From the Department of Cell Biology, University of
Cincinnati College of Medicine, Vontz Center for Molecular Studies,
Cincinnati, Ohio 45267-0521, the
Department of Pathology and
Laboratory Medicine, University of North Carolina Chapel Hill, North
Carolina 27599, and ** The Lineberger Comprehensive Cancer Center,
University of North Carolina, Chapel Hill, North Carolina 27599
Received for publication, October 25, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Aberrant regulation of CD44, a transmembrane
glycoprotein, has been implicated in the growth and metastasis of
numerous tumors. Although both CD44 overexpression and loss have been
implicated in tumor progression, the mechanism of CD44 down-regulation
in these tumor types is not known. By immunoblot and reverse
transcription-polymerase chain reaction analysis we determined that a
cervical carcinoma cell line, C33A, lacks CD44 expression. To determine
how CD44 is down-regulated in C33A cells, we utilized cell fusions of
C33A cells with a CD44-expressing cell line (SAOS-2). We found that SAOS-2 fusion restored CD44 expression in C33A cells, suggesting that a
trans-acting factor present in SAOS-2 cells promotes CD44 production.
C33A cells are BRG-1-deficient, and we found that CD44 was absent in
another BRG-1-deficient tumor cell line, indicating that loss of BRG-1
may be a general mechanism by which cells lose CD44. Reintroduction of
BRG-1 into these cells restored CD44 expression. Furthermore,
disruption of BRG-1 function through the use of dominant-negative BRG-1
demonstrated the requirement of BRG-1 in CD44 regulation. Finally, we
show that Cyclin E overexpression resulted in the attenuation of CD44
stimulation, which is consistent with previous observations that Cyclin
E can abrogate BRG-1 action. Taken together, these results suggest that
BRG-1 is a critical regulator of CD44 expression, thus implicating
SWI/SNF components in the regulation of cellular adhesion and metastasis.
The CD44 family of transmembrane glycoproteins has been implicated
in cell-cell and cell-matrix adhesion, cell growth, and metastasis
(1-3). A number of different CD44 proteins are produced through
alternative RNA splicing, and these proteins are extensively modified.
Many tumors express higher than normal levels of total CD44 protein as
well as splice variants that do not occur in normal cells (1, 3, 4).
How CD44 expression is regulated in normal cells and in tumors is
poorly understood.
A role for CD44 in tumor progression has been documented in numerous
clinical and experimental studies (1, 2). Ectopic expression of some
forms of CD44 can enhance metastasis and tumor growth both in
vitro and in vivo (5-7). It is believed that CD44 expression in some tumors increases as the tumor becomes more proliferative and invasive (1). These findings suggest that CD44 might
be regulated by environmental or genetic factors that have been shown
to contribute to cancer progression. The expression of activated
oncogenes like v-Ras, v-Src, and v-Fos, which promote transformation
and invasion, have been reported to induce CD44 expression (8-10). In
addition, the epidermal growth factor receptor has also been shown to
stimulate CD44 (10, 11).
In contrast to studies that correlate CD44 overexpression with cancer
progression, a significant number of reports also indicate that loss of
CD44 expression can contribute to tumorigenesis (12). Specifically, it
has been shown that loss of CD44 in cervical carcinomas,
neuroblastomas, prostate carcinomas, melanomas, and small cell lung
carcinomas correlates with increased aggressiveness of these tumors
(13-17). It has been hypothesized that loss of CD44 may facilitate the
abrogation of epithelial-mesenchymal interactions in some tumors,
leading to a more metastatic phenotype (4, 12). Unlike positive
regulators of CD44, no activity has been identified that leads to loss
of CD44 expression.
In the present study, we have identified the
Brahma-related gene,
BRG-1,1 as a critical
regulator of CD44 expression. BRG-1 is a component of the SWI/SNF
complex that regulates gene transcription through ATPase-dependent remodeling of chromatin (18-21). BRG-1
has been shown to inhibit the transcription of a subset of genes
involved in cancer such as Cyclin A and c-fos (22-25). In
addition, BRG-1 can facilitate estrogen receptor, glucocorticoid
receptor, c-Myc, and BRCA1-mediated transcription (26-30).
