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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4798-4807
An Activating Mutation in the Kit Receptor Abolishes the Stroma
Requirement for Growth of ELM Erythroleukemia Cells, But Does Not
Prevent Their Differentiation in Response to Erythropoietin
By
Nick R. Leslie,
Jim O'Prey,
Chris Bartholomew, and
Paul R. Harrison
From the Beatson Institute for Cancer Research, CRC Beatson
Laboratories, Glasgow, Scotland.
 |
ABSTRACT |
We have previously shown that murine ELM erythroleukemia cells can
only be grown in vitro in the presence of a stromal feeder layer, or
alternatively stem cell factor (SCF), without which they differentiate.
When grown in the presence of SCF, ELM cells can still differentiate in
response to erythropoietin (Epo), but growth on stroma prevents this.
We previously isolated a stroma-independent ELM variant, ELM-I-1, that
is also defective in Epo-induced differentiation. We show here that
this variant has an activating mutation in the Kit receptor, converting
aspartic acid 814 to histidine. Expression of the mutant receptor in
stroma-dependent ELM-D cells causes growth factor-independent
proliferation and also gives the cells a selective advantage, in terms
of proliferation rate and clonegenicity, compared with ELM-D cells
grown in optimal amounts of SCF. Expression of the mutant receptor in
ELM-D cells also prevents spontaneous differentiation, but not
differentiation induced by Epo. Analysis of mitogenic signaling
pathways in these cells shows that the mutant receptor induces
constitutive activation of p42/p44 mitogen-activated protein kinases.
It also selectively inhibits the expression of p66Shc but not the
p46/p52 Shc isoforms (as did treatment of ELM cells with SCF), which is
of interest, because p66Shc is known to play an inhibitory role in
growth factor signaling.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
OVERCOMING THE GROWTH factor dependence
of cellular proliferation is an important step in the development of
many cancers, and many oncogenes encode proteins that cause the
abnormal activation of growth factor stimuli. Many growth factors act
through cell surface receptors of the receptor tyrosine kinase (RTK)
family that induce intrinsic tyrosine kinase activity and downstream intracellular signaling pathways upon binding of ligand.1
Abnormal activation of RTK family members may be caused by mutations
and is causally implicated in the development of human
malignancies.2-6
The Kit receptor tyrosine kinase is the cellular receptor for stem cell
factor (SCF) and a member of the type III RTK subfamily.7 The study of Kit and SCF has been aided by the natural occurrence in
mice of mutations in their corresponding genetic loci, W
(dominant white spotting) and Sl (Steel),
respectively.7 Loss of function of Kit or SCF leads to lack
of skin and hair pigmentation, lack of intestinal pacemaker activity,
sterility, and anemia. These effects correlate with the normal
expression of Kit in melanocytes,8,9 the interstitial cells
of Cajal in the gut,10 germ cells,8,11 and many
hematopoietic cell lineages, including stem cells and erythroid
progenitors,12 and demonstrate a requirement for Kit activity in the development of these cell lineages.
The potency of the growth signal induced in many cell types by the
Kit/SCF interaction is confirmed by the frequent deregulation of this
signal in cancer. The HZ4 feline sarcoma virus encodes the transforming
oncogene, v-kit, a mutated and truncated viral homologue of
c-kit.13 Also, activation of Kit through autocrine expression of SCF has been described in small cell lung
cancers,14 colorectal carcinomas,15 breast
carcinomas,16 gynecological tumors,17 and
neuroblastomas.18 Finally, receptor mutations resulting in
constitutive activation of Kit have been identified in mast cell
leukemic cell lines and samples derived from patients with
mastocytosis.19-23
Leukemia progression is generally associated with changes in growth
factor requirements or responsiveness, differentiation arrest, and loss
of stroma cell dependence. We have used the murine ELM erythroleukemia
model to investigate the mechanisms of leukemia progression, because it
is unusual in having a less abnormal phenotype than most other murine
erythroleukemias, in that it has not progressed to stroma independent
growth and retains the capacity to undergo erythroid differentiation in
response to erythropoietin (Epo).24-26 We previously
isolated variants that were able to grow without stroma. One of these
variants, ELM-I-1, was not only growth factor independent, but had also
lost the capacity to differentiate in response to Epo. We show here
that ELM-I-1 posesses an activating mutation in c-kit and
analyze how the mutant Kit receptor may have contributed to the
phenotype of the ELM leukemic cells by testing its effects on the
growth factor requirements, differentiation, and cellular signaling of
erythroleukemic cells.
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MATERIALS AND METHODS |
Cells and tissue culture.
