Radiation Research Society
Elected Biology Councilor
Chairman, Web-Site Committee
Chairman, Michael Fry Research Award Selection Committee
Sigma Xi
Theobald Smith Society
Stephen A. Marino
Member
Columbia University Radiation Safety Committee
Radiation Research Society
Sigma Xi
Guest Scientist
Brookhaven National Laboratories, Upton, NY
Dr. Tej K. Pandita
Member
American Association for the Advancement of Science.
American Association of Cancer Research.
Radiation Research Society
The American Society of Microbiology
NIH Study section
142
Reviewer
Cancer Research
Clinical Cancer Research
Cytogenetics and Cell Genetics
Carcinogenesis
FASEB Journal
International Journal of Radiation Biology
Mutation Research
Oncogene
Oncology Reports
Proceedings of National Academy of Science, USA.
Radiation Research
143
THE COLUMBIA COLLOQUIUM AND LABORATORY SEMINARS
At intervals of approximately one month during the academic year, a regular
colloquium has been held to discuss ongoing research. Dr. Tej Pandita organized them
and scheduled the speakers. These have been attended by the professional staff, graduate
students, and senior technical staff of this Laboratory and RARAF, as well as by scientists
from other departments of the College of Physicians & Surgeons interested in
collaborative research. Attention has focused on recent findings and future plans, with
special emphasis on the inter-disciplinary nature of our research effort.
During the year, we have been pleased to welcome a number of visitors who have
presented formal seminars and/or spent time discussing ongoing research with various
members of the Laboratory. These have included Drs. Carol Griffin, Medical Research
Council, Harwell, UK, Brian Ponnaiya, University of California, San Francisco, Yuxin
Yin, Princeton University, Princeton, NJ, Adayabalam Balajee, National Institutes of
Health, Bethesda, MD, R. Kucherlapati, Albert Einstein College of Medicine, NY, Joel
Bedford, Colorado State University, Ft. Collins, CO, Terry Ashley, Yale University
School of Medicine, New Haven, CT, Sally Amundson, National Cancer Institute,
Bethesda, MD, Tracy Ruscetti, Los Alamos National Laboratory, Los Alamos, NM, JunJie Chen, Dana Farber Cancer Institute, Boston, MA, Jai Parkash, Haverford College,
Haverford, PA, Simon J. Hall, The Mount Sinai Medical Center, New York, Srikumar
Chellappan, Columbia University, New York, Carmel Hensey, Dept. of Genetics &
Development, Columbia University, Jan K. Kitajewski, Dept of Pathology, Columbia
University, and Basil Worgul, Dept. of Ophthalmology, Columbia University.
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PUBLICATIONS
Azbaid AH, Dymnikov AD, and Martinez, G. The optimal construction of an
electrostatic quadruplet as focusing microprobe system. Nucl. Instr.and Meth.
B158, 61-65 (1999).
Brenner DJ, Does fractionation decrease the risk of breast cancer induced by low-LET
radiation? Radiat. Res. 151:225-9 (1999).
Brenner DJ, The relative effectiveness of exposure to 131I at low doses. Hlth. Phys.
76:180-185 (1999).
Brenner DJ and Hall EJ. Fractionation and protraction for radiotherapy of prostate
carcinoma. Int. J. Radiat. Oncol. Biol. Phys. 43:1095-101 (1999).
Brenner DJ, Leu C-S, Beatty JF, Shefer RE, Clinical relative biological effectiveness of
low-energy x-rays emitted by miniature x-ray devices. Phys. Med. Biol. 44:323-33
(1999).
Brenner DJ and Sachs RK, A more robust biologically based ranking criterion for
treatment plans. Int. J. Radiat. Oncol. Biol. Phys. 43: 697 (1999).
Calaf G, Russo J, Tait L, Estrada S, and Alvarado ME. Morphological phenotypes in
neoplastic progression of human breast epithelial cells. J. Submicroscopy Cytology
and Pathology. Vol. 32, n.1 (January 2000).
Dymnikov AD. Matrix methods in periodic focusing systems. Nucl. Instr. and Meth.
A427:6-11 (1999).
Dymnikov AD and Garcia G. High-frequency focusing system for nuclear microprobes.
Nucl. Instr.and Meth. B158:85-89 (1999).
Hall EJ, Miller RC, and Brenner DJ. Radiobiological Principles in Intravascular
Therapy. Cardiovasc. Radiat. Med. 1:42-47 (1999).
Hall EJ, Schiff PB, Hanks GE, Brenner DJ, Russo J, Chen J, Sawant SG, and Pandita
TK. A preliminary report: Frequency of A-T heterozygotes amongst prostate cancer
patients with severe late responses to radiation therapy. Can J Sci Amer 4: 385-389
(1998).
Hei TK, Liu SX, and Waldren CA. Mutagenicity of arsenic in mammalian cells: Role
of reactive oxygen species. Proc. Natl. Acad. Sci., 95: 8103-8107 (1998).
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Hei TK, Piao CQ, Wu LX, Willey JC, and Hall EJ. Genomic instability and
tumorigenic induction in immortalized human bronchial epithelial cells by heavy
ions. Advances in Space Research 22: 1699-1707 (1999).
