BRCA1 Interaction with Human Papillomavirus Oncoproteins
2005; Elsevier BV; Volume: 280; Issue: 39 Linguagem: Inglês
10.1074/jbc.m505124200
ISSN1083-351X
AutoresYiyu Zhang, Saijun Fan, Qinghui Meng, Yongxian Ma, Pragati Katiyar, Richard Schlegel, Eliot M. Rosen,
Tópico(s)Ovarian cancer diagnosis and treatment
ResumoPreviously, we reported that BRCA1 strongly represses the transcriptional activity of estrogen receptor-α (ER-α) in human breast and prostate cancer cells but only weakly inhibits ER-α in cervical cancer cells. We now report that introduction of the human papillomavirus E7 or E6 oncogenes into human papillomavirus-negative cells rescues the BRCA1 repression of ER-α activity and that the E7 and E6 oncoproteins interact directly with BRCA1 in vitro and associate with BRCA1 in vivo in cultured cells. This interaction involves at least two contact points on BRCA1, one within an N-terminal site shown previously to interact with ER-α and the other in a C-terminal region of BRCA1 containing the first BRCA1 C-terminal domain. Point mutations within the zinc finger domains of E7 and E6 inactivated the binding to the N terminus of BRCA1 and reduced their ability to rescue BRCA1 inhibition of ER-α. E6 and E7 also antagonized the ability of BRCA1 to inhibit c-Myc E-box-mediated transactivation and human telomerase reverse transcriptase promoter activity, in a manner dependent upon the zinc finger domains. Finally, the ability of E6 and E7 to antagonize BRCA1 did not involve proteolytic degradation of BRCA1. These findings suggest functional interactions of BRCA1 with E7 and E6. The potential significance of these findings is discussed. Previously, we reported that BRCA1 strongly represses the transcriptional activity of estrogen receptor-α (ER-α) in human breast and prostate cancer cells but only weakly inhibits ER-α in cervical cancer cells. We now report that introduction of the human papillomavirus E7 or E6 oncogenes into human papillomavirus-negative cells rescues the BRCA1 repression of ER-α activity and that the E7 and E6 oncoproteins interact directly with BRCA1 in vitro and associate with BRCA1 in vivo in cultured cells. This interaction involves at least two contact points on BRCA1, one within an N-terminal site shown previously to interact with ER-α and the other in a C-terminal region of BRCA1 containing the first BRCA1 C-terminal domain. Point mutations within the zinc finger domains of E7 and E6 inactivated the binding to the N terminus of BRCA1 and reduced their ability to rescue BRCA1 inhibition of ER-α. E6 and E7 also antagonized the ability of BRCA1 to inhibit c-Myc E-box-mediated transactivation and human telomerase reverse transcriptase promoter activity, in a manner dependent upon the zinc finger domains. Finally, the ability of E6 and E7 to antagonize BRCA1 did not involve proteolytic degradation of BRCA1. These findings suggest functional interactions of BRCA1 with E7 and E6. The potential significance of these findings is discussed. Mutations of the breast cancer susceptibility gene 1 (BRCA1) 2The abbreviations used are: BRCA1, breast cancer susceptibility gene 1; BRCA2, breast cancer susceptibility gene 2; BRCT, BRCA1 C-terminal domain; E2, 17β-estradiol; ER-α, estrogen receptor-α; ERE-TK-Luc, estrogen-responsive luciferase reporter plasmid; GST, glutathione-sulfotransferase; hTERT, human telomerase reverse transcriptase (catalytic subunit of telomerase); HPV, human papillomavirus; IP, immunoprecipitation; IVT, in vitro transcribed and translated; LXCXE, consensus retinoblastoma family protein-binding site; RB1, retinoblastoma susceptibility gene 1; wt, wild type; DMEM, Dulbecco's modified Eagle's medium; HPV, human papillomavirus; siRNAs, small interfering RNAs.2The abbreviations used are: BRCA1, breast cancer susceptibility gene 1; BRCA2, breast cancer susceptibility gene 2; BRCT, BRCA1 