Establishment and characterization of a granulocyte

Establishment and characterization of a granulocyte

Establishment and Characterization o{ a (;ranulocyle-l\Iacrophage
Colony-Stimulating l'actor-Dependent Human \lyeloid Cell Line
By Sadik Oez, H.nne T tre
ba.h
m!nd Fahso d, nol.id S.h.elz, Chr stoph Bührer,
Fa
Jens Atzpöd
e.,
and Joach m R. Ka den
A n6w hum.n
myeloid cell line has been established
recently lrom ihe bone marrow c€ls of a patisnr wtrh
chronic myologenous l€!k€mia in bläsr crisis. Th€ äctiv€
proliierat on and survivalof rhe c€lls in FPMll640medrum
containing fetal calf serum are clearly dependenr on rh€
pr€ssnc€ of either natural or re6ombinanl humän granulocyte macrophage colony-stimularing läctor (rhGM CSF).
bur nor cD3, cD7, CDl0, CD'I1b, CO14, CD2o, or CD42b.
oespite permanenl culturing in rhcM CSF (100 U/nL), the
cells do noi difi6r6ntiar6 and boar the myelomonocytic
surrace markers CD34, CD13, CD36, as well as HLA DR,
additional hybridizing rsstrictiontr.gm€nts ora.6and 11.0
lr .l\lircru , s oj.
..1e boo.o pro\r '1.rbr .,.
ro,,
,,d 9 e .r'
t'.i io1
\!.i.u\ ceLL liies hale been rcporrcd from nouse or hum.n
oriCiis ibr inrcrlcukin-2 llL-:). tunror ne.rosis lactof o
l.r
\-
(TNFd).r lL 6,igm.ulocyle colonJ stinulalinB facto. (G
CSI),r and gmnuloclrc-na.roph!ge CSF (GM-CSF) "
GV-CSF is one of the trellchafucrcrizcd hendtopoler'c
gro*Lh facloß Lhat acts on m)eloid progenitor cells al
värious naLunLi.n sLaSes and induccsproliferation, ditreren
rirrion..nd funclionrl stimularion."' The p..Liferarive
ef-
fccl ofCNl'CSF is nor absolurelt restricLed ro mreloid cclh,
,1. . j ' \. llo, rdc_o.. .'1or. d
':n.? l. rr' '.
celL Liies.' and huhai e.dotheliaL cells' ah. havc bccn
rcportcd 1o .cspond to G\f CSF
ln rhß rcporr M dcscnbc rhc chamctcristicsofa CM-CSF
deJrendeir n,lclold ccll llne. shich h!5 recentlr been de.,!ed
ftom Lhe bone nrroE cells of a prtient trith chronlc
ürtelog.rous leukenia (CN{L) in blasr c.isis Althoügh
seveful!s|)ects rk stilL under ii!enigation, se rhink rlat this
ne\l)_ $ublished cell line, desiemred GM/SO. $illconlribuLe Lo undeßr.id Lhe biolo-qic acrion of GNI-CSF on the
c.lls Bl
usine rheeficct Df
G\'I CSFon
this cellline.
! 'ery
ren\iri1e bioassr! has dLrcadr been delelol'ed fo. measurin8
rhc C\.1-CSF concenlfurions i. cell culLure supernarrnrs or
in human scr. (Ocz S. Bührer C, Atzpodien J, Kalden JR.
The predominanr karyotype, apart rrod tetraploidy
several
lder t[9;22]), der {13q1, wirh rhrss addiiional marker
f.oh which one was observed in the pa
tienfs l€ukemic cells. On A9lll disesred ONA, Southern
blot anälysis with bcr 5 as the probe detected two
chromosomes,
a
|
99O
l!mphoctrcs)i $r.nelr d.cicrscd xLkaline leukoc!le phosphatse (3
score): as *ell as crlolo-qic aod hnblogic eriminr!io. ol the bone
mano* specrnren oblaried lbrouCh rlir. crei tü..ttrre AllhoLgh
HLA tJ-pinghrdbeen penormed, nokart.llte.nxllsn h!dbecn
undenake. $ith r.rtccr tolhe Ph .hrono$nc.
