Caveolin-1 controls BRCA1 gene expression and cellular localization in human breast cancer cells

FEBS Letters 580 (2006) 5268–5274

Caveolin-1 controls BRCA1 gene expression and cellular localization in human breast cancer cells Chen Glaita, Dana Ravidc, Sam W. Leeb, Mordechai Liscovitchc, Haim Wernera,* a

Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA c Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel

b

Received 1 June 2006; accepted 31 August 2006 Available online 11 September 2006 Edited by Varda Rotter

Abstract The role of caveolae and the caveolin proteins in cancer has been the subject of extensive research. BRCA1 participates in multiple biological pathways including DNA damage repair, transcriptional control, cell growth, apoptosis, and others. Little information, however, is available regarding the cellular mechanisms that control BRCA1 gene expression. The present study examined the potential regulation of BRCA1 gene expression and subcellular localization by Cav-1. Results of Western blots, RT-PCR, and transfection experiments showed that Cav-1 enhances BRCA1 protein and mRNA levels via a mechanism that involves transactivation of the BRCA1 promoter and which is p53-dependent. In addition, immunostaining experiments demonstrate that Cav-1 induced the cytoplasmic sequestration of BRCA1. Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Caveolin-1; Caveolae; BRCA1; Gene expression; Breast cancer

1. Introduction Caveolae are non-clathrin-coated flask-shaped invaginations of the plasma membrane which were first identified as endocytic structures involved in the transport of macromolecules across endothelial cells [1]. One of the main structural components of caveolae are the caveolins, the products of a mammalian gene family comprised of Cav-1, -2 and -3. Cav-1 and -2 are coexpressed in many cell types, and they form both homooligomeric and heterooligomeric complexes. Cav-3, on the other hand, is a muscle-specific protein [2]. Cav-1, a 22-kDa protein, constitutes an integral part of the striated coat of caveolae [3,4]. Upon Cav-1 integration into the microenvironment of a lipid raft, these microdomains invaginate and form caveolae [5]. Furthermore, by virtue of its capacity to act as a scaffolding protein able to organize and concentrate specific lipids and lipid-modified signaling molecules within caveolar membranes, Cav-1 participates in vesicular trafficking events and also in a number of signal transduction processes [1,2]. The role of caveolae and the caveolin proteins in cancer has been the subject of extensive research and continuing debate. Disruption of caveolar integrity appears to be a common theme * Corresponding author. Fax: +972 3 6406087. E-mail address: [email protected] (H. Werner).

in oncogenic transformation. Furthermore, malignant transformation is usually associated with a reduction in the cellular level of caveolin [5–7]. In addition, Cav-1 has been shown to inhibit the activity of several caveolae-associated signaling molecules, including Src tyrosine kinase family members, Ha-Ras, endothelial nitric oxide synthase, heterotrimeric G proteins, etc. Given that many of these proteins display oncogenic types of activities, it has been suggested that Cav-1 possesses a transformation-suppressing activity [8]. Consistent with this notion, antisense-mediated downregulation of Cav-1 in NIH-3T3 fibroblasts led to anchorage-independent growth and hyperactivation of the MAPK pathway [9]. In addition, forced expression of Cav-1 in the human breast cancer cell line MCF7 suppressed proliferation, cell viability, and anchorage-independent growth [10–12]. Furthermore, a dominant-negative mutant Cav-1 molecule (P132L) was reported to be present in about 16% of the cases out of a collection of 92 primary human breast cancers [13]. Finally, genetic knockout of Cav-1 results in mammary gland ductal epithelial cell hyperplasia [14] and greatly accelerates tumorigenesis in MMTV-PyMT mice prone to development of mammary gland tumors [15,16]. On the other hand, evidence in support of an oncogenic role for Cav-1 was provided by studies showing that Cav-1 promotes cell survival in metastatic prostate cancer cells [17,18] and inhibits anoikis in MCF7 breast cancer cells [12]. In addition, Cav-1 gene knockout was recently shown to promote apoptosis in the TRAMP mouse prostate cancer model [19]. Indeed, Cav-1 expression is upregulated in numerous cancer cell lines and tumor specimens, and Cav-1 expression often correlates with cancer cell grade and tumor progression stage (reviewed in Liscovitch et al. [20]). The familial breast cancer susceptibility gene, BRCA1, encodes a 220-kDa phosphorylated protein with putative tumor suppressor activity. The human BRCA1 gene has been mapped to chromosome 17q21 [21]. Mutated forms of BRCA1 were correlated with the appearance of familial breast and/or ovarian cancer at very young ages [21,22], although BRCA1 has been also implicated in cases of sporadic breast cancer [23–27]. BRCA1 participates in multiple biological pathways, including DNA damage repair, transcriptional control, cell growth, and apoptosis [27]. BRCA1 is normally a nuclear protein which is targeted to the nucleus via two nuclear localization signal (NLS) sequences [28–30]. Many breast and ovarian cancer cell lines and primary tumors, on the other hand, exhibit a predominantly cytoplasmic pattern of distribution [31,32]. Little information, however, is available regarding the cellular mechanisms that are responsible for BRCA1 gene expression and cellular localization.