C-terminal domain; E2, 17β-estradiol; ER-α, estrogen receptor-α; ERE-TK-Luc, estrogen-responsive luciferase reporter plasmid; GST, glutathione-sulfotransferase; hTERT, human telomerase reverse transcriptase (catalytic subunit of telomerase); HPV, human papillomavirus; IP, immunoprecipitation; IVT, in vitro transcribed and translated; LXCXE, consensus retinoblastoma family protein-binding site; RB1, retinoblastoma susceptibility gene 1; wt, wild type; DMEM, Dulbecco's modified Eagle's medium; HPV, human papillomavirus; siRNAs, small interfering RNAs. (chromosome 17q21) are linked to a high risk for breast and ovarian cancers in hereditary early onset breast and breast-ovarian cancer families (1Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W. Bell R. Rosenthal J. Hussey C. Tran T. McClure M. Frye C. Hattier T. Phelps R. Haugen-Strano A. Katcher H. Yakumo K. Gholami Z. Shaffer D. Stone S. Bayer S. Wray C. Bogden R. Dayananth P. Ward J. Tonin P. Narod S. Bristow P.K. Norris F.H. Helvering L. Morrison P. Rosteck P. Lai M. Barrett J.C. Lewis C. Neuhausen S. Cannon-Albright L. Goldgar D. Wiseman R. Kamb A. Skolnick M.H. Science. 1994; 266: 66-71Crossref PubMed Scopus (5289) Google Scholar, 2Rowell S. Newman B. Boyd J. King M.C. Am. J. Hum. Genet. 1994; 55: 861-865PubMed Google Scholar). These mutations also confer and increased risk for these cancer types in Ashkenazi Jewish women unselected for a family history of cancer (3Struewing J.P. Hartge P. Wacholder S. Baker S.M. 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An earlier and smaller study of cancer incidence in the relatives of BRCA1 and BRCA2 mutation carriers revealed about a 4-fold increased risk of cervical cancer in BRCA2-associated families, although the risk in BRCA1 families was not similarly elevated (9Johannsson O. Loman N. Moller T. Kristoffersson U. Borg A. Olsson H. Eur. J. Cancer. 1999; 35: 1248-1257Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Most interestingly, loss of heterozygosity at chromosome 17q, a site that contains the BRCA1 gene, appears to be a common event in cervical cancer (10Miyai K. Furugen Y. Matsumoto T. Iwabuchi K. Hirose S. Kinoshita K. Fujii H. Gynecol. Oncol. 2004; 94: 115-120Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Previously, we found that the overexpression of BRCA1 inhibits the estrogen (E2)-induced transcriptional activity of the estrogen receptor-α (ER-α) and inhibits E2-stimulated gene expression (11Fan S. Wang J. Yuan R. Ma Y. Meng Q. Erdos M.R. Pestell R.G. Yuan F. Auborn K.J. Goldberg I.D. Rosen E.M. Science. 1999; 284: 1354-1356Crossref PubMed Scopus (418) Google Scholar, 12Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Oncogene. 2001; 20: 77-87Crossref PubMed Scopus (228) Google Scholar, 13Xu J. Fan S. Rosen E.M. Endocrinology. 2005; 146: 2031-2047Crossref PubMed Scopus (44) Google Scholar). Most unexpectedly, the ability of BRCA1 to repress ER-α activity was found to be cell type-specific. Thus, BRCA1 virtually abolished E2-stimulated ER-α activity in four of four human breast cancer (MCF-7, T47D, MDA-MB-231, and MDA-MB-453) and four of four human prostate cancer (DU-145, LNCaP, PC-3, and TsuPr-1 (now known to be derived from a bladder cancer)) cell lines; however, BRCA1 caused only a modest or no reduction of ER-α activity in four of four human cervical cell lines (HeLa, SiHa, CaSki, and C33A) (11Fan S. Wang J. Yuan R. Ma Y. Meng Q. Erdos M.R. Pestell R.G. Yuan F. Auborn K.J. Goldberg I.D. Rosen E.M. Science. 1999; 284: 1354-1356Crossref PubMed Scopus (418) Google Scholar). Moreover, BRCA1 overexpression inhibited the E2-stimulated activity of activation function-2, the ligand-inducible transcriptional activation domain of ER-α in breast and prostate cancer cells but not in cervical cancer cell lines (11Fan S. Wang J. Yuan R. Ma Y. Meng Q. Erdos M.R. Pestell R.G. Yuan F. Auborn K.J. Goldberg I.D. Rosen E.M. Science. 