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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 1934-1946
Myb and Ets Proteins Are Candidate Regulators of
c-kit Expression in Human Hematopoietic Cells
By
Mariusz Z. Ratajczak,
Danilo Perrotti,
Paola Melotti,
Mark Powzaniuk,
Bruno Calabretta,
Kuzufumi Onodera,
David A. Kregenow,
Bogdan Machalinski, and
Alan M. Gewirtz
From the Departments of Pathology and Internal Medicine, University
of Pennsylvania School of Medicine, Philadelphia, PA and the Department
of Microbiology and Immunology and Kimmel Cancer Center, Thomas
Jefferson University, Philadelphia, PA
 |
ABSTRACT |
Kit is a tyrosine kinase receptor that plays an
important role in human hematopoietic cell growth. The promoter
elements that modulate the gene's expression have not been extensively
studied. Because of c-kit's acknowledged importance in
hematopoiesis, we sought to address this issue in more detail. To
perform these studies we analyzed a human c-kit 5
flanking fragment ~1 kilobase in length. Deletion constructs showed a
region ~139 nucleotides upstream from the translation initiation site
that was critical for promoter activity. A region containing a
potential silencing element was also identified. Sequence analysis
indicated several potential Myb- and Ets-binding sites.
The functional significance of these sites was explored by showing that
both wild-type Myb and Ets-2 protein, but not a DNA
binding-deficient Myb mutant protein, bound to distinct
5 flanking fragments that included these sites. Furthermore,
binding of recombinant Myb and Ets-2 protein to these
fragments could be competed with an excess of double stranded
oligodeoxynucleotides containing canonical, but not mutated,
Myb- or Ets-binding sites. We also showed that the 5 flanking region of c-kit exhibited promoter activity
in nonhematopoietic cells only when the cells were transfected with
c-myb or ets-2 expression vectors. Moreover,
Myb and Ets-2 coexpression in such cells augmented
transactivation of c-kit promoter constructs in comparison to
that observed in cells transfected with either construct alone.
Promoter constructs lacking various Myb and Ets sites
deleted were much less effective in this same system. Finally,
Myb and Ets-2 mRNA expression was detected in
CD34+, Kit low as well as
CD34+, Kit bright cells. In aggregate,
these data further define the human c-kit promoter's
functional anatomy and suggest that Myb and Ets
proteins play an important, perhaps cooperative, role in regulating
expression of this critical hematopoietic cell receptor.
| |
INTRODUCTION |
THE C-KIT PROTO-ONCOGENE encodes
a tyrosine kinase receptor, Kit1 whose cognate
ligand2-5 is now commonly referred to as Steel Factor (SF).
Stimulation of Kit by SF is required for development of normal
neural crest melanocytes, gametocytes, and hematopoietic progenitor
cells.6 In the latter cell type, binding of SF by the early
stem/progenitor cells that express the c-kit encoded receptor
is likely very important for cell survival.7,8 Binding of
SF may also play a role in a number of critical events, including stromal adhesion and cell proliferation.9,10 In vitro, SF synergizes with other growth factors to stimulate in vitro colony formation by colony-forming units of mixed lineages (CFU-Mix), burst-forming units of erythrocytes (BFU-E), colony-forming units of
granulocytes and macrophages (CFU-GM), and colongy-forming units of
megakaryocytes (CFU-Meg).9 Erythropoiesis, in particular, appears critically dependent on the Kit/SF
interaction.11-14 In vivo, mice with significant mutations
at either the Kit encoding locus, w, or the SF locus,
sl, are infertile, white coated, and anemic.15 In
humans, defects in Kit expression result in amelanotic skin
changes known as piebaldism.16,17 Major disturbances of human hematopoiesis resulting from defects of the Kit/SF axis have not yet been described.
Despite the importance of Kit in regulating hematopoietic cell
development, relatively little is known about the factors that regulate
its expression. Cytokines such as TNF- and TGF- are known to
transiently decrease Kit expression, perhaps by destabilizing the mRNA.18-23 Factors that operate at the transcriptional
level are even more obscure. We have hypothesized that the Myb
transcription factor may regulate c-kit expression because when
c-myb expression is perturbed with antisense
oligodeoxynucleotides, c-kit mRNA levels
decline.12,24 We have also noted that the lethal anemia suffered by c-myb "knockout" mouse embryos25
resembles a w-like hematopoietic defect and speculated that
this might be the direct result of an inability of hematopoietic cells
to express c-kit. Recent results from one of our
laboratories,26 and from Vandenbark et al,27
also suggest a role, likely complex, for Myb protein in the
regulation of c-kit receptor expression. Nevertheless, although
the above noted data strongly suggest a link between Myb and
Kit expression, it is not entirely certain if Myb
directly regulates c-kit by binding in its promoter region, if
it cooperates with other transcription factors in this regard, or if
intermediate events are also required.
To pursue these fundamental questions, and to learn more about the
functional organization of the c-kit promoter region, we isolated and sequenced ~1kb of genomic DNA in the 5 flanking region of the human c-kit gene. In support of our hypothesis
that Myb might play a direct role in regulating c-kit
expression, sequence analysis of the region showed several potential
Myb-binding sites. Of interest, a number of potential
Ets family protein binding sites were also uncovered. Because
Myb and Ets have been reported to cooperate in the
regulation of other hematopoietic genes, such as CD34,28,29
CD4,30 and mim-1,31 the possibility
that Myb and Ets might regulate Kit expression
in a cooperative manner was also explored. By using a variety of
complementary strategies, we found that wild-type, but not mutant,
Myb and Ets protein bound specifically to expected
sites within the c-kit promoter, and that cotransfection of
wild-type, but not mutated, proteins could upregulate activity of
chloramphenicol acetyltransferase (CAT) expression constructs driven by
the c-kit promoter in a variety of cell types. These results
suggest that Myb and Ets may play a physiologically
important cooperative role in regulating c-kit expression. They
therefore add to our understanding of the biology of these critical
hematopoietic cell genes.
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MATERIALS AND METHODS |
Cell lines.