0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.08.071

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In view of the important roles of Cav-1 and BRCA1 in breast cancer development, we examined in the present study the potential regulation of BRCA1 gene expression by Cav1. The results obtained showed that Cav-1 upregulates BRCA1 protein and mRNA levels by enhancing transcription of the BRCA1 promoter in a p53-dependent fashion. In addition, Cav-1 expression had a significant impact on BRCA1 subcellular localization, with Cav-1-expressing cells displaying a primarily cytoplasmic pattern of distribution compared to its predominantly nuclear localization in Cav-1-null cells. Interactions between Cav-1 and BRCA1 may have important consequences in terms of breast cancer biology.

2. Materials and methods 2.1. Cell cultures Human breast adenocarcinoma MCF7 cells were kindly provided by Dr. Merril E. Goldsmith (National Cancer Institute, Bethesda, MD, USA). The generation of the Cav-1-transfected MCF7/Cav-1 cell line was described previously [12]. MCF7/pSUPER-p53 cells, expressing a siRNA against p53, and control MCF7/pSUPER cells were kindly provided by Dr. Reuven Agami (Netherlands Cancer Institute, Amsterdam, The Netherlands). All of the cell lines were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, and 50 lg/ml gentamicin sulfate. MCF7/Cav-1 cells were routinely supplemented with 400 lg/ml G418 (InVitrogen, Carlsbad, CA, USA), and transferred to drug-free medium before the experiments. MCF7/pSUPER and MCF7/pSUPER-p53 were selected with 1 lg/ml puromycin (Sigma–Aldrich, Saint Louis, MO, USA). 2.2. Luciferase reporter plasmids, transfections and promoter activity measurements For transient transfection experiments, the pGL3-I plasmid, containing the full-length BRCA1 promoter region (nucleotides 1–2696) subcloned upstream of a luciferase reporter gene in the pGL3-basic vector, was employed [33]. A full-length wild-type Cav-1-expression vector was generated by subcloning a myc-tagged murine Cav-1 cDNA into pcDNA3. To normalize for transfection efficiency, cells were cotransfected with a b-galactosidase expression vector (pCMVb; Clontech, Palo Alto, CA, USA). Each 60-mm dish received 1 lg of reporter plasmid, 0.2 lg of pCMVb, and variable amounts of Cav-1 expression vector using the jetPEIä transfection reagent (Poly-Plus, Illkirch, France). The total amount of DNA transfected was kept constant with empty pcDNA3 vector. Luciferase and b-galactosidase activities were measured after 48 h, as previously described [34]. Promoter activities were expressed as luciferase values normalized for b-galactosidase activity. 2.3. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was prepared using the AquaPure RNA Isolation kit (Bio Rad, Hercules, CA, USA). BRCA1 mRNA levels were measured by semiquantitative RT-PCR. Total RNA (2600 ng) was used to synthesize a single strand cDNA using Superscriptä III Reverse Transcriptase (InVitrogen). BRCA1 mRNA levels were measured using the following primers: sense, 5 0 -GGTGGTACATGCACAGTTGC3 0 ; antisense, 5 0 -TGACTCTGGGGCTCTGTCTT-3 0 . The size of the amplified BRCA1 mRNA fragment was 241-bp. For control purposes, levels of GAPDH mRNA were measured in the same tubes using the following primers: sense, 5 0 -ACCACAGTCCATGCCATCAC-3 0 and antisense, 5 0 -TCCACCACCCTGTTGCTGTA-3 0 [35]. The size of the amplified GAPDH mRNA fragment was 452-bp. 2.4. Western blot analysis Cells were harvested with ice-cold phosphate-buffered saline (PBS) and lysed in a lysis buffer containing 50 mM Tris–HCl, pH 7.4–7.6 and 2% SDS in the presence of a protease inhibitor cocktail. Protein content was determined using the BCA Protein Assay method (Pierce, Rockford, IL, USA) using bovine serum albumin as a standard. Sam-

