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Mol Cell Biol, January 1998, p. 566-575, Vol. 18, No. 1
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Role for the Putative Tumor Suppressor Bin1 in
Muscle Cell Differentiation
Robert J.
Wechsler-Reya,
Katherine J.
Elliott, and
George C.
Prendergast*
The Wistar Institute, Philadelphia,
Pennsylvania 19104
Received 15 May 1997/Returned for modification 11 July
1997/Accepted 20 October 1997
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ABSTRACT |
Bin1 is a Myc-interacting protein with features of a tumor
suppressor. The high level of Bin1 expression in skeletal muscle prompted us to investigate its role in muscle differentiation. Significant levels of Bin1 were observed in undifferentiated C2C12 myoblasts, a murine in vitro model system. Induction of differentiation by growth factor withdrawal led to an upregulation of Bin1 mRNA and to
the generation of higher-molecular-weight forms of Bin1 protein by
alternate splicing. While Bin1 in undifferentiated cells was localized
exclusively in the nucleus, differentiation-associated isoforms of Bin1
were found in the cytoplasm as well. To examine the function of Bin1
during differentiation, we generated stable cell lines that express
exogenous human Bin1 cDNA in the sense or antisense orientation. Cells
overexpressing Bin1 grew more slowly than control cells and
differentiated more rapidly when deprived of growth factors. In
contrast, C2C12 cells expressing antisense Bin1 showed an impaired
ability to undergo differentiation. Taken together, the results
indicated that Bin1 expression, structure, and localization are tightly
regulated during muscle differentiation and suggested that Bin1 plays a
functional role in the differentiation process.
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INTRODUCTION |
The processes of proliferation,
differentiation, and tumorigenesis are intricately related. In normal
tissues, immature cells proliferate until environmental signals and
intrinsic genetic programs trigger irreversible withdrawal from the
cell cycle and terminal differentiation (29, 33). Tumor
cells, in contrast, are unable to withdraw from the cell cycle and lack
many of the characteristics of differentiated cells (11).
This relationship is clinically important, because the degree of
dedifferentiation of a tumor cell typically correlates with a poorer
prognosis (31). Moreover, interventions that promote
differentiation retard tumor growth or even induce tumor regression
(7, 9). Thus, proliferation and differentiation are mutually
exclusive fates of a cell, and unraveling the mechanisms that control
them has clear implications for cancer therapy.
In recent years, many aspects of the genetic programs controlling
proliferation and differentiation have been elucidated. In general,
these cellular responses are regulated by the opposing actions of two
groups of genes, one which promotes cell growth (proto-oncogenes) and
the other which opposes it (tumor suppressors) (26). During
normal cellular proliferation, growth-promoting genes that control cell
cycle entry, DNA synthesis, and cell division are activated by growth
factors and by extracellular matrix proteins (4, 32).
Inappropriate activation of these genes due to mutation or
dysregulation can induce abnormal proliferation and thereby contribute
to tumorigenesis (24, 25). During differentiation, many
growth-promoting genes (e.g., Myc and cyclin D1) are repressed (36, 43) while many growth-inhibitory genes (e.g., those
encoding the retinoblastoma protein and the cyclin-dependent kinase
inhibitor p21WAF1) are activated (21, 22). Significantly,
differentiation can be inhibited either by forced expression of
growth-promoting genes or by inactivation of growth inhibitors
(27, 37, 39, 41). Thus, whether a cell grows or
differentiates is determined, in large part, by the balance between
proto-oncogenes and tumor suppressors.
Bin1 is a novel gene whose features suggest that it may
influence this balance (34). Originally identified as a
protein that interacts with the N terminus of the Myc oncoprotein, Bin1 is structurally similar to RVS167, a negative regulator of the cell
cycle in the yeast Saccharomyces cerevisiae (5).
Consistent with the notion that it might play a role in regulating cell
growth, Bin1 was found to suppress the cell transforming activity of
Myc as well as that of the adenovirus E1A and mutant p53 proteins (19, 34). In addition, Bin1 expression is reduced in
carcinoma cells derived from malignancies of the breast and other
tissues, and introduction of Bin1 into tumor cell lines lacking
endogenous expression reduces their proliferative capacity. Finally,
the human Bin1 gene maps to chromosome 2q14 (28),
a locus within the mid-2q region that is deleted in >40% of
metastatic prostate carcinomas (13). Together, these
observations lend strong support to the hypothesis that Bin1 is a tumor
suppressor.
Interestingly, analysis of the tissue distribution of Bin1 indicated
that the highest levels of expression were in skeletal muscle and
brain, tissues which are abundant in postmitotic, terminally differentiated cells (34). Since Bin1 has features of
a tumor suppressor, we hypothesized that it might contribute to the
regulation of differentiation in these tissues. To investigate this
hypothesis, we analyzed Bin1 in an in vitro murine model for muscle
differentiation, C2C12 myoblasts (6). In this report, we
demonstrate that Bin1 plays a critical role in C2C12 differentiation.
After induction of differentiation, Bin1 message and protein levels are
dramatically increased and there is a change in the structure of the
Bin1 protein due to alternative RNA splicing. This splicing results in
a larger form of the protein that localizes in the cytoplasm as well as the nucleus, suggesting a Myc-independent role(s) for Bin1 in differentiated cells. Increased expression appears to be crucial for
differentiation, because overexpression of Bin1 promotes myotube formation and upregulation of myosin heavy chain while interference with Bin1 expression significantly impairs these processes.
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MATERIALS AND METHODS |
Cell culture.
C2C12 cells (kindly provided by David
Goldhamer) were carried in growth medium (GM; Dulbecco's modified
Eagle medium supplemented with 15% fetal bovine serum and
penicillin-streptomycin). Cells were grown to approximately 70%
confluence and then passaged or induced to differentiate.
Differentiation was induced by removing the GM, washing the cells with
phosphate-buffered saline (PBS), and then culturing the cells in
differentiation medium (DM; Dulbecco's modified Eagle medium
supplemented with 5% horse serum and penicillin-streptomycin) for 5 days (or as indicated).
Cells were transfected by using a calcium phosphate precipitation
protocol that has been described previously (12). Briefly, 2 × 105 cells seeded in 10-cm-diameter dishes were
transfected overnight (18 h) with 15 µg of the appropriate plasmid
and 10 µg of pBS+ (Stratagene). The next day, the cells were washed
and refed; after an additional 24 h, they were trypsinized and
passaged at a 1:25 ratio into new dishes. The following day, G418 was
added to 0.8 mg/ml for selection of stable transfectants. The medium
was changed every 2 to 3 days, and after 7 to 8 days, individual
colonies were ring cloned and expanded into cell lines.
