From The Burnham Institute, Cancer Research Center, La Jolla, California 92037
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ABSTRACT |
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BAG-1 (also known as RAP46) is an anti-apoptotic protein, which has been shown previously to interact with a number of nuclear hormone receptors, including receptors for glucocorticoid, estrogen, and thyroid hormone. We show here that BAG-1 also interacts with retinoic acid receptor (RAR). Gel retardation assays demonstrated that in vitro translated BAG-1 protein could effectively inhibit the binding of RAR but not retinoid X receptor (RXR) to a number of retinoic acid (RA) response elements (RAREs). A glutathione S-transferase-BAG-1 fusion protein also specifically bound RAR but not RXR. Interaction of BAG-1 and RAR could also be demonstrated by yeast two-hybrid assays. In transient transfection assays, co-transfection of BAG-1 expression plasmid inhibited the transactivation activity of RAR/RXR heterodimers but not RXR/RXR homodimers. When stably expressed in breast cancer cell lines, BAG-1 inhibited binding of RAR/RXR heterodimer to a number of RAREs and suppressed RA-induced growth inhibition and apoptosis. In addition, RA-induced suppression of Bcl-2 expression was abrogated by overexpression of BAG-1. These results demonstrate that BAG-1 can regulate retinoid activities through its interaction with RAR and suggest that elevated levels of BAG-1 protein could potentially contribute to retinoid resistance in cancer cells.
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INTRODUCTION |
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Development of a multicellular organism requires tightly regulated cellular processes, such as proliferation, differentiation, and cell death. Failure to maintain the balance among these fundamental and mechanistically related processes may result in abnormal cell growth, as seen in cancer cells where cell death is often inhibited (1, 2). Retinoids, a group of natural and synthetic vitamin A derivatives, are currently used to treat epithelial cancer and promyelocytic leukemia and are being evaluated for prevention and therapy of other human cancers (3, 4). The anti-cancer effects of retinoids are mainly due to their inhibition of cell proliferation, induction of cell differentiation, and promotion of apoptosis. Retinoids alone or in combination with other stimuli induce apoptosis during normal development and in different types of cancer cells in vitro (5-11). However, it remains largely unknown how retinoid-induced apoptosis is regulated.
The effects of retinoids are mainly mediated by two classes of nuclear
receptors, the retinoic acid receptors
(RARs)1 and retinoid X
receptors (RXRs). RARs and RXRs are encoded by three distinct genes
(,
, and
) and are members of the steroid/thyroid/retinoid hormone receptor superfamily that function as ligand-activated transcription factors (12-14). 9-cis RA is a high affinity
ligand for both RARs and RXRs, whereas all-trans-RA
(trans-RA) is a ligand for only RARs. RARs and RXRs
primarily function as RXR/RAR heterodimers that bind to a variety of RA
response elements (RAREs) and regulate their transactivation
activities.
Regulation of gene expression either positively or negatively by nuclear hormone receptors is modulated by additional factors. Some of them appear to provide a direct link to the core transcriptional machinery and to modulate chromatin structure (15), such as SRC-1 (16), SUG-1 (17), TIF-1 (18), RIP-140 (19), N-CoR (25), SMRT (26), TIF-2 (20), GRIP-1 (21), p160 (22), CBP (23), AIB1 (8), and ACTR (24), whereas a number of other cellular proteins, such as AP-1, have been implicated in the regulation of nuclear hormone receptor activity, probably through their interaction with receptors (27).
The involvement of retinoid receptors in retinoid-induced apoptosis has
been demonstrated in several studies. Expression of RAR may be
involved in the apoptosis of mesenchyme of the interdigital regions
during mouse limb development (28). RAR
is required for RA-induced
apoptosis of breast cancer (5) and lung cancer (7) cells, whereas
activation of RXR is essential for RA-induced HL-60 cell apoptosis
(29). In 4-HPR-induced apoptosis, activation of RAR
may be involved
(30, 31), whereas regulation of activation-induced apoptosis of T-cells
by 9-cis RA requires activation of both RARs and RXRs
(32).
Although much interest has been directed to the role of
retinoid-induced apoptosis in both physiological and pathological processes, very little is known regarding regulation of the process. It
is believed that apoptosis, once triggered, proceeds through a central
death pathway in which specific cellular proteases and endonucleases
are activated (1, 2, 33). Members of the Bcl-2 family play an important
role in the regulation of the central death pathway. Bcl-2 can suppress
induction of apoptosis in many systems, whereas Bax promotes apoptosis.