CHO (Chinese hamster ovarian cancer), COS (green monkey
renal cancer cells), TK-ts13 (hamster fibroblasts), NIH 3T3
(mouse fibroblasts), K562, and HEL (human erythroleukemia) cells
were obtained from the American Type Culture Collection
(Bethesda, MD). K562 and HEL cells were maintained in RPMI (Gibco BRL,
Grand Island, NY) with 10% bovine calf serum (BCS; Hyclone, Logan,
UT). The other cell types were maintained in alfa MEM (Gibco BRL)
supplemented with 10% BCS (Hyclone), 100 U/mL of penicillin G, 100 µg/mL of streptomycin, and 250 ng/mL of Fungizone (JRH
Biosciences, Lenexa, KS).
Isolation of the 5 flanking region of human c-kit
gene.
A 4.2 kb EcoRI fragment of human genomic DNA known to contain
the first exon of c-kit (a kind gift from R. Spritz, University of Wisconsin) was digested with various frequent cutting restriction enzymes. The digestion products were visualized on a 1% agarose gel
and then transferred to a nitrocellulose filter. c-kit gene fragments were identified by hybridization with a 32 P-end-labeled c-kit receptor antisense oligodeoxynucleotide
(ODN) complementary to codons 1 to 6 with standard methods. An
RsaI digest of the original fragment generated suitably sized
fragments for sequencing. A 1064 nt fragment was verified by
hybridization and selected for further analysis.
Sequencing reactions.
DNA fragments to be sequenced were subcloned into pT7 Blue Vector
(Novagen, Madison, WI). Reactions were then performed by using a
Sequenase V 2.0 Kit essentially as directed by the manufacturer (USB, Cleveland, OH).
Plamid expression constructs.
Myb expression constructs were generated as follows: A
full-length human c-myb cDNA (kindly provided by Dr E. Prochownik, University of Pittsburgh, Pittsburgh, PA) was digested with
NcoI/DraI, yielding a 2116 nt fragment containing the
entire open reading frame. The NcoI site was blunt ended with
Klenow, allowing ligation of EcoRI adapters (Stratagene, La
Jolla, CA) to both ends. The adapter ends were phosphorylated with T4
polynucleotide kinase (Promega, Madison, WI) and then subcloned into
the pcDNA3 expression vector, thereby confering neomycin resistance to
transfected cells (InVitrogen, San Diego, CA). This construct was named
pCMV-myb. Construction of a plasmid engineered to express a
mutated Myb protein lacking the carboxy terminal 46 amino acids
of the R1 binding domain and the aminoterminal 23 amino acids of the R2 domain, has recently been reported.32
An Ets-2 expression construct was generated in a similar manner
with pSVets-2, a plasmid kindly provided by Dr S. Reddy
(Allegheny University of Health Sciences, Philadelphia, PA). The
plasmid was digested with EcoRI, releasing a fragment
containing the full-length human ets-2 cDNA (2269 bp). This
fragment was gel-purified and then subsequently subcloned in a sense or
antisense orientation into EcoRI-digested pcDNA3 (Invitrogen
Inc). The former construct was designated pCMV ets-2.
Synthesis of recombinant Myb protein.
TK-ts13 fibroblasts (2 × 106 ) were
transfected with pCMV-myb (20 µg) by using a Calcium
Phosphate Transfection Kit (InVitrogen, San Diego, CA), as directed by
the manufacturer. After 24 hours, G-418 selection (2 mg/mL) (GIBCO BRL,
Grand Island, NY) was performed. Medium was exchanged every 2 to 3 days
and after 7 to 10 days resistant clones were replated. After 2 weeks,
surviving clones were screened for Myb protein expression by
Western blot analysis (see below). A single clone expressing a high
level of Myb was expanded and maintained.
Western blot detection of Myb protein.
TK-ts13 cells (2 × 106) were detached
from culture dishes with ethylenediaminetetraacetic acid [EDTA] (0.53 mmol/L)-trypsin (0.05%) in phosphate-buffered saline (PBS), washed
twice in PBS, resuspended in 1× sodium dodecyl sulfate (SDS)
gel-loading buffer [50 mmol/L Tris-Cl (pH 6.8), 100 mmol/L
dithiothretiol (DTT), 2% SDS, 0.1% bromophenol blue, 10% glycerol],
boiled for 2 minutes, and then chilled on ice. Resulting cell extracts
were centrifuged briefly (14,000g for 1 minute) and then
analyzed for Myb protein content. Clarified cell extracts,
protein size markers, and a positive Myb protein control were
loaded onto gels and then electrophoresed in the Pharmacia LKB Phast
gel apparatus (Pharmacia, Uppsala, Sweden). Separated proteins were
transferred to a nitrocellulose membrane filter (Gibco BRL,
Gaithersburg, MD), which were probed with murine antihuman Myb
monoclonal antibodies (MoAbs) (UBI, Lake Placid, NY). Binding of the
anti-Myb antibody was detected by enhanced chemiluminescence
(ECL Western blot Kit, Amersham, Buckinghamshire, UK) by
using antimouse IgG (L+H) (Boehringer-Mannheim, Mannheim, Germany)
conjugated with peroxidase. Positive reactions were visualized by
exposure to high-sensitivity radiograph film (DuPont, Boston, MA).
DEAE-dextran-mediated transient transfections.
Briefly, TK-ts13 cells were plated in 10 cm plastic Petri
dishes and grown to 70% to 80% confluence. The medium was exchanged 2 hours before transfection. Four mL of culture medium containing 40 µL
of DEAE-Dextran (50 mg/mL stock) (Sigma, St. Louis, MO), 5 µg of
reporter pSV2pap plasmid (kind gift from Dr T. Kadesh, University of
Pennsylvania, Philadelphia, PA), and 50 µg of different CAT construct
plasmids was added to each plate. After 4 hours of incubation at
37°C, the transfection medium was removed. Cells were then shocked
with 10% DMSO (Sigma, St. Louis MO) for 2 minutes, and then washed
three times with fresh medium. After 48 hours of incubation (37°C,
5% CO2) in a fully humidified incubator, the cells were
detached by using TEN buffer (40 mmol/L Tris-HCl pH 7.5, 1 mmol/L EDTA,
15 mmol/L NaCl), harvested, and analyzed for PAP and CAT activity.
Lipofectamine-mediated transient transfections.
