Constitutively Activating Mutations of c

From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
Constitutively Activating Mutations of c-kit Receptor Tyrosine Kinase
Confer Factor-Independent Growth and Tumorigenicity of
Factor-Dependent Hematopoietic Cell Lines
By Hitoshi Kitayama, Yuzuru Kanakura, Takuma Furitsu, Tohru Tsujimura, Kenji Oritani, Hirokazu Ikeda,
Hiroyuki Sugahara, Hideki Mitsui, Yoshio Kanayama, Yukihiko Kitamura, and Yuji Matsuzawa
The c-kit receptor tyrosine kinase (KIT) is activated upon
ligand binding, therebyleading t o a varietyofsignaling
events that play a fundamental role in hematopoiesis. In
addition t o ligand-dependent activation, we have previously
shown that KIT is constitutively activated in a ligand-independent manner bytwopoint
mutations, Val-559 --t
Gly (G5591 mutation in the juxtamembrane domain and
Asp-814 + Val (V8141 mutation in the phosphotransferase
domain. To investigate the biochemical consequence and
biologic significance of these mutations, retroviral vectors
encoding KIPs9 or KITV8" were introduced into murine proB-type Ba/F3 cells and myeloidFDC-P1 cells, both of which
require interleukin-3 (IL-3) for their growth and survival. In
the cells,
or KITV814werefound t o beconstitutively
phophorylated on tyrosinein the absence ofstem cell factor
(SCF)that isa ligand forKIT. Chemical cross-linking analysis
showed that a substantial fraction of the phosphorylated
KITG559
underwent dimerization even in the absence of SCF,
whereas the phosphorylated KITV814did not, suggesting the
distinct mechanisms underlying constitutive activation of
KIT by G559 and V814 mutations. Furthermore, the cells expressing either KIT0559or KITV8" were found t o show a factor-independent growth, whereas the cells expressing wildtype KIT ( K I F ) proliferated in response t o SCF as well as
IL-3. Moreover, subcutaneous injection of Ba/F3 cells expressing
or KITV8" into nudemiceresulted in production of large tumors at all sites of the injection within 2
weeks, and all nude mice quickly succumbed t o leukemia
and died. These results suggest that, although the mechanisms
underlying constitutive
activation of
or KITV814
may be different, both of the activating mutations have a
function t o induce a factor-independent and tumorigenic
phenotype. Also, the data of this study raise the possibility
that the constitutively activating mutations
of c-kit may
play
a causal role in development of hematologic malignancies.
0 1995 by The American Society of Hematology.
T
tion isknown toplay acrucial rolein proliferation, differentiation, migration, and survival of hematopoietic stem cells,
mast cells,neuralcrest-derived
melanocytesandgerm
For example, dominant-negative or loss-of-function mutations at KITIW locus impair the receptor function
and thereby give rise to hypoplastic anemia and a deletion
of mast cells in hematopoietic s y ~ t e m . ~ In
, ~ addition
~ - * ~ to a
fundamental role of KIT in hematopoiesis, KIT may potentially function as an oncogenic protein, because c-kit was
originally defined as a cellular homologue of feline sarcoma
virus kit).'.'^ Furthermore, activating mutations of other
RTKs such as c-fms and c - e r b l 3 2 I n e ~ ~ have
. ~ " ~been
~ shown
to have transforming activity.
To test this possibility, we have previously examined the
expression and state of tyrosine phosphorylation of KIT in
a series of leukemia cells,'""' and have found that KIT is
constitutively activated in a ligand-independent manner in a
human mast-cell leukemia cell line, HMC-I." Sequencing
analysis of c-kit gene in HMC- 1 cells showed thepreviously
unidentified two point mutations, the Val-560 --t Gly ( ( 3 6 0 )
mutation in the juxtamembrane domain and the Asp-816
Val (V816) mutationin the phosphotransferase domain.3'
or c-kjtVa-811"(VRi4)
muTransfection of murine c-kitG'y~ss9'Gss9'
tants, correspondingtohuman c-kitGs'" or c-kit""'" genes,
intoa humanembryonic kidney cellline (293T)resulted
in aligand-independentactivation
of KITGssyor
suggesting the presence of two constitutively activating mutations in c-kit gene.j' In addition, we have recently found
that both a murine mastocytoma cell line (P-815) and a rat
mast cell leukemia cell line (RBL-2H3) carry constitutively
activating mutations of c-kit gene in the same Asp codon in
the phosphotransferase d ~ m a i n .These
~ ~ . ~results
~
suggested
that the activating mutations of c-kit gene could be involved
in some aspect of neoplastic transformation of KIT-positive
hematopoietic cells, including mast cells. However, the biologic significance and biochemical consequence of these activating mutations have yet to be determined.
HE PROTO-ONCOGENEc-kit is allelic with the white
spotting (W) locus on mouse chromosome S,'.2 and it
encodes a receptor tyrosine kinase (RTK) that is a member
of the same RTK subfamily (typeI11 RTK) as the receptors
for platelet-derived growthfactorand
colony-stimulating
factor 1 (CSF-1).3.4This RTK subfamily is characterized by
the presence of five Ig-like repeats in the extracellular
domain and an insert that splits the cytoplasmic kinase domain
into an adenosine triphosphate (ATP)-binding region and
the phosphotransferase d ~ m a i n . "The
~ ligand for c-kit product has been genetically mapped at
the steel (Sl) locus on
mouse chromosome 10, and is variously designated as stem
cell factor (SCF),mast cell growth factor, kit ligand, or steel
factor.'-''
Binding of SCF to c-kitreceptortyrosine kinase (KIT)
triggers a series of rapid events, including KIT dimerization
and activation, association of KIT with certain cytoplasmic
enzymes,and phosphorylation of othercellular proteins,
thereby initiating a signaling
cascade that leads to various
cellular response^.^^^^^^-^' This KIT-mediated signal transduc-
+
From the Second Department of Internal Medicine and Department of Pathology, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osuka 565, Jupan.
Submitted June 20, 1994; accepted October 17, 1994.
Supported in part by grunts from the Ministry of Education. Science and Culture, the Inumori Foundation, Senri L$e Science Foundation, and Mochidu Memorial Foundation.
Address reprint requests to Yuzuru Kanakura, MD,PhD,The
Second Department of Internal Medicine, Osaka University Medical
School, 2-2, Yamadaoka, Suita, Osaka 565, Japan.
The publication costsof this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with l 8 U.S.C. section 1734 solely to
indicate this fact.
0 l995 by The American Society of Hematology.