Northern analysis.
Total cytoplasmic RNA was isolated from
C2C12 cells as described in reference 35. For
Northern analysis, 15 µg of RNA was fractionated on an agarose gel
and transferred onto a nylon membrane (Duralon-UV; Stratagene). After
UV cross-linking, membranes were prehybridized in Church buffer
(35) for 4 h at 65°C and then hybridized overnight
with a 32P-labeled human Bin1 cDNA probe (generated by
random priming) or with an exon 10-specific oligonucleotide probe,
5'-GGAGAATTCGTTGTCACTGTTCTTCTTTCTG (47), labeled
by using T4 polynucleotide kinase (Boehringer Mannheim Biochemicals).
Membranes were washed twice in 0.1% sodium dodecyl sulfate
(SDS)-0.2% saline sodium citrate for 10 min at 50°C and then
exposed to film.
Antibodies and blocking proteins.
The anti-Bin1 monoclonal
antibodies (MAbs) 99D and 99F, generated by immunization of mice with a
glutathione S-transferase (GST) fusion polypeptide
containing amino acids 189 to 398 of human Bin1 (GST-99Pst), are
described in detail elsewhere (46). For some
immunoprecipitation and Western blotting experiments, 99D was blocked
by incubation with a molar excess of a GST fusion polypeptide
containing a fragment of murine Bin1 (GST-ATG99) with high affinity for
this antibody. Anti-immunoglobulin D (IgD) MAbs (AMS 9.1), used as a
negative control for immunoprecipitation and flow cytometry, were a
gift from J. Erikson (Wistar Institute). A polyclonal rabbit antiserum
to mouse c-Myc (06-213) was obtained from Upstate Biotechnology Inc.
MAbs specific for myosin heavy chain (MF20), developed by D. A. Fischman (3), were obtained from the Developmental Studies
Hybridoma Bank (Iowa City, Iowa). Fluorescein isothiocyanate
(FITC)-coupled goat anti-mouse IgG antiserum, used as a secondary
antibody for flow cytometry and immunofluorescence, and horseradish
peroxidase (HRP)-coupled goat anti-mouse and anti-rabbit IgGs, used for
Western blotting, were obtained from Boehringer Mannheim Biochemicals.
For flow cytometry, Western blotting, and immunofluorescence, hybridoma
supernatants were diluted 1:20 and secondary antibodies were diluted
1:1,000 (FITC conjugates) or 1:15,000 (HRP conjugates). All antibodies were diluted in PBS plus 0.1% Tween 20 (PBST).
Immunoprecipitation.
C2C12 cells were metabolically labeled
by incubation for 4 h in methionine- and cysteine-free medium
(Life Technologies, Gaithersburg, Md.) containing 100 µCi of
[35S]methionine-[35S]cysteine (EXPRESS
label; NEN) per ml and then lysed in 1 ml of Nonidet P-40 (NP-40)
buffer (50 mM Tris, pH 8.0; 150 mM NaCl; 1% NP-40) containing
aprotinin, antipain, leupeptin (2 µg/ml each), and
phenylmethylsulfonyl fluoride (100 µg/ml). Lysates were spun in a
microcentrifuge (Eppendorf) for 15 min at maximum speed to remove
insoluble matter, and protein (0.5 mg per sample) was precleared by
incubation for 1 h at 4°C with 40 µl of protein G-Sepharose beads. Proteins were immunoprecipitated by incubating lysates for
2 h with 20 µl of protein G-Sepharose beads that had been precoated with 100 µl of hybridoma supernatant (anti-IgD, 99D or 99F
plus blocking proteins, added where indicated in the figures). Immunoprecipitates were washed four times in NP-40 buffer, resuspended in 2× SDS-polyacrylamide gel electrophoresis (PAGE) gel loading buffer, boiled for 5 min, and fractionated on a 10% polyacrylamide gel. Labeled proteins were visualized by fluorography.
Western analysis.
Cells were lysed in NP-40 buffer, and the
lysate was centrifuged to remove insoluble material. Protein (50 µg
per sample) was then resuspended in 2× SDS-PAGE gel loading buffer,
boiled for 5 min, and fractionated on a 10% polyacrylamide gel.
Proteins were transferred onto nitrocellulose membranes (Hybond-ECL;
Amersham), which were subsequently blocked overnight in PBST containing
5% nonfat dried milk. To detect Bin1 and myosin heavy chain, membranes were incubated for at least 1 h in primary antibody (99D or MF20) and 1 h in secondary antibody (HRP-conjugated goat anti-mouse IgG). To detect c-Myc, membranes were incubated similarly except that
anti-c-Myc antibody and HRP-conjugated goat anti-rabbit IgG were used.
Membranes were then incubated for 5 min in a chemiluminescent HRP
substrate (Pierce) and exposed to film.
Flow cytometry.
Proliferating C2C12 cells (106
per sample) were trypsinized and washed with PBS. Cells were fixed in
PBS containing 0.25% paraformaldehyde for 1 h at 4°C and
permeabilized in PBST for 15 min at 37°C. Cells were then stained
with primary antibody (99D) for 1 h at 4°C and with secondary
antibody (FITC-conjugated goat anti-mouse IgG) for 1 h at 4°C.
After being stained, cells were washed three times in PBST and analyzed
on a Becton Dickinson FACScan using CellQuest software.
Immunofluorescence.
For immunofluorescence analysis, cells
were grown on glass coverslips in GM or DM, as indicated in the
figures. At the end of the culture period, cells were fixed for 10 min
with ice-cold PBS containing 1% paraformaldehyde and then
permeabilized for 10 min with ice-cold PBS containing 0.2% Triton
X-100. After being washed with PBS, cells were stained for 1 h (at
room temperature) with primary antibody (99D or MF20) and for 1 h
with secondary antibody (FITC-conjugated goat anti-mouse IgG).
Coverslips were washed three times in PBS after each staining step and
then mounted on slides with VectaStain mounting medium. Slides were
examined and photographed by using a Leitz microscope.
RT-PCR.