1999; 284: 1354-1356Crossref PubMed Scopus (418) Google Scholar, 14Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Cancer Res. 2002; 62: 141-151PubMed Google Scholar). Three of the four cervical cancer cell lines that we studied (HeLa, SiHa, and Caski) are known to contain integrated oncogenic human papillomavirus (HPV) genomes: HPV-16 for SiHa and CaSki and HPV-18 for HeLa (15Meissner J.D. J. Gen. Virol. 1999; 80: 1725-1733Crossref PubMed Scopus (158) Google Scholar, 16Liang X.H. Mungal S. Ayscue A. Meissner J.D. Wodnicki P. Hockenbery D. Lockett S. Herman B. J. Cell. Biochem. 1995; 57: 509-521Crossref PubMed Scopus (69) Google Scholar). The two major HPV oncoproteins, E7 and E6, function in part by inactivating host cell tumor suppressor proteins, retinoblastoma 1 (RB1) (and other retinoblastoma family proteins) and p53 (17Scheffner M. Whitaker N.J. Semin. Cancer Biol. 2003; 13: 59-67Crossref PubMed Scopus (137) Google Scholar). Taken together, these findings raised the possibility that HPV proteins may functionally inactivate BRCA1 in cervical cancer cells. The purpose of this study was to evaluate the hypothesis that the HPV oncoproteins E6 and E7 can interact with and inactivate the function of BRCA1. Human breast cancer (MCF-7 and T47D), prostate cancer (DU-145), and cervical cancer (SiHa, Caski, and HeLa) cell lines and 293T human embryonal kidney cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured as described before (11Fan S. Wang J. Yuan R. Ma Y. Meng Q. Erdos M.R. Pestell R.G. Yuan F. Auborn K.J. Goldberg I.D. Rosen E.M. Science. 1999; 284: 1354-1356Crossref PubMed Scopus (418) Google Scholar, 12Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Oncogene. 2001; 20: 77-87Crossref PubMed Scopus (228) Google Scholar, 13Xu J. Fan S. Rosen E.M. Endocrinology. 2005; 146: 2031-2047Crossref PubMed Scopus (44) Google Scholar, 14Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Cancer Res. 2002; 62: 141-151PubMed Google Scholar). Briefly, the cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 or 10% (v/v) fetal calf serum, l-glutamine (5 mm), nonessential amino acids (5 mm), penicillin (100 units/ml), and streptomycin (100 μg/ml). All cell culture reagents were obtained from BioWhittaker, Walkersville, MD). Expression Vectors for GST Proteins—The GST-E7 (HPV16, wild-type, full-length) expression plasmid was a gift from Dr. Karl Munger (Dept. of Pathology, Harvard Medical School, Boston). The GST-E6 (HPV16, wild-type, full-length) plasmid was a gift from Dr. Peter M. Howley (Dept. of Pathology, Harvard Medical School) (18Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1969) Google Scholar). The GST-E7-(2–38), -(16–98), and -(38–98) vectors and the GST-E6-(2–40), -(16–83), and -(80–151) vectors were generated by PCR cloning. Briefly, BamHI and EcoRI digestion sites were designed on the 5′ and 3′ primers, respectively; and the BamHI and EcoRI double digestion products were inserted into BamHI and EcoRI site of the pGEX2T vector (Amersham Biosciences). The GST-E7 C91G expression vector, which encodes a full-length chimeric E7 protein with an inactivating point mutation of the C-terminal zinc finger domain, was provided by Dr. Tony Kouzarides (Wellcome/CR UK Gurdon Institute, Cambridge University, Cambridge, UK) (19Brehm A. Nielsen S.J. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. EMBO J. 1999; 18: 2449-2458Crossref PubMed Scopus (270) Google Scholar). The GST-E6 C66,136G vector, which encodes a full-length chimeric E6 protein with an inactivating point mutation in both zinc finger domains, was generated by PCR cloning, using the pBS-E6 C66,136G plasmid (a gift from Dr. Denise A. Galloway, Fred Hutchinson Cancer Research Center, Seattle, WA (20Foster S.A. Demers G.W. Etscheid B.G. Galloway D.A. J. Virol. 