0006-4971/95/8S03-0024$3.00/0
790
Blood, Vol 85, No 3 (February l),
1995:
pp 790-798
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
ROLE OF c-kit MUTATIONS IN TUMORIGENESIS
In this study, we introduced wild-type KIT (KITw),
KIPSS9and KITV814
into cells of two murine interleukin-3
(IL-3)-dependent cell lines, Ba/F3 (pro-B type)35and FDCP1 (myeloid type).36Expression of
or KITVs14with
constitutive tyrosine kinase activity was found not only to
abrogate IL-3 requirement of the cells, but also to cause
them to become tumorigenic in nude mice. Furthermore,
chemical cross-linking analysis showed that KITG559 was
organized at the plasma membrane in a dimerized form,
whereas KITV8l4was not. Thus, the results of these studies
provide new insight into potential role of the activating mutations of c-kit proto-oncogene in receptor activation and oncogenesis.
MATERIALS AND METHODS
Reagents. Recombinant murine (rm) SCF and IL-3 were generous gifts of Kirin Brewery COLtd (Tokyo, Japan). Antiphosphotyrosine
a murine monoclonal antibody (MoAb) generated
against phosphotyramine, was generously supplied by Dr B. Drucker
(Oregon Health Science University, Portland, OR). Rat-antimouse
c-kit MoAb (ACK2) and full length of murine c-kit cDNA were
kindly donated from Dr S.-I. Nishikawa (Kyoto University, Kyoto,
Japan)." Anti-c-kit polyclonal antibody against synthetic peptide
of C-terminal of human KIT was purchased from Oncogene Science,
Inc (New York, NY); this antibody was found to react with murine
KIT as well as human KIT. G418 (geneticin) and Polybrene were
purchased from Sigma Chemical CO (St Louis, MO), and chemical
cross-linker BS3 from Pierce (Rockford, IL).
Cells and mice. Ba/F3,3s a murine IL-3-dependent pro-B
lymphoid cell line, was cultured inRPM1 1640 medium (Nakarai
tesque, Kyoto, Japan) supplemented with 10%fetal calf serum (FCS;
Flow Lab, North Ryde, Australia) and d L - 3 at the concentration
of 10 ng/mL. FDC-P1,36a murine IL-3-dependent myeloid cell line,
was maintained in the Fischer's medium (GIBCO, Grand Island,
NY) supplemented with 20% horse serum (GIBCO) in the presence
of rmIL-3 (10 ng/mL). A helper virus-free packaging cell line,"' $2,
was maintained in Dulbecco's modified essential medium (Nakarai
tesque) supplemented with 10% FCS. SUSl-3T3, a fibroblast cell
line derived from SVS1-mutant mice lacking in SCF expression, was
BALB/c-nu/
established in our laboratory as described previo~sly.~~
nu (nude) mice were obtained from Nippon SLC (Shizuoka, Japan);
all mice were female and were 8 weeks of age at the time of cell
injection.
Construction and retroviral transfer of mutated c-kit gene. A
retroviral vector pM5Gneo;' a derivative of myeloproliferative sarcoma virus (MPSV), was a kind gift from Dr W. Ostertag (Universitat Hamburg, Hamburg, Germany). The mutated murine c-kit genes
or
9KITV8I4were constructed by site-directed mutaencoding KIFss
genesis as described p r e v i ~ u s l yThe
. ~ ~ full length of wild-type (WT)
or mutated c-kit cDNAs were inserted into the EcoRI site of
pM5Gneo. Retroviral vector (pM5Gneo) alone or retroviral vectors
containing c-kitw, c-kitGSs9or c-kitV8I4genes were transfected into
packaging cell line ($2) by calcium phosphate method, and G418resistant clones of each packaging cell line were isolated. The virus
titer was determined by infecting SVSZ-3T3 fibroblasts with serial
dilutions of retrovirus-containing supernatant from each clone and
by counting the numbers of G418-resistant colonies. The resultant
viruses were termed pM5Gne0, pM5Gneo-KITWT, pM5GneoKIFss9,
and ~ M ~ G ~ ~ O - Kand
I Thad
~ " approximately
~,
comparable
viral titers ranging from 2 to 5 X 10' colony-forming units/mL of
supernatant. For gene transfer, BaF3 and FDC-P1 cells (2 x 10'
celldeach cell line) were cocultured with the virus-producing packaging cells for 48 hours in the presence of rmIL-3 (10 ng/mL) and
791
Polybrene (8 pg/mL). After infection, the cells expressing neomycinresistant gene were selected by cultivation in a medium containing
G418 (0.6 mg/mL) and rmIL-3 (10 ng/mL).
Flow cytometry. The cells ( 5 X 10') were washedwithphosphate-buffered saline (PBS) and resuspended inPBS containing
0.5% bovine serum albumin (BSA; Sigma Chemical CO, St Louis,
MO) and 0.1% NaN3. Cells were incubated with rat antimurine c-kit
MoAb (ACK2) or rat monoclonal IgGl control (Becton Dickinson,
Mountain View, CA) at 4°C for 30 minutes, and then washed three
times with the buffer. Cells were subsequently incubated with fluorescein-conjugated goat-antirat IgG antibody at 4°C for 30 minutes,
and washed two times before analysis using a FACScan flow
cytometer (Becton Dickinson).
Stimulation with factors and cell lysis. Exponentially growing
cells were washed free of serum and growth factors and incubated
in serum-free medium (Cosmedium 003; Cosmobio, Tokyo, Japan)
for 12 hours at 37°C to factor-deprive the cells. The cells (lo7cells
suspended in 1 mL of Cosmedium 003) were exposed to SCF (100
ng/mL) at 37°C for 15 minutes. After stimulation with factors, cells
were washed with cold PBS and lysed in lysis buffer (20 mmom
Tris-HC1,pH 8.0, 137 mmom NaC1, 10% glycerol, 1% Nonidet
P-40) containing protease and phosphatase inhibitors as described
p r e v i o ~ s l y . ~In~soluble
~ ~ ~ material
. ~ ~ . ~ was
~ removed by centrifugation
at 10,000g for 15 minutes at 4°C.
Immunoprecipitation and immunoblotting. The procedures of
immunoprecipitation, gel electrophoresis, and immunoblotting were
performed according to the methods described p r e v i o u ~ l y . ~ ~ ~ ~ ~ ~
Briefly, the lysates were precleared with protein-G Sepharose beads
(Pharmacia AB, Uppsala, Sweden) for 2 hours at 4°C.The precleared
lysates were incubated with ACK2 and protein-G Sepharose beads
to collect antigen-antibody complexes. The immunoprecipitates were
washed fivetimes with lysis buffer containing protease and phosphatase inhibitors, and subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred from the gel onto a polyvinylidene difluoride
membrane (Immobilon, Millipore Corp, Bedford, MA). After
blocking residual binding sites onthe filter by incubation in TBS
(10 mmollL Tris-HCI pH 8.0, 150 mmom NaCl) containing 1%
gelatin (Bio-Rad Laboratories, Richmond, CA), immunoblotting was
performed with an antiphosphotyrosine MoAb or anti-c-kit polyclonal antibody.