A murine Bin1 cDNA has been described previously
(40). DNA sequences from this cDNA were used to generated
the following primers for analysis of the endogenous Bin1 message in
C2C12 cells: mNTsen1 (5'-CAGTGCGTCCAGAATTTC) and mNTanti1
(5'-AACACCTTCTGGGCTTTG), mMIDsen1
(5'-AAGCCCAGAAGGTGTTCGAG) and 5'ATG99
(5'-TGGCTGAGATGGGGACTT), and mCTsen1
(5'-CTGAGATCAGAGTGAACCATG) and mCTanti1
(5'-CACCCGCTCTGTAAAATTC). To detect exogenous Bin1 in
transfected cells, the human Bin1-specific primers hX7.1
(5'-GCCAAAATTGCCAAGGCCGAG) and hAntiNLS2
(5'-GTTGTCACTGTTCTTCTTTCTGC) were used. Reverse
transcription-(RT)-PCR was performed as follows. Two micrograms of
total cytoplasmic RNA was mixed with 50 pmol of the appropriate primer,
heated to 70°C, and cooled rapidly on wet ice. RNA and primers were
added to a mixture of Moloney murine leukemia virus reverse
transcriptase (Life Technologies) and reaction buffer provided by the
manufacturer and incubated at 42°C for 1 h to allow first-strand
synthesis. From this reaction mixture, 3 µl was removed and added to
a solution containing fresh primers, PCR buffer, and Taq
polymerase. Samples were subjected to 30 cycles of denaturation (30 s
at 94°C), annealing (45 s at 55°C) and polymerization (60 s at
72°C). For each reaction, 10 µl of the product was removed, mixed
with sample buffer, and separated on an agarose gel containing ethidium
bromide. For further analysis, bands were subcloned into the vector
pCR+ (Invitrogen). The DNA sequences of subcloned fragments were
determined and analyzed with MacVector and AssemblyLIGN software.
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RESULTS |
Expression of Bin1 in C2C12 myoblasts.
Previous work had
indicated that Bin1 mRNA levels in murine skeletal muscle were higher
than those in most other tissues (34), suggesting that Bin1
might have a role in this tissue. To begin to address this issue, we
examined Bin1 expression in C2C12 cells, a nontransformed myoblast cell
line derived from murine skeletal muscle (6). In serum-rich
medium, C2C12 cells proliferate rapidly, but when cultured at high
density in growth factor-deficient medium, the cells stop dividing,
align with one another, express muscle-specific genes, and fuse into
multinucleate myotubes (2, 6). Bin1 was immunoprecipitated
from extracts of metabolically labeled, proliferating C2C12 cells with
99D, a MAb raised against a human Bin1-GST fusion protein
(46). Samples of lysate were also immunoprecipitated with a
control antibody (anti-IgD) or with 99D that had been preincubated with
a molar excess of nonspecific or specific blocking proteins. Immunoprecipitates were subjected to SDS-PAGE and fluorography (Fig.
1A). 99D specifically recognized a
polypeptide of ~65 kDa, similar in size to that generated by in vitro
translation of a full-length Bin1 cDNA (34). The ~65 kDa
protein from C2C12 cells was not recognized by isotype-matched control
antibodies or by 99D that was preincubated with the GST-Bin1 fusion
protein (incubation with unfused GST had no effect). We concluded that
99D recognized murine Bin1 in C2C12 cells.
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FIG. 1.
Expression of Bin1 in C2C12 cells. (A)
Immunoprecipitation. C2C12 cells were metabolically labeled with
[35S]methionine-[35S]cysteine and lysed in
NP-40 lysis buffer. Lysates were precleared and then subjected to
immunoprecipitation with anti-IgD antibodies (control [Ctrl]), with
99D (anti-Bin), or with 99D that had been preincubated with GST
(anti-Bin+gst) or a protein consisting of GST fused to a murine Bin1
polypeptide (GST-Bin [anti-Bin+gst-Bin]). Immunoprecipitates, along
with a 35S-labeled human Bin1 polypeptide generated by in
vitro translation (IVT), were analyzed by SDS-PAGE and visualized by
fluorography. The positions of molecular mass markers (in kilodaltons)
are shown on the left. (B) Fluorescence-activated cell sorter analysis.
C2C12 cells were trypsinized to generate a cell suspension and then
stained with anti-IgD (control) or 99D (anti-Bin1) antibodies followed
by FITC-coupled goat anti-mouse IgG. Cells were then washed and
analyzed by flow cytometry. FL1-H, fluorescence channel 1 (FITC).
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To determine whether Bin1 was expressed throughout the C2C12
population, cells stained with 99D were examined by flow cytometry.
A
suspension of proliferating cells was generated by trypsinization,
then
fixed, permeabilized, and stained with 99D or control antibodies
followed by fluorescein-conjugated secondary antibodies. Flow
cytometric analysis of the stained cell suspension demonstrated
that
essentially all cells in the population fluoresced above
background
(Fig.
1B). We concluded that proliferating C2C12 cells
universally
expressed Bin1 protein.
Bin1 is upregulated during C2C12 differentiation.
We next
investigated whether Bin1 expression was affected by differentiation.
C2C12 cells grown to near confluence and then shifted to DM undergo a
pronounced change in morphology; cells elongate, align with one
another, and fuse into myotubes (Fig. 2A). In our cultures, morphological
differentiation (alignment and fusion) typically began 2 to 3 days
following addition of DM, biochemical differentiation (expression of
myosin heavy chain; see below) was detectable by days 3 to 4, and
myotube generation was maximal (50 to 70% fusion) by day 5 to 6. To
assess Bin1 expression during this period, RNA was isolated from cells
at various times and subjected to Northern analysis. As shown in Fig.
2B, the level of Bin1 message in C2C12 cells increased dramatically
during differentiation (days 1, 3, and 5). Expression began to increase
as early as day 2 and reached its highest level at 5 days, when cell
differentiation was maximal.
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FIG. 2.
Bin1 message is upregulated during differentiation. (A)
Differentiation of C2C12 cells. Cells were photographed after being
cultured in medium containing 15% FCS (Growth) or after 3 or 5 days of
culture in medium containing 5% horse serum (Differentiation). (B)
Expression of Bin1 mRNA during differentiation. Total cytoplasmic RNA
from proliferating (day 0) or differentiating (days 1, 3, and 5) C2C12
cells was separated by agarose gel electrophoresis and blotted onto a
nylon membrane. The membrane was probed with a 32P-labeled
human Bin1 cDNA and then exposed to film (top panel). RNA integrity and
quantity were confirmed by ethidium bromide (EtBr) staining of the gel
before transfer (bottom panel).