K562 cells were transfected with pCAT constructs by using
Lipofectamine, as directed by the manufacturer (GIBCO BRL). Briefly, exponentially growing K562 cells were washed once in the serum-free OPTI MEM (GIBCO BRL), and thereafter plated in six-well culture plates
at a density 2 × 106 cells/mL in 0.8 mL. Two hundred
µL of OPTI MEM containing 10 µL of lipofectamine and 11 µg of
plasmid DNA (10 µg of PSV2pap and 1 µg of pCAT) was then added to
the cells that were subsequently incubated for 12 hours at 37°C in
a humidifed atmosphere containing 5% CO2 . The cells were
then diluted in 4 mL of RPMI containing 15% BCS. After an additional
48-hour incubation, the cells were harvested for PAP and CAT assays, as
described below.
PAP assays.
PAP assays were performed to evaluate transfection efficiencies in a
given set of experiments, thereby allowing better standardization of
subsequently performed CAT assays. Target cells were cotransfected with
a given pCAT construct and the pSVpap plasmid that expresses human
placental alkaline phosphatase.33 Briefly, 1/15 of the extracts prepared from transfected cells were heated to 65°C for 15 minutes, then resuspended in 1 mL of reaction buffer (0.5 mmol/L p-nitro-phenyl-phosphate, 1mol/L diethanolamine), and incubated at
37°C for 15 minutes. Postincubation, the reaction mixture was clarified by centrifugation and accumulation of dye product quantitated spectrophotometrically at a wavelength of 405 nm. Preparations containing equal levels of PAP activity were then used for CAT assays,
as described below.
CAT assay.
CAT assays were employed to map potential promoter elements in the ~1
kb of DNA upstream of the c-kit translational start site. DNA
fragments of varying sizes were generated by using the following
restriction enzyme combinations: RsaI+BamHI [929 nt fragment]; XhoI+BamHI [461 nt fragment];
SmaI+BamHI [158 nt fragment]; and
NaeI+BamHI [118 nt fragment]. Fragments generated by
these digestions were initially subcloned into pGEM4Z or pGEM7Z
(Promega) in order to facilitate cloning of the fragments into the
multiple cloning site of the pCAT basic (pCAT B) vector (Promega). An
RsaI fragment with a BamHI/NaeI deletion was
also subcloned into the pCAT B vector in a similar manner.
Kit promoter constructs containing Myb or
Myb/Ets binding site deletions were constructed as
follows: A 910bp and a 731bp fragment were generated by PCR
amplification on the c-kit-XhoI/BamHI plasmid
by using two different upstream primers located inside the
c-kit 5 flanking region, and a common downstream
(3) primer mapping inside the pCAT basic vector at nts [+2819
to +2840]. The 5 primers employed were complementary to nts
[-405 to -385], and nts [-224 to -205]. The former allowed
amplification of a c-kit promoter fragment missing the
Myb binding site located between nts -420 and -410 that we
designated M1, whereas the latter 5 primer amplified a
c-kit promoter fragment missing M1, an additional downstream
Myb binding-site designated M2, and two putative
Ets-binding sites designated E1 and E2
(Fig 1). The amplified products were subcloned into the pCR2.1 vector by using the TA cloning kit
(Invitrogen Corp) to generate constructs called pCR D M1 and pCRE3,
respectively. These plasmids were digested with
HindIII/NcoI restriction endonucleases and the
fragments released (containing the c-kit flanking region from
nucleotide -405, or from -224, to the BamHI site in the
c-kit 5UTR plus the indicated portion of the CAT gene)
and subcloned into pCAT basic vector previously digested with the
HindIII/NcoI restriction enzymes. The
resulting plasmids were designated pc-kit D M1, and pc-kit-E3.
Plasmids sequences were confirmed by automated sequencing with the Taq
Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster
City, CA).
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| Fig 1.
Nucleotide sequence of human c-kit gene spanning
929 bases upstream of the initiating methionine to 69 bases within the
first intron. Coding sequence is indicated by capital letters and is underlined. Potential Myb-binding sites, on the + or  strand, in the region upstream of the ATG are enclosed by solid line
boxes. Potential Ets-binding sites, on the + or strand, are enclosed by dashed boxes. Location of restriction sites are
indicated below the sequence.
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CAT assays were performed according to the manufacturer's protocol
(Promega). Products of the CAT assay, 14 C-chloramphenicol,
and its acetylated derivatives, were detected by thin layer
chromatography. After drying, gels were exposed to high-sensitivity
films (NEF 496, DuPont, Boston, MA). Reaction intensity, a direct
function of the amount of 14 C-labeled product present in
the gel, was quantitated by densitometry with a Personal Densitometer
(Molecular Dynamics, Sunnyvale, CA).
Nuclear extracts and electrophoretic mobility shift (EMSA)
assay.
Nuclear protein extracts and EMSA assays were performed, as previously
described.34  32 P-labeled double-stranded
DNA probes (~5 × 104 cpm) corresponding to three
different segments of the human c-kit promoter from nucleotides
-724 to -480; -465 to -220; and -196 to -20 upstream from the
translation start site (Fig 1) were synthesized by PCR.
Oligonucleotides corresponding to the canonical MYB-binding sites (M1
[nucleotides from -426 to -404] and M2 [nucleotides from -367 to
-343]; or to Ets-binding sites (E1 [nucleotides from -384 to
-360], E2 [nucleotides from -257 to -233] and E3 [nucleotides from
-203 to -179] were synthesized in an Applied Biosystems automated synthesizer and end labeled with -[32 P] ATP.
Five hundred ng of GST-MYB fusion protein35 or 15 µg of
bacterial lysate from HB101 cells transformed with a
pFlag-c-myb vector36 or a pFlag-ets-2
vector37 were used in EMSA, as described,36 with some modifications. Briefly, proteins (purified fusion protein or
bacterial lysate) in binding buffer (10 mmol/L Tris-HCl [pH 7.5], 50 mmol/L NaCl, 1 mmol/L EDTA, 10% glycerol, and 1 mmol/L dithiothretiol)
were incubated with 0.12 mg/mL of poly (dI-dC) for 10 minutes on ice.