Unless otherwise specified, all reagents for tissue culture were
supplied by GIBCO-BRL (Paisley, UK). Murine ELM leukemic cells and MS5
stromal cells were maintained in minimum essential medium  supplemented with 10% horse serum (Sigma, Poole, UK). Serum
deprivation was conducted for 18 hours in 0.5% horse serum without
added growth factors. Q2BN quail fibroblasts27
were maintained in Dulbecco's modified Eagle medium supplemented with 8% fetal bovine serum (TCS Biologicals, Buckingham, UK) and 2% chicken serum. Recombinant murine SCF (rmSCF) and recombinant human Epo
(rhEpo) were purchased from R&D Systems (Abingdon, UK) and were
routinely used at 10 ng/mL and 5 U/mL, respectively.
The growth factor-dependent ELM cells used were ELM-D6
cells,24,26,28 routinely passaged in coculture with the
bone marrow-derived stromal cell line MS5.29,30 Leukemic
cells were separated from stroma for experiments by vigorous agitation
and three rounds of purification by settling and gentle
agitation.28 Where specified, ELM-D6 subclone
20.5D28 was used and grown in 10 ng/mL rmSCF. The precise
derivation of the growth factor-independent ELM-I cell lines ELM-I-1,
I-2, and I-5 is described in Nibbs et al.26
Proliferation and colony assays.
Proliferation assays were performed over 7 days in 96-well microtiter
plates, inoculating 200 cells into 100 µL medium with 0, 1, or 10 ng/mL SCF. Cell metabolic activity was assayed using the Promega MTT
kit (Southampton, UK). An initial control plate (5,000 cells/well, 10 ng/mL SCF) was assayed after 4 hours to verify equal starting
populations. Colony assays were performed using MethoCult M3230
methylcellulose colony medium (StemCell Technologies, Vancouver,
British Columbia, Canada) supplemented with no growth factors, SCF (10 ng/mL), Epo (5 U/mL), or both SCF and Epo. Five hundred growing cells
were inoculated into 1.5 mL medium in 30-mm dishes and scanned and
counted unstained after 3 weeks. Scanned images were recorded using an
Agfa Arcus PDI 420oe scanner (Agfa-Gevaert, Brentford, UK)
and analyzed using the NIH Image program.
Transfections.
ELM-D6-20.5D cells were stably transfected by electroporation using a
Bio-Rad gene pulser (Bio-Rad, Hemel Hemstead, UK). Cells (5 × 106) in 250 µL medium (20% serum + 10 ng/mL rmSCF) were
mixed with 10 µg linearized expression vector in 4-mm electroporation
cuvettes (Equibio, Kent, UK) and pulsed at 960 µF and 240 V. Cells
were then diluted into 10 mL of fresh medium (10% serum and 10 ng/mL SCF) and, 48 hours after electroporation, transferred into microtiter wells with Geneticin Sulphate (800 µg/mL; GIBCO BRL) and SCF for isolation of transfected cell clones. Growth factor independence of
transfectants was assessed by seeding 10,000 cells in 1 mL of
medium without SCF into 24-well tissue culture dishes and providing fresh medium every 3 to 4 days. Quail fibroblasts were transiently transfected by calcium phosphate coprecipitation and maintained in
fresh medium for 24 hours before lysis and Western blot analysis.
Antibodies and protein analysis.
Two antibodies against the murine Kit receptor were used, a rat
monoclonal antibody (ACK231) for immunoprecipitation and a
goat polyclonal antiserum (M-14; Santa Cruz, Santa Cruz,
CA; used at 200 ng/mL) for immunoblotting. A polyclonal
antiserum against Shc was purchased from Transduction Laboratories
(Lexington, KY; used at 50 ng/mL), and antiphosphotyrosine antibodies
PY20 and 4G10 (both used at 200 ng/mL) were purchased from Santa Cruz and Upstate Biotechnology (Lake Placid, NY), respectively. Analysis of
the level of cellular MAPK activation used a polyclonal antibody preparation with specific activity against the dually phosphorylated forms of MAPK p44 and p42 (Promega; used at 50 ng/mL). An antibody against total p44 and p42 (sc-093; Santa Cruz; used at 50 ng/mL) was
used as a loading control.
For immunoprecipitation of the Kit receptor, cells were lysed in buffer
(1% NP40, 0.5% deoxycholate, 150 mmol/L NaCl, 50 mmol/L Tris, pH 8.0, 10% glycerol, 2 mmol/L EDTA, 1 mmol/L sodium orthovanadate, 1 mmol/L
phenylmethyl sulfonyl fluoride [PMSF], 10 µg/mL
leupeptin, 20 µg/mL aprotinin), precleared by centrifugation at
15,000 rpm for 1 hour, and incubated with 2 µg/mL ACK2 antibody.
Immune complexes were collected with a rabbit antirat antibody (Sigma;
25 µg/mL) and protein A/G sepharose (Sigma). Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
immunoblotting using polyvinylidene fluoride (PVDF) membrane
(Millipore, Watford, UK) followed standard procedures and the
manufacturer's instructions. Proteins were detected using specific
primary antibodies, horseradish peroxidase-conjugated secondary
antibodies, and ECL chemiluminescence (Amersham, Little Chalfont, UK).