We demonstrate that loss of BRG-1 correlates with a lack of CD44
expression in several cell lines. Reintroduction of BRG-1 restored
expression of CD44 in BRG-1-deficient cells, suggesting that BRG-1
regulates basal CD44 levels. Moreover, we show that functional BRG-1 is
required for stimulating CD44, because dominant-negative BRG-1 inhibits
CD44 production. In addition, we show that Cyclin E can antagonize
BRG-1 regulation of CD44. As such, this report provides the first
evidence that the SWI/SNF complex regulates genes involved in cell
adhesion and cancer metastasis and suggests that BRG-1 loss in human
tumors contributes to the formation of a metastatic phenotype.
Cells, Cell fusions, Plasmids, and Transfection--
C33A,
SAOS-2, MCF-7, U87, PC3, U2OS, RD, and SW13 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated fetal bovine serum, 100 units/ml
penicillin-streptomycin, and 2 mM L-glutamine
at 37 °C in 5% CO2. 3T3 cells (BO5-1) were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated calf serum (Calbiochem), 100 units/ml
penicillin-streptomycin, 2 mM L-glutamine, 350 units of Hygromycin B, and 0.12 mg/ml Geneticin either in the presence
or absence of 2 µg/ml tetracycline (28). Plasmids were transfected
using the calcium phosphate method (31). Antibiotic-resistant C33A and
SAOS-2 cells were generated as previously described (32). The following
plasmids: CMV-NEO, pBabe-Puro, Cyclin E, PSM-RB, and BRG-1 have been
previously described (33-36). The cell fusions were generated as
previously described (32).
Immunoblotting--
Approximately 1 × 106 C33A
and SW13 cells were plated in 10-cm dishes 24 h before
transfection. C33A and SW13 cells were cotransfected with effectors and
the puromycin-selectable plasmid (pBabe-Puro). Transfected cells were
subsequently selected with 2.5 µg/ml puromycin (Sigma) for 48-72 h
and then harvested for immunoblot analysis. To isolate BRG-1, total
protein was isolated from subconfluent cultures using an 8 M urea extraction (8 M urea, 0.1 M
NaH2PO4, 10 mM Tris, pH 8). For
immunoblotting, cells were trypsinized and subsequently washed with
PBS. C33A and SW13 cell pellets were resuspended in radioimmune
precipitation buffer supplemented with the following protease inhibitor
mixture (10 mg/ml, 1,10-phenanthroline, 10 mg/ml aprotinin, 10 mg/ml
leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and
incubated for 15 min on ice. The lysates were briefly sonicated and
then centrifuged for 10 min at 20,000 × g at 4 °C. The lysates were then separated via SDS-polyacrylamide gel
electrophoresis and then transferred onto Immobilon-P membranes
(Millipore). The membranes were then incubated with either of the
following antibodies: anti-CD44 (Hermes 3), anti-Cdk4 (clone H22, Santa
Cruz Biotechnology), anti-Cyclin E (clone C19, Santa Cruz),
anti-actin (Sigma A-2066), anti-p53 (Dr. K. Fukasawa,
University of Cincinnati, College of Medicine, Cincinnati, OH),
anti-pRB (Dr. J. Wang, University of California at San Diego, 851 polyclonal antibody), and anti-BRG-1 (Dr. Weidong Wang, National
Institutes of Health, Baltimore, MD). The blots were then incubated
with either horseradish peroxidase-conjugated anti-rabbit or anti-mouse
for 1 h at room temperature. The antibody-antigen complex was
detected by enhanced chemiluminescence (ECL, Amersham Pharmacia
Biotech). The levels of Cdk4 and CD44 were quantitated using Metamorph
software (Universal Imaging Corp.).