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To confirm that the upregulation of Bin1 message was associated with an
increase in Bin1 protein, lysates from proliferating
or differentiating
C2C12 cells were analyzed by Western blotting
with 99D (Fig.
3A). Proliferating cells contained a
~65-kDa polypeptide
similar to that observed after
immunoprecipitation. Following
induction of differentiation, the level
of this protein increased
a few fold. In addition, differentiated cells
contained higher-molecular-weight
proteins (68 to 70 kDa) that were
recognized by 99D. These proteins
appeared to be Bin1 related, since
they were also observed in
immunoprecipitates from differentiated cells
(see below) and they
were not detected when blots were probed with an
isotype-matched
control antibody or with 99D that had been preincubated
with a
GST-Bin1 blocking protein (data not shown).
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FIG. 3.
Induction of novel isoforms of Bin1 and downregulation
of Myc during differentiation. (A) Western analysis of Bin1. NP-40
lysates from proliferating (day 0) or differentiating (days 1, 3, and
5) C2C12 cells were separated by SDS-PAGE and then transferred onto a
nitrocellulose membrane. The membrane was probed with the anti-Bin1
antibody 99D followed by HRP-conjugated goat anti-mouse IgG. Proteins
were detected by chemiluminescence. (B) Western analysis of Myc.
Lysates from proliferating (day 0) or differentiating (days 2.5 and 5)
cells were analyzed as above, except that a rabbit anti-Myc antibody
and HRP-conjugated anti-rabbit IgG were used. The bottom panel shows
Bin1 induction on a parallel blot of the same lysates. The positions of
molecular mass markers (in kilodaltons) are shown to the left of both
panels A and B.
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Since we initially identified Bin1 through its ability to interact with
Myc (
34), we examined Myc expression in a second
population
of C2C12 cells that were growing or that had been subjected
to serum
withdrawal for 2.5 or 5 days. As observed in other cell
systems, Myc
was rapidly downregulated after induction of differentiation,
such that
it was undetectable at 2.5 days after serum withdrawal
(Fig.
3B). In
contrast, it was at this time that one could begin
to detect the
altered forms of Bin1 that were induced by serum
withdrawal. Thus, the
increased expression and apparent alteration
of Bin1 occurred in cells
lacking Myc. We concluded that during
C2C12 differentiation, Myc levels
decreased whereas Bin1 mRNA
and protein levels increased and novel Bin1
species were generated.
Bin1 mRNA is subject to alternate splicing.
Although the
larger polypeptides that appeared during C2C12 differentiation were
immunologically related to Bin1, their structural relationship to Bin1
was not clear. If they represented alternate forms of Bin1, rather than
related proteins, the larger and smaller species would be expected to
have similar peptide maps. To examine this, 99D immunoprecipitates were
fractionated by SDS-PAGE and the larger and smaller species were
isolated from gels and subjected to V8 protease mapping. We observed
that the different species had virtually identical peptide maps (data
not shown), suggesting that they represented different isoforms of
Bin1.
Since one explanation for the different sizes of Bin1 was alternate RNA
splicing, we compared Bin1 mRNA structure in proliferating
and
differentiated cells by RT-PCR. Segments representing the
5' end,
middle, and 3' end of the Bin1 RNA were amplified with
separate sets of
primers. The results are shown in Fig.
4A. RT-PCR
with the 5'-end primers,
corresponding to the N-terminal region
of the polypeptide, generated a
single band of ~450 bp from RNA
from both proliferating and
differentiated cells. In contrast,
RT-PCR with the midsection primers
yielded fragments of ~400 bp
from proliferating cells and of 445 bp
from differentiated cells.
Finally, RT-PCR with the 3'-end primers,
corresponding to the
C-terminal region of the polypeptide, yielded
products of 425
and 515 bp that were present at similar levels in both
proliferating
and differentiated cells.
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FIG. 4.
Differentiation-associated isoforms are generated by
alternate splicing. (A) Detection of splicing by RT-PCR. Total
cytoplasmic RNA from growing (G) and from differentiated (D) C2C12
cells was reverse transcribed, and the resulting cDNA was amplified by
PCR with primers designed to hybridize to the 5' end, middle (MID), or
3' end of the murine Bin1 mRNA (see Materials and Methods). PCR
products were separated by agarose gel electrophoresis, stained with
ethidium bromide, and photographed. The positions of molecular size
markers are shown on the left. (B) Summary of RT-PCR results. The PCR
products shown in panel A were sequenced, and the sequences from
growing and differentiated cells were compared. 5' fragments from each
population were identical to one another (data not shown). Midregion
(MID) fragments from differentiated cells contained a 45-bp sequence
(homologous to human exon 10) that was absent in fragments from
proliferating cells. Each cell population contained two 3' fragments;
these differed from one another in the presence or absence of a 60-bp
sequence homologous to human exon 13. UTR, untranslated region. (C)
Detection of alternate splicing by Northern blotting. RNA from growing
and from differentiated cells was separated by electrophoresis,
transferred onto a nylon membrane, and probed with a
32P-labeled oligonucleotide fragment derived from exon 10 of human Bin1 (top panel) or with a full-length human Bin1 cDNA probe
(bottom panel). Membranes were exposed to film for 1 week. No exon
10-positive RNA was detected on film exposed for up to ~3 weeks.
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DNA sequence analysis of the 5' and 3' RT-PCR products indicated no
change in the structures of these regions in proliferating
and
differentiated cells. The two 3' products (detected in RNA
from both
sources) differed in the presence or absence of a 90-bp
segment
encoding part of the Myc-binding domain (MBD) of Bin1
(
19,
34). Significantly, this 90-bp fragment corresponded
exactly to
an exon conserved in the human gene, exon 13 (
47).
This
result strongly suggested that a murine exon corresponding
to human
exon 13 was subject to alternate splicing in both proliferating
and
differentiated C2C12 cells.
A similar analysis of the RT-PCR products amplified with the midsection
primers showed that the 400- and 445-bp products found
in proliferating
and differentiated cells, respectively, were
identical except for the
presence of an additional 45-bp segment
in the latter. This segment is
absent from a murine Bin1 cDNA
isolated from an embryo library
(
40) but is present in a human
cDNA isolated from a skeletal
muscle library (
34). As had been
observed with the 3'
segment, the 45-bp segment spliced into the
midsection was found to
correspond to a discrete exon in the human
gene, exon 10. Thus, a
murine exon corresponding to human exon
10 is alternately spliced into
Bin1 mRNA, and this event is regulated
during C2C12 differentiation.