In addition, several other proteins that modulate Bcl-2 activity by
interacting with Bcl-2 have been described (1, 2, 33). One of these
genes, BAG-1 (for Bcl-2-associated
anti-death ene 1), was cloned from a
murine embryo cDNA library using a protein-protein interaction technique (34). Two differently localized BAG-1 isoforms, the long
BAG-1 isoform and the short BAG-1 isoform, generated by alternative translation initiation are expressed in mammalian cells (35). Whether
two isoforms act differently remains to be determined. Recent studies
demonstrated that co-expression of BAG-1 and Bcl-2 in Jurkat lymphoid
cells, NIH 3T3 fibroblasts, and melanoma cells promoted the survival of
these cells in response to a variety of apoptotic stimuli (34, 36, 37).
In addition to Bcl-2, BAG-1 also interacts with Raf-1 (38), resulting
in activation of its kinase activity. Furthermore, BAG-1 can interact
with hepatocyte growth factor receptor and platelet-derived growth
factor receptor and enhance the ability of these receptors to transduce
signals for cell survival (39). These observations suggest that BAG-1 may function as an adaptor to mediate the interaction between survival
factors and apoptotic machinery and may also play a role in regulating
cellular proliferation. The recent observation that BAG-1 binds tightly
to Hsp70/Hsc70-family proteins and modulates their chaperone activity
(40-42) suggests that the ability of BAG-1 to alter the activities of
diverse groups of proteins involved in cell growth control may be
attributed to its effects on Hsp70/Hsc70 proteins.
Interestingly, the human BAG-1 homolog (also known as RAP46) was cloned from a human liver cDNA library by virtue of its interaction with the glucocorticoid receptor (43). In vitro, RAP46 interacts with a number of nuclear hormone receptors, including estrogen receptor and thyroid hormone receptor (TR) (43). Since molecular chaperones are known to play an important role in controlling the activity of many members of the steroid/thyroid/retinoid receptor family (44), it is possible that BAG-1 could alter the function of these transcriptional regulators. Before this report, however, it was unknown whether BAG-1 regulates the activities of the nuclear hormone receptors and whether BAG-1 interacts with retinoid receptors.
Here we demonstrate that short BAG-1 isoform interacts with the RAR but not the RXR both in vitro and in vivo. GST pull-down and the yeast two-hybrid assays show that BAG-1 directly interacts with RAR but not RXR. Moreover, BAG-1 inhibits RAR/RXR heterodimer DNA binding and suppresses RA-induced transactivation activity of RARs on various RAREs. Overexpression of BAG-1 in MCF-7 and ZR-75-1 breast cancer cells reduces the ability of trans-RA to inhibit the growth and induce apoptosis, as well as its modulation of Bcl-2 expression. Taken together, our results demonstrate that BAG-1 can physically interact with RARs and is an important component in the retinoid response pathway. Our findings suggest that this protein-protein interaction may play a role in the regulation of retinoid-induced growth inhibition and apoptotic processes, potentially contributing to retinoid resistance in cancer.
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MATERIALS AND METHODS |
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Cell Culture-- Monkey kidney CV-1 cells and breast cancer MCF-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, and ZR-75-1 breast cancer cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum.
Growth Inhibition Assay-- Cells were seeded at 1,000-2,000 cells/well in 96-well plates and treated 24 h later with various concentrations of trans-RA for 7 days. Media and trans-RA were changed every 48 h. Relative viable cell number was determined using the MTT assay (52).
Apoptosis Analysis--
For the terminal deoxynucleotidyl
transferase assay (5), cells were treated with or without
106 M trans-RA. After 48 h,
cells were trypsinized, washed with phosphate-buffered saline, fixed in
1% formaldehyde in phosphate-buffered saline, washed with
phosphate-buffered saline, resuspended in 70% ice-cold ethanol, and
stored at
20 °C overnight. Cells were then labeled with
biotin-16-dUTP by terminal transferase and stained with
avidin-fluorescein isothiocyanate (Boehringer Mannheim). Fluorescently
labeled cells were analyzed using a FACScater-Plus. Representative
histograms are shown.