Immunohistochemistry--
Cells were fixed in 3.7% formaldehyde
for 15 min and then either permeabilized (PBS, 0.3% Triton X-100, 5 mg/ml bovine serum albumin) for p53 staining or directly blocked with
5% normal goat serum for CD44 staining. The p53 antibody (1:100
dilution, ab-6, Calbiochem) was detected with a rhodamine-conjugated
anti-mouse antibody. Human CD44 was probed with Hermes 3 (1:25,
monoclonal antibody) and detected with a fluorescein-conjugated
anti-mouse antibody. Mouse CD44 was identified with IM7 (1:100,
Zymed Laboratories Inc.) and visualized with a
fluorescein-conjugated anti-rat antibody.
RNA Isolation and RT-PCR--
To analyze CD44 mRNA
expression levels, reverse transcription-polymerase chain reaction
(RT-PCR) was performed. First, ~1 × 106 C33A cells
were seeded into a 10-cm dish and transfected with either Vector-,
PSM-RB-, or BRG-1-encoding plasmids along with pBabe. After 48 h
of selection with puromycin, total mRNA was isolated using TRIzol
(Life Technologies, Inc.) according to the manufacturer's protocol.
cDNAs were then synthesized using ~1 µg of isolated total
mRNA and the ThermoScript RT-PCR system (Life Technologies, Inc.)
following the manufacturer's instructions. To amplify human CD44,
cDNAs were subjected to PCR using the forward primer:
5'-CAGACCTGCCCAATGCCTTTGATGGACC-3' and the reverse primer: 5'-CAAAGCCAAGGCCAAGAGGGATGCC-3'. As a control, human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified
using the forward primer: 5'-GGTCATCAATGGGAAACCCATCAC-3' and the
reverse primer: 5'-ACAAACTCTGCTACTTCTGG-3'. PCR was performed with the
following conditions: 94 °C, 1 min; 62 °C, 1 min; and 72 °C,
2.5 min, for 30 cycles. Approximately 10 µl of the PCR products were
then separated by electrophoresis in a 1% agarose gel. The PCR
products were visualized by ethidium bromide staining and then
photographed. The levels of GAPDH and CD44 were quantitated using
Metamorph software (Universal Imaging Corp.) from two independent PCR reactions.
CD44 Expression Is Down-regulated in the C33A Cervical Carcinoma
Cell Line--
CD44 is lost some in cervical carcinomas, and this loss
correlates with increased metastatic potential (13, 14). We identified a human cervical carcinoma cell line, C33A, which demonstrates loss of
CD44 expression as determined by immunoblot analysis with a
CD44-specific antibody (Fig.
1A, lane 1). By
contrast, the osteogenic sarcoma cell line, SAOS-2, readily expresses
detectable CD44 (Fig. 1A, lane 2). Blotting for
Cdk4 showed that equal total protein was loaded (Fig. 1A).
To examine whether loss of CD44 expression in C33A occurred at the RNA
level, RT-PCR analysis was used to examine the levels of CD44 mRNA
in C33A cells versus the CD44-positive SAOS-2 cells. In C33A
cells, the levels of CD44 mRNA were ~20-fold less than that found
in SAOS-2 (Fig. 1B, lanes 3, 4,
5, and 6). Thus, CD44 expression in C33A is lost
at the level of RNA, suggesting that C33A cells either possess a factor
important for repressing CD44 expression or alternatively lack a factor
required for stimulating CD44 expression.