InniaLl!, Lhc taticnr had bccn üearöd wirh busLltun onlll until
Vly 1937: bur busulfan srs inelleclive, a.d {as disconlhued lnd
replaced br nldroxrurea. Th. pali.nfs famill doctor thcn tookover
artendance in June 1987 bul acrinh3d to referhertolhe hosprtalrn
December 1988 becxüseola blasl incrcascin periphüalblood Two
dr'\ l!ter, she 9a! sert l. our clinic
Cn rxrfrinalid.lhd pdtient appeared aculely ill and somnolenr,
lcrnper.ture! g.eärer lhln J0'C, dJ_spnea, aDd lach!cardix
an
"lLh coünr was
$BC
A
50
rer" old Lmrl.
p i.rt tC V.l srs rclci..d ro our crLnic
t a bhn crGis ola CVL. Fhich had been
t
t
Phenollpeeuminationof rh.teriFheral blood leukocyressaveclear
eliden.e that rhe blas cells bor. nlcloid a.tilcos su.h !s 5fr CD3
(oKTr),1t cD7 {lAr). 2? CDt0 (Bt). l6q. cD?4 (BA r), t6%
CDll (\1!7),and20'.CDr3 (M)9) Crrochcdicalnrinrneol bo.c
nr.ro* cells sho\red Ihat 25tf oirhc blans *ere po:irivc ior cncnsc
but wüc nsgxhve rorperoxidase andPAS
Despn. tf.armcnr sirl ai!ibiotics. the patieot nnaUy died ofan
oveit pn.umonia.nd sspsis on the lhhd hospilalda)
MATEN ALS
1
Jüuatt 2 l9qA;..t?ptttl 1tri1 I 1990
Addrrs rcprhi r.qr?esta Sddik 4... MD M.dizinß.h. Xliaik
I1l. Unireßitbt Ldaaten Närnb.ry, Ktatk.nhaurt !2 852A
Srbtrttted
Erldrgen, FRG
| tu r,bli.atiar.ais afthit
atti.l.rti leii.)!d i" r)dn br tag.
thrt!. . rtn.)t Thi! ani.lt klue th.rafot. he h.r.bt nnrked
''rd!.{ßcJncia ,,..r./,,..\i1,
It L S a tedt.n li la t.lel) ta
i990
[)
Th?.4hr?ti..h S.rilt |
htr.i.!l!\
AND METHODS
\\rrh tle paticifs Fridr infornre{j.onsent.
maro* .ells {&c obili.ed br
means
ol iliac .resr
puncture.
\l.J.onucl*r cells 1rü. sctrrlrcd by d€nsnr cLedi.nr .c.hfuea
iion.n Frcoll lNlcomed
Osl.. N..sar) 1nd culrured in RP\ll
'\S,
l6,10ncdiu6 \trlh 10'? retll caliscrum (fCS) and Bro$rb laDtors.
refe presened br frc.znre in liquid nnro8cn si(h
10?dimelhrlsulln\ide (D\lSO) and RPI\'l l640mediuncontarn
rie,10t FCS.rid lhese \rc.e üsed Uterlorkrtorypernabsn
Clr,kr,.r Rc.onbnranr huna. G-CSF (rhC-CsF), rhcv
CSF, nrlL-1. rhlL.,1, and rhlL-6 {ere Folid.d bJ- L Souza
(AI\'!Ceo, Thousand Oaks, C^), \rh-e!s rhlL.la aid rhlL 2 {ere
obid,ned lioD Boehri0ser\IanDhcim lFRG)
,.1,rtboltcr Rabbil ami GNI.CSF anliserun ras also a giir
Part ol lha cells
FrDh th. ltl.dt:intt.h. Klint]: llI tn.l tlatjtrl Jüt uhla\aü
k, L:nt !^ r.t E rl r nge r-Nii rhbetg: dhd M?tlt 2 i nt.h? Hnt 11s.ht 1!
.