Antibodies and Immunoblotting-- Cells were lysed in 150 mM NaCl, 10 mM Tris, pH7.4, 5 mM EDTA, 1% Triton X-100 and protease inhibitors phenylmethylsulfonyl fluoride, aprotinin, leupeptins, and pepstatin. Equal amounts of lysates (50 µg) were boiled in SDS sample buffer, resolved by SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membrane. After transfer, the membranes were blocked in TBST (50 mM Tris, pH7.5, 150 mM NaCl, 0.1% Tween 20) containing rabbit anti-Bcl-2 serum. The membranes were then washed three times with TBST and then incubated for 1 h at room temperature in TBST containing horseradish peroxidase-linked anti-rabbit immunoglobulin. After three washes in TBST, immunoreactive products were detected by chemiluminescence with an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).
Transient and Stable Transfection Assay--
For CV-1 cells,
1 × 105 cells were plated per well in a 24-well
plates 16-24 h before transfection as described previously (45). For
ZR-75-1 cells, 5 × 105 cells/well were seeded in
6-well culture plates. A modified calcium phosphate precipitation
procedure was used for transient transfection (45). For CV-1 cells, 100 ng of reporter plasmid, 150 ng of -galactosidase expression vector
(pCH 110, Amersham), and various amounts of BAG-1 expression vector
that expresses short BAG-1 isoform (35) were mixed with carrier DNA
(pBluescript) to 1,000 ng of total DNA/well. Reporter plasmids
RARE-tk-CAT, TREpal-tk-CAT, and TREMHC-tk-CAT have been
previously described (45-48). For stable transfection, the
pRc/CMV-BAG-1 plasmid (34) that expresses short BAG-1 isoform was
stably transfected into MCF-7 or ZR-75-1 cells using calcium phosphate
precipitation method, followed by selection using G418 (Life
Technologies, Inc.) as described (5).
Preparation of Receptor, BAG-1, and Nuclear
Protein--
cDNAs for RAR, RXR
, estrogen receptor, and
BAG-1, which expresses short BAG-1 isoform cloned into pBluescript
(Stratagene), were transcribed by using T7 or
T3 RNA polymerase, and the transcripts were translated in
the rabbit reticulocyte lysate system (Promega) as described previously
(45). The relative amounts of the translated proteins were determined
by separating the [35S]methionine-labeled proteins on
SDS-polyacrylamide gels, quantitating the amount of incorporated
radioactivity and normalizing it relative to the content of methionine
residues in each protein. To synthesize receptor fusion protein, RAR
or RXR
cDNAs were cloned in-frame into the bacterial expression
vector pGex.2T (Amersham) as described (45). Preparation and
purification of GST-BAG-1 and GST-BAG-1(
172-218) fusion proteins
has been described (41). Preparation of nuclear extract was described
previously (6).
Gel Retardation Assay--
Analysis of in vitro
synthesized or bacterially expressed receptor proteins or nuclear
proteins by gel retardation was described previously (45). To analyze
the effect of BAG-1 protein, in vitro synthesized BAG-1
protein was preincubated with receptor protein at room temperature for
10 min before the gel retardation assay. The oligonucleotides
used for gel retardation assays were RARE
(TGTAGGGTTCACCGAAAGTTCAGTC) (46); TREpal (TGAGGTCATGACCTGA) (45);
DR-5-RARE(TGTAGGGTTCACACTGAGTTCACTCA); and
DR-2-RARE(AGGTCAAAAGGTCAG).
GST Pull-down Assay-- To analyze the interaction between BAG-1 and RAR, GST-BAG-1 fusion protein was immobilized on glutathione-Sepharose beads as described (52). As a control, GST prepared under the same conditions was also immobilized. The beads were preincubated with bovine serum albumin (1 mg/ml) at room temperature for 5 min. 35S-Labeled in vitro synthesized receptor proteins (2 to 5 µl, depending on translation efficiency) Bcl-2 or Hsc70 were then added to the beads. The beads were then continuously rocked for 1 h at 4 °C in a final volume of 200 µl in EBC buffer (140 mM NaCl, 0.5% Nonidet P-40, 100 mM NaF, 200 µM sodium orthovanadate, and 50 mM Tris, pH 8.0). After washing five times with NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, 0.5% Nonidet P-40), the bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis.