Factors Expressed by SAOS-2 Cells Act in-trans to Restore CD44
Signaling in C33A Cells--
To examine the underlying difference in
CD44 signaling between C33A and SAOS-2, we utilized C33A-SAOS-2 (C-S)
cell fusion lines that have been previously characterized (32). Four
independent C-S fusion clones (#1-4) exhibited robust
expression of CD44 comparable to that observed in parental SAOS-2 cells
(Fig. 2A, lanes
1-6). Immunohistochemical analysis confirmed that CD44 expression
was restored and appropriately localized in the cell fusions (Fig. 2B, right panel). As a control, p53 staining
confirmed that the fusion expressed p53 protein (contributed from C33A
nuclei), because SAOS-2 are p53-negative (Fig. 2B,
left and middle panel). These findings suggest
that C33A cells lack a factor(s) required for CD44 signaling, which is
restored after nuclear fusion with SAOS-2.
Loss of BRG-1 Correlates with Reduced CD44 Expression--
A known
difference between SAOS-2 cells and C33A cells is that SAOS-2 cells
express BRG-1, a component of the SWI/SNF complex involved in both gene
regulation and chromatin remodeling, whereas C33A lack BRG-1 (36). To
examine whether loss of BRG-1 correlated with lack of CD44 expression,
we analyzed several tumor lines that were either positive or negative
for BRG-1 expression. Immunoblot analysis revealed that those tumor
lines that were positive for BRG-1 were also positive for CD44
expression (Fig. 3A,
upper and middle panel, lanes 2,
3, 5, 6, and 7). However,
the SW13 adenocarcinoma cell line, which is BRG-1-negative, was
deficient in CD44 protein expression, similar to that observed in C33A
cells (Fig. 3A, upper and middle
panel, lanes 1 and 4) (36). As a control,
the detection of actin shows the relative amount of protein loaded per
lane (Fig. 3A, lower panel, lanes
1-7). These data demonstrate that loss of BRG-1 correlates with a
lack of CD44 protein, suggesting that BRG-1 is required for CD44
expression.
BRG-1 Restores CD44 Expression in C33A and SW13 Cells--
To
determine whether BRG-1 is sufficient to induce CD44 expression, BRG-1
was restored in BRG-1-negative (C33A and SW13) cells. C33A and SW13
cells were cotransfected with vector, a phosphorylation site mutant of
RB (PSM-RB) as controls, or BRG-1 along with a puromycin resistance
plasmid (pBabe-Puro). After selecting the transfected cells with
puromycin, cells were harvested and subjected to immunoblot analysis.
In C33A and SW13 cells, transfection of either vector or PSM-RB did not
alter CD44 levels. However, ectopic expression of BRG-1 caused a
significant increase in CD44 protein levels (Fig.
4A, upper panel,
lanes 1-6). Immunoblotting for BRG-1 and PSM-RB confirmed
their expression in C33A and SW13 cells, whereas Cdk4 levels indicate
that equal total protein was loaded per lane (Fig. 4A,
middle and lower panels, lanes 1-6).
Because RB has been shown to recruit BRG-1 for transcriptional
repression, we also examined whether expression of PSM-RB could alter
BRG-1 stimulation of CD44. To test this, we cotransfected PSM-RB with BRG-1 into C33A cells and found that PSM-RB did not alter the ability
of BRG-1 to modulate CD44 production (data not shown).
Because BRG-1 is known to mediate its function through transcriptional
regulation, we investigated whether BRG-1 stimulates CD44 mRNA
levels (37). C33A cells were transfected with either vector or PSM-RB
along with pBabe-Puro and selected in puromycin. RT-PCR analysis
revealed that the level of CD44 mRNA quantitated from
vector-transfected cells was equal to that of PSM-RB-transfected cells
after normalization to GAPDH (Fig. 4B, upper and
lower panels, lanes 4, 5,
8, and 9). In contrast, C33A cells transfected
with BRG-1 showed an ~40-fold induction in CD44 mRNA when
compared with that of vector- or PSM-RB-transfected cells and
normalized against GAPDH (Fig. 4B, upper and
lower panels, lanes 4-9). PCR analysis of the
RNA prior to reverse transcribing revealed no amplification, thus
eliminating the possibility of DNA contamination (Fig. 4B,
upper and lower panels, lanes 1-3).