Protein loading and transfer during blotting was finally verified by
staining with Amido Black (Sigma).
The Kit receptor in vitro kinase assay was adapted from the method of
Furitsu et al.19 The Kit receptor was immunoprecipitated as
described above; washed at 4°C once each in lysis buffer, PBS, 500 mmol/L LiCl, and kinase buffer (10 mmol/L MnCl, 20 mmol/L Tris pH 7.4);
and then resuspended in 30 µL kinase buffer containing 10µCi
[ -32P] ATP (Amersham) and 2 µmol/L unlabeled ATP,
before incubation for 20 minutes at 25°C. Proteins were separated
by SDS-PAGE and blotted onto PVDF membrane. Radioactive incorporation
was determined by autoradiography and the relative amount of Kit
receptor protein present in the immune complex was determined by
immunoblotting.
RNA analysis.
Total cellular RNA was purified using the RNAzol B reagent and
manufacturer's protocol (Biogenesis, Poole, UK). Resolution of total
RNA samples in formaldehyde-agarose gels and Northern blotting onto
Hybond-N membrane (Amersham) was performed using standard protocols and
manufacturers instructions. DNA probes were labeled using random primed
DNA labeling (Boehringer Mannheim, Lewes, UK).
cDNA synthesis and sequencing.
Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed
using a Gene-amp RNA PCR Core Kit (Perkin Elmer, Norwalk, CT). The oligonucleotide primers used for the
amplification of full-length c-kit cDNAs were KitS1
5 -GAGTCTAGCGCAGCCACC-3 and KitA1
5-CTGTTGGACTTGGGTTTCTG-3. Oligonucleotide primers used for PCR sequencing were based on the published sequence of the murine
c-kit gene.33 The amplification procedure used a
Perkin Elmer Cetus DNA thermal cycler, including a manual hot-start, and 35 cycles of 1 minute of denaturation at 95°C, 1 minute of annealling at 58°C, and 2 minutes of synthesis at 72°C. The
72°C synthesis step of the cycle was extended by 4 seconds per
cycle. Amplification products were purified from agarose gels using
geneclean (Bio 101 Inc, La Jolla, CA) and ligated into the pCR2.1
cloning vector (Invitrogen, NV Leek, The Netherlands). All cloned cDNAs investigated further were obtained from separate PCR amplifications. These full-length cDNAs were then sequenced in both orientations using
an Applied Biosystems 373A sequencer (Foster City, CA). Shorter fragments of the c-kit sequence were also amplified by RT-PCR using only 1 minute of synthesis cycles at 72°C,
gel-purified, and sequenced directly.
Expression vector construction.
cDNAs of the full-length coding sequence of the wild-type murine
c-kit gene and c-kitD814H were excised from the pCR2.1
vector with Xba I and Kpn I restriction enzymes and
ligated into these sites of the expression vector pcDNA3 (Invitrogen).
Expression vector cDNA inserts and ligation junctions were fully
sequenced.
| |
RESULTS |
The ELM-I-1 cell line displays constitutive activation of the Kit
receptor.
We initially set out to identify cellular changes associated with the
conversion from stroma dependence to independence by comparison of
stroma-dependent ELM erythroleukemia cells (ELM-D) and
stroma-independent variants (ELM-I), particularly
ELM-I-1.26
We have recently shown that growth of ELM leukemic cells on stromal
cells relies on a complex array of signals provided by stroma,
including integrin-mediated signaling, insulin-like growth factor-1
(IGF-1), and SCF.28 However, under optimal conditions, SCF
alone (but not IGF-1) is able to replace stroma in supporting long-term
cell growth without affecting its stroma/SCF dependence or ability to
differentiate in response to Epo.28
Experiments were therefore performed to investigate the possible
stimulation of ELM-I cells by autocrine growth factor production, either using blocking antibodies or detecting growth factor mRNAs by
RT-PCR. This showed that none of the ELM-I cell clones investigated produced detectable quantities of autocrine growth factors for which
they were known to express corresponding receptors, SCF, IGF-1, or Epo
(O'Prey et al28 and data not shown). As a next step, we
investigated the activation of tyrosine kinase signaling pathways known
to be activated by many mitogenic growth factor signals and oncogenes.
Initial experiments showed that several ELM-I cell lines displayed
constitutive phosphorylation and activity of the p42/p44
mitogen-activated protein kinases (MAPKs) in the absence of
stimulation, with levels being highest in the ELM-I-1 cell line (data
not shown). The ELM-I-1 cell line was also found to exhibit
constitutive tyrosine phosphorylation of the p46 and p52 Shc signaling
proteins and a greatly reduced level of expression of the p66 isoform
of Shc (data not shown). Because recent work has shown that, in
contrast to the p46 and p52 Shc isoforms, p66 is able to play an
inhibitory role in growth factor signaling,32,33 this may
be significant.