1994; 68: 5698-5705Crossref PubMed Google Scholar)) as a template. The BamHI and EcoRI double digestion product was inserted into the BamHI and EcoRI site of pGEX2T vector. Expression vectors for GST-BRCA1 protein fragments corresponding to BRCA1 amino acids 1–324, 260–553, 502–802, 758–1064, 1005–1313, and 1314–1863 were kindly provided by Dr. Toru Ouchi (Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York). These constructs have been described earlier (21Ouchi T. Lee S.W. Ouchi M. Aaronson S.A. Horvath C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5208-5213Crossref PubMed Scopus (187) Google Scholar). Expression Vectors for in Vitro Translation and/or Expression within Mammalian Cells—The wild-type BRCA1 expression vector (wtBRCA1) was created by cloning the BRCA1 cDNA into the pcDNA3 vector (Invitrogen) by using artificially engineered 5′ HindIII and 3′ NotI sites (22Fan S. Wang J.A. Yuan R.Q. Ma Y.X. Meng Q. Erdos M.R. Brody L.C. Goldberg I.D. Rosen E.M. Oncogene. 1998; 16: 3069-3082Crossref PubMed Scopus (99) Google Scholar). cDNAs for BRCA1-(1–1313), -(1–771), -(1–302), -(34–300), -(67–300), -(101–300), and -(134–300) within the pcDNA3 or pCMV-Tag2 vector (Stratagene, La Jolla, CA) have been described earlier (12Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Oncogene. 2001; 20: 77-87Crossref PubMed Scopus (228) Google Scholar, 23Fan S. Yuan R. Ma Y.X. Meng Q. Goldberg I.D. Rosen E.M. Oncogene. 2001; 20: 8215-8235Crossref PubMed Scopus (66) Google Scholar, 24Ma Y.X. Tomita Y. Fan S. Wu K. Tong Y. Zhao Z. Song L.N. Goldberg I.D. Rosen E.M. Oncogene. 2005; 24: 1831-1846Crossref PubMed Scopus (47) Google Scholar). The BRCA1-(1–320) cDNA fragment plasmid was generated by PCR cloning, and the BamHI and XbaI double digestion product was inserted into the BamHI and XbaI site of the pcDNA3 vector. The BRCA1-(310–806), -(802–1314), -(1314–1863), and -(1532–1749) cDNA fragments were generated by PCR cloning, followed by BamHI and EcoRI double digestion and insertion into the BamHI and EcoRI site of pcDNA3 vector. The pcDNA3 FLAG-E7 expression vector was generously provided by Dr. Tony Kouzarides. The pcDNA3-E6 expression vector has been described earlier (25Peng S. Ji H. Trimble J. He L. Tsai Y.-C. Yeatermeyer J. Boyd D.A.K. Hung C.-F. Wu T.-C. J. Virol. 2004; 78: 8468-8476Crossref PubMed Scopus (103) Google Scholar). The p3XFLAG-E6 and p3XFLAG-E6-(C66,136G) expression vectors were generated by PCR cloning of the FLAG-E6 cDNAs, followed by HindIII and XbaI double digestion and insertion into the HindIII and XbaI site of the p3XFLAG vector (Sigma). The FLAG-E7-(C91G) expression plasmid was created in a similar manner. The correct insertion of the cDNAs was confirmed by sequencing of each of the subcloned plasmids. The ER-α expression vector pCMV-ER-α was used to express ER-α in transient transfection assays of estrogen receptor transcriptional activity (12Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Oncogene. 2001; 20: 77-87Crossref PubMed Scopus (228) Google Scholar). Expression vectors encoding the wt adenovirus E1A-(243R) protein and various mutant or truncated E1A-(243R) proteins (including single point mutations RG2 and 928m; the double point mutants RG2/928m and YH47/928m; and the deletion mutants Δ 15–35 and Δ 38–67) were provided by Dr. Richard G. Pestell (Lombardi Comprehensive Cancer Center, Georgetown University, Washington, D. C.) (26Pestell R.G. Albanese C. Lee R.J. Watanabe G. Moran E. Johnson J. Jameson J.L. Cell Growth & Differ. 1996; 7: 1337-1344PubMed Google Scholar). Expression vectors encoding the wild-type SV40 large T oncoprotein (SV40T) and a mutant protein defective for RB family protein binding (mut-SV40T) (27Zhu J. Rice P.W. Gorsch L. Abate M. Cole C.N. J. Virol. 