-32 P-end labeled double-stranded oligonucleotide probes
were added to the binding reactions, which were incubated for an
additional 15 minutes at room temperature. When indicated, EMSA were
also performed in the presence of a 100-fold molar excess of
double-stranded oligonucleotides used as specific or nonspecific
competitors. Binding reactions were electrophoresed in native 5%
polyacrylamide gel electrophoresis (PAGE) in low ionic strength (0.25 × Tris borate EDTA). Gels were dried and exposed to radiograph
films for autoradiography. Double-standed oligonucleotides of the human cdc2 promoter containing an Ets binding site37 (wt
Ets binding site= gagagggggaAGGAAagaacaagaa) and of the chicken
mim-1 promoter containing a Myb-binding site (wt
Myb tcgacacattaTAACGGttttttagc) were employed as specific
competitors in EMSA. Double-stranded oligonucleotides carrying
mutations (mut) in the core of either MYB (mut MYB binding site:
tcgacacattaTGGGGGttttttagc) or Ets (mut Ets binding
site: gagagggggcGTAGCagaacaagaa) consensus sequence, were used as
nonspecific competitors in EMSA.
Fluorescence-activated cell sorting (FACS) analysis and cell
sorting.
FACS analysis and cell sorting were performed as previously
reported.8 In brief, MNC (~3 to 6 × 107
) were simultaneously labeled with fluorescein conjugated anti-CD34 MoAb (Becton-Dickinson, Mountain View, CA), and a phycoerythrin-labeled antihuman Kit receptor MoAb (kind gift of Dr V. Broudy,
University of Washington, Seattle, WA). Cells categorized
as CD34+, Kit low (defined as the
dimmest 50% of labeled cells) and CD34+, Kit
bright (defined as the brightest 50% of labeled cells)
were isolated by FACS. RNA was extracted from these cells, reverse
transcribed, and then PCR amplified to detect Ets, Myb,
and -actin mRNA expression, as previously reported.8,12
| |
RESULTS |
Sequence analysis of the 5 flanking region of the human
c-kit gene.
RsaI digestion of a 4.2 kb genomic fragment containing the
c-kit 5 flanking region generated a number of fragments
that were suitably sized for sequencing. One of these, ~1064 nts in
length, hybridized with a c-kit oligonucleotide probe
complementary to codons 1 to 6. This fragment was cloned into the pT7
blue sequencing vector for complete sequencing. Comparison of the
sequence derived with those previously reported by Giebel et
al,38 and Yamamoto et al39 showed that the
fragment contained the entire first exon of c-kit flanked
downstream by a small part of the first intron, and upstream by 925 nts
of sequence (Fig 1). In the areas in which the reported sequences
overlap, they are virtually identical. As reported,38 we
too noted the presence of numerous potential binding sites for
transcription factors including two AP 2-binding sites (nts 218- and
nts 420-), several SP 1 sites, and a number of cis elements for
melanocyte-specific genes including melanocyte-specific upstream
(MEL-US) and downstream (MEL-DS) elements. In addition, we have also
identified several potential Myb (Py-AAC G/T
G)40,41 and Ets family protein
(AGGAA)42 consensus binding sites (Fig 1).
Functional analysis of c-kit 5-flanking
region.
CAT assays were employed to map potential promoter elements in the ~1
kb of DNA upstream of the c-kit translational start site. As
noted, the 1064 nts RsaI fragment was restriction digested with
various enzyme combinations to yield a 909 nts
RsaI/BamHI fragment, a 461 nts
XhoI/BamHI fragment, a 158 nts
SmaI/BamHI fragment, and a 118 nts
NaeI/BamHI fragment. The fragments positions relative
to the translational start site are shown in
Figure 2. They were subcloned into pCAT B,
which contains no promoter or enhancer sequence, and then transiently
transfected into K562 cells. FACS analysis of these human leukemia
cells shows that ~70% of the cells employed in our laboratory
express Kit at low level (data not shown). Reporter constructs
were also transfected into nonhematopoietic NIH3T3, TKts-13, CHO, and
COS cells. Activity in each case was compared with that obtained with a
pCAT positive control vector driven by the SV40 promoter and enhancer,
which was arbitrarily assigned a value of 100%.
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| Fig 2.
Restriction fragments used to functionally characterize
the c-kit promoter. Fragments were derived from a 1064 nts
RsaI fragment as detailed in the methods section and ultimately
subcloned into pCAT vectors to assess their promoter activity. Length
and position of the various fragments relative to the translational
start site (+1) is indicated in the cartoon.
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In K562 cells, no CAT activity was detected after transfection of the
empty pCAT B vector (Fig 3A). However, when the
RsaI-BamHI fragment was subcloned into the pCAT B
vector, CAT activity approximately one third that of the positive
control was observed suggesting that promoter elements were contained
within this region. Assays with additional constructs were also
performed in hopes of deriving a more detailed functional map of the
promoter region. The XhoI-BamHI fragment had nearly
twice the activity of the longer RsaI-BamHI fragment,
suggesting that an unidentified transcription inhibitory protein might
bind upstream of the XhoI site. The SmaI-BamHI
fragment gave promoter activity similar to the
XhoI-BamHI fragment, but the NaeI-BamHI
fragment missing the intervening 40 nucleotides between the
SmaI and NaeI sites (NaeI-BamHI
[del]) lost ~50% of the promoter activity. Because the
NaeI/BamHI fragment had the weakest measureable
activity, we then determined if this fragment might represent a minimal
promoter unit. To test this possibility, the RsaI/BamHI
fragment was restriction digested with NaeI. This yielded a 790 nt fragment missing the proximal (relative to the start site) 118 nt
piece (Fig 2). When the 790 nt fragment was subcloned into pCAT B and
expressed in K562 cells, no promoter activity was detected in the CAT
assay (Fig 3A). Therefore, the promoter and
transcription initiation site likely reside within this fragment of
genomic DNA.
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| Fig 3.
(A) CAT activity in K562 cells transiently transfected
with a pCAT positive control vector, pCAT B negative control vector, and pCAT B containing RsaI-BamHI,
XhoI-BamHI, SmaI-BamHI, NaeI- BamHI, and NaeI-BamHI (del) inserts. (B) CAT
activity in CHO cells transiently transfected with a pCAT positive
control vector, pCAT B-negative control vector, and pCAT B containing
RsaI-BamHI, XhoI-BamHI, Sma
I-BamHI, NaeI-BamHI, and
NaeI-BamHI (del) inserts. Transfection reactions were
normalized by use of PAP assays, as detailed in Materials and Methods.
