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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
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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
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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
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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.
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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.
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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.
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cross-linking analysis using BS3 showed
that a dimeric form of KITV814was scarcely detectable in
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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
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