Chemical cross-linking experiments. Dimerization of KITwas
analyzed by chemical cro~s-linking."~
Ba/F3 cells expressing KITWT,
KITGss9or KITV*l4were washed twice and suspended in PBS containing 1 mg/mL BSA. The cells were stimulated with or without
rmSCF (300 ng/mL) for 90 minutes at 4"C, and were washed five
times in ice-cold PBS. The cells were then incubated in PBS containing 1 mmollL BS3 for 30 minutes at 22"C, and the reaction was
terminated by washing in ice-cold PBS, followed by the incubation
in 150 mmollL glycine-HC1 (pH 7.5) for 5 minutes at 4°C. In these
buffers used after stimulation with or without rmSCF, sodium orthovanadate (Na3V04, 100 pmoVL) was included to prevent dephosphorylation of KIT. The cells were lysed in lysis buffer, and immunoprecipitations were performed with anti-c-kit polyclonal antibody
as described above. For the detection of KIT that wasphosphorylated
on tyrosine, KIT-containing immunoprecipitates were subjected to
SDS-PAGE (4% acrylamide) and immunoblotting with an antiphosphotyrosine MoAb and developed with horseradish-peroxidase-conjugated antimouse IgG (Promega, Madison, WI) and chemiluminescence reagent (Renaissance; DuPont, Boston, MA).
Cellproliferation assay. To quantitate cell proliferation, we used
tetrazolium broan M T I [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
mide, Sigma] rapid colorimetric assay as previously de~cribed.~'.~'
Briefly, quadruplicate aliquots of cells (1 X IO4 Ba/F3 cells or 2 x
lo4 FDC-PI cells suspended in 100 pL of the medium supplemented
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
792
KITAYAMA ET AL
with 10% FCS) were cultured in 96-well microtiter plates for 72
hours at 37°C in the presence or absence of various concentrations
of mSCF or rmLL-3. M l T (10 pL of 5 mg/mL solution of M'IT in
PBS) was added to all wells for the final 4 hours of culture. Acid
isopropanol (100 pL of 0.04 N HCl in isopropanol) was added to
all wells, and mixed thoroughly to dissolve the dark blue crystals.
The optical density (OD) was then measured on a Microelisa plate
reader (Corona Electric CO, Ibaragi, Japan) with a test wavelength
of 540 nm and a reference wavelength of 620 m. This assay was
found to give equivalent results obtained by 3H-thymidineincorporation or cell enumeration as described p r e v i o ~ s l y . ~ ~ * ~ ' ~ ~ ~
Tumorigenicity assay. BaR3 cells expressing KITw,
or K I T V s l 4 were washed and resuspended in serum-free RPMI-1640
media. In addition, Ba/F3 cells transfected with pM5Gneo were used
as a negative control. Cells (3 X lo6 injected site; two sites per
mouse) were subcutaneously injected into the posterior flank ofnude
mice that had received 2.5 Gy (250 rads) of x-ray irradiation 1 day
before the injection. Mice were carefully monitored for their signs
of palpable or visible tumors at the sites of injection. The tumor
tissues were fixed by 10% formamide overnight and embedded in
paraffin. The sections (4-pm thick) were histopathologically analyzed after staining with hematoxylin and eosin.
A
V&l4
B
BdF3
FDC-P1
I
RESULTS
Retroviral transfer of wild-type and mutant KIT into murine IL-3-dependent cell lines. To investigate the role of
constitutively activating mutations of c-kit proto-oncogene
in factor-independent growth and tumorigenicity, we infected two murine IL-3-dependent cell lines, B e 3 (pro-B
type) and FDC-P1 (myeloid type), with replication-defective
retroviruses containing c-kitw,c-kitGSs9 or c-kitV8l4genes,
which were termed pM5Gneo-KITw, pM5Gneo-KI'F9 or
pM5Gneo-KITVSL4,
respectively (Fig 1A). As a control, viLogFluorescence
ruses carrying pM5Gneo retroviral vector alone (pM5Gneo)
were infected into the cells. After selection in a G418-conFig l. (AI Schematic representationof murine K I F , K I P w , and
Locations of transmembrana (TMI,kinase insert (Kt) and tyrotaining medium for 2 weeks, surface expression of KIT on
sine kinase (W)domains are indicated. KIPssscarries a point mutathe infected cells was examined with a rat antimouse c-kit
tion in codon 559 (Val Gly), whereas KITV8" has a point mutation
MoAb ACK2, which recognizes the extracellular domain of
in codon 814 (Asp -Val). (B) Flow cytometric analysis of the surface
murine KIT.Flow cytometric analysis showed that, although
binding of a monoclonal anti-c-kit antibody (ACK2) to Balm and
infection of B e 3 cells with pM5Gneo vector alone (BaF43FDC-P1 cells infected with raplicntion-defective retroviruses encodor KIT"'. Cells were incuing vector alone (Vector), K F , KIVector) resulted in no expression of KIT, BalF3 cells inbated in either negative control antibody l- - -1 orACK2 I-),
fected with pM5Gneo-KITw (BaF3-KITw) or pM5Gneowashed, incubatedwith fluorescein-conjugated goat-antirat IgG antiKIFs
(BaF3-KIFss9)
59
showed abundant surface expression
body, and analyzed on a FACScan.
of KITw or
on their surface, respectively (Fig 1B).
In the cells infected with ~ M S G ~ ~ O - K(BaF3-KITV8l4),
IT~"~
surface expression of KITV814was also detectable, albeit to
KIT was then immunoprecipitated and assayed by immua lesser degree (Fig 1B). Similar results were obtained
noblotting with either antiphosphotyrosine MoAb or antifrom flow cytometric analyses of FDC-P1 cells infected
with pM5Gne0, pM5Gneo-KITw, ~ M ~ G ~ ~ Oor- K c-kit
I ~ polyclonal
~ ~ ~ , antibody. As shown in Fig 2 (lower panel),
all of KITw,
and KITV8I4were found to be com~ M S G ~ ~ O - (Fig
K I T1B):
~ ~ FDC-P1
~~
cells infected with
pM5Gneo-KITw (FDCP1-KITWT) or pM5Gneo-KITGSs9 posed of 145-kD (mature) and 125-kD (immature) forms of
(FDCP1-KITG559)exhibited high levels of KITw or KIPss9 KIT protein in the infected BalF3 cells. Immunoblotting with
expression; the cells infected with pM5Gneo-KITV8I4 an antiphosphotyrosine MoAb showed that increased phosphotyrosine was observed in WWT,
particularly in the 145(FDCPi-KITv8") did moderate KITV8I4expression; and little
kD form of KITw, after treatment with rmSCF (Fig 2, upper
expression of KIT was observed on the cells infected with
panel). By contrast, both 145-kD and 125-kD forms of
pM5Gneo (FDCP1-Vector).