The splice forms of Bin1 identified
in this analysis are summarized in
Fig.
4B.
Two additional experiments were performed to verify that exon 10 was
expressed only in differentiated C2C12 cells. First,
total cytoplasmic
RNA from proliferating and differentiated cells
was subjected to
Northern analysis with an oligonucleotide probe
specific for exon 10 sequences. While a full-length cDNA probe
recognized Bin1 mRNA from
either population, the exon 10-specific
probe detected message only in
differentiated cells (Fig.
4C).
Second, to confirm this difference at
the protein level, we used
a Bin1 MAb, 99F, that had been determined to
recognize an exon
10-encoded epitope (
46). 99F was found to
bind in vitro-translated
Bin1 polypeptides that included exon 10 sequences but not those
that lacked such sequences. Moreover, 99F
failed to detect Bin1
protein present in a variety of tumor cell lines,
suggesting that
the exon 10 epitope was masked or absent in these
cells. We employed
99F as a probe to examine the exon 10-containing
Bin1 species
identified in differentiated C2C12 cells. As shown in Fig.
5,
immunoprecipitation of extracts from
35S-labeled C2C12 cells indicated that 99D recognized Bin1
proteins
from both proliferating and differentiated cells. In contrast,
99F failed to detect Bin1 in proliferating cells but recognized
the
larger Bin1 species in differentiated cells. Both the smaller
and
larger species detected in differentiated cells were heterogeneous.
The
reason for this was unclear but might reflect differences
in
phosphorylation states since Bin1 has been shown to be a phosphoprotein
(
46). We concluded that exon 10 sequences were spliced into
Bin1 message during differentiation and that the
higher-molecular-weight
species of Bin1 protein observed in
differentiated cells were
due to the expression of exon 10-encoded
residues.
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FIG. 5.
Differentiation-associated Bin1 proteins can be detected
with an exon 10-specific antibody. Growing and differentiated C2C12
cells were metabolically labeled with
[35S]methionine-[35S]cysteine, lysed in
NP-40 buffer, and subjected to immunoprecipitation with anti-IgD
antibodies (Ctrl), 99D, or the exon 10-specific antibody 99F.
Immunoprecipitates were separated by SDS-PAGE and visualized by
fluorography. The proliferation-associated (Exon 10 ) and
differentiation-associated (Exon 10+) forms of Bin1 are
indicated; note that 99F recognizes only the Exon 10+ form.
The positions of molecular mass markers are shown on the left.
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Changes in Bin1 structure correlate with changes in cellular
localization.
To begin to assess the significance of alternate
splicing of exon 10 in differentiated cells, we used 99D and 99F to
compare the localization of Bin1 in C2C12 cells before and after
differentiation (Fig. 6). Consistent with
the results described above, in proliferating cells (top panels), Bin1
was detected by 99D but not by 99F. In these cells, as had been
observed in other human and rodent cells (34, 46), Bin1 was
localized exclusively in the nucleus. In contrast, in differentiated
myotubes (bottom panels), Bin1 was detected by 99D as well as 99F, and
the pattern of staining with each of these MAbs was distinct. 99D
staining was observed in both the nucleus and cytoplasm, while 99F
staining appeared predominantly in the cytoplasm, in a fibrous pattern
along the length of the myotube. These staining patterns were specific
for Bin1, because they were not observed with isotype-matched control
antibodies and because they were blocked by preincubation with specific
blocking proteins (data not shown). In addition, staining with an
antibody specific for myosin heavy chain confirmed that extensive
differentiation had taken place in these cultures. Taken together,
these results indicated that the low-molecular-weight form of Bin1 in
proliferating C2C12 cells was confined to the nucleus whereas the
high-molecular-weight, differentiation-associated Bin1 species were
found predominantly in the cytoplasm.
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FIG. 6.
Localization of Bin1 changes during differentiation.
C2C12 cells were plated onto glass coverslips and cultured in GM for 1 day or in GM for 5 days. Cells were then stained with the anti-Bin1
antibody 99D, the exon 10-specific antibody 99F, or the antimyosin
antibody MF20, in each case followed by FITC-conjugated goat anti-mouse
IgG antibodies. Cells were photographed by using a Leitz microscope.
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Bin1 is necessary for C2C12 differentiation.
The complex
regulation of Bin1 structure and localization during differentiation
suggested that it might play a role in the differentiation process. To
test this hypothesis, we investigated the effects of overexpressing
sense and antisense forms of human Bin1 cDNA in C2C12 cells. Since
alternate splicing of exon 10 (but not exon 13) sequences was tightly
associated with differentiation, we also examined the effects of
overexpressing a Bin1 species lacking exon 10 sequences to distinguish
whether exon 10-encoded information, rather than upregulation of Bin1
expression per se, might be important. Cells were transfected with an
expression vector encoding a neomycin resistance gene or the same
vector containing a full-length human Bin1 cDNA (sense or antisense). Cell lines derived from G418-resistant colonies were screened for
expression of exogenous Bin1 by RT-PCR, using primers specific for the
human cDNA that was introduced. To rule out any effects of clonal
variation, at least 10 cell lines derived from each vector were
generated. A summary of the phenotypes exhibited by each set of cell
lines is depicted in Fig. 7.
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|
FIG. 7.
Phenotypes of C2C12 cell lines. This figure summarizes
the phenotypes of clonal cell lines generated by transfection of the
indicated vectors. Cells were incubated for 5 to 6 days in DM and then
assessed for the phenotypic characteristics noted in the key. Each
triangle represents a single cell line. The total number of cell lines
examined (n) is indicated beneath the type of vector
transfected. The range phenotypes represent clonal variation in the set
of cell lines examined; the trend on the y axis represents a
greater or lesser tendency toward a differentiated character following
incubation in DM.  , phenotype of parental cells in GM; #, phenotype
of parental cells in DM.
|
|
We observed that sense and antisense lines differentiated better and
worse, respectively, than the vector control lines. Only
a limited
number (10 to 20%) of the cell lines derived from sense
cDNA-transfected cells showed elevated expression of Bin1. In
addition,
cells showing exogenous Bin1 expression grew more slowly
than control
cell lines, both during and after the selection period
(data not
shown). These observations argued that Bin1 overexpression
might
interfere with the growth of C2C12 cells, consistent with
results in
other cell lines (
19,
34). Notably, lines overexpressing
Bin1-10 did not show this growth deficit, although they shared
with the
sense lines a propensity to differentiate more strongly
than controls
(see below). To further examine the effects of Bin1
overexpression on
differentiation, several sense lines were selected
for further analysis
(from a total of 41 lines generated and phenotypically
examined), two
of which are reported here (Fig.