\itb i6? bla$s, l6rt, neutrophils. 5{
m.io.!res, xid the pl.telet counr *as 46.000
16.1,000
Irnrphoc]'tes. and raa
Bate
trom aoorhü bospiraldu.
dirai.5ed in Februx.Y 1986
by The American Society ol Hematology,
lh. d,\ sc hrd been dirsno\ed tb.re trin rir no rhe basis of
clinicrl ac !..: olair..s. *hnc blood.cLl (wtsc) coün1 l:3.000
($irh 99 blrns.l9i troFlrloqies. I89 .rr-.1..rle\. 9'1 metrnr)el..j-r.s, lSL Lrdd, -10r'. .cutopliils, 6a .osinophils. ,1.; brsophiLs, zt
ninuscripr sübn,irrdl
CASE BEPORT
in
cells,is45,XX, 9, 17, 19, 22,7p ,9q+
.! Hü,üb|.si
r.06 L
Souza (AI\'Cen). MonocLondl dntibodies (l{oAbt aeainn
CDr4 (HPCA l).CDi (Leu 9). CDl0 (CAl l-A).CDl6 (Lcu I tb),
.D.'0. 1 o..l \DP
mouse werc purchased
0
'o".1,.
fton lecton-Dicki.son
Lg
r.d.o..
(Mountain Vies,'
c^) cDl6 (oKM il. cDf b (oKM l). cDl] (oKNt t3), cDl4
(oKNl lll and CDr (OKT l) lrom Ofrho-Dirgdo{,c Sr-$enr
i\eckrrt.dihd. FRG): and CD,1lb (AN jl) fron Dakopatß
0406 a9:)i90i76aJ 00 t 1s3 aaia
srood
vo 76
No 3lAusust
I
i990
pp
5r3
532
6M-CSF DEPENDENT HUMAN MYELOO CELL
LINE
Praliletulian aßa\. Afrc. washine tne cells ihree tincs in
:
phosphale-bü|löred saline (rBs), ihe celts { 1 I01 celh/sell) w*c
incubated for 72 hoüa in flat bouon 96-we1l microriter plaFs in a
nn,l voluhe o1200 /]L RPMI 1640 mediün containi.g lo% FCS
and various cltokines. rH-rhlnidine (Aneshan, Brlunschweie,
FRC) ras,dded at I aci (r7 kDq) Fer well for 4 houß before the
end.rtlecullure Thecelhweretnenhanestedon6bergld$fiheß
blolted onroa nt10. n.nbrane (cenescreen PlüsiNE\. duponrde
Nemou$- lad Honbure, FRG). Thc b./-5' prcbe (proridcd by C.R.
Barhao, Uln. FRC) was tabeled by .ick-translation and hybrid
izedaccordinetonandardprotocols.L. Staiüi,slor
Mat-Grtinwald and cytochenicat stain
ings torPAS, nycloporoxidase, and d-naplryl acerarc esLerase were
pertnrmed in our roütine iematoloct laboraroiy
hi.ota
and deasurcd by liqüid scintillation countin8. Al1 assays were
perlorned in tdplicate and dala are expre$ed as tne nea. counß
iH-thynidine ircorporarcd
ter ninule ol
Im,tunophenat]tihs Cell surlace aniigens sere detec&d by
nandard i.direci innünofluorescence .say. ln briel thc cels were
incubated al 4oC lor 30 ninuts üith nouse MoAbs, washed llrcc
tif,es by cenlrifugltion witn PBS. followed bt .ddins FITCco.Juglted soat anl1-nouse antibody aor r0 ninutes at 4oc. shined
cclLs sere üen evalüated with ! Uv-nicroscope The cells qere
co.sidered positire for ücanligen in question shen tne nuorcscence
i.lensny on inlact cells was clearly visible in comparison silh the
cells treated only witn FITC-mnjügaLed eoar !iri-mouse airibody
Chronasane
analrtß.
Krryotyping oaCM/SO c.lh and fyobt sandard techniqucs,
pr€served bone narrow cells rvas perforned
includin8 Ciensa fypsin bdnding.
'
ahdlttü. High noleculd $eight DNAftoncells
rnhrg.lll, elecrophoresed on a 0.8% agarosegel, and
Southühblot
*as djgested
BESULTS
.ellulü charucte^ti.s.
Estdblishment and
bone
lnitially,
maros nononuclear celh
were cultured in the prcs
ence of rhG'CSF. .hGM'CSF, and IL-3 (1,000 U/mL cach)
$i1h the intertion ofstudyi!81!e ditre.entjarion capacity of
the blasl cells. Asüsual.1lc cnlture nedium used rvas RPVI
1640. supplemefued
$ilh
L0% FCS.