Fig. 1. Regulation of BRCA1 gene expression by Cav-1. (A) RT-PCR analysis of BRCA1 mRNA levels in MCF7 and MCF7/Cav-1 cells. Total RNA was isolated from the MCF7 and MCF7/Cav-1 cell lines and semi-quantitative RT-PCR analysis of BRCA1 and GAPDH mRNA levels was performed as described in Section 2. The positions of BRCA1 mRNA and GAPDH mRNA are indicated by the arrows. (B) BRCA1 mRNA levels were quantitated by densitometric scanning and expressed as BRCA1 mRNA values normalized to the corresponding GAPDH mRNA band. Results are means ± S.E.M. of three independent experiments. *, P < 0.05 versus MCF7 cells. (C) Western blot analysis of BRCA1 levels. MCF7 and MCF7/Cav-1 cells were lysed as indicated in Section 2. Equal amounts of protein (80 lg) were resolved by 8% SDS–PAGE and transferred onto nitrocellulose membranes. Membranes were blotted with a polyclonal BRCA1 antibody, followed by incubation with a horseradish peroxidase conjugated secondary antibody. Membranes were re-blotted with anti-tubulin. The positions of the 220-kDa BRCA1 and 50-kDa tubulin bands are indicated by arrows. Shown is a typical experiment repeated 4–6 times with similar results.

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ples containing equal amounts of protein (80 lg) were subjected to 8% SDS–PAGE and transferred to a nitrocellulose membrane. After blocking with 3% skim milk, membranes were blotted with a polyclonal anti-BRCA1 antibody (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA). To measure p53 levels, blots were incubated with a mixture of two p53 antibodies (DO-1 and Pab1801, Santa Cruz Biotechnology). Blots were re-probed with anti-tubulin. The secondary antibodies were anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Proteins were detected using the SuperSignalä West Pico Chemiluminescent Substrate (Pierce). 2.5. Immunofluorescence microscopy Cells were seeded on glass coverslips the day before immunostaining. Following washing with cold sterile PBS, fixation was performed in 2% formaldehyde diluted in sterile PBS for 20 min. Cells were then incubated for 1 h with anti-BRCA1 C-20 (dilution 1:300) or Ab-1 (Oncogene Science, Cambridge, MA, USA). Cells were then permeabilized and blocked with 0.1% Saponine (Sigma–Aldrich) in 1.5% FBS in PBS. After washing with PBS, slides were incubated for 1 h with Cy TM3 conjugated Affinitipure goat anti-rabbit IgG (Jackson Immuno Research) or with AlexaFluor 488 goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA). Nuclei were stained briefly with 5 lg/ml DAPI (4,6-diamidino2-phenylindole). The subcellular localization of BRCA1 was obtained by scoring cells with a Leica TCF SP2 confocal microscope.

3. Results 3.1. Regulation of BRCA1 mRNA and protein levels by Cav-1 BRCA1 functions as a tumor suppressor capable of arresting growth of mammary-derived cells [36]. In addition, recent experiments have shown that stable expression of Cav-1 in

Fig. 2. Regulation of BRCA1 promoter activity by Cav-1. MCF7 and MCF7/Cav-1 cells were cotransfected with the full-length BRCA1 promoter luciferase reporter construct (pGL3-I), along with a pCMVb control plasmid, as described in Section 2. Promoter activities are expressed as luciferase values normalized for b-galactosidase levels. A value of 100% was given to the promoter activity in MCF7 cells. Bars are means ± S.E.M. of a typical experiment repeated three times. *, P < 0.05 versus MCF7 cells.