Johnson KL, Brenner DJ, Geard CR, Nath J, Tucker JD, Chromosome Aberrations of
Clonal Origin in Irradiated and Unexposed Individuals: Assessment and
Implications. Radiat Res 152:1-5 (1999).
Kharbanda S, Pandey P, Morris PL, Whang Y, Xu Y, Sawant S, Zhu L, Kumar N, Yuan Z,
Weichselbaum R, Sawyers CL, Pandita TK, and Kufe D. Functional role for the c-Abl
tyrosine kinase in meiosis. Oncogene 16: 1773-1777 (1998).
Martinez G, Azbaid AH, Dymnikov AD. The numerical synthesis of an optimal
microprobe focusing system. Nucl. Instr.and Meth. A 427, 344-349 (1999).
Miller RC, Randers-Pehrson G, Geard CR, Hall EJ, and Brenner, DJ. The oncogenic
transforming potential of the passage of single alpha particles through mammalian
cell nuclei. Proc. Natl. Acad. Sci. USA 1999: 18-22 (1999).
Okayasu R, Takashashi S, Yamada S, Hei TK, and Ullrich RL. Asbestos and DNA
double strand breaks. Cancer Research 59:298-300 (1999).
Okayasu R, Wu LJ, and Hei TK. Biological effects of naturally occurring and manmade fibers: In vitro cytotoxicity and mutagenesis in mammalian cells. British J.
Cancer 59: 298-300 (1999).
Pandita TK, Westphal CH, Anger M, Sawant SG, Geard CR, Pandita RK, and
Scherthan H. Atm inactivation results in aberrant telomere clustering during
meiotic prophase. Mol. Cell. Biol. 19:5096-5105 (1999).
Piao CQ, Willey JC, and Hei TK. Alterations of p53 in tumorigenic human bronchial
epithelial cells correlate with metastatic potential. Carcinogenesis 20:1529-1533
(1999).
Rakovitch E, Mellado W, Hall EJ, Sawant SG, Geard CG, Newman RN, and Pandita
TK. Penclomedine-induced DNA fragmentation and p53 accumulation correlates
with reproductive cell death in colorectal carcinoma cells with different status of
p53. Oncology Reports 6:161-165 (1999).
Rakovitch E, Mellado W, Hall EJ, Pandita TK, Sawant SG, and Geard CR. Pacitaxel
sensitivity correlates with p53 status and DNA fragmentation but not G2/M
accumulation. Int. J. Radiat. Oncol. Biol. Phys. 44:1119-1124 (1999).
Sawant SG, Gregiore V, Umbricht CB, Cvilic S, Sukumar S, and Pandita TK.
Telomerase activity as a measure for monitoring radiocurability of tumor cells.
FASEB J. 13:1047-1054 (1999).
146
Smilenov LB, Dhar S, and Pandita TK. Altered telomere nuclear matrix interactions and
nucleosomal periodicity in cells derived from individuals with ataxia telangiectasia before
and after ionizing radiation treatment. Mol. Cell. Biol. 19:6963-6971 (1999).
Smilenov LB, Mellado W, Roa PH, Umbricht CB, Sukumar S, and Pandita TK. Cloning
and chromosomal localization of Chinese hamster telomeric protein chTRF1: A
potential role in chromosomal instability. Oncogene 17: 2137-2142 (1998).
Smith LG, Miller RC, Richards BS, Brenner DJ, Hall EJ, Investigation of
hypersensitivity to fractionated low-dose radiation exposure. Int. J. Radiat. Oncol.
Biol. Phys. 45:187-192 (1999).
Waldren C, Vannais D, Drabek R, Gustafson D, Kraemer S, Lenarczek M, Kronenberg,
A, Hei TK, and Ueno A. Analysis of mutant quantity and quality in human hamster
hybrid AL cand AL-179 cells exposed to gamma-rays and HZE-Fe ions. Advances
in Space Research 22(4): 579-585 (1998).
Vaziri H, Squire JA, Pandita TK, Bradley G, Kuba RM, Nolan GP, Zhang H, Gulyas S,
Hill RP, and Benchimol S. Analysis of genomic integrity and p53 dependent G1
checkpoint in telomerase induced extended life span fibroblast (TIELF) cells. Mol.
Cell. Biol 19: 2373-2379 (1999).
Wu LJ, Randers-Pehrson G, Waldren CA, Geard CR, Yu ZY, and Hei TK. Targeted
cytoplasmic irradiation by alpha particles induces gene mutations. Proc. Natl.
Acad. Sci. U.S.A. 96: 4959-4964 (1999).
Xu A, Wu LJ, Santella R, and Hei TK. Role of oxyradicals in mutagenicity and DNA
damage induced by asbestos in mammalian cells. Cancer Research 59:5922-5926
(1999).
Zhou HN, Zhu LX, Li KB, and Hei TK. Radon, tobacco-specific nitrosamine, and
mutagenicity. Mutation Research 870(430):145-153 (1999).
147