02 frmol/L
exposed to rhG\,I'CSF. In subsequent srcls, liree slable
subclones sere establlshed bl usire liniling dilution tech-
w
)
g
fr
e
F
G
&
0ä
t.w
rG
"Q
\h
Fis 1. Morphol6sy,nd morphotosic difl6r6nti
ation or GM/so ool|3r cM/So c6tts growins in
m€dium containins loo U /bL rhcM-CSF .tono {A),
dditionat PMA {1oO
24 hours {3), Th..d66r6nr cotls w6r€
cuiturod on st€rit. slid6s and .n-dri6d beror€
staining {May Grünw3ldl.
ns/mllror
s*#
!
,
-!
t
q"
t
B
L-
Slulamine, ard antibiotics. Wlthin ih€ fis1 2 weeks rle.e
sere no d flerence ro be.erec..d. e .he in pro re% or '. .e
or i! malu.dtion of lle cells in the presence of rhese lhree
hemaropoietic gro*lh factoß. However. afte. several pa!
sages an active prollfe.alion sas süstailed only by the celh
-!k"'
E
Fig 2, Prolir.rativ6 r6
cponss of GM/SO c6lt3 io
rhGM-CSF, rhc-csF, rhll 1a,
rhlL 2, rhll-3, rhll-4, or rhll 6
€irhor alono ai conc€ntr.iions
b6rw6.n 1 a.d 1,0OO U/mL or
in combin.rion wirh 1 U/mL
rhGM CSF| mo8lr.n6nt an€r
72 houra. Th. n6urrarizing 6nti
bodY wls to3t€d in l:4o linal
dirurion, and th€ curv€3 f.pf6,
a
rique. These subclones din'er in their norpholos] and
generarion time, but thet, still retain their .equkement for
external supplJ_ of rbGM CSF. The dependencr can be
circumvented if the cells are cultnred by carefully reducing
GM-CSF fo. 2 or mo.e wceks. As a conscqü€nce, celh tlat
have once becone independent do no1 require GM'CSF for
their survival and prolile.ation. whereas they süstain to
.espold to GM-CSF ivitb increased proliferalion (data nol
sho{n). To avoid overly complexda!a. onll thecloncrermed
as GM/SO is discused in this reporr.
^
r{
Although tlc.c is a colsiderable variation in cell size, the
tlpical mo.pholosy of GM/SO cells in May-Grü.Nald
stainine is a large, rourd, of slighrLl jr.egular nuclcus, three
to six proni.en! nucleoli. ard deep blue cytopLasn wjih a
perinu.lear pale zone (Fis lA). There is no s.llnula present
and the cytochemical stainiig tor PAS. m)eloperoxidase.
ard a-napht_vl acetate esrerase r.e neeative. Moreorer. 1he
cells grosing in 100 U/mL ibGM'CSF (unless othe.ivise
ltinulated) rclain rhe; norpholoe] and display no olerr
dillerentiation jn liquid cultlre. HoNever. a rapid change
,*{-d-}t-
"-fi-!;
tf
" f{-l f
i:
$-lr
10
D
1l-f
E h-r
!-tt-
t6
13
F
_r
!i
12
I
s€
17
1a
15
_-l.l
G
-r a-r-
20
21
,17
Fig3. Sr.nrin. ka'yorypo of tin€ cM /SO: 4s, xX, - 9,
ohromGom€s lM1, M2, and M3)rhat oould not b6 €x!cdv cla$ili6d,
22
,9q-
dor t{S:221 (q3ajq
1 1
l, dor (13q1, otusihr€6 mark6r
GM.CSF DEPENDENT HUMAN MYELOO CELL LNE
rnlo a monocyte-nacrophagc morphologl aid
increascd
adhe.encc!oplasLicsurla.ecanbcinducedb) theddditionof
phorboln!rishrc acerale (P\,1A) (Siema, St Louis. MO) at
concentratio.s bcrween 1.000 ng,lml- and I ne/ml (FiB
I
A
B
c
D
B).
Inrmunophenor!I)e anllysis sho\led rhar rhe CM/SO ccUs
clea.ly bear surlace dntiecns of mlelononocllic cel15 CD3.1
(Sreater llan ?5%), CDlS (g.catü than 967.), and CD36
{gieaLer thrn 92:Z), bur iot rhe oller typical granuloc}re,
nacrophage anlisens such as CDllb. CDl.1. or CDl6.