The effect of ectopic BRG-1 expression on the CD44 promoter was
assessed by reporter analysis in transient transfection assays. Ectopic
expression of BRG-1 did not stimulate CD44 reporter activity, and high
basal activity of the reporter was observed in cells either expressing or deficient in BRG-1 (data not shown). This finding is consistent with
the idea that BRG-1 stimulates transcription through the remodeling of
chromatin, because we show that BRG-1 clearly stimulates the expression
of endogenous CD44 RNA and protein.
Functional BRG-1 Is Required for Stimulating CD44
Production--
Because the data presented imply that BRG-1 may have a
role in maintaining the production of CD44, we investigated whether BRG-1 is required for this process. To test this we utilized an NIH3T3
cell line, called BO5-1, that inducibly expresses dominant-negative BRG-1 (dnBRG-1) (28). The mutant BRG-1 protein that is induced in BO5-1
cells contains a mutation in the ATP-binding site that renders it
catalytically inactive, and in the presence of tetracycline, BO5-1
cells fail to demonstrate detectable levels of dnBRG-1 (Fig. 5A, lanes 1 and
2). However, after incubation in the absence of tetracycline
for 72 h, a strong induction of dnBRG-1 was observed. Immunostaining of the BO5-1 cells grown in the absence of tetracycline revealed that expression of dnBRG-1 results in the reduction of CD44
expression when compared with uninduced BO5-1 cells (Fig. 5B). Microscopic analysis revealed that expression of
dnBRG-1 significantly inhibits CD44 staining in ~90% of induced
cells when compared with BO5-1 cells grown in the presence of
tetracycline (Fig. 5C). These data imply that functional
BRG-1 is required for stimulating CD44 expression.
Cyclin E Antagonizes BRG-1 Transcriptional Activity--
It has
been previously reported that Cyclin E can bind to and antagonize the
ability of BRG-1 to induce growth arrest (38). Based on this, and the
findings that Cyclin E is also overexpressed in tumors, we examined
whether expression of Cyclin E could disrupt the ability of BRG-1 to
signal to CD44 (39). To test this, C33A cells were cotransfected with
vector, BRG-1, or BRG-1 and Cyclin E together with pBabe-Puro. After
rapid puromycin selection the cells were harvested and analyzed for
CD44 expression via immunoblotting. As expected, ectopic expression of
BRG-1 in C33A stimulated CD44 expression when compared with vector
(Fig. 6, upper panel,
lanes 1 and 2). However, when Cyclin E was
cotransfected with BRG-1 in two independent transfections, CD44
expression was inhibited by ~62% as determined after normalization
to Cdk4 (Fig. 6, upper and lower panels,
lanes 1, 3, and 4). Immunoblotting for
BRG-1 and Cyclin E confirms their expression while Cdk4 levels were monitored as a loading control (Fig. 6, middle and
lower panels, lanes 1-4). These findings show
that Cyclin E can antagonize the ability of BRG-1 to stimulate CD44
production.
Aberrant expression of CD44 is associated with advanced tumor
progression and poor clinical outcome (1-3). However, little is known
about what factors are required for maintaining CD44 expression. Here
we show that BRG-1 plays a critical role in regulating CD44
transcription. Reintroduction of BRG-1 restored CD44 expression in
BRG-1-negative (C33A and SW13) cells, whereas expression of dominant-negative BRG-1 in NIH3T3 cells inhibited CD44 production. Furthermore, ectopic expression of Cyclin E inhibited the ability of
BRG-1 to stimulate CD44, consistent with the notion that at least some
components of the cell cycle machinery could also mediate CD44
expression. These results indicate that BRG-1 is required for
regulating CD44 expression, suggesting that loss of BRG-1 may
contribute to the invasive and metastatic potential of certain cancers.