Two-hybrid Assay--
For the yeast two-hybrid assay, the yeast
two-hybrid system from CLONTECH Inc. (Palo Alto,
CA) was used (52). BAG-1 cDNA was cloned into the yeast expression
vector pGAD424 to generate an in-frame fusion with the Gal4 activation
domain. RAR or RXR
cDNAs were cloned into pGBT11 or pGBT9,
respectively, to produce an in-frame fusion with Gal4 DNA binding
domain. RAR
was also cloned into pGAD426 that contains Gal4
activation domain to study the interaction between RAR
and RXR
.
The yeast reporter strain Y190 containing a LacZ reporter plasmid with
Gal4 binding sites was used for transformation.
-Galactosidase
activity was determined following the conditions provided by the
manufacturer.
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RESULTS |
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Inhibition of Retinoid Receptor DNA Binding by BAG-1--
We
investigated whether BAG-1 could interact with retinoid receptors by
studying the effect of BAG-1 protein on binding of retinoid receptors
to their target DNA sequences. In vitro synthesized RAR and
RXR formed a strong RAR/RXR heterodimer complex with the TREpal as
described previously (45). When increasing amounts of in
vitro synthesized short BAG-1 isoform protein were incubated with
RAR and RXR, the binding of RAR/RXR heterodimers was inhibited in a
BAG-1 concentration-dependent manner (Fig.
1a). At a 5 M excess of BAG-1 protein relative to RAR/RXR, the binding was almost completely inhibited. The effect of BAG-1 on RAR/RXR binding was specific because similar amounts of estrogen receptor did not show any
effect. BAG-1 also effectively inhibited the binding of TR/RXR to the
TREpal probe (Fig. 1b), consistent with a prior report that
BAG-1 can interact with TR (43). To study whether the inhibitory effect
of BAG-1 on RAR/RXR heterodimer binding is specific to the TREpal, we
used another RA responsive element (RARE), which is derived from the
RAR
promoter (46). As shown in Fig. 1c, binding of
RAR/RXR on the
RARE was also inhibited by the addition of BAG-1
protein. Similar results were obtained with other RAREs, including
CRBPI-RARE and ApoAI-RARE (data not shown). Thus, inhibition of RAR/RXR
binding to their target DNA sequences by BAG-1 is independent of
RAREs.
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Interaction of BAG-1 with Retinoid Receptors--
To further study
the interaction between BAG-1 and RAR, we used an in vitro
GST pull-down assay. A GST-BAG-1 fusion protein was expressed in
bacteria and immobilized on glutathione-Sepharose beads. The beads were
then incubated with in vitro synthesized 35S-labeled RAR or RXR protein. After extensive washing,
the mixtures were analyzed on a SDS-polyacrylamide gel. In comparison
to the input lane, significant amounts of labeled RAR but not RXR were retained by GST-BAG-1-Sepharose beads but not by GST control beads (Fig. 2). For control, Bcl-2, a known
BAG-1 interacting protein (34), bound strongly to GST-BAG-1 beads. We
also employed a BAG-1 mutant protein in which the last 47 amino acid
residues are deleted from its C-terminal end (41). This mutant protein (BAG-1/c) can interact with Bcl-2 but not with Hsc70. Interestingly, labeled RAR but not RXR was also retained by the mutant BAG-1, thus
implying that the interaction of BAG-1 with RAR is independent of its
binding to Hsc70.
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Inhibition of Transactivation Activity of Retinoid Receptors by
BAG-1--
To further examine the BAG-1-RAR interaction, we studied
the effects of the short BAG-1 isoform on RAR transactivation activity on a number of RAREs by transient transfection assay. When CV-1 cells
were transiently transfected with RAR expression vector together
with either TREpal-tk-CAT (Fig.
4a) or
RARE-tk-CAT (Fig. 4b), trans-RA-induced reporter gene activity was
markedly inhibited by co-transfection of BAG-1 expression plasmid in a
concentration-dependent manner. The effect is specific to
BAG-1 because co-transfection of similar amounts of empty expression
vector (pcDNA3) did not inhibit trans-RA-induced gene
expression. BAG-1 also showed inhibitory effect on
trans-RA-induced RAR
activity on the TREpal. However, the
trans-RA-independent RAR
activity was not affected. This suggests that ligand-dependent RAR activity is more
sensitive to the inhibitory effect of BAG-1. A similar inhibitory
effect of BAG-1 was also obtained when RAR
expression vector was
used (data not shown). We also studied the effects of BAG-1 on thyroid hormone (T3)-induced TR
activity, and we observed a
significant inhibitory effect of BAG-1 on TR
(Fig. 4d),
consistent with the ability of BAG-1 to bind the TR protein (Fig.