Having found activation of kinase signaling pathways in the ELM-I-1
cell line, antiphosphotyrosine Western blotting of whole cell lysates
was performed to identify activated kinases. The pattern of protein
tyrosine phosphorylation in unstimulated ELM-I-1 cells showed a protein
of approximately 145 kD that comigrated with a protein phosphorylated
in ELM-D cells stimulated with SCF and with the immunoprecipitated Kit
receptor (data not shown). This suggested constitutive phosphorylation
and activation of the Kit receptor in ELM-I-1 cells. This was confirmed
directly by immunoprecipitation of the Kit receptor and Western
blotting with antiphosphotyrosine and anti-Kit antibodies
(Fig 1A) and also by an in vitro assay to
detect Kit receptor kinase activity (Fig 1B). These experiments show
that the Kit receptor is tyrosine phosphorylated in unstimulated
serum-starved ELM-I-1 cells in contrast to two other ELM-I variants,
ELM-I-2 and ELM-I-5 (Fig 1A), and that Kit receptor immunoprecipitates
from ELM-I-1 cells have an elevated level of constitutive kinase
activity (Fig 1B). We also found that, although ELM-I-1 cells express
similar levels of Kit receptor protein as other ELM cells, there were
changes in the relative abundance of the two receptor forms (immature and mature34) and in the apparent electrophoretic mobility
of the larger mature form (Fig 1A and B and data not shown).
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| Fig 1.
Tyrosine phosphorylation and kinase activity of the Kit
receptor in serum-starved ELM-I-1 cells. (A) Kit protein was
immunoprecipitated from serum-starved cells and from ELM-D cells
subsequently stimulated with 10 ng/mL SCF for 10 minutes before cell
lysis. Immunoprecipitated proteins were then analyzed by Western
blotting using antiphosphotyrosine and anti-Kit receptor antibodies. A
control antibody was used in the immunoprecipitation loaded into the
right-hand lane. Size markers are shown in kilodaltons. (B) Kit protein
was immunoprecipitated from unstimulated ELM-D and ELM-I-1 cells and
its autophosphorylation activity was assayed in vitro as described in
Materials and Methods.
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ELM-I-1 cells contain a point mutation in the c-kit gene in
the region encoding the kinase domain.
Because normal Kit receptor activation is believed to occur by
ligand-induced dimerization and autophosphorylation and it is known
that ELM-I-1 cells do not express detectable levels of SCF, these
changes suggest that ELM-I-1 cells express a constitutively active
mutant form of the Kit receptor. To investigate this possibility, Kit
receptor mRNAs present in ELM-I-1 cells were cloned by RT-PCR and
compared with those cloned from growth factor-dependent ELM-D cells.
Each of the three independent cDNAs from ELM-I-1 cells sequenced showed
a single G to C change at nucleotide 2468 compared with the sequence
from growth factor-dependent ELM-D cells. This point mutation causes a
change in the predicted amino acid sequence from aspartic acid to
histidine at codon 814 (Fig 2). In
addition, one cDNA from ELM-I-1 cells also contained a previously
described 12 nucleotide insertion caused by differential
splicing.35 Subsequent experiments using expression vectors
with the mutant Kit cDNA without the insertion showed that it is not
required for constitutive receptor activation; therefore, the effects
of this insertion were not investigated further. There were also three
previously described strain-specific polymorphisms36 in the
c-kit nucleotide sequence of both ELM cell lines, derived from
a C3H mouse, that differed from the published BALB/c mouse c-kit
sequence.37 RT-PCR amplification products of the
c-kit kinase domain region were also gel-purified and sequenced
directly. These showed only the wild-type sequence expressed in ELM-D
cells, but both wild-type and mutant sequences at nucleotide 2468 in
ELM-I-1 cells, showing that ELM-I-1 cells retain expression of the
wild-type Kit receptor in addition to the KitD814H mutant.
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| Fig 2.
A mutation in c-kit expressed in ELM-I-1 cells.
Nucleotide 2468 was a G both in each c-kit cDNA clone from
ELM-D cells and the published wild-type c-kit
sequence,37 but a C in each c-kit cDNA clone from
ELM-I-1. This change results in an aspartic acid to histidine mutation
at codon 814 in the predicted amino acid sequence of the mutant Kit
receptor kinase domain. Direct sequencing of RT-PCR products shows that
ELM-I-1 cells express both mutant and wild-type sequences.
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Expression of c-kitD814H abrogates the SCF dependence of
ELM-D cells.
To determine whether the mutation in c-kit is responsible for
the growth factor independence of ELM-I-1 cells, we introduced the
c-kitD814H mutation into ELM-D cells. Expression constructs for
c-kit and c-kitD814H were created using the vector
pcDNA3 (Invitrogen). These were found to give efficient expression of the Kit receptor, as verified by Western blotting of transiently transfected Quail fibroblasts (Fig 3A).