KIFss9
or KITV8I4were strikingly phosphorylated on tyroConstitutive tyrosine phosphorylation of mutant KIT in
sine regardless of m S C F stimulation (Fig 2, upper panel),
factor-dependent cell lines. To examine the state of KITsuggesting constitutive activation of KIFss9
and K I T V 8 l 4 In
'
tyrosyl phosphorylation in the infected cells, the cells were
BaF3-KIPSS9and BaF3-KITVSL4
cells, respectively. Also in
deprived of serum and growth factors for 12 hours and stimuFDC-P1 cells, KITw was found to be phosphorylated on
lated with or without rmSCF (100 ng/mL) for 15 minutes.
+
mss9
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
ROLEOF
c-kit MUTATIONS IN TUMORIGENESIS
Fig 2. Tyrosine phosphorylation of KIT in BalF3
cells stably expressing KITm, KITG5", and KIT."*" KIT
was
immunoprecipitated
withanti-c-kitMoAb
(ACK2) from lysates of the indicatedcells before and
after stimulation with rmSCF (100 nglmL). The immunoprecipitates were divided into t w o aliquots,
separated by SDS-PAGE, and subjected t o immunoblottingwithantiphosphotyrosine(anti-P-Tyr)
MoAb (upperpanel) or with anti-c-kitpolyclonal antibody
(lower
panel). The cells infected with
pM5Gneo vector alone (Vector) were used as a negative control. The mobilities of the mature (145 kD)
and immature(125 kD) forms ofKIT are indicated at
right.
793
n n n n
mSCF - + - + - + - +
anti-P-Tyr
- 145kD
anti-c-kit
tyrosine in a ligand-dependent manner, whereas KIFss9and
KIT'"'
showed ligand-independent, constitutive tyrosine
phosphorylation (data not shown).
Dimerization of KIT. All of RTKs have been shown to
undergo ligand-dependent dimerization that plays animportant role in activation of the intrinsic protein kinase activit^.'.^ To determine if the activating mutations of c-kit led
to receptor dimerization regardless of stimulation with SCF,
we performed cross-linking analysis of wild-type and mutant
KIT (Fig 3). BalF3 cells expressing KITw, KIF"' , or
KITV81'4 were stimulated with or without rmSCF (300 ng/
mL) and treated with the water-soluble cross-linker BS' that
hardly enters the cytosol. Lysates for the cells were then
immunoprecipitated with anti-c-kit polyclonal antibody and
were subjected to immunoblotting with antiphosphotyrosine
MoAb. As expected, rmSCF induced tyrosine phosphorylation of KITw in BaF3-KITw cells, and the phosphorylated
form of KITw was detected in proteins at an approximate
molecular mass of 330 kD that represented a cross-linked
dimer of KITw and rmSCF ( K I T w / ~ S C F )(Fig 3, left
panel). By contrast, a substantial fraction of tyrosine-phosphorylated KIF5s9was detectable in an -290-kD homodimeric form even in the absence of rmSCF, although tyrosine
phosphorylation of dimerized K I F ' " / I ~ S C F (-330 kD)
was slightly intensified by the treatment with rmSCF (Fig
3, middle panel). Notably, KITVX1'was barely detectable in
a -290-kD dimeric form without the addition of rmSCF,
whereas an -330-kD cross-linked form of KITV*"/rmSCF
was observed after stimulation with rmSCF (Fig 3, right
panel). These results suggest that G559 mutation may result
in dimerization of K P s s 9 ,whereas V814 mutation may not
induce receptor association at least in the extracellular domain.
Role of constitutively activating mutations in factor-independent growth. To determine if KIFss9and KITVRi4
could
induce factor-independent growth, BalF3 and FDC-P1 cells
expressing KITw, KIFss9,or KITVR1'were cultured in the
presence of 0 to 10 ng/mL rmIL-3 or 0 to 500 ng/mL rmSCF
for 72 hours, followed by measurement of cell proliferation
using an M7T colorimetric assay (Fig 4). As was the case for
each parental cell line,gs.gh
rmIL-3 induced a dose-dependent
- 125kD
proliferation of pM5Gneo-infected BalF3 and FDC-P1 cells;
and rmSCF did not have any effects on proliferation of the
cells. In addition to the expected proliferative response to
rmIL-3, BaE3 or FDC-P1 cells expressing KITWTshowed
a dose-dependent proliferation in response to rmSCF over
the range of 0.1 to 100 ng/mL, indicating functional expression of KITw. By contrast, BaF3 or FDC-P1 cells express-
n
mSCF
BS3
n
n
+ - + + - -k
- ++ -++
t - +
- ++
c
c
c
210-
105Fig 3. Chemical cross-linking analysis of K I F , K I P 9and
, KIT"'
phosphoproteins expressed on Ba/F3cells. The cells expressing
K I F (left panel),
(middle panel) and KITM" (rightpanellwere
incubated in the presence or absence of rmSCF (300 nglmL) at 4°C
for 90 minutes, and were treated with the
water-soluble cross-linker
BS3 ( l mmollL) for30 minutes at 22°C. The reaction was stopped by
the incubation in 150 mmollL glycine-HCI (pH 7.5) for 5 minutes at
4°C. and the cells were washed and lysed. Lysates of the cells were
then immunoprecipitated with anti-c-kit polyclonal antibody and
were subjected t o immunoblotting with antiphosphotyrosine
MoAb.
The mobilities of KIT-monomer (-145 kDI, KIT-homodimer (-290 kD)
and dimerized KlTlrmSCF (-330 kDI are indicated at right of each
panel.