Fig. 3. Effect of p53 on the regulation of BRCA1 gene expression by Cav-1. Untransfected MCF7/pSUPER and MCF7/pSUPER-p53 were harvested, lysates were electrophoresed through 8% SDS–PAGE, and endogenous p53 (A) and BRCA1 (B) levels were measured by Western blot. The positions of the p53 and BRCA1 bands are denoted by the arrows. Membranes were re-probed with a tubulin antibody. (C) MCF7/pSUPER and MCF7/pSUPER-p53 cells were transfected with 0.5 lg of a Cav-1 expression vector (or empty pcDNA3) and, after 48 h, endogenous BRCA1 and tubulin levels were measured by Western blots. (D) MCF7/pSUPER and MCF7/pSUPER-p53 cells were transiently cotransfected with 1 lg of the full-length BRCA1 promoter-luciferase reporter plasmid (pGL3-I) and 0.5 lg of a Cav-1 expression vector (solid bar), or empty pcDNA3 expression vector (open bar), along with 0.2 lg of pCMVb, using the jetPEIä reagent. After 48 h, luciferase and b-galactosidase levels were measured. Promoter activities are expressed as luciferase normalized for bgalactosidase levels. A value of 100% was given to the basal BRCA1 promoter activity, in the absence of Cav-1. Bar graphs represent smean ± S.E.M. of three independent experiments. *, P < 0.05 versus pcDNA3-transfected cells.

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MCF7 mammary adenocarcinoma cells attenuates proliferation and blocks anchorage-independent growth [12]. To investigate whether Cav-1 regulates BRCA1 gene expression, total RNA was obtained from untreated MCF7 and MCF7/Cav-1 cells, and BRCA1 and GAPDH mRNA levels were measured by semi-quantitative RT-PCR. As shown in Fig. 1A and B, endogenous BRCA1 mRNA levels were 2.7 ± 0.8-fold higher in MCF7/Cav-1 than in MCF7 cells. No changes were detected in GAPDH mRNA levels. To assess whether the Cav-1-induced increase in BRCA1 mRNA levels was associated with a corresponding elevation in BRCA1 protein levels, lysates were prepared from MCF7/ Cav-1 and control MCF7 cells, electrophoresed through 8% SDS–PAGE gels and immunoblotted with a BRCA1 antibody (C-20). Results of multiple Western blot analyses showed that BRCA1 levels were approximately 40% higher in MCF7/Cav-1 than in native MCF7 cells (Fig. 1C). 3.2. Effect of Cav-1 on BRCA1 promoter activity To determine whether the effect of Cav-1 on BRCA1 mRNA levels was mediated at the level of transcription, a luciferase reporter construct containing the entire 2.7-kb BRCA1 promoter sequence (pGL3-I) was transiently transfected into both MCF7 and MCF7/Cav-1 cell lines, along with the pCMVb control plasmid. After 48 h, cells were harvested and luciferase and b-galactosidase values were measured. Results of transfection experiments showed that BRCA1

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promoter activity was 3-fold higher (297.4 ± 17.0%) in MCF7/Cav-1 cells compared with control MCF7 cells, suggesting that the mechanism responsible for the augmented BRCA1 mRNA levels in Cav-1-expressing cells involves transactivation of the BRCA1 promoter (Fig. 2). 3.3. Involvement of p53 in transcriptional regulation of the BRCA1 promoter by Cav-1 Since previous studies have established that BRCA1 works in conjunction with tumor suppressor p53 [37], we investigated the potential participation of p53 in regulation of BRCA1 gene expression by Cav-1. For this purpose we employed an MCF7derived cell line in which p53 expression was suppressed by stable transfection of a p53-RNAi vector (MCF7/pSUPER-p53) [38] and control, empty vector-transfected, cells (MCF7/pSUPER) expressing a wild-type p53 (Fig. 3A). Western blot analysis with a BRCA1 antibody revealed no distinct changes in basal BRCA1 expression levels between p53-expressing and p53-null cells (Fig. 3B). MCF7/pSUPER and MCF7/pSUPER-p53 cells were transiently transfected with a Cav-1 expression vector, and after 48 h endogenous BRCA1 levels were measured by Western blots. Results obtained showed that Cav-1 expression induced a 33% increase in BRCA1 levels in p53-containing cells, whereas it reduced BRCA1 levels by more than 90% in p53-null cells (Fig. 3C). To assess whether the effect of p53 was mediated at the level of transcription of the BRCA1 gene, MCF7/pSUPER and MCF7/pSUPER-p53