8A).
Relative
to control lines, these cells had significant amounts of
exogenous
Bin1 mRNA detectable by RT-PCR (top panel). Western analysis
of
extracts derived from these cells showed two- to fourfold-higher
levels of Bin1 protein, as detected with 99D (second panel). Despite
the presence of elevated levels of Bin1, however, their morphology
in
GM was similar to that of control cells (data not shown), with
no
evidence of premature alignment or fusion. We concluded that
Bin1
overexpression impeded C2C12 proliferation to some extent
through a
mechanism requiring exon 10-derived sequences but that
on its own, Bin1
was insufficient to drive differentiation in
GM.
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|
FIG. 8.
Overexpression of Bin1 accelerates differentiation. (A)
Bin1 and myosin expression. (Top panel) Stable cell lines transfected
with empty vector (Neo-1 and Neo-2) or with human Bin1 cDNA (Sense-1
and Sense-2) were selected in antibiotic-containing medium and analyzed
by RT-PCR with primers specific for human (exogenous) Bin1. (Second
panel) Cells cultured in GM were analyzed for Bin1 protein expression
by Western blotting with 99D. (Third panel) Cells were cultured in DM
for 3 days, and then Bin1 protein levels were assessed by Western
blotting. (The high-molecular-weight forms of Bin1 are presumably
generated by alternate splicing of the endogenous mRNA.) (Bottom panel)
Differentiated cells were analyzed for myosin heavy chain expression by
Western blotting with MF20. (B) Morphology of control and
Bin1-overexpressing cells after 3 days in DM. Note the extensive cell
fusion in Bin1-overexpressing (Sense-1 and Sense-2) cells compared to
controls (Neo-1 and Neo-2).
|
|
After 3 days in DM, control cells became elongated and aligned but
showed limited fusion into myotubes (Fig.
7 and
8B). Consistent
with
these observations, only modest increases in expression of
differentiation-associated isoforms of endogenous Bin1 and myosin
heavy
chain were observed (Fig.
8A, third and fourth panels).
Although
control cells showed increased alignment and fusion after
longer
culturing in DM (see below), they seldom displayed the
rate or degree
of differentiation observed in parental (nontransfected)
cells. This
blunted differentiation response in transfected cells
might have been
due to the high density at which cells were cultured
during the drug
selection period.
In comparison to control cells, cells overexpressing Bin1 underwent a
more rapid and pronounced differentiation in DM (Fig.
7 and
8A). An
examination of 12 cell lines overexpressing Bin1
10 showed a similar
response trend (data not shown), suggesting
that it was the
overexpression of Bin1 rather than exon 10-encoded
sequences per se
that mediated the effect. Notably, cells expressing
sense Bin1 or
Bin1-10 differentiated even more vigorously than
parental C2C12 cells.
Within 2 to 3 days of culture in DM, cells
exhibited sharp increases in
their overall level of Bin1 protein
(due to increases in endogenous
expression), with significant
accumulation of the high-molecular-weight
differentiation-associated
species (Fig.
8A, third panel). In parallel
with this upregulation,
there was a dramatic increase in myosin heavy
chain levels (Fig.
8A, fourth panel), efficient cell alignment, and
extensive cell
fusion into myotubes (Fig.
8B). This rapid and efficient
differentiation
was not vector dependent, because similar phenomena
were observed
in cells that were transfected with two other Bin1
vectors (data
not shown). We concluded that elevation of the levels of
Bin1,
either containing or lacking exon 10-encoded sequences, was
insufficient
to induce C2C12 differentiation but accelerated or
enhanced the
differentiation program once it was initiated.
An examination of antisense cDNA-expressing cell lines suggested that
Bin1 was a necessary component of the differentiation
program (Fig.
7).
Unlike sense transfectants, a significant proportion
(50 to 60%) of
the G418-resistant cell lines transfected with
the antisense vector
exhibited expression of the exogenous construct
by RT-PCR (Fig.
9A, top
panel). Moreover, whereas the sense cDNA-expressing
cells were observed
to grow more slowly than controls, antisense
cDNA-expressing cells
proliferated somewhat more rapidly, such
that more frequent passaging
was necessary to avoid confluence.
Several antisense cell lines were
selected for further analysis
(from a total of 29 lines generated and
phenotypically examined),
two of which are reported here (Fig.
9A). Western blotting revealed
a two- to
fourfold decrease in basal levels of Bin1 protein in
these cell lines
relative to controls (second panel). Similar
to sense cDNA-expressing
cell lines, the morphology of antisense
cDNA-expressing cells in GM was
indistinguishable from that of
control cells, and these cells did not
show an increased tendency
to undergo alignment or fusion.
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|
FIG. 9.
Antisense Bin1 expression impairs differentiation. (A)
Bin1 and myosin expression. (Top panel) Stable cell lines transfected
with empty vector (Neo-1) or with antisense Bin1 (Anti-5 and Anti-11)
were selected in antibiotic-containing medium and analyzed by RT-PCR
for expression of exogenous Bin1. (Second panel) Bin1 protein
expression in cells cultured in GM (detected by Western blotting with
99D). (Third panel) Bin1 protein levels in cells cultured in DM for 6 days. (Bottom panel) Myosin heavy chain expression (detected by Western
blotting with MF20) in cells cultured in DM for 6 days. (B) Morphology
of control (Neo-1) and antisense Bin1 cDNA-expressing (Anti-5 and
Anti-11) cells after 6 days in DM. Antisense cDNA-expressing cells
appear round and unfused, while control cells show substantial
alignment and fusion at this stage.
|
|
The effects of antisense cDNA expression on differentiation were
determined by examining the same set of biochemical and morphological
features as before, in cells cultured in DM for up to 6 days (a
time
point at which control cells exhibited maximal morphological
differentiation). Compared to control lines, antisense lines showed
significantly less upregulation of differentiation-associated
Bin1
species (Fig.