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
794
KITAYAMA ET AL
A. Ba/F3
0.5,
0.51
0.0
0
,001 .01 .l
1
10
0
mlL-3 ( n g h l )
.l
1
10
100 1000
infiltration of BaF3-KITGss9and BaF3-KITV8'4cells into
bone marrow, spleen, and liver (Fig 5). The tumor cells
isolated from bone marrow and spleen were similar to the
injected cells in the expression patterns of KIT and B-220
antigen, suggesting that the tumors were originated from the
injected cells (data not shown). In the mice injected with
BaF3-KITWTcells, no detectable tumors were noted within
2 weeks, but small nodules developed at 3 of 8 injection
sites atday 21 (Table 1). Tumors developed at onlytwo
thirds of the BaF3-KITWT-injectedsites after an extended
latency (3 to 6 weeks), and the mice were all alive within
the 6-week observation period.
mSCF ( ng/ml )
DISCUSSION
B. FDC-P1
0.4,
0.4,
' l
J
0.2
0
.l
1
mlL-3 ( ng / ml )
10
0
.l
1
10
100 1000
mSCF ( ng/ml )
Fig 4. Proliferationof BalF3 (A) and FDC-P1(B) cells in responseto
various concentrationsof rmlL-3 (left panel) and rmSCF (right panel).
Quadruplicatealiquots of the cells expressing vector alone
(01,KITw
(B),
(0).
and KIT"*" ( 0 )were cultured with each
factor
and
cell proliferationwas measured using an
MTT colorimetricassay. The
results are shown as mean 2 SEM for one of three similar experiments.
ing either KITGss' or KITV8I4proliferated in the absence of
exogenous rmIL-3 or rmSCF; and rmIL-3 or rmSCF had a
minimal effect on proliferation of the cells. To exclude the
possibility that factor-independence could be caused by autocrine stimulation by IL-3 or SCF synthesized by the KITGss9,
KITV*'4-expressingcells th'emselves, we assayed conditioned
media from these cells for growth supporting activity. The
conditioned media could not induce proliferation or survival
of parental IL-3-dependent cell lines or SCF-responsive
KITwT-positivelines (data not shown), suggesting ligandindependent growth of Ba/F3 and FDC-P1 cells by KITGss9
and KIT""'.
Effects of wild-type and mutant KIT on tumorigenicity.
To further analyze the malignant potential of the KIT-infected cell lines, each was inoculated subcutaneously into
five nude mice (3 X lo6cells/injected site; two sites/mouse).
BaF3-vector cells that were used as a control did not yield
any detectable tumors within the 6-week observation period
(Table 1, Fig 5). By contrast, both BaF3-KITGS5'and BaF3KITV814
cells produced large tumors at all sites of inoculation
within 10 days; andall nude mice quickly succumbed to
leukemia and died within 2 to 3 weeks (Table 1). Furthermore, pathologic analysis of these mice showed massive
It is well established that a large number of structural
alterations of KIT identified so far in mice, rats, and humans
cause either the diminished levels of KIT expression or the
qualitative defects inKIT tyrosine kinase activity, thereby
leading to a decrease in tyrosine kinase activity ofIn
addition to the loss-of-function mutations, we have recently
provided evidence thatKITcanbe
activated in a ligandindependent manner by two activating mutations resulting
in intracellular amino acid substitutions of Gly-559 for Val
and Val-814 for Asp." However, the importance of these
activating mutations in cell transformation has not beenclarified.
In this study, wehave investigated the effects of these
mutations on the state of KIT expression and also on the
cell growth and tumorigenesis. The results show that G559
and V814 mutations of the c-kit proto-oncogene result not
only in constitutive tyrosine phosphorylation of KIT proteins
without the addition of exogenous ligand, but also in ligandindependent growth of IL-3-dependent murine hematopoietic cell lines, B e 3 and FDC-PI, at an almost similar level.
In addition, both of these mutations were found to render
Ba/F3 cells tumorigenic innude mice. This almost equal
ability of KITGss9and KITVX14
to induce a factor-independent
and tumorigenic phenotype waspartially unexpected, be-
Table l . Tumorigenicity of BalF3 Cells Expressing Wild-Type
and Mutant KIT
No. of TumorsINo. of Injection
Sites
Type of Cells
Day 14
Day 21
BaF3-Vector
BaF3-KlfNT
BaF3-KITG559
BaF3-KITvs'4
0110
0/10
10/10
10/10
0/8*
318,
NDt
NDt
Each type of cells (3 x lo6 cellslinjection site) was inoculated subcutaneously into the posterior flank of five nude mice (two sitednude
mouse).
Abbreviation: ND, not determined.
* A t days 14 and 21,mouse was killed and pathologically analyzed.
Pathological analysis performed at day 21 showed that BaF3-KITWT
cells were observed in tumors at the site of injection, but not in bone
marrow or spleen, whereas BaF3-Vector cells were not detected at
any sections.
t All mice died within 2 to 3 weeks.
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
ROLE
OF c-kit MUTATIONS IN TUMORIGENESIS
795
Fig 5. Hrtopathologic anah/sis ofnude mice injected
with Bal
F3 transfectants. The skin (A and
B),spleen (C and D),andbone
marrow (E and F ) of the mice injected with BaF3-Vector (A, C, E1
andBaF3-KITV8"(B, D, F) were
fixed by 10% formamide and embedded in paraffin. The histologic
sections were stained with hematoxylin and eosin. BaF3-KIT"8'4-injectedmiceshowedmassiveinfiltration ofBaF3-KIT"""cellsin
the skin (B), spleen(D), and bone
marrow IF), whereas the tissues
of BaF3-Vector-injected mice had
normalmorphologicappearance
(A, C, E). (Original magnification:
A and B, x 100; C and D, x 40; E
and F, x 400.)
cause our previous study showed that KITVXl4
exhibited a
significantly higher level of tyrosine phosphorylation and
activation than
in a transient expression system using
293T cells.32However, the results of this study using a stable
expression system suggest thatboth KITC'"' and KITV8"
have potential to function as oncogenic proteins.
We also provide the data suggest that high or stable expression of KIT^^ may play a role in excessive proliferation
or transformation of hematopoietic cells. Although proliferation of BaF3-KITW cells was not supported in vitro in the
absence of IL-3 or SCF, the subcutaneous inoculation of
BaF3-KITWTcells into nude micewas found to result in
development of skin tumors, albeit to a much lesser degree
than that of BaF3-KIPS5' or BaF3-KITVX"
cells. Nude mice
are known to lack T cells that are a major source of IL-3.
but to exhibit normal number of mast cells in the connective
tissue^^^^^' indicating the absence of IL-3, but the presence
of SCF in nude mice. Therefore, it is suggested that the slow
but detectable growth of BaF3-KITWT
cells may be generated
by their interaction with SCF that is presumably producedby
certain mesenchymal cells as a membrane-bound or soluble
form." Also, it is suggested that the enhanced expression of
KITWTby itself is not sufficient to cause cell transformation,
and the continuous stimulation of KITWTby ligand is necessary for exhibiting the transformed phenotype.