Fig. 4. Immunofluorescence staining of BRCA1 with anti-BRCA1 C-20. MCF7 (A–C) and MCF7/Cav-1 (D–F) cells were grown on glass coverslips and maintained as described in Section 2. Following fixation in 2% formaldehyde, immunostaining was performed using a polyclonal BRCA1 antibody (C-20), diluted in 0.1% Saponine with 1.5% FCS in PBS. Cells were permeabilized in the presence of 0.1% Saponine with 1.5% FCS diluted in PBS and incubated with Cy TM3 conjugated Affinitipure, goat anti-rabbit IgG [red; (C,F)]. Nuclear localization was assessed by DAPI staining [blue; (B,E)]. The composite images of two-color staining are presented in A and D. The scale bars denote 20 lm.

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Fig. 5. Immunofluorescence staining of BRCA1 with anti-BRCA1 Ab-1.MCF7 (A–C) and MCF7/Cav-1(D–F) cells were grown on glass coverslips and maintained as described in Section 2. Cells were processed as described in the legend to Fig. 4 and incubated with AlexaFlour 488, goat antimouse IgG [green; (C,F)]. Nuclear localization was assessed by DAPI staining [blue; (B,E)]. The composite images of two-color staining are presented in A and D.

cells were transiently cotransfected with the Cav-1 expression vector (or empty pcDNA3), along with the full-length BRCA1 promoter-luciferase reporter vector (pGL3-I), or empty pGL3basic promoter plasmid as a negative control. As shown in Fig. 3D, Cav-1 induced a 174.73 ± 0.77% increase in BRCA1 promoter activity in MCF7 pSUPER cells, whereas it had a small inhibitory effect in p53-depleted cells (86.87 ± 3.33% decrease). These results suggest that the stimulatory effect of Cav-1 on BRCA1 promoter activity is p53-dependent. Cav-1 had no effect on the empty pGL3-basic promoter (data not shown).

3.4. Effect of Cav-1 on BRCA1 subcellular localization Immunofluorescent staining of BRCA1 was performed to investigate whether Cav-1 can modulate BRCA1 subcellular localization. To this end, MCF7 and MCF7/Cav-1 cells were seeded on glass cover slips and BRCA1 distribution was analyzed using a polyclonal anti-BRCA1 antibody (C-20) raised against the C-terminal portion of human BRCA1 (Fig. 4) and a monoclonal anti-BRCA1 antibody (Ab-1) raised against the N-terminus of BRCA1 (Fig. 5). In MCF7 cells, BRCA1 exhibited a typical immunoreactivity pattern which consisted of a punctuated nuclear staining, with cytoplasmic staining (A–C) [32]. In contrast, in MCF7/Cav-1 cells BRCA1 was predominantly localized in the cytoplasm (D–F). The DAPI counter staining confirmed the identity of nuclei in both cell lines (B,E). Similar results were obtained with a polyclonal antiBRCA1 antibody (D-20) raised against the N-terminal portion of human BRCA1 (data not shown).