Mean ±SD of six different experiments, each performed in duplicate,
are shown. CAT activity was normalized to the CAT control transfected
cells, which were arbitrarily set at 100%.
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To determine tissue specificity of the putative promoter unit, the CAT
constructs were also expressed in nonhematopoietic NIH3T3 cells and
TK-ts13 fibroblasts. Neither of these cell types express
the Kit receptor. In distinct contrast to results obtained with
K562 cells, none of the pCAT B constructs showed promoter activity in
3T3 or TK-ts13 fibroblasts (not shown). Slightly different
results were obtained when the same constructs were transfected into
COS and CHO (Fig 3B) cells, both of which constitutively express
c-kit at low levels. All of the fragments cloned into the pCAT
B vector showed weak promoter activity in these cells. Again, the
NaeI/BamHI fragment appeared to have the weakest
activity, suggesting it might represent the minimal promoter unit.
Effect of Myb and Ets protein on c-kit
promoter activity.
Our previous studies,12 and the presence of several
potential Myb-binding sites in the c-kit 5
flanking region (Fig 1), suggested that Myb might play a role
in regulating Kit expression. To address this question, we
first sought to determine if Myb protein expression induced
transactivation of the Kit promoter. TK-ts13 cells
were therefore transfected with Myb protein expression constructs. Stable lines were screened for protein expression and a
positive clone was expanded. Cells in this clone were then transiently
cotransfected with the various pCAT B-kit promoter constructs.
In distinct contrast to results obtained with Myb negative
TK-ts13 cells, transient transfection of the various
c-kit promoter constructs into the Myb-positive
TK-ts13 cells led to clearly detectable, albeit weak, CAT
activity in the cell extracts. A histogram of the densitometric
quantitation of five separate CAT assays, each performed in duplicate,
with each of the constructs is shown in
Figure 4. As previously observed, the
XhoI/BamHI fragment had greater activity than the
intact RsaI-BamHI fragment. This result again supports
the possibility that a negative regulatory element might reside
upstream of the XhoI site between nts -929 and -481. Finally,
as an additional proof that Myb may regulate c-kit
promoter activity, wild-type TK-ts13 cells were
cotransfected with a pCDNA3 construct expressing either the wild-type
Myb protein or a DNA binding-deficient mutant,35 as
well as each of the various pCAT B c-kit promoter constructs (Fig 2). CAT activity was only observed in cells expressing the wild-type protein (data not shown), again strongly suggesting that a
functional Myb protein was necessary for the observed
Kit promoter activity .
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| Fig 4.
Densitometric quantitation of CAT activity in stable,
MYB-expressing TK-ts13 cells cotransfected with a pCAT B
plasmid containing various portions of the c-kit 5
flanking region. CAT activity was normalized to the CAT
positive-control transfected cells, which were arbitrarily set at
100%. Mean ±SD of five different experiments, each performed in
duplicate are shown. Transfection reactions were normalized by use of
PAP assays, as detailed in Materials and Methods.
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As noted in Figure 1, in addition to Myb-binding sites, a
number of potential Ets-binding sites were also identified in
the c-kit flanking region under study. The potential
significance of these sites was evaluated with the experimental
approach employed to determine Myb's role in regulating
Kit expression. The same sets of pCAT B-Kit promoter
constructs were cotransfected into TK-ts13 cells along with
an Ets-2 expression vector, pCMV-ets-2. Ets-2 was
chosen for these studies because Myb/Ets-2 cooperation has previously been shown important in regulating promoter activity of
the CD34 and mim-1 genes.28,31 As shown in
Figure 5, no CAT activity was detectable in
the TK-ts13 cells transfected with the pCAT B
RsaI-BamHI construct alone. However, when cells were cotransfected with an Ets expression construct, weak
transactivation relative to the pCAT positive control, of the
RsaI/BamHI promoter fragment, and even stronger
activity with the XhoI/BamHI fragment was observed. Of
interest, in contrast to results obtained with Myb expressing
cells, Ets-2 did not transactivate the promoter units contained
within the smaller SmaI/BamHI and
NaeI/BamHI fragments. These latter results were
expected because neither of these fragments contain Ets-binding
sites. As an additional control, cells were cotransfected with the pCAT
B RsaI-BamHI construct and pCMV ets-2 in which
the Ets expression cassette was cloned into pCMV in an antisense orientation [pCMV ets-2 (as)]. CAT activity was
undetectable in extracts prepared from these cells (Fig 5).
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| Fig 5.
Densitometric quantitation of CAT activity in
TK-ts13 cells cotransfected with pCMV-Ets-2 and
pCAT B constructs containing various portions of the c-kit
5 flanking region. pCAT B RsaI-BamHI + pCMV
ets-2 in antisense (as) orientation was employed as an additional
control. Transfection reactions were normalized by use of PAP assays,
as detailed in Materials and Methods. Mean ±SD of five different
experiments, each performed in duplicate, are shown. CAT activity was
normalized to the CAT control transfected cells that were arbitrarily
set at 100%.
|
|
Myb and Ets-2 protein have additive effects on
c-kit promoter activity.
We also investigated if Myb and Ets-2 could cooperate
in transactivating the Kit promoter by using several different
strategies. TK-ts13 fibroblasts were transfected with
Myb (pCMV-myb), Ets-2 (pCMV-ets-2), or
Myb + Ets expression vectors, along with the pCAT B
RsaI/BamHI fragment construct
(Fig 6 A). No CAT activity was detected in extracts from cells transfected with the pCAT B
RsaI-BamHI construct alone, or in cells cotransfected
with this Kit promoter construct and an insertless
Myb/Ets expression vector. As noted previously, CAT
activity was detected in cells cotransfected with pCAT B
RsaI-BamHI and either the Myb or Ets-2
expression vector. Cotransfection of pCAT B RsaI-BamHI
with both the Myb and Ets-2 expression vectors into the
TK-ts13 cells led to CAT activity that was approximately threefold
greater then that observed with the Myb or Ets
expression constructs alone.