Despite the dramatic effects of G559 and V814 mutations
on the factor-independent and tumorigenic phenotypes, the
precise mechanisms by whichthec-kit mutations activate
KIT tyrosine kinase and transmit oncogenic signals into cells
are not completely understood. Although some evidence has
been presented that epidermal growth factor receptor can be
activated through an intramolecular mechanism anddoes not
necessarily require receptor dimerization,"',s" a large number
of studies have suggested that receptor dimerization plays
an essential role in the activation of intrinsic protein kinase
activity and also in signal
Furthermore, there
is increasing evidence that the transphosphorylation between
the dimerized receptor kinase domains can occur after ligand
binding and the receptor cross-phosphorylation may underlie
the natural mechanism of receptor activation.u.s'.s2In this
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
796
KITAYAMA ET AL
study, we have found that a substantial fraction of
results in production of a factor-independent and tumorigenic
exists as a dimerized form even in the absence of rmSCF.
phenotype and suggest that the constitutively activating muThis result suggests that G559 mutation may yield receptor
tations of c-kit gene may be involved in some aspect of
dimerization resulting in enzymatic activation that leads to
neoplastic transformation of hematopoietic cells. Identificacell transformation. This finding is comparable with the pretion of molecular mechanisms responsible for the receptor
vious results showing that a point mutation of c-erbBlneu,
activation and signaling events of the KIT mutants will be
in which glutamic acid substitute for valine at codon 664 in
important in understanding the role of KIT in normal and
the transmembrane domain, leads to constitutive dimerizaabnormal growth of hematopoietic cells.
tion and activation of the tyrosine kinase recept0r.2~In conREFERENCES
trast to
cross-linking analysis using BS3 showed
that a dimeric form of KITV814was scarcely detectable in
1. Chabot B, Stephenson DA, Chapman VM, Besmer P, Berntein
A: The proto-oncogene c-kit encoding a transmembrane tyrosine
the absence of rmSCF, whereas a 3 3 0 - 0 cross-linked form
kinase receptor maps to the mouse W locus. Nature 33538, 1988
of the KITV8'4/rmSCFwas observed after stimulation with
2. Geissler EN, RyanMA, Housman DE: The dominant-white
rmSCF. Therefore, it is possible that V814 mutation may be
spotting
(W) locus of the mouse encodes the c-kit proto-oncogene.
a unique activating mutation that induces factor-independent
Cell 55:185, 1988
growth and tumorigenesis independently of receptor dimer3. Yarden Y,Ullrich A: Growth factor receptor tyrosine kinases.
ization. Also, despite little evidence for dimerization of RTK
Annu Rev Biochem 57:443, 1988
in the cytoplasmic domain, it is possible that association of
4. Ullrich A, Schlessinger J: Signal transduction by receptors with
KITV814
receptor may be mediated by the cytoplasmic dotyrosine kinase activity. Cell 61:203, 1990
main, thereby leading to receptor activation and cell transfor5. Yarden Y, Kuang WJ, Yang-Feng T, Coussens L, Munemitsu
mation. The experiments to test these possibilities are curS, Dull TJ, Chen E, Schlessinger J, Francke U, Ullrich A: Human
proto-oncogene c-kir: A new cell surface receptor tyrosine kinase
rently underway.
for an unidentified ligand. EMBO J 6:3341, 1987
In addition to KIT, constitutively activating point muta6. Qiu F H , Ray P, Brown K, Barker PE, Jhanwar S, Ruddle FH,
tions have been found in hematopoietic growth factor recepBesmer P Primary structure of c-kit: Relationship with the CSF-I/
tors such as CSF-1 receptor (CSF-1R; the product of c-fis
PDGF receptor kinase family-oncogenic activation of v-kif involves
p r o t o - o n ~ o g e n e )and
~ ~ ,erythropoietin
~~
receptor ( E ~ o R ) ? ~ , ~ ~
deletion of extracellular domain and C terminus. EMBO J 7:1003,
Activating mutations in CSF-1R were shown to induce mor1988
phologic transformation, anchorage-independent growth,
7. Reith AD, Bernstein A: Molecular Biology of the W and Steel
and tumorigenicity in mouse NM3T3
activating
Loci. Genome Analysis v01 3: Genes and Phenotypes. Cold Spring
mutations have been detected in a fraction of myelodysplasHarbor, N Y , Cold Spring Harbor Laboratory, 1991, p 105
8. Zsebo KM, Wypych J, McNiece IK, Lu HS, Smith KA, Kartic syndrome and acute myelocytic leukemia.55Furthermore,
kare SB, Sachdev RK, Yuschenkoff VN, Birkett NC, Williams LR,
despite no kinase sequences in the cytoplasmic domain, actiSatyagal VN, Tung W, Bosselman RA, Mendiaz EA, Langley KE.
vating mutations in EpoR were reported to initiate the develIdentification,
purification, and biological characterization of hemaopment of erythroleukemia in murine experimental models.54
topoietic stem cell factor from buffalo rat liver-conditioned medium.
However, CSF-1 and Epo, respectively, are thought to act
Cell 63:195, 1990
primarily on cells of monocyte/macrophage and erythroid
9. Martin FH, Suggs SV, Langley KE, Lu HS, Ting J, Okino KH,
lineages, and neither of them are very potent growth factors
Moms CF, McNiece IK, Jacobsen F W , Mendiaz EA, Birkett NC,
for hematopoietic stem ~ e l l s .By
~ ~contrast,
, ~ ~ KIT is known
Smith KA, Johnson MJ, Parker VP, Flores JC, Pate1 AC, Fisher EF,
to express on various types of hematopoietic stedprogenitor
Erjavec HO, Herrera CJ, Wypych J, Sachdev RK, Pope JA, Leslie
cells as well as mast ce11s7.23,3y,58,59
and SCF acts as a potent
I, Wen D, Lin C, Cupples RL, Zsebo KM: Primary structure and
functional expression of rat and human stem cell factor DNAs. Cell
mitogen to stimulate directly the growth of the cells particu63:203, 1990
larly in combination with other growth factors like Epo,
10. Zsebo K M , Williams DA, Geissler EN, Broudy VC, Martin
granulocyte-macrophage CSF, granulocyte CSF, IL-3, and
FH, Atkins HL, Hsu RY, Birkett NC, Okino KH, Murdock DC,
1~-7.7.Y.I3.l4.60.62Therefore, it is possible that the activating
m559,
mutations in KIT may contribute to aberrant cell growth and
leukemogenesis of mast cells and various types of hematopoietic stemlprogenitor cells. This possibility may be supported, at least in part, by our recent findings that constitutively activating mutations of c-kit gene are detectable in the
same Asp codon within the phosphotransferase domain in
both a murine mastocytoma cell line (P-815) and a rat mast
cell leukemia cell line (RBL-2H3).33*34
Also, the possibility
may be additionally supported by our preliminary study
showing that retroviral transfer of the c-kif mutant genes
into normal hematopoietic stem cells results in formation of
various types of colonies in the absence of hematopoietic
growth factors.