Whilc HLA DR (greaiü than 98.:i) is slronel) exprcsed on
Lhe cell\. luphoclric hukers such as CDl, CDr, CDl0.
aD'i)
"nd
As shosn
I olb., . i. n.d-l(r ( D-)b . e . d.nl
.c..
Fig 2. the p.olifer.livc cfccl ol cNI CSF on
r'(.( .ll . 'ndo'eorFde-."ndr e'' u'
r's oo
\re-.
11.O
to be b\' 100 U/i1l-. Hoseve., b) nsing a pol,raalent rabbit
anliscrxm a-qainst GN{-CSF at neutralizing titeß. the lrolifcrarion could be conpleteLr abolished in ihe\e assxys Tlis
inhibilion rppareitl] is no1 due to anr lo\ic effecl in
8.6
7.2
antise.um. sincc lhe neutralizine elTecr could in ruin be
enrnel! supp.essed bl exces of rhGNI CSF in culture
nedium In eddilion. $c simullaneously lested rhc CSF and
rhlL-3, as qcll as rhIL-la. rhlL-2. rll]--.l. and rhll-6 to
'.le | .n\ Do.' b. r-P. r .1 rL . .e l l.-e .\' rLc g \c
exanples demonstratej there {as io deteclable prolileralive
eflict of lhese cytokines a1 conccrtrations betseen I and
1,000 ll/nL, either aloie or in combinarion with 1 U/mL
rhGl\f-csF.
5.5
t
*
qse
5.O
C),tagereti. antl DNA andlr:sts To find ou1 xhellc.
GN1/SO celis possess thc typical characrerislicr of .) CML
ceLl, eg. PI' chrohosome or ,.F r/ rearangefreil. se
anxl)zed the karyorype with a C-bandin-s rech.ique and
cramined the Dr.\A hybridization will a ,..-5 probe on
Southern blot\. Tle srcmline karyoL\pe rls determined as
XX. 9. 17. 19,
22. 7p . 9q- (der t[9i22]
tq34:ql ll), de. ( l.lq). and tlree additiondl ndrke. ch.omo-
45,
somcs (Fis 3). Since v!rious numerical chronosona I anonalies $ereobserved in the bonenanox bl.sl cells, ar leasr one
oftnesc markeß
(Ml),
toCether
sith
the 9q+ chromosome,
seenr\ to bccharacLe.isticforlhepalienfs leukemictransfor
maüor because it was also presenr in all neraphases of the
bone marol cells freshl! frozen during rhe btasl crisis. t!
thc couße of establishneir. an incrcasing raLe oirelraploid
karyotypes Na\ found in sübsequen1.nal!ses Soulhern blot
analtsl! oft!/11dieested DNA wirh bcr 5' as h)bridization
probesho{ed tso AglII reslriction fraemenrs ofE.6 and I1.0
kilobasepair (kbp) size, girineclcarcvidencctbr aicaffance'
ment ltpical fo. CNfL (Fig 4. lane
ÄddirionalL), tlc
sisnalof tle 8.6 kbp fraghent. ifcompared
^). 10 1le 5.0 kbp, js
m.n) tlme\ st.ongcr, and in rlis case indicares a. anplifica'
tion proces. The occurence oftwo aberrani fragnents $as
obse.ved rn scyüal blouing a.d h)bridiz.tion cxperinenrsl
thus,.n artifact due to p.fiial dieesrions is ercluded.
D SCUSSION
Thecelllines Lhat eere cstabiished in.ecenl dccades have
be.one an c$ential pa.1 ol our conre porar) biomcdicat
.csearch Their conlribulions ro d;coveri!8 laflous iew
4,
Sourhsrn hybridizaiion of DNA from cM/so .s tin€
5 pfob*. Each rans conlainod 5 ps ol ,g/ digoslod DNA
from GM/so cs s (a), K562 c€ tino (a), bon€ marow csrrs of
Fig
u3ins bcr
anorh€r pati€ni wirh cML lcl,.nd p.riphsrat btood mononuctdaf
corls or a h€a thy p€13on lDl.
diaenoslic and therapeuric a-qenrs can actxallt be exempli
äed $i1h 11. developmelt of Mo^bs,,i plriltcarion ol
c)tokines,tr! bioasays, :.r'and so fo.ll. ]n Lhis con!e:r, the
Iictor deFndent cell
lines, bcing an ideal sub.jecr ior srudy-
'-8,'. fh\'oog. p e .n.c or 'he a or que..io....
undoubtedh oi paniculai inleresr.