1b; Ref. 43). To determine the effect of BAG-1 on RXR
homodimer activity, we co-transfected TREpal-tk-CAT reporter plasmid
and RXR
expression vector. However, the 9-cis-RA-induced
RXR
homodimer activity was not affected by co-transfection of BAG-1
(Fig. 4e), consistent with our observation that BAG-1 does
not interact with RXR
in vitro (Figs. 1d, 2,
and 3).
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Overexpression of BAG-1 Inhibits Trans-RA-induced Cancer Cell Apoptosis-- The above data suggest that BAG-1 may function as a modulator of trans-RA-induced biological responses. We previously showed that trans-RA effectively inhibits the growth and induces apoptosis of some human breast cancer cell lines (5). We, therefore, stably expressed BAG-1 in human breast cancer cell lines MCF-7 and ZR-75-1. MCF-7/BAG-1(#3), 75-1/BAG-1(#5), and 75-1/BAG-1(#6) that stably expressed high levels of the transfected BAG-1 plasmid (data not shown) were chosen to examine the effect of BAG-1 overexpression on RA activities. As shown in Fig. 5a, trans-RA effectively inhibited the growth of parental MCF-7 and ZR-75-1 cells. However, the BAG-1-overexpressing clones displayed resistance to the growth inhibitory effects of trans-RA. The growth of the MCF-7/BAG-1(#3) was even stimulated by trans-RA (Fig. 5a). Although trans-RA did not stimulate the growth of 75-1/BAG-1(#5) and 75-1/BAG-1(#6) cells, its inhibitory effect on the growth of these cells was significantly reduced as compared with its effect on ZR-75-1 cells (Fig. 5b). The effect on trans-RA activity observed above was specific because clones stably transfected with the empty vector, MCF-7/neo and 75-1/neo, exhibited similar responses to trans-RA as that observed with the parental cell lines. Thus, BAG-1 partially abrogates the growth inhibitory effects of trans-RA on human breast cancer cells.
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Overexpression of BAG-1 Abrogates Down-regulation of Bcl-2 by Trans-RA-- Retinoids have been shown to down-regulate the expression of Bcl-2 in leukemia (11). We therefore studied whether BAG-1 affected RA-regulated expression of Bcl-2 in MCF-7 cells. Bcl-2 was highly expressed in MCF-7 cells, and its expression level was significantly reduced by trans-RA as determined by immunoblotting. In MCF-7/BAG-1(#3)-stable transfectant, however, treatment of trans-RA had little or no effect on Bcl-2 expression (Fig. 7). These data suggest that BAG-1 may inhibit trans-RA-induced apoptosis at least in part through its effects on trans-RA-regulated genes, such as Bcl-2.
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DISCUSSION |
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Previously, it was reported that BAG-1 (RAP46) can interact with several steroid hormone receptors (43). However, the biological effects of the interaction are unknown. In this report, we show that the short BAG-1 isoform that is known to be predominantly expressed in cytoplasm (35) can antagonize RAR activity through its direct interaction with RAR. Using gel retardation assays, we observed that BAG-1 inhibits binding of RAR/RXR heterodimers to several RAREs (Fig. 1). In GST-pull down assay, we found that BAG-1 directly interacts with RAR in solution (Fig. 2). By using yeast two-hybrid assays (Fig. 3), we showed that the BAG-1-RAR interaction could occur in vivo. Moreover, a functional interaction was demonstrated by our observation that co-transfection of BAG-1 inhibits trans-RA-induced RAR transactivation activities on several RAREs (Fig. 4). Furthermore, RAR/RXR RARE binding was abrogated in MCF-7 cells that express transfected BAG-1 (Fig. 8). Thus, the interaction of BAG-1 with steroid hormone receptors can be extended to RAR. We also present evidence here that BAG-1 can similarly prevent TR DNA binding and transactivation activity. However, BAG-1 does not interact with all nuclear hormone receptors, as shown here by the failure of BAG-1 to bind to and modulate the activity of RXR (Figs. 1d, 2, 3, and 4e).