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| Fig 6.
Myb and Ets-2 transactivate c-kit
promoter in a cooperative manner. (A) CAT activity in
TK-ts13 fibroblasts cotransfected with Myb
(pCMV-myb), Ets-2 (pCMV-ets-2), or
Myb+Ets expression vectors, plus a pCAT
B-RsaI/BamHI fragment construct. Transfection of all
three vectors into the TK-ts13 cells led to CAT activity ~threefold
greater than that observed with the Myb or Ets
expression constructs alone, and ~140% that of the pCAT positive
control (not shown). (B) Ability of Myb and Ets protein
to transactivate wild-type, or mutated forms of the XhoI-BamHI Kit promoter fragment in TK-ts13
cells. In the absence of Myb or Ets, the construct was
without CAT activity (lane 1). Cotransfection of Myb or
Ets-2 expression constructs along with the pCAT B
XhoI-BamHI promoter construct significantly increased CAT activity (lanes 2 to 3). Deletion of a single Myb binding site (c-kit-D M1) resulted in loss of Myb's (lane 5),
but not Ets's (lane 6) ability to augment CAT activity.
Deletion of both Myb sites and two of three Ets sites
diminished, but did not entirely abolish the ability of Ets to
transactivate the promoter construct (lane 9). Neither c-kit-D
M1, nor c-kit-E3 alone elicited CAT activity in the TK-ts13
cells (lanes 4 and 7, respectively). See text for complete details.
(Panel C) Schematic representation of Myb and
Ets-2-binding sites within the XhoI c-kit
promoter region (-481/-24).
|
|
We then examined the ability of Myb and Ets protein to
transactivate wild-type, or mutated forms of the
XhoI-BamHI Kit promoter fragment in TK-ts13
cells (Fig 6B). As was shown above, in the absence of Myb or
Ets, the construct was without significant activity (Fig 6B,
lane 1). Again, cotransfection of Myb or Ets-2
expression constructs along with the pCAT B XhoI-BamHI
kit promoter fragment construct led to a significant increase
in CAT activity (Fig 6B, lanes 2 to 3). Deletion of a single
Myb binding site (c-kit-D M1) located within nts -427 to -402 of the promoter fragment (see M1, Fig 6C) resulted in almost a
complete loss of the ability of Myb protein to augment CAT
activity (Fig 6B, lane 5). This Myb binding site appears then
to be crucial for transactivation of this promoter fragment.
Nevertheless, in spite of this deletion, Ets-2 protein remained
active as a transactivating factor (Fig 6B, lane 6). When both
Myb sites (nts -427 to -402; -367 to -390) and two (-384 to
-360 and -332 to -309) of three Ets sites contained within this
fragment were deleted (c-kit-E3; see Fig 6C- M1, M2, E1, and
E2), only minimal CAT activity was observed in the presence of
Myb (Fig 6B, lane 8), whereas activity could be detected in cells cotransfected with the Ets-2 construct (Fig 6B, lane 9), presumably due to the remaining Ets-2 binding site (E3).
c-kit-D M1, and c-kit-E3 elicited only minimal CAT
activity in the TKts-13 cells (Fig 6B, lanes 4 and 7, respectively).
The specificity of these interactions was further confirmed by
competition experiments performed in intact cells. In these studies,
TK-ts13 cells were again cotransfected with the Myb
(pCMV-myb) or Ets-2 (pCMV-ets) expression
vectors previously shown capable of transactivating the pCAT B
XhoI/BamHI Kit expression construct (Fig 7A, lanes 2 and 5 respectively; Fig 7B
as labeled). CAT activity in cells in which a 200-fold molar excess of
double-stranded mutated or wild-type Myb binding site
oligonucleotides had been added was either unchanged or significantly
reduced, respectively (Fig 7A, lanes 3 and 4; Fig 7B, as labeled).
Similar results were obtained when this experiment was repeated with a
200-fold molar excess of mutated or wild-type double-stranded
Ets-2 binding site oligonucleotides Ets-2 (Fig 7A,
lanes 6 and 7; Fig 7B, as labeled).
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| Fig 7.
(A) CAT activity competition assay of double stranded
oligomers containing wild-type (WT) or mutated (mut) Myb or
Ets-2 binding motifs in the transient transfections experiments
in TK-ts13 fibroblasts. pCAT B XhoI-BamHI
(lane 1), pCAT B XhoI-BamHI + pCMV-myb (lane
2), pCAT B XhoI-BamHI + pCMV-myb + mut
oligomers (lane 3), pCAT B XhoI-BamHI + pCMV-myb + wt oligomers (lane 4), pCAT B
XhoI-BamHI + pCMV-ets-2 (lane 5), pCAT B
XhoI-BamHI + pCMV-ets-2 + mut oligomers
(lane 6), pCAT B XhoI-BamHI + pCMV-ets-2 + wt oligomers (lane 2; lane 7), pCAT B XhoI-BamHI + pCDN3
(lane 9). (B) Densitometric quantitation of CAT activity in
transiently, MYB and Ets-2 expressing TK-ts 13 cells cotransfected with a pCAT B plasmids containing the XhoI-BamHI c-kit 5 flanking region and
200 molar excess of wt or mut oligomers. CAT activity present in cells
transfected with the pCAT B XhoI-BamHI + pCMV-myb constructs was arbitrarily given a value of 100%.
Mean ±SD of six different experiments, each performed in duplicate,
are shown.
|
|
Myb and Ets-2 protein bind to their predicted sites in
the Kit promoter region.
The above studies clearly suggest a role for the Myb and
Ets proteins in the regulation of c-kit promoter
activity. They do not indicate, however, whether this effect is a
direct one. This issue was explored by electrophoretic mobility shift
assays (EMSA) designed to determine if these respective proteins could
bind to their predicted sites within the promoter region, and whether such binding was specific. Our initial experiments employed three different 32 P-labeled fragments of the c-kit
flanking region that were incubated with bacterial lysates that either
contained, or did not contain, Myb protein
(Fig 8A). With each fragment tested, one
complex was retarded in the gel by lysates that contained Myb
protein. In contrast, there was no retardation in the lysates that did
not contain Myb protein. The specificity of these reactions was
confirmed in competition experiments. When a 100-fold molar excess of
an unlabeled specific competitor (a 36 bp fragment of the CD34 gene 5flanking region containing two Myb-binding sites
[20]) was added to the Myb containing binding mixture, the
previously noted band shifts were no longer observed. When the two
Myb-binding sites were mutated, and a 100-fold molar excess of
this nonspecific competitor was added to the mixtures, the shifted
bands were noted again. Finally, in the presence of bacterial lysate
containing mutated Myb protein lacking portions of the R1 and
R2 DNA binding domains, no band shift could be detected (Fig 8B). These
results provided highly suggestive evidence that Myb protein
bound specifically to sites within the promoter region.