However, only the immature 125kD Kit glycoprotein was detected in
cells transfected with the mutant c-kit cDNA, in contrast with
those transfected with the wild-type vector, which expressed both 125- and 145-kD forms of Kit. As expected, the transiently expressed mutant
Kit was found to be constitutively phosphorylated on tyrosine, unlike the wild-type receptor (Fig 3A).
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| Fig 3.
Expression and tyrosine phosphorylation of Kit and
MAPK in ELM cells expressing KitD814H and wild-type Kit. (A) pcDNA3
vector (Invitrogen) and expression constructs for c-kit and
c-kitD814H based on pcDNA3 were transfected into the Q2BN Quail
fibroblastic cell line and expression of Kit protein was analyzed by
Western blotting with anti-Kit antibodies. This blot was then stripped
and an antiphosphotyrosine antibody was used. (B) Phosphorylation of
the Kit receptor in ELM-I-1 cells and ELM-D cells stably transfected
with wild-type c-kit or c-kitD814H expression vectors.
Phosphorylation of the Kit receptor in unstimulated serum-starved cells
was examined by immunoprecipitation with anti-Kit antibodies and
antiphosphotyrosine Western blotting. The membrane was stripped and an
anti-Kit antibody was used to verify Kit immunoprecipitation. (C) MAPK
phosphorylation was analyzed in serum-starved ELM-I-1, ELM-Kit, or two
clones of ELM-KitDH cells (i and ii) and in serum-starved ELM-Kit cells
either stimulated for 10 minutes with 10 ng/mL rmSCF before lysis or
continuously maintained in 10 ng/mL rmSCF. Whole cell lysates were
analyzed by Western blotting using an antibody against the activated
form of MAPK and a nonspecific antibody against p42/p44.
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The Kit expression constructs were then stably introduced by
electroporation into stroma/SCF-dependent ELM-D cells that already express the wild-type Kit receptor. To obviate the need for isolating transfectants on antibiotic-resistant stroma, ELM-D cells were maintained in 10 ng/mL SCF instead of stroma, because SCF has been
shown to support ELM cells without altering their growth factor
dependence or differentiation capacity.28 After
electroporation, antibiotic resistant clones were selected in the
continuous presence of SCF, expression of introduced constructs was
investigated by RT-PCR, and the SCF-dependence of the clones was tested
by removal of SCF. As previously described,26 growth
factor-independent variant cells arise spontaneously during selection:
1 of 12 or 2 of 16 ELM cell clones transfected with empty expression
vector or the wild-type Kit cDNA expression vector, respectively, gave rise to cells capable of growth in medium without SCF. In contrast, 10 of 27 clones transfected with the KitD814H mutant cDNA were growth
factor independent. However, only 8 of 18 of the latter clones tested
showed detectable expression of the mutant KitD814H vector by RT-PCR
(data not shown), and all of these 8 expressing clones continued to
proliferate in the absence of SCF. Thus, in all cases, expression of
KitD814H resulted in SCF-independent growth. Two of the expressing
ELM-KitDH clones (i and ii) were chosen for further characterization:
both showed constitutive phosphorylation of the Kit receptor, in
contrast to cells expressing the wild-type receptor (ELM-Kit cells; Fig
3B). As expected, direct sequencing of RT-PCR products obtained from
one of these expressing clones using c-kit primers showed that
both wild-type and mutant Kit receptors were expressed, as is the case
also with ELM-I-1 cells (data not shown). These ELM-KitDH clones also
showed a constitutively high level of MAPK phosphorylation after
serum-starvation, in contrast to ELM-Kit cells (Fig 3C). The extent of
constitutive MAPK activation in ELM-KitDH cells was similar to that in
ELM-I-1 cells and, interestingly, consistently higher than that seen in ELM-Kit cells maintained in 10 ng/mL SCF (Fig 3C), the growth factor
concentration shown to induce optimal growth of these cells in
short-term assays (O'Prey et al28 and data not shown).
Effects of the c-kitD814H mutation on the proliferation and
clonegenicity of ELM-D cells.
To determine the effects of the c-kitD814H mutation on the
proliferation and survival of ELM cells, the growth of ELM-Kit, ELM-KitDH, and ELM-I-1 cells was compared in a microtiter well proliferation assay and a semisolid medium colony assay in the presence
and absence of additional growth factors. These experiments showed that
the KitD814H mutation not only allows ELM cells to continue to
proliferate in the absence of SCF, but also gives ELM-KitDH cells a
growth advantage compared with ELM-Kit cells maintained in the
concentration of SCF we have previously shown to be optimal for ELM
growth28 (10 ng/mL; Fig 4A).