Activation or repression of gene transcription by nuclear hormone receptors requires their interaction with multiple cellular co-regulatory factors. These include receptor co-activators, which exert their effect on receptor transactivation activity by mediating transcription-initiation complex formation and affecting chromatin structure (15), and receptor co-repressors, which bind to receptors in the absence of ligand and actively repress target gene transcription by impairing the activity of the basal transcription machinery (25). BAG-1 appears to function differently from receptor co-activators or co-repressors. It does not induce ligand-dependent gene activation nor does it cause repression of target gene transcription in the absence of ligand (Fig. 4a). Instead, interaction of RAR and BAG-1 resulted in inhibition of RAR DNA binding (Fig. 1) and trans-RA-induced RAR transactivation activities (Fig. 4). Thus, it may function as a modulator of RAR activities through the mechanisms that resemble the effect of AP-1, which was previously shown to inhibit trans-RA-induced RAR activity by preventing RAR binding to target DNA sequences in promoters (27). However, whether BAG-1 affects recruitment of co-activator or co-repressor by RAR remains to be determined.
The mechanism by which BAG-1 inhibits RAR binding to DNA and transactivation activity remains to be elucidated. Recently, BAG-1 was reported to bind tightly with Hsp70/Hsc70-family proteins and modulate their activity (40-42). Although the role of molecular chaperones in transcriptional activation by retinoid receptors remains controversial (49, 50), Hsp70/Hsc70 and other heat shock proteins are known to participate in the regulation of several other steroid hormone receptors (44). It is therefore tempting to speculate that BAG-1 may also influence RAR activity through Hsp70/Hsc70-mediated conformational changes that prevent it from binding DNA and transactivating retinoid-responsive target genes. Interestingly, a deletion mutant of BAG-1 lacking its C-terminal 47 amino acids, which does not bind to Hsc70, was capable of binding to RAR in vitro (Fig. 2). Thus, the domains in BAG-1 required for interactions with RAR and Hsp70 appear to be separable. Unfortunately, when expressed in mammalian cells, the BAG-1 mutant protein was unstable, precluding functional evaluation of its effects on RAR-mediated gene expression. Thus, the relevance of BAG-1 interactions with Hsp70-family proteins to its function as inhibitor of RAR remains to be determined.
One of the interesting features of BAG-1 is its ability to promote cell survival (34, 36, 37). The effect was previously attributed to its interaction with Bcl-2 (34). In addition, BAG-1 interacts with platelet-derived growth factor and hepatocyte growth factor receptors, enhancing their ability to transduce signals that promote cell survival (39). BAG-1 can also bind to Raf-1 and activate its kinase activity (38). Our observations that overexpression of BAG-1 inhibits trans-RA-induced apoptosis (Fig. 6) and prevents trans-RA-induced down-regulation of Bcl-2 expression (Fig. 7) suggest that interaction with RAR may represent another mechanism by which BAG-1 promotes cell survival. These observations also suggest that overexpression of BAG-1 may contribute to retinoid resistance in certain malignancies. Taken together with recent observations that BAG-1 protein levels are elevated in breast and prostate cancers (51), BAG-1 may represent an important regulator of cell survival and growth, which may contribute in multiple ways to tumorigenesis and resistance to therapy.
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ACKNOWLEDGEMENT |
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We thank S. Waldrop for preparation of the manuscript.
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Note Added in Proof |
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The longer isoform of BAG-1, BAG-1L, has been reported recently to enhance androgen receptor transactivation, implying that BAG-1 family proteins may either potentiate or suppress the actions of specific nuclear receptors (Froesch, B. A., Takayama, S., and Reed, J. C. (1998) J. Biol. Chem. 273, 11660-11666).
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA60988 and CA67329, the United States Army Medical Research Program Grant DAMD17-4440, the California Breast Cancer Research Program Grant 3BP-0018, and the California Tobacco-related Disease Research Program Grant 6RT-0168.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.
To whom correspondence should be addressed: The Burnham Institute,
Cancer Research Center, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3141; Fax: 619-646-3195; E-mail: xzhang{at}ljcrf.edu.
1 The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response elements; TR, thyroid hormone receptor; MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; GST, glutathione S-transferase; tk, thymidine kinase; MHC, myosin heavy chain; CAT, chloramphenicol acetyl- transferase.
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REFERENCES |
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