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| Fig 8.
Purified Myb protein binds in the c-kit
promoter and can be specifically competed. Three different fragments of
the c-kit promoter, with nt positions as indicated, were
labeled with -32 P and then used as probes in EMSA
assays performed with bacterial lysates containing, or not containing,
Myb protein. (A) Lanes 3, 8, and 13 contain Myb
protein. Lanes 1, 2, 6, 7, 11, and 12 contain no Myb protein.
Lanes 4, 9, and 14 contain 100 times excess of cold Myb-binding
site competitor (CD34 promoter). Lanes 5, 10, and 15 contain a
competitor oligonucleotide with mutated Myb-binding sites. (B)
Lanes 1 and 2, no Myb protein in lysate. Lane 3, lysate with
Myb protein. Lane 4, lysate with mutated Myb protein
(R1 and R2 partially deleted).
|
|
Additional experiments were also performed to further determine if
Myb and Ets-2 protein could bind to specific motifs
identified in the 5 flanking region of c-kit gene (Fig
1). Because the strongest promoter activity appeared to reside in the
XhoI/BamHI fragment, and because this fragment also
contained three Ets-2 and two Myb canonical binding
sites, we selected this fragment for analysis. EMSA assays employing
oligonucleotides corresponding to the Myb- (M1 and M2)
(Fig 9A) and Ets (E1, E2, E3) (Fig
9B)-binding sites in the XhoI/BamHI promoter fragment
are shown in Figure 9. Binding of bacterially expressed Myb
(Fig 9, Panel A) or Ets proteins (Fig 9, Panel B) was
specifically competed by wild-type, but not mutated, double-stranded
oligonucleotides. Moreover, EMSA assays performed with nuclear proteins
derived from K562 cells also showed the formation of specific complexes
with Myb M1- and M2-binding sites (Fig 9C), as well as the E1
and E2 Ets-2-binding sites (Fig 9D).
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| Fig 9.
Myb and Ets-2 proteins bind specifically
to their predicted sites in the kit promoter. -32 P-end
labeled double-stranded oligonucleotide probes corresponding either to
the canonical Myb (M1 and M2) or Ets-2 (E1, E2 and E3) binding sites within the XhoI-SmaI promoter region
fragment (nts-461 to -24) (see Fig 6C) were employed in EMSA. Binding
of bacterially expressed Myb (A) or Ets proteins (B)
was competed with either wild-type (wt) or mutated (mut)
double-stranded oligonucleotides as indicated above each lane. Similar
experiments were also performed with nuclear extract derived from K562
cells. Fifteen µg of K562 nuclear extract was incubated in binding
buffer with -32 P-end labeled double-stranded
oligonucleotide probes (lane 1; [C and D]) corresponding either to
the canonical Myb (M1 and M2; [C]) or Ets-2- (E1,
E2; [D]) binding sites as described above. Specificity of binding was
assessed using wild type (lane 2; [C and D]) or mutated (lane 3; [C
and D]) double-stranded oligonucleotides as indicated above each lane.
Sequences of the double-stranded oligodeoxynucleotides for both mutated
and wild-type Myb and Ets-2 binding sites are given in
the Materials and Methods section.
|
|
Myb and Ets-2 mRNA expression in primitive
hematopoietic cells.
Because c-kit is expressed on the most primitive of human
hematopoietic cells, one might expect to find that Myb and
Ets are also expressed in these cells if they play an important
physiologic role in regulating Kit expression. Accordingly, we
FACS isolated normal human CD34+ Kit
bright cells and CD34+ Kit
dull cells (Fig 10A) and
determined whether c-myb and ets-2 mRNA could be
detected in these primitive cells by using reverse-transcriptase polymerase chain reaction (RT-PCR). As shown in Figure 10B, both mRNA
species, as anticipated, were detected in these very primitive human
hematopoietic cells.
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| Fig 10.
(A) FACS histogram of bone marrow MNC double-labeled for
expression of CD34 antigen (FITC) and c-kit receptor (PE).
Cells in region R2 are defined as being Kit "bright,"
whereas cells in the region labeled R3 are defined as Kit
"low." (B) RT-PCR analysis of FACS isolated cells for Ets
and Myb mRNA expression. Molecular weight marker lanes are
indicated by ø. Lanes 1 and 2 show Ets-2 mRNA expression,
lanes 4 and 5 c-myb expression, and lanes 7 and 8, -actin
expression in Kit "bright" and Kit "dull"
cells respectively. Lanes 3, 6, and 9 represent water control lanes for
the PCR reactions.
|
|
| |
DISCUSSION |
The human c-kit gene is located on the long arm of chromosome 4 [4q11 - 12], close to the locus of the platelet-derived growth factor
receptor-alpha (PDGF-R) gene.43 Several groups have reported genomic analyses of the c-kit gene43-45
and a few have begun to study the promoter region as
well.27,39,44 By providing additional functional analyses
of the human c-kit promoter we usefully extend these earlier
works and provide new insights into the regulation of this critical
hematopoietic gene. For example, in agreement with the previously
published studies, we have shown that the ~1kb genomic fragment we
analyzed contained weak promoter activity akin to the 5 flanking
region of the c-fms gene to which c-kit is highly
related. Also in agreement with previously published reports,27,39 we found that CAT activity in Kit
expressing cells was highly dependent on the presence of a relatively
small region of the promoter immediately upstream of the translational start site. Our experiments showed that deletion of the distal NaeI/BamHI fragment (nts -138 to -20) resulted in
almost complete loss of promoter activity in K562 cells. This fragment
was required for Kit promoter activity in nonhematopoietic
cells as well, even if, like the K562 cells employed in our laboratory,
they expressed Kit at low level. Whether distal genomic
elements modulate Kit expression in a cell type specific
manner, as has been suggested by Vandenbark et al,27 was
not evaluated.