I. this rcpon we descrjbc a nes
lunan myeloid cell tine
Gltl/SO. of xhictr sxrvival and p.oliferarion in vnro ß
p.inarily dependent on the presence oi hGM-CSF. eiller
natural or reconbinanl.
f.om exchding p.obable
^part
eiTecls of rhl]- 1a. rhIL-2,
rhIL-3. rhIL.1. .hIL,6, and
rhG-CSF on C\1/SO b) lesljng thcm direclly, *e provid€d
evide.ce
bl
using a neurralizing anribody rhar rhe laclor
fof the detecred proliierarion is reslricred 10
G\'I-CSF. ln contast to 1le reporled synereistic efecl ol
.espoisible
rhc-CSF.rr IL-6,11xnd IL-II on leukefric blasrs or no.fral
CD3,1- ceLlr. respectivel). {e tailed 10 observe such }
lyncrgisnonCM/SOcells.rl\{ CSFand lL-5travenor!e1
In addition. $e conlirned rhrough inmunophenotrping
OEZ ET AL
and dilit.cntiatio. induclion lbat GM/SO cells posess
exclusivelJ myelogcnous, bu1!o lymphoid, features. This is
indeed the najo. differenc€ in conparison with the othcr cell
ofthe 1{o Philad to be secondarily rearanged a1lhc 5 b./
lines reporled to be dependent on the prescnce of external
GM CSF.r6 Another charrclerislic to be noled is rhe addilio.al second sienal \irh D./ 5' probe in Soulhern blot
analysis ofDNA f.om GM/SO cells. This nnding is actually
coisistent wilh lhc obsematio! made bl Bartran e1 all
TLE colld ,l.o dere. . n
\eL { bc -tnemen. in
"dd,u 1-l 1
parient
blast cells ofa
with CNIL. and the) suggest that one
GV/SO ccllline
I Gillis S, Fern l'l, Ou W. Snitn KA T celi gtu*th factor:
Pannetcs ofFrodüction a.d quanlitative nicrcassay lor activitt. J
Vaious human nenatoFoietic gro*lh lactos (interlcukin-1, cl\l
CSF, C-CSF) sinulate clonal grosLh of nonhenatopoierc tunor
lnnunol120:2007,19?8
2. EsperikT, Nissen-Maycr J: Ä nignlt scnsnivc c.llline, WEHI
16.1clonc ll lor ncasurinC cltoloxic iactor/tunor ne{osis factor
ironhunanmonocytes. JlmnunolMelhods95:99, 1986
3. Shißizu S, Yoshioka R, Hnose Y, Sugai S, Tachibana J,
Konda S: Eslablishnent of two inte.leuki. 6 (B ccll slrnulaloN
lactor 2/interldo. t2)-dependent human bone naro* deived
dlel.ma ccll lincs. I ExF Mcd 169 139,1989
t Weinstein Y, Ihle JN, Lavü S, Reddy El: Truncation of the
c-nyb gene by a retovnal i.regration in aninterleukin 3-dependenl
nvoloid lcukcnir colllinc. Proc Natl Acdd Sci USA 83 5010, 1986
5. Lanee B, valtieri M, Santoli D, Caracciolo D, Vavilio F.
CcmFcncin I, Cri$n C, Enlnuel B, Fina. J. \o*cllP, Rovera G
Crowth laclor requirene.ß olchildhood acntc leukenia: Eslablnbme.t ol CV-CSF lepe.dent cell lines. Blood l0:19:, 1987
6. Valrieri M. Santoli D, Crracciolo D, Kreid.r BL. Altnann
Sw, Tseardy DJ, Gcnpcrlcin I. Mavilio F, LrnCe B. Rovera C
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iioi
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f,
!,
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rlpid ba.dine techniqüe loriuhdn chrofro-
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ee
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