BRG-1 plays a critical role in transcriptional regulation and chromatin
remodeling and can both stimulate and repress the transcription of
specific genes (22, 26-30, 32). BRG-1 is an ATP-dependent
chromatin remodeler that regulates transcription through its ability to
modify nucleosome structure (37). Activation of CD44 expression is
apparently dependent on chromatin remodeling, because the
dominant-negative BRG-1 used in this study specifically disrupts the
ATPase activity of BRG-1. In the case of CD44, BRG-1 likely converts
the CD44 promoter from a transcriptionally inactive state to an active
one. The finding that transiently transfected CD44 reporter constructs
maintained high basal activity in the absence of BRG-1 suggests that
the plasmid-borne CD44 promoter construct already exists in a
transcriptionally active state, thus masking any BRG-1 stimulatory
effect at the level of chromatin (data not shown).
The loss of CD44 expression by some tumors correlates with increased
metastatic potential (1). The mechanism of CD44 loss in these tumors is
largely unknown; however, in prostate carcinomas, CD44 expression is
repressed through the methylation of the 5'-CpG islands in the CD44
promoter (40). Interestingly, loss of heterozygosity mapped to the 19p
locus (which overlaps with BRG-1) also occurs as a late-stage event in
prostate cancer (41). There are numerous ties between transcriptional
regulation, chromatin structure, and methylation. Three SWI2/SNF2-like
proteins, Mi-2, ATRX, and DDM1, have been implicated in DNA methylation
(42, 43). Specifically, DDM1 maintains genomic methylation through its
ability to either modify histones or increase the accessibility of DNA
methyl-transferase. The chromatin-remodeling activity of Mi-2 and ATRX
is thought to be targeted specifically to DNA to initiate or maintain
methylation-mediated transcriptional control. However, it remains to be
determined whether the recruitment of SWI/SNF components to chromatin
stimulates methylation or if methylation is causative for the
recruitment of specific chromatin-remodeling activities. Therefore,
determining whether BRG-1 influences the methylation of the CD44
promoter would be important in defining its mechanism of gene regulation.
The importance of SWI/SNF in cancer is becoming increasingly apparent.
Recently, BRG-1 has been linked to breast cancer by the finding that
the coactivator function of BRCA1 on p53 transcription is mediated
through BRG-1 (30). In addition, it has been shown that BRG-1 is
required for RB signaling to specific cell cycle targets (22, 32).
Alteration of another SWI/SNF subunit was identified after a genetic
screen in malignant rhabdoid tumors, where the hSNF5/INI1
gene located on the long arm of chromosome 22, is either mutated or
deleted in rhabdoid tumors (44, 45). In addition, there is evidence
that loss of other SWI/SNF subunits like Baf 47, Baf 57, and Baf 155 occur in a variety of tumors; however, it remains to be determined
whether loss of these other subunits affect tumorigenesis or CD44
expression (46).2 This study
shows that BRG-1 is required for enabling CD44 expression and provides
a mechanism through which CD44 is lost in cancer. These findings
suggest that BRG-1 loss may contribute to the formation of the
metastatic phenotype.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
C33A cells have reduced CD44 expression.
A, C33A (lane 1) and SAOS-2 (lane 2)
cells were harvested and lysed, and equal total protein was resolved by
SDS-PAGE and immunoblotted for CD44 and Cdk4. B, C33A cells
were harvested as in A, total RNA was prepared, and RT-PCR
was performed utilizing 1 µg of total RNA as described under
"Materials and Methods." As a control, PCR analysis of isolated RNA
was carried out from C33A (lane 1) and SAOS-2 (lane
2). The level of CD44 RNA was normalized to that of GAPDH. The
results are representative of two independent RT-PCR experiments.
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Fig. 2.
Genetic determinants in SAOS-2 restore CD44
signaling in C33A. A, parental C33A and SAOS-2
cells along with four independent C-S fusion clones were harvested and
lysed, and equal total protein was resolved by SDS-PAGE and
immunoblotted for CD44 and Cdk4. B, immunohistochemical
staining of both parental lines and a C-S fusion clone for p53 and CD44
expression. The results of this fusion clone are representative of all
other fusion clones tested.