Perhaps of greater importance we have also begun to identify important
regulatory factors that govern the gene's expression (Fig 1).
Previously, a TATA-like element (-ATTAA-; nts -232 to -228) was
identified by Giebel et al38 but its functional
significance was not explored. Our deletion experiments suggest that it
is not critical for Kit expression. For example, the data
presented in Figure 4 show that expression constructs devoid of this
element do not lose significant promoter activity relative to those
that contain it. Whereas the more quantitative data presented in
Figures 4 and 7 do suggest some weakening of activity in the absence of this element, a deletion construct that contains the TATA-like element,
but not the proximal ~118 nts of the putative promoter, has no
activity at all in our CAT reporter assays. Further analysis of the
available 5 flanking sequence suggests that a number of other
potentially important regulatory sites, both positive and negative,
have been identified. For example, deletions from the 5 end of
the RsaI fragment appeared to yield fragments with stronger promoter activity. Potential negative regulatory elements present in
the deleted region include an MZF1 binding site at nucleotides -820 to
-808.30 Studies to directly confirm the importance of these
sites in the expression of the Kit receptor in human
hematopoietic cells are presently ongoing in our laboratory.
We have also explored the potential functionality of several putative
Myb-and Ets-2-binding sites in the 5 flanking
region of the c-kit gene. This was of great interest to us
because we have previously shown that when Myb expression is
perturbed in human CD34+ hematopoietic cells exposed to
c-myb antisense oligodeoxynucleotides12 or murine
embryonic stem cells,24 c-kit mRNA levels also
decline. In addition, Vandenbark et al27 identified and
evaluated the effect of two potential Myb-binding sites,
beginning at nts -900 (MYB2) and -1329 (MYB1), on Kit promoter
activity in K562 cells. They found that only one of these sites (MYB1)
actually bound c-Myb protein and further analysis suggested
that this site functioned as a transcriptional repressor. Although MYB2
appeared to be transcriptionally active, it could not be shown that
c-Myb protein actually bound to the site. Accordingly, this
work did not establish a direct effect of c-Myb protein on
upreguation of Kit expression. We now provide experimental data
that supports the hypothesis that Myb is a direct, positive
regulator of c-kit expression and that it may function in this
capacity through an Ets-2 cooperation. In this regard, we have
shown by gel retardation assays that Myb and Ets-2 bind
to segments of the c-kit 5 flanking region containing putative Myb and Ets-2-binding sites. We have also
shown that CAT constructs driven by the 5 flanking region of
c-kit have increased activity in the presence of functional
Myb and Ets-2 proteins. Moreover, we showed that both
proteins have a cooperative effect on transactivation of the human
c-kit promoter. Additional supporting studies showed that
mutated Myb protein does not bind to the c-kit
promoter, nor does it have activity in the reporter assays. Finally,
although c-myb and ets have previously been shown to be
expressed in CD34+ cells, we now show that the mRNAs for
these respective genes are also expressed in very primitive
CD34+, Kit+ and CD34+,
Kit- cells. Accordingly, whereas it remains
possible that Myb and/or Ets-2 indirectly
regulate Kit expression, we consider this possibility less
likely in view of the overwhelming evidence that these transcription factors interact directly with c-kit's promoter.
The finding that Myb and Ets-2 may play a role in
regulating Kit expression is of interest from several points of
view. First, because c-kit expression is clearly important for
hematopoietic cell development6,12-15 another critical
function for Myb, in addition to its role in regulating
hematopoietic cell development,25,46 G1/S
transition,47 transactivating CD4,30,48
CD13/APN,49 and CD34,28,29,50 may now have been
discerned. In this regard, it is interesting to postulate that the
anemia noted in Myb "knock-out" mice, which
phenotypically resembles that observed in w or sl mutations of c-kit and SF, respectively, may have been
mechanically caused by failure of these developing embryos to express
Kit. Second, and perhaps more speculative, is Myb's
potential role in the regulation of apoptosis via its effects on
Kit receptor expression. A number of laboratories, including
our own, have suggested that Kit's ligand, SF, prevents
hematopoietic cells from undergoing apoptosis.7,8 There are
also suggestive data that the mechanism for this effect involves
upregulation of bcl-2 because in at least one system,
expression of bcl-2 decreases when Kit expressing cells
are deprived of SF.51 If Myb proves to be
physiologically required for Kit expression, it may then assist
in early hematopoietic cell survival by permiting the Kit receptor/ligand interaction to occur. Of equal interest, we have reported that malignant hematopoietic cells are less tolerant of
transient interruption of Myb expression than normal
cells.52,53 Downregulation of Kit expression in
cells that are dependent on Kit ligand for viability may be at
least a partial explanation of this observation. Finally, although
recent studies suggest that c-ets and c-myb may have
opposing function in the regulation of c-fms
expression,54 their activities appear to be entirely cooperative in regulating Kit expression.
Besides Myb and Ets, additional transcription factors,
such as one encoded by the mi locus in mice, may also be
important regulators of c-kit expression.55 It is
therefore clear that knowledge of the factors that regulate Kit
expression in hematopoietic cells remains incomplete. Because the basic
scientific and translational implications of these studies continues to
grow, we continue to pursue these investigations as important
corollaries of understanding the developmental biology of normal and
malignant human hematopoietic stem cells.
| |
FOOTNOTES |
Submitted June 17, 1997;
accepted November 5, 1997.
Supported in part by grants from the National Institutes of Health to
A.M.G. and B.C.
Address reprint requests to Alan M. Gewirtz, MD, Room
513B, Stellar-Chance Laboratories, University of Pennsylvania School of
Medicine, 422 Curie Blvd, Philadelphia, PA, 19104.
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.
| |
ACKNOWLEDGMENT |
We thank R. Spritz for c-kit genomic DNA fragment employed for
sequencing, Dr H. Hung (Department of Pathology, University of
Pennsylvania, Philadelphia, PA) for constructive comments, and E.R.
Bien for editorial assistance.
| |
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Abstract
Introduction
Abstract