However, the proliferation of ELM-KitDH cells was not as great as that of ELM-I-1 cells (Fig 4A). These findings were supported by cell cycle
analysis showing that ELM-Kit cells starved of SCF for 24 hours
accumulate in the G0/G1 phase of the cell cycle, in contrast to
ELM-KitDH cells, which maintain a large proportion of cells in S and
G2/M phases in the presence and absence of SCF (data not shown). The
ELM-KitDH cells also cloned at high efficiency without SCF, in contrast
to ELM-Kit cells, and produced larger colonies than ELM-Kit cells
cloned in the presence of optimal SCF concentrations (Fig 4B). Thus,
the KitD814H mutation clearly confers a selective advantage over the
wild-type Kit in terms of proliferation and clonegenicity.
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| Fig 4.
Expression of c-kitD814H in ELM cells causes
SCF-indpendence and enhanced proliferation and cloning efficiency. (A)
The proliferation of ELM-Kit and ELM-KitDH cells over 7 days was
compared after seeding 500 cells in 96-well plates in different
concentrations of SCF. Cells were assayed using a colorimetric MTT
proliferation assay and each data point shown is an average of 28 cultures, each read twice, with standard deviations. (B) Colony
formation by ELM-Kit, ELM-KitDH, and ELM-I-1 cells in response to SCF.
Five hundred cells were inoculated in the presence or absence of SCF
(10 ng/mL) in semisolid medium.
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Effects of the c-kitD814H mutation on the differentiation
of ELM-D cells.
One of the obvious characteristics of the ELM-I-1 cells in which the
KitD814H mutation was identified is that they do not undergo erythroid
differentiation in response to Epo or upon removal from SCF or stroma,
as ELM-D cells do.26,28 We therefore investigated the
effects of the KitD814H mutation on the balance of proliferation and
differentiation of ELM cells, either after removal of SCF or in
response to Epo. The first approach adopted was to investigate the
effects of SCF and/or Epo on colony formation of ELM-KitDH cells and ELM-Kit cells in clonogenic assays and measuring the colony
size distributions by computer analysis (see Materials and Methods for
details). Cloning in either SCF or Epo (or both) increased the total
number of colonies of ELM-Kit cells, but few of them reached a size of
300 µm (a few hundred cells; Fig 5A). In
contrast, a greater number of ELM-KitDH colonies were formed whether or
not SCF or Epo were present, of which 40% to 50% of these grew to
sizes greater than 300 µm (Fig 5A). However, the numbers of
large colonies were reduced by treatment with Epo even in the presence
of SCF (Fig 5A), suggesting that these ELM-KitDH cells can still
differentiate in response to Epo, whereas ELM-I/1 cells are unaffected
(Fig 5A).
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| Fig 5.
The effect of KitD814H on the proliferation and
differentiation of ELM cells in response to SCF and/or Epo. (A)
ELM-KitDH or ELM-Kit cells were cloned in methocel in the presence or
absence of 10 ng/mL SCF and/or 5 U/mL Epo as described in Fig
4B and the colonies were analyzed after 2 weeks. To analyze the numbers
and sizes of colonies, the dishes were recorded using a scanner, and
colonies were counted and sized using the NIH image program. The
representative plotted data are shown as the average colony number from
triplicate plates, with error bars showing maximum and minimum numbers.
(B) To examine the extent of erythroid differentiation, the cells were
incubated for 3 days in the presence or absence of 2 U/mL Epo or 10 ng/mL SCF, as indicated. Total RNA was then analyzed by Northern
blotting using labeled cDNA probes encoding -globin or 7S ribosomal
RNA.
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To measure the differentiation responses directly, the expression of
-globin mRNA in ELM-KitDH and ELM-Kit cells was first determined
after treatment with SCF and/or Epo (Fig 5B). Expression of
globin mRNA correlated with the development of a red color in cell
pellets. This experiment confirmed that ELM-KitDH cells did not undergo
spontaneous differentiation upon removal of SCF, in contrast to ELM-Kit
cells; nevertheless, ELM-KitDH cells still differentiated in response
to Epo (Fig 5B).
Effects of the KitD814H mutation on expression of p66Shc, Fli-1, and
Bcl-2.
Besides the c-kitD814H mutation, a number of other differences
have been identified between ELM-I-1 and ELM-D cells: they express high
levels of the ets protein Fli-126 and the regulator of
apoptosis Bcl-2 (J. Qiu and P. Harrison, unpublished
observations), and they also show greatly reduced levels
of the p66 Shc protein (data not shown and
Fig 6A). We therefore tested whether any of these other changes in ELM-I-1 cell gene expression are induced by
expression of the c-kitD814H mutant.
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| Fig 6.