4. Discussion Tumor suppressor BRCA1 has been shown to be involved in the regulation of a number of biological processes in various cellular and animal models [27]. Cav-1, a major component of the caveolar membrane, modulates the activity of several signaling molecules, including G proteins, members of the MAPK cascade, and others [39,40]. The present study was designed to examine the potential regulation of BRCA1 gene expression by Cav-1. The rationale for our emphasis on a functional interaction between these two proteins lies in the fact that Cav-1 and BRCA1 were identified as tumor suppressors with important roles in the etiology of breast cancer. Little information, however, is available regarding the cellular factors involved in regulation of BRCA1 gene expression. This study identifies the BRCA1 gene as a novel downstream target for Cav-1 action in breast cancer cells. Semiquantitative RT-PCR analysis of BRCA1 mRNA levels in Cav-1-overexpressing MCF7 cells revealed a 2.7-fold increase in transcript abundance compared to native MCF7 cells. This increase was associated with an elevation in endogenous BRCA1 protein levels, although the extent of BRCA1 increase at the protein level was lower than that seen at the mRNA level. Results of transient transfection experiments using a full-length BRCA1 promoter reporter construct are consistent with the notion that the enhanced BRCA1 mRNA values in MCF7/Cav-1 cells are the result of augmented BRCA1 gene transcription. The Cav-1-induced transcription factors that are directly responsible for BRCA1 gene transactivation, however, remain to be identified.

C. Glait et al. / FEBS Letters 580 (2006) 5268–5274

BRCA1 was previously shown to be present predominantly in the nucleus, although it can shuttle between the nucleus and cytoplasm [41]. Our results demonstrate a clear difference in BRCA1 subcellular localization between Cav-1-expressing and Cav-1-null cells. Specifically, MCF7 cells exhibited a typical nuclear dotted pattern of expression whereas MCF7/Cav-1 cells displayed mainly a cytoplasmic distribution. Mislocalization of BRCA1 in the cytoplasm of Cav-1-expressing cells may result from aberrations in BRCA1 nuclear transport. BRCA1 includes two NLS in the central portion of the molecule [29,30], although BRCA1 can be also targeted to the nucleus in an NLS-independent manner [42]. In addition, the BRCA1 RING domain, located in the N-terminal portion of the protein, participates in nuclear targeting via interaction with BARD1 [43,44]. BARD1 binding was shown to mask the BRCA1 nuclear export signal, thus leading to nuclear accumulation of BRCA1 [45]. The BRCA1/BARD1 complex is also known to possess auto-ubiquitination activity [46–48]. It has been suggested that upon dissociation from BARD1, ubiquitinated BRCA1 is exported to the cytoplasm and degraded by the proteasome complex [42]. We may speculate that the relatively small increase in BRCA1 protein levels in MCF7/Cav-1 cells, in comparison to the more pronounced increase in mRNA levels, results from the Cav-1-induced cytoplasmic sequestration of BRCA1. The net consequence of this event is an enhanced cytoplasmic degradation of BRCA1. Various lines of evidence suggest that BRCA1 and p53 act synergistically in the control of cellular proliferation. Tumor suppressor p53 has a central role in the control of the apoptotic process and in the regulation of the DNA damage response. Specifically: (i) BRCA1 and p53 were shown to physically interact [37]; (ii) BRCA1 stimulated promoter constructs containing specific p53-response elements [49]; (iii) p53 regulated BRCA1 expression in response to DNA damage at the transcriptional level [33]; and (iv) BRCA1 is a potent activator of p53-dependent transcription [37]. Consistent with these findings, we show here that upregulation of BRCA1 promoter activity by Cav-1 requires an intact p53 signaling pathway. In summary, we have demonstrated that a novel mechanism of action of Cav-1 in breast cancer cells involves the p53dependent transcriptional activation of the BRCA1 gene, with ensuing increase in BRCA1 mRNA levels. In addition, Cav-1 expression leads to BRCA1 exclusion from the nucleus. Elevated BRCA1 levels may account for the Cav-1-induced reduction in proliferation rate and anchorage-independent growth in MCF7 cells. Aberrant regulation and/or expression of Cav-1 may lead to pathologic dysregulation of BRCA1 levels and localization, with potentially important consequences in breast cancer development. Acknowledgements: This work was performed in partial fulfillment of the requirements for a M.Sc. degree by Chen Glait in the Sackler Faculty of Medicine, Tel Aviv University. We thank Drs. M.E. Goldsmith and R. Agami for providing cell lines. We thank Drs. K. Hirschberg, I. Tzarfati, and R. Sarfstein (Tel Aviv University), and Mrs. L. Tencer (Weizmann Institute of Science) for assistance with some of the experiments.

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