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Fig. 3.
Loss of BRG-1 correlates with a lack of CD44
expression. Thirty micrograms of lysate was isolated from
subconfluent SW13, U87, PC-3, C33A, SAOS-2, U2OS, and RD cells using an
8 M urea extraction (8 M urea, 0.1 M NaH2PO4, 10 mM Tris,
pH 8). The lysates were then separated via SDS-PAGE and blotted for
BRG-1, CD44, and actin.
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Fig. 4.
BRG-1 stimulates CD44 expression in C33A and
SW13 cells. A, C33A and SW13 cells were transfected
with (1 µg) pBabe-Puro and (15 µg) of vector (lane 1),
PSM-RB (lane 2), or BRG-1 (lane 3). Following
puromycin selection, equal total protein was resolved by SDS-PAGE and
then immunoblotted for CD44, BRG-1, PSM-RB, and Cdk4. B,
C33A cells were transfected with vector (lanes 4,
5), BRG-1 (lanes 6 and 7), and PSM-RB
(lanes 8 and 9) and then selected as performed in
A. Total RNA was prepared, and RT-PCR was performed
utilizing 1 µg of total RNA as described under "Materials and
Methods." As a control, PCR analysis of isolated RNA was carried out
from the vector (lanes 1)-, BRG-1 (lane 2)-, and
PSM-RB (lane 3)-transfected cells. The level of CD44 RNA was
normalized to that of GAPDH. The results are representative of two
independent RT-PCR experiments.
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Fig. 5.
BRG-1 is required for CD44 production.
A, BO5-1 cells were washed three times with PBS and then
split into plates containing media supplemented with and with out
tetracycline (2 µg/ml). After 3 days, the cells were harvested and
resolved by SDS-PAGE, and the expression of dnBRG-1 was detected with
an anti-Flag antibody. B, immunohistochemical staining for
CD44 in BO5-1 cells cultured either in the presence or absence of
tetracycline. C, quantitation of the immunohistochemical
staining in B.
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Fig. 6.
Cyclin E antagonizes BRG-1 transcriptional
regulation of CD44. C33A cells were transfected with (1 µg)
pBabe-Puro and (15 µg) of vector (lane 1), BRG-1
(lane 2), or BRG-1 + Cyclin E (lanes 3 and
4). Following puromycin selection, equal total protein was
resolved by SDS-PAGE and then immunoblotted for CD44, BRG-1, Cyclin E,
and Cdk4. The level of CD44 protein was normalized to that of Cdk4. The
results are representative of two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Karen Knudsen, Dr. Kenji Fukasawa, Steve Angus, and Yelena Whetherhill for critical reading of the manuscript. We are also grateful to Dr. S. Goff for supplying us with the BRG-1 plasmid and Dr. A. Imbalzano for providing the BO5-1 cells.
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FOOTNOTES |
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* This work was supported in part by Grant CA-82525 from the NCI, National Institutes of Health (NIH), by grants from The Ruth Lyons Cancer Foundation and the Sidney Kimmel Cancer Foundation (to E. S. K.), by NIH Grant NS-39550 (to L. S. S.), and by NIH Grants DE-12355 and CA-63176 (to B. E. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by training grants from the National Institutes of Health.
¶ To whom correspondence should be addressed: Dept. of Cell Biology, University of Cincinnati College of Medicine, Vontz Center for Molecular Studies, 3125 Eden Ave., Cincinnati, OH 45267-0521. Tel.: 513-558-6849; Fax: 513-558-4454; E-mail: shobecm@email.uc.edu.
Published, JBC Papers in Press, December 6, 2000, DOI 10.1074/jbc.M009747200
2 B. Weissman, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: BRG-1, Brahma-related gene; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; C-S, C33A-SAOS-2 cell fusion line; dnBRG-1, dominant-negative BRG-1; PAGE, polyacrylamide gel electrophoresis.
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