Comparison of the effects of wild-type c-kit and
c-kitD814H expression in ELM-D cells on (A) the pattern of Shc
isoforms expression, (B) bcl-2 mRNA, and (C) Fli-1 protein. (A)
Relative expression of Shc isoforms was investigated by Western
blotting of whole cell lysates of serum-starved cells and ELM-Kit cells
serum-starved in the presence of 10 ng/mL SCF. (B) Expression of bcl-2
mRNA in ELM-I-1, ELM-Kit maintained in 10 ng/mL SCF, and ELM-KitDH
cells (clones i and ii) was analyzed by Northern blotting of total RNA
from growing cells and probing with bcl-2 cDNA and 7S rRNA
probes. Size markers are shown in kilobases. (C) Whole cell lysates
from ELM-KitDH and ELM-I/1 cells or ELM-Kit cells maintained in SCF
were analyzed by Western blotting with an anti-Fli-1 antibody.
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Expression of p66Shc was compared in ELM-KitDH (clones i and ii),
ELM-Kit, and ELM-I-1 cells. Western blotting of serum-starved cells
showed that removal of SCF from ELM-Kit cells expressing the wild-type
Kit receptor caused a marked increase in p66 Shc expression (Fig 6A).
Starved ELM-KitDH cells expressed a low level of p66 Shc, similar to
ELM-Kit cells grown in SCF, although this was not as low as in ELM-I-1
cells. No change in p46/p52 isoforms was evident. Thus, Kit signaling,
either from the wild-type receptor and SCF or constitutively from the
mutant KitDH receptor, suppresses the expression of p66 Shc. Finally,
the expression of Bcl-2 and Fli-1 in ELM-Kit, ELM-KitDH, and ELM-I-1
cells was compared by Northern and Western blotting, respectively.
However, neither wild-type Kit nor KitD814H induced expression of Bcl-2
or Fli-1 in ELM cells, compared with the ELM-I-1 control (Fig 6B and
C).
| |
DISCUSSION |
We have identified an activating mutation in the Kit receptor of the
ELM-I-1 growth factor-independent erythroleukemic cell line and, by
expression of this mutant receptor in growth factor-dependent ELM
cells, defined how it may have contributed to the phenotype of the
leukemic cells in which it arose. Thus, we have demonstrated that this
mutation gives the ELM cells a selective advantage in three respects:
(1) SCF or stroma independence; (2) enhanced proliferation/cloning efficiency compared with an exogenous SCF signal; and (3) a reduction in spontaneous differentiation on stroma or SCF withdrawal. However, activation of Kit by mutation is obviously not the only mechanism whereby ELM leukemic cells acquire stromal independence, because two
other stroma-independent ELM lines, ELM-I-2 and ELM-I-5, do not display
constitutive Kit phosphorylation (Fig 1A). Collectively, these changes
would be expected to increase the leukemogenic potential of the cells.
We have tested whether expression of the mutant Kit receptor in
stroma-dependent ELM-D leukemic cells enables them to grow in
nonhematopoietic sites, eg, subcutaneously, as is the case with other
aggressive retrovirus-induced erythroleukemia lines. However, none of
the ELM-D, ELM-Kit, or ELM-Kit DH cell lines tested formed tumors after
subcutaneous inoculation in nude mice after a period of 4 months (data
not shown).
Because expression of the mutant Kit does not prevent ELM cells
differentiating in response to Epo, this implies that some other
genetic event is responsible for the total lack of response of ELM-I/1
cells to Epo (Fig 5B). Because ELM-I-1 cells also do not differentiate
in response to the chemical inducers, dimethyl sulfoxide
(DMSO) or N,N-hexamethylene bisacetamide (HMBA; data not
shown), it seems likely that this differentiation block is not simply
caused by a defect in Epo signaling, but is more general. We have
previously shown that growth of ELM cells on stroma, but not in soluble
SCF, inhibits Epo-induced differentiation. This might be due to the
stromal expression of the membrane bound form of SCF causing a more
persistent intracellular Kit phosphorylation signal than the soluble
form of the ligand.38 Our data here would
argue against this, because the KitD814H mutation produces a constantly
elevated level of receptor signaling and yet does not inhibit
Epo-induced differentiation. This suggests that a different signal
supplied by stroma inhibits the erythroid differentiation of ELM-D
cells.