In summary, the results presented here show that introduction of the c-kit mutant genes into IL-3-dependent cell lines
Jacobsen F W , Langley KE, Smith KA, Takeishi T, Cattanach BM,
Galli SJ, Suggs SV: Stem cell factor is encoded at the S1 locus of
the mouse and is the ligand for the c-kit tyrosine kinase receptor.
Cell 63:213, 1990
11, Williams DE, Eisenman J, Baird A, Rauch C, Ness VK,
March CJ, Park LS, Martin U, Mochizuki DY, Boswell HS, Burgess
GS, Cosman D, Lyman SD: Identification of a ligand for the c-kit
proto-oncogene. Cell 63:167, 1990
12. Copeland NG, Gilbert DJ, Cho BC, Donovan PJ, Jenkins NA,
Cosman D, Anderson D, Lyman SD, Williams D E Mast cell growth
factor maps near the steel locus on mouse chromosome 10 and is
deleted in a number of steel alleles. Cell 63:175, 1990
13. Anderson DM, Lyman SD, Baird A, Wignall JM, Eisenman
J, Rauch C, March CJ, Boswell HS, Gimpel SD, Cosman D, Williams DE: Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms.
Cell 63:235, 1990
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
ROLE OF c-kit MUTATIONSINTUMORIGENESIS
14. Nocka K, Buck J, Levi E, Besmer P: Candidate ligand for
the c-kit transmembrane kinase receptor: KL, a fibroblast derived
growth factor stimulates mast cells and erythroid progenitors. EMBO
J 9:3287, 1990
15. Huang E, Nocka K, Beier DR, Chu TY, Buck J, Lahm H W ,
Wellner D, Leder P, Besmer P: The hematopoietic growth factor
KL is encoded by the S1 locus and is the ligand of the c-kif receptor,
the gene product of the W locus. Cell 63:225, 1990
16. Witte ON: Steel locus defines new multipotent growth factor.
Cell 635, 1990
17. Koch CA, Anderson D, Moran MF, Ellis C, Pawson T: SH2
and SH3 domains: Elements that control interactions of cytoplasmic
signaling proteins. Science 252:668, 1991
18. Rottapel R, Reedijk M, Williams DE, Lyman SD, Anderson
DM, Pawson T, Bernstein A: The steeUW transduction pathway:
kit autophosphorylation and its association with a unique subset of
cytoplasmic signaling proteins is induced by the steel factor. Mol
Cell Biol 11:3043, 1991
19. Reith AD, Ellis C, Lyman SD, Anderson DM, Williams DE,
Bemstein A, Pawson T: Signal transduction by normal isoforms and
W mutant variants of the Kit receptor tyrosine kinase. EMBO J
9:2451, 1991
20. Yi T, Ihle JN: Association of hematopoietic cell phosphatase
with c-kif after stimulation with c-kit ligand. Mol Cell Biol 13:3350,
1993
21. Fantl WJ, Johnson DE, Williams LT: Signalling by receptor
tyrosine kinases. Annu Rev Biochem 62:453, 1993
22. Russel ES: Hereditary anemias of the mouse: A review for
geneticists. Adv Gen 20:357, 1979
23. Kitamura Y: Heterogeneity of mast cells and phenotypic
change between subpopulations. Ann Rev Immunol 759, 1989
24. Galli SJ, Kitamura Y: Genetically mast-cell-deficient W W
and SVSld mice. Am J Pathol 127:191, 1987
25. Besmer P, Murphy JE, George PC, Qiu F, Bergold PJ, Lederman L, Snyder H W Jr, Brodeur D, Zuckerman EE, HardyWD:
A new acute transforming feline retrovirus and relationship of its
oncogene v-kif with protein kinase gene family. Nature 320:415,
1986
26. Woolford JW, McAuliffe A, Rohrschneider LR: Activation
of the feline c-fms proto-oncogene: Multiple alterations are required
to generate a fully transformed phenotype. Cell 55965, 1988
27. Roussel M, Downing J, Rettenmier CW, Sherr CJ: A point
mutation in the extracellular domain of the human CSF-1 receptor
(c-fms proto-oncogene product) activates its transforming potential.
Cell 55:979, 1988
28. Bargmann CI, Hung MC, Weinberg RA: Multiple independent activations of the neu oncogene by a point mutation altering
the transmembrane domain of p185. Cell 45:649, 1986
29. Weiner DB, Liu J, Cohen JA, Williams WV, Greene MI: A
point mutation in the neu oncogene mimics lligand induction of
receptor aggregation. Nature 339:230, 1989
30. Kuriu A, Ikeda H, Kanakura Y, Griffin JD, Druker B, Yagura
H, Kitayama H, Ishikawa J, Nishiura T, Kanayama Y, Yonezawa
T, Tarui S : Proliferation of human myeloid leukemia cell line associated with the tyrosine-phosphorylation and activation of the protooncogene c-kif product. Blood 78:2834, 1991
3 1. Ikeda H, Kanakura Y, Tamaki T, Kuriu A, Kitayama H, Ishikawa J, Kanayama Y, Yonezawa T, Tarui S, Griffin JD: Expression
and functional role of the proto-oncogene c-kit in acute myeloblastic
leukemia cells. Blood 78:2962, 1991
32. Furitsu T, Tsujimura T, Tono T, Ikeda H, Kitayama H, Koshimizu U, Sugahara H, Butterfield JH, Ashman LK, Kanayama Y,
Matsuzawa Y, Kitamura Y, Kanakura Y: Identification of mutations
in the coding sequence of the proto-oncogene c-kit in a human mast
797
cell leukemia cell line causing ligand-independent activation of ckif product. J Clin Invest 92:1736, 1993
33. Tsujimura T, Furitsu T, Morimoto M, Isozaki K, Nomura
S, Matsuzawa Y, Kitamura Y, Kanakura Y: Ligand-independent
activation of c-kif receptor tyrosine kinase in a murine mastocytoma
cell line P-815 generated by a point mutation. Blood 83:2619, 1994
34. Tsujimura T, Furitsu T, Morimoto M, Kanayama Y, Nomura
S, Matsuzawa Y, Kitamura Y, Kanakura Y: Substitution of an aspartic acid in c-kit receptor tyrosine kinase results in constitutive activation not only in human and mouse mastcytoma cell lines but also
in a rat mastocytoma cell line RBL-2H3. Int Arch Allergy Immunol
(in press)
35. Palacios R, Steinmetz M: IL 3-dependent mouse clone that
express B-220 surface antigen, contain Ig genes in germ-line configration, and generate B lymphocytes in vivo. Cell 41:727, 1985
36. Dexter TM, Garland J, Scott D, Scolnick E, Metcalf D:
Growth of factor dependent precursor cell lines. J Exp Med
152:1036, 1980
37. Kanakura Y, Druker B, Cannistra SA, Furukawa Y, Torimoto
Y, Griffin JD: Signal transduction of the human granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors involves
tyrosine phosphorylation of a common set of cytoplasmic proteins.