9, third panel). In addition, while control
cells
exhibited increased levels of myosin heavy chain, antisense
cDNA-expressing cells showed little upregulation of this marker
(Fig.
9, bottom panel). Finally, while control cells showed substantial
alignment and some fusion after 6 days in DM, antisense lines
showed
little if any alignment, instead retaining the rounded
morphology that
is characteristic of undifferentiated cells. Taken
together with the
sense results, these data led us to conclude
that upregulation of Bin1
is necessary for differentiation of
C2C12 cells.
| |
DISCUSSION |
Many genes originally identified through their action in cancer
cells have since been shown to play a role in regulating normal cellular growth and differentiation (1, 23, 38). Bin1 was originally identified through its interaction with the N terminus of
the Myc oncoprotein (34). Bin1 inhibits the oncogenic and transcriptional properties of Myc but also displays the ability to
inhibit cell growth by at least two other Myc-independent mechanisms (19, 34). In this study, we demonstrated that the
differentiation of C2C12 myoblasts is accompanied by upregulation and
alternate splicing of Bin1 mRNA. This splicing results in the
generation of differentiation-specific isoforms of the Bin1 protein
which are characterized by their higher molecular weights and distinct patterns of cellular localization. By modulating the amount of Bin1
protein in C2C12 cells, we also demonstrated that Bin1 has an integral
role in the muscle differentiation program.
Regulation of Bin1 structure and expression during muscle cell
differentiation.
Interest in Bin1 in muscle cells was initially
stimulated by our observation that murine skeletal muscle expressed
higher levels of Bin1 mRNA than most other tissues (34).
Consistent with this observation, we found that C2C12 cells contain at
least 10-fold-higher levels of Bin1 protein than other cell lines that have been examined. It is noteworthy that these cells express relatively high levels of Bin1 even before myotube differentiation. One
possible reason for this comes from studies of the human Bin1 promoter,
which have revealed that Bin1 transcription is activated by the
myogenic transcription factor myoD (47). Since C2C12 cells
are committed to the muscle lineage and already express myoD
(1), they may express relatively higher amounts of Bin1 for
this reason. Whether Bin1 has a distinct role in the early stages of
myogenic commitment in addition to its role in differentiation remains
to be determined.
In examining the expression of Bin1 during C2C12 differentiation, we
found that Bin1 mRNA levels were dramatically upregulated
within 2 days
of growth factor withdrawal, at approximately the
same time as
morphological differentiation was first detectable.
Thereafter, Bin1
expression continued to increase as greater numbers
of cells aligned
and fused into myotubes. In addition to changes
in mRNA levels, we
observed changes in mRNA splicing during differentiation,
with an exon
corresponding to human exon 10 (
47) being introduced
into
Bin1 message in differentiated cells. Notably, upregulation
and
splicing of Bin1 mRNA did not take place when cells were allowed
to
reach confluence in GM or when growth factors were withdrawn
from
subconfluent cultures, conditions that do not promote complete
morphological or biochemical differentiation (
45). Thus,
upregulation
of Bin1 is intimately linked to activation of a
differentiation
program.
Several species of Bin1 were found to be generated in C2C12 cells by
alternate splicing. Approximately half of Bin1 mRNAs
in both
proliferating and differentiated cells contained a 3'
sequence
corresponding to human exon 13 (
47). In differentiated
cells, several Bin1 bands were detected by immunoprecipitation
and
Western blotting, and it is possible that these species differ
from one
another in expression of exon 13. In proliferating cells,
such
heterogeneity is not readily apparent; however, longer gels
offering
higher resolution have revealed closely spaced Bin1 bands
that are also
consistent with an exon 13 splicing event (
45).
Interestingly, exon 13 forms part of the MBD of Bin1, which allows
it
to antagonize Myc-mediated transcriptional activation and cell
transformation (
34). The fact that exon 13 is subject to
alternate
splicing suggests that not all Bin1 polypeptides in the cell
have
Myc-binding capability. Since Bin1 is known to have a
Myc-independent
as well as a Myc-dependent growth-inhibitory capacity
(
19,
34),
these studies raise the possibility that different
functions of
Bin1 are mediated by separate species within a cell.
The larger species of Bin1 identified in differentiated cells were
shown to result from alternate splicing of a sequence corresponding
to
human exon 10 (
47). While the functional significance of
exon 10 splicing remains unclear, its correlation with cytosolic
localization suggests that exon 10 sequences may be responsible
for
targeting of Bin1 to the cytosol. Counterintuitively, exon
10 encodes a
highly basic segment which resembles nuclear localization
signal motifs
(
8,
34). In the context of Bin1, however, this
motif is
neither necessary nor sufficient for nuclear localization,
since Bin1
species that lack exon 10 are found in the nucleus
of C2C12 cells, as
well as other human and rodent cell lines (
46),
and species
that contain exon 10 are present in the cytoplasm
of C2C12-derived
myotubes. An alternative function for exon 10
may be revealed by an
ongoing analysis of a recently identified
Bin1-interacting protein
whose binding appears to depend on exon
10-encoded sequences
(
32a).
Although alternate splicing explains some of the major differences
observed in Bin1 species in C2C12 cells, additional complexity
of Bin1
structure exists in these and other cells. Work in human
cell lines has
provided evidence for alternate splicing of another
exon in the central
region of the Bin1 gene, exon 12A (
47),
and additional exons
are spliced into brain-specific forms of
Bin1 (
10,
30,
42,
47). While we have not detected any
of these exons in mRNA from
either human muscle cells or C2C12
cells, they may be relatively rarer
and/or spliced at other stages
of muscle differentiation or in other
cell lineages. Posttranslational
modification may also contribute to
structural variation, because
Bin1 has been found to be phosphorylated
in both proliferating
and differentiated C2C12 cells (
46).
In future work, it will
be important to analyze the various isoforms of
Bin1, since this
would provide insights into Bin1 function and into the
significance
of alternative splicing events in C2C12 and other cell
types.
Requirement for Bin1 in muscle cell differentiation.