The activating point mutation in c-kit converts the aspartic
acid at codon 814 to a histidine. Although a spontaneous D814H mutation
has not been previously described, aspartic acid 814 has been
identified as a hot spot for c-kit activating mutations, being
mutated to a valine or a tyrosine in mast cell lines and mastocytosis.19-21 Mutation of aspartic acid 814 to
tyrosine has also been shown to alter the substrate specificity of the
Kit receptor kinase.39 Aspartic acid 814 lies within the
activation loop of the cytoplasmic kinase domain. Studies with the
insulin and fibroblast growth factor RTKs have suggested that this loop inhibits access to the active site for substrate peptides and ATP until
tyrosine residues within the loop are phosphorylated upon ligand
binding.40,41 It seems possible that mutation of aspartic
acid 814 interferes with this autoinhibition, increasing accessibility
of the active site. Mutagenesis has shown that many amino acids
introduced in place of aspartic acid 814 allow some level of
ligand-independent kinase activity.42 In these studies, the
D814H mutation was described as modestly activating and displayed accelerated ligand-independent degradation relative to the wild-type receptor when expressed in kidney 293 cells. The identification of the
KitD814H mutation in an erythroleukemia is also significant, because it
appears that Kit codon 814 mutations are only transforming in some cell
types, as KitD814V is unable to transform fibroblasts, in contrast to
the wild-type receptor stimulated with SCF.43
Activating mutations have been shown to interfere with the normal
cellular processing of the Kit receptor, normally present as 125-kD and
145-kD glycoproteins. We show that expression of mutant KitD814H in ELM
cells results in the reduced expression of p145 and its complete
absence when expressed in Quail fibroblasts. This correlates with the
rapid ligand-independent receptor degradation and reduced cell surface
expression seen in studies with other activating
mutants,42,44 the rapid polyubiquitination and degradation of wild-type Kit after ligand stimulation,45,46 and the
inefficient receptor maturation seen with some activated mutants of the
related CSF-1-R.47
Activating mutations have also been identified in the kinase domain
activation loop in other RTKs. For example, they have been identified
in the Ret and Met receptors, associated with hereditary multiple
endocrine neoplasia 2B (MEN2B) and papillary renal carcinoma,
respectively,5,6 and in the
CSF-1-R.44 Indeed, mutations in Met and
CSF-1-R were found in the homologous aspartic acid residue to the Kit
D814, at D1246 in Met, and at D802 in the CSF-1-R.
It appears that the Kit receptor mediates its cellular effects through
association with a wide range of signaling molecules and activation of
many different cellular signaling pathways, depending on cell type (eg,
PKC isoforms,48 the JAK-STAT pathway,49 Lyn,50 Vav,51 Chk,52
CRKL,53 and Shp154). However, activation by Kit
of PI3 kinase and the Ras-Raf-MAPK pathway is
widespread.9,35,55 We have therefore analyzed
the phosphorylation of p42/p44 MAPK as an example of a downstream
target of Kit signaling believed to mediate profound effects in many
cells. Our data show constitutively high levels of MAPK phosphorylation
in unstimulated ELM cells expressing KitD814H and in the
stroma-independent ELM-I-1 cells from which the mutant c-kit
cDNA was cloned. This degree of activation was consistently higher than
that induced by continuous incubation of cells expressing similar
levels of the wild-type receptor in the optimal SCF concentration for
growth of these cells. Because there is evidence that a quantitatively
different level or duration of MAPK activation can result in a
qualitatively different cellular response,56 the constant
level of MAPK activation caused by the KitD814H mutation may be
significant. This could generate a different range of responses in ELM
cells to the short-term signal produced by SCF interacting with the
wild-type Kit receptor.
Finally, our data show specific changes in the level of expression of
the p66 isoform of the signaling molecule Shc in response to Kit
signaling. Recent data show that, unlike the p46 and p52 Shc isoforms,
p66 does not transform fibroblasts and inhibits, rather than enhances,
EGF-induced activation of MAPK and the c-fos promoter.32,33 This implicates loss of p66 expression as a potential mechanism that may increase the sensitivity of cells expressing the KitD814H mutant to extracellular stimuli, and this may
have been a factor in the selection of growth factor-independent ELM-I-1 cells during ELM erythroleukemia progression. Increased expression of p66Shc on SCF removal correlates with cells spontaneously differentiating, and a similar increase in p66 expression has been
observed when myoblasts are induced to differentiate by serum starvation.57 However, increased p66Shc expression is not
found in ELM cells induced to differentiate with Epo and, therefore, does not appear to be a marker of differentiation per se and may be
more directly a consequence of growth factor withdrawal and cell cycle
arrest.
| |
ACKNOWLEDGMENT |
The authors are grateful to Prof W. Ostertag (Heinrich-Pette Institute,
Hamburg, Germany) and Dr K. Itoh (University of Kyoto, Kyoto, Japan)
for the original gift of the ELM-D and ELM-I/1 cell lines and for many
helpful discussions. We also thank Margaret Frame, Dave Gillespie, and
John Wyke (all at the Beatson Institute) for their comments on this
manuscript and Robert McFarlane and Peter McHardy for expert assistance
with sequencing and FACS analysis, respectively.
| |
FOOTNOTES |
Submitted January 23, 1998;
accepted August 19, 1998.
Supported by the Cancer Research Campaign.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Paul R. Harrison, PhD, Beatson
Institute for Cancer Research, CRC Beatson Laboratories, Garscube
Estate, Switchback Road, Glasgow, G61 1BD, Scotland.
| |
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Abstract
Introduction