Blood 76:706, 1990
38. Kanakura Y, Drucker B, Decarlo J, Cannistra SA, Griffin
JD: Pholbor 12-mytistate acetate 13-acetate inhibits granulocytemacrophage colony-stimulating factor-dependent cell line. J Biol
Chem 266:490, 1991
39. Ogawa M, Matsuzaki Y, Nishikawa S, Hayashi S-I, Kunisada
T, Sudo T, Kina H, Nakauchi H, Nishikawa S-I: Expression and
function of c-kif in hematopoietic progenitor cells. J Exp Med
174:63,1991
40. Mann R, Mulligan RC, Baltimore D: Construction of a retrovirus packaging mutant and its use to produce helper-free defective
retrovirus. Cell 33:153, 1983
41. Fujita J, Onoue H, Ebi Y, Nakayama H, Kanakura Y: In vitro
duplication and in vivo cure of mast cell deficiency of SVSld mutant
mice by cloned 3T3 fibroblasts. Proc Natl Acad Sci USA 86:2888,
1989
42. Laker C, Stocking C, Bergholz U, Hess N, DeLamarter JF,
Ostertag W: Autocrine stimulation after transfer of the granulocyte/
macrophage colony-stimulating factor gene and autonomous growth
are distinct but interdependent steps in the oncogenic pathway. Proc
Natl Acad Sci USA 84:8458, 1987
43. Blume-Jensen P, Claesson-Welsh L, Siegbahn A, Zsebo KM,
Westermark B, Heldin CH: Activation of the human c-kif product
by ligand-induced dimerization mediates circular actin reorganization and chemotaxis. EMBO J 10:4121, 1991
44. Schlessinger J, Ullrich A: Growth factor signaling by receptor
tyrosine kinases. Neuron 9:383, 1992
45. Tsujimura T, Koshimizu U, Katoh H, Isozaki K, Kanakura
Y, Tono T, Adachi S , Kasugai T, Tei H, Nishimune Y, Nomura S,
Kitamura Y: Mast cell number in the skin of heterozygotes reflects
the molecular nature of c-kit mutation. Blood 81:2530, 1993
46. Ruitenberg ET, Elgersma A: Absence of intestinal mast cell
response in congenically athymic mice during Trichihellu spiralis
infection. Nature 264:258, 1976
47. Kanakura Y, Sonoda S, Nakano T, Fujita J, Kuriu A, Asai
H, Kitamura Y: Formation of mast-cell colonies in methylcellulose
by mouse skin cells and development of mucosal-like mast cells
from the cloned cells in the gastric mucosa of W W mice. Am J
Pathol 129:168, 1987
48. Flanagan JG, Chan DC, Leder P Transmembrane form of
the kif ligand growth factor is determined by alternative splicing and
is missing in the Sld mutant. Cell 64:1025, 1991
49. Koland JG, Cerione RA: Growth factor control of epidermal
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
798
growth factor receptor kinase activity via an intramolecular mechanism. J Biol Chem 263:2230, 1988
50. Northwood IC, Davis RJ: Activation of the epidermal growth
factor receptor tyrosine protein kinase in the absence of receptor
oligomerization. J Biol Chem 263:7450, 1988
51. Honegger AM, Kris RM, Ullrich A, Schlessinger J: Evidence
that autophosphorylation of solubilized receptors for epidermal
growth factor is mediated by intermolecular cross-phosphorylation.
Proc Natl Acad Sci USA 86:925, 1989
52. Ueno H, Colbert H, Escobedo JA, Williams LT: Inhibition of
PDGFB receptor signal transduction by coexpression of a truncated
receptor. Science 252:844, 1991
53. Yoshimura A, Longmore G, Lodish HF: Point mutation in
the exoplasmic domain of the erythropoietin receptor resulting in
hormone-independent activation and tumorigenicity. Nature 348:
647, 1990
54. Longmore GD, Lodish HF: An activating mutation in the
murine erythropoietin receptor induces erythroleukemia in mice: A
cytokine receptor superfamily oncogene. Cell 67:1089, 1991
55. Ridge SA, Wonvood M, Oscier D, Jacobs A, Padua RA: FMS
mutations in myelodysplastic, leukemic, and normal subjects. Proc
Natl Acad Sci USA 87:1377, 1990
KITAYAMA ET AL
56. Metcalf D: Control of granulocytes and macrophages: Molecular, cellular, and clinical aspects. Science 254:529, l991
57. Krants SB: Erythropoietin. Blood 77:419, 1991
58. Papayannopoulou T, Brice M, Broudy VC, Zsebo KM: Isolation of c-kit receptor-expressing cells from bone marrow, peripheral
blood, and fetal liver: Functional properties and composite antigenic
profile. Blood 78:1403, 1991
59. Ashman LK, Cambareri AC, To LB. Levinsky RJ, Juttner CA:
Expression of the YB5.B8 antigen (c-kiz proto-oncogene product) in
normal human bone marrow. Blood 78:30, 1991
60. Bemstein ID, Andrews RG, Zsebo KM: Recombinant human
stem cell factor enhances the formation of colonies by CD34 ' and
CD34'lin- cells, and the generation of colony-forming cell progeny
from CD34'lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor. Blood 77:2316, 1991
61. McNiece IK, Langley KE, Zsebo KM: Recombinant human
stem cell factor synergises with GM-CSF, G-CSF, IL-3, and EPO
to stimulate human progenitor cells of the myeloid and erythroid
lineages. Exp Hematol 19:226, 1991
62. Billips LG, Petitte D, Dorskind K, Narayanan R, Chiu CP.
Landreth KS: Differential roles of stromal cells, interleukin-7, and
kif ligand in the regulation of B lymphopoiesis. Blood 79: 1 185, 1992
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
1995 85: 790-798
Constitutively activating mutations of c-kit receptor tyrosine kinase
confer factor-independent growth and tumorigenicity of
factor-dependent hematopoietic cell lines
H Kitayama, Y Kanakura, T Furitsu, T Tsujimura, K Oritani, H Ikeda, H Sugahara, H Mitsui, Y
Kanayama and Y Kitamura
Updated information and services can be found at:
http://www.bloodjournal.org/content/85/3/790.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.