We found
that perturbing Bin1 expression in C2C12 cells altered their growth and
their susceptibility to induction of differentiation. Expression of
exogenous Bin1 (in the sense orientation) interfered with cell growth
and promoted cell differentiation. The effects of Bin1 on growth were
inferred from the fact that only a small proportion of G418-resistant
Bin1 sense cDNA-transfected cells showed overexpression of the
exogenous gene by RT-PCR. One interpretation of this finding was that
cells expressing high levels of Bin1 had a growth disadvantage and were
diluted out during the selection period by cells that expressed lower
levels of the protein. Consistent with this notion, the lines that did
survive selection expressed only moderate levels of exogenous Bin1
(two- to fourfold-higher levels of expression relative to controls) and
grew more slowly than empty-vector control lines. The ability of Bin1
to inhibit cell growth has been documented previously (34),
and as noted above, exon 10-encoded sequences may contribute to this
property in certain cell lineages, such as muscle cells.
Notably, exogenous Bin1 expression did not promote differentiation of
C2C12 cells in GM but dramatically accelerated and enhanced
expression
of the differentiation program induced by growth factor
withdrawal.
This accelerated differentiation was observed both
morphologically (in
terms of cell alignment and fusion) and biochemically
(in terms of
increased expression of myosin heavy chain and of
endogenous Bin1). The
fact that Bin1-expressing cells cultured
in DM showed more rapid
upregulation of differentiation-associated
Bin1 isoforms than control
or parental cells suggested that Bin1
may positively regulate its own
expression, a possibility which
needs further investigation.
The analysis of antisense cDNA-expressing cells also strongly supported
a role for Bin1 in differentiation. In these cells,
the morphological
and biochemical features of differentiation
were diminished
significantly compared to those of control cells.
Although we did not
determine precisely where Bin1 acts in the
differentiation pathway, the
facts that Bin1 upregulation occurs
relatively quickly (within 2 days
of serum withdrawal) and that
antisense Bin1 inhibits the earliest
morphological signs of differentiation
suggest that it may function
rather early. Taken together, the
data argued that Bin1 upregulation
may be a rate-limiting step
in the differentiation program.
Although the exact mechanism(s) by which Bin1 acts is unclear, its
ability to promote differentiation may reflect both Myc-dependent
and
Myc-independent activities. Studies of Bin1 structure and
function in
cell transformation (
19,
34) prompt several testable
hypotheses. First, as discussed above, Bin1 can interact with
the Myc
oncoprotein and can inhibit Myc-mediated transcription
and
transformation. At very early times, before Myc is effectively
removed
by downregulation, it is possible that Bin1 directly antagonizes
Myc's
growth-promoting effects and thereby relieves cells of one
barrier to
differentiation. Previous studies on the role of Myc
in muscle
differentiation indicate that its overexpression can
interfere with
biochemical differentiation and/or fusion (
15,
16,
27,
29).
In this light, Bin1 may directly antagonize
the growth-promoting
activity of Myc at early times after induction
of differentiation,
thereby relieving cells of one barrier toward
this process. At later
times, when Myc is absent, Bin1 would have
to act by a Myc-independent
mechanism(s). Although we did not
define the exact point(s) where Bin1
acts, the altered splicing
and relocalization that it undergoes at
later times suggests some
other role, possibly one affecting cell
alignment or fusion. The
question of whether Bin1 would be dominant to
Myc in C2C12 is
somewhat moot because enforced Myc expression is
compatible with
differentiation (though not cell fusion) in this cell
system (
15).
In addition to its Myc-related functions, Bin1 also can act in a
Myc-independent manner. For example, Bin1 can inhibit transformation
of
primary rat embryo fibroblasts by the adenovirus gene product
E1A, in a
manner independent of the MBD (
34). Since E1A can
inhibit
differentiation of myoblasts and reactivate the cell cycle
in
differentiated myotubes (
41,
44), it is possible that Bin1
may counteract these effects as well. In this scenario, Bin1 may
function in differentiation by directly or indirectly affecting
known
targets of E1A, such as the retinoblastoma protein, p107,
and p300/CBP
(
17,
18,
20,
41). Similarly, we have observed
that Bin1 can
inhibit cell transformation by a dominant inhibitory
mutant of p53
(
19). Although the mechanism of this effect is
not clear,
the fact that p53 function is required for C2C12 differentiation
(
39) raises the possibility that Bin1 also exerts its
effects
on differentiation via p53. Future analysis of stable C2C12
lines
that overexpress Bin1 mutants defective in E1A and/or p53
inhibition
may shed light on the pathways involved in BIN action.
Our studies provide strong evidence that Bin1 can regulate muscle
differentiation. Since Bin1 is expressed ubiquitously (
34),
it may also contribute to the control of differentiation programs
in
other cell types. Consistent with this possibility, we have
noted that
during induction of the monocytic differentiation program
in the
promyelocytic cell line HL-60 (
14), Bin1 expression and
splicing patterns are altered in a manner similar to that observed
in
C2C12 cells (
45). Thus, Bin1 may have a general role in cell
differentiation. If so, the frequent loss of Bin1 may contribute
to
malignant development both via the loss of processes required
for
terminal differentiation and by contributing to Myc deregulation.
| |
ACKNOWLEDGMENTS |
We are grateful to David Goldhamer for providing C2C12 cells,
helpful discussions, and criticism of the work. We thank Wei Du for
providing a human Bin1 cDNA lacking exon 10 sequences. Anti-IgD
antibodies were a gift of Jan Erikson. We thank Rudi Grosschedl and
Paul Stein for critically reading the manuscript. MAbs specific for
myosin heavy chain (MF20), developed by D. A. Fischman (Cornell
University, New York, N.Y.), were obtained from the Developmental
Studies Hybridoma Bank maintained by the University of Iowa Department
of Biological Sciences, Iowa City, IA 52242, under contract
NO1-HD-7-3263 from the National Institute of Child Health and Human
Development.
This work was supported by grants CN-160 from the American Cancer
Society and DAMD17-96-1-6324 from the U.S. Army Breast Cancer Research
Program to G.C.P. R.W.-R. is a fellow of the Medical Research
Council of Canada. K.J.E. was supported by an NIH training grant.
G.C.P. is the recipient of an American Cancer Society Junior Faculty
Award and is a Pew Scholar in the Biomedical Sciences.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: The Wistar
Institute, 3601 Spruce St., Philadelphia, PA 19104. Phone: (215)
898-3792. Fax: (215) 898-2205. E-mail:
prendergast{at}wista.wistar.upenn.edu.
Present address: Department of Neurobiology, Stanford University
School of Medicine, Stanford, CA 94305.
| |
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Abstract
Introduction