1992; 66: 2780-2791Crossref PubMed Google Scholar) were also provided by Dr. Pestell. Reporters—The estrogen-responsive reporter ERE-TK-Luc is composed of the vitellogenin A2 estrogen-responsive enhancer (ERE), controlling a minimal thymidine kinase promoter (TK81) and luciferase, in plasmid pGL2 (28Henttu P.M. Kalkhoven E. Parker M.G. Mol. Cell. Biol. 1997; 17: 1832-1839Crossref PubMed Scopus (192) Google Scholar). Assays of ER-α transcriptional activity utilizing the ERE-TK-Luc reporter are described below. The E-box-Luc reporter contains a canonical c-Myc E-box upstream of a minimal promoter and luciferase (29Xiong J. Fan S. Meng Q. Schramm L. Wang C. Bouzahza B. Zhou J. Zafonte B. Goldberg I.D. Haddad B.R. Pestell R.G. Rosen E.M. Mol. Cell. Biol. 2003; 23: 8668-8690Crossref PubMed Scopus (77) Google Scholar). Its activity is stimulated by c-Myc and inhibited by the c-Myc/Max repressor Mad1 (29Xiong J. Fan S. Meng Q. Schramm L. Wang C. Bouzahza B. Zhou J. Zafonte B. Goldberg I.D. Haddad B.R. Pestell R.G. Rosen E.M. Mol. Cell. Biol. 2003; 23: 8668-8690Crossref PubMed Scopus (77) Google Scholar). The hTERT-Luc reporter consists of the core human telomerase reverse transcriptase (hTERT) promoter upstream of the luciferase gene, within the pGL3 plasmid (29Xiong J. Fan S. Meng Q. Schramm L. Wang C. Bouzahza B. Zhou J. Zafonte B. Goldberg I.D. Haddad B.R. Pestell R.G. Rosen E.M. Mol. Cell. Biol. 2003; 23: 8668-8690Crossref PubMed Scopus (77) Google Scholar). Subconfluent proliferating cells in 24-well dishes were incubated overnight with 0.25 μg of each indicated vector in serum-free DMEM containing Lipofectamine™ (Invitrogen). The total transfected DNA was kept constant by addition of the appropriate control vectors. The cells were washed, incubated in phenolphthalein-free DMEM containing 5% charcoal-stripped serum (obtained from the Tissue Culture Core Facility of the Lombardi Comprehensive Cancer Center) (0.2 ml per well) ± 17β-estradiol (E2, 1 μm or 10 nm, as indicated) for 24 h, and harvested for luciferase assays. To control for transfection efficiency, plasmid pRSV-β-gal was co-transfected to allow normalization of luciferase values to β-galactosidase activity in the same sample. Values are means ± S.E. of four replicate wells and are representative of two or more independent experiments. To study the association of the endogenous BRCA1 and oncoprotein E7, proliferating SiHa cells at about 80% of confluence in 100-mm plastic dishes were harvested; and whole cell extracts were prepared as described before (12Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Oncogene. 2001; 20: 77-87Crossref PubMed Scopus (228) Google Scholar, 14Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Cancer Res. 2002; 62: 141-151PubMed Google Scholar, 24Ma Y.X. Tomita Y. Fan S. Wu K. Tong Y. Zhao Z. Song L.N. Goldberg I.D. Rosen E.M. Oncogene. 2005; 24: 1831-1846Crossref PubMed Scopus (47) Google Scholar), using RIPA buffer. Each IP was carried out as described before by using 10 μg of antibody (anti-BRCA1 (I-20, Santa Cruz Biotechnology, Santa Cruz, CA), anti-E7 (ED-17, Santa Cruz Biotechnology), or nonimmune rabbit or mouse IgG (negative control)) and 500 μg of total cell extract protein. The precipitated proteins were collected using protein A/G Plus-agarose beads (Santa Cruz Biotechnology) and eluted using boiling Laemmli sample buffer. The eluted proteins were then subjected to Western blotting to detect the BRCA1 or E7 proteins, as described below. To study the interaction of BRCA1 with E6, subconfluent proliferating 293T cells were transfected overnight with the p3XFLAG-E6 and wtBRCA1 expression vectors (see above) using LipofectamineTM, washed, post-incubated for 24 h to allow gene expression, harvested, and then immunoprecipitated using anti-BRCA1 (as above) or nonimmune rabbit IgG (control). The precipitated proteins were collected using protein A/G Plus-agarose beads or anti-FLAG M2 affinity gel (Sigma), washed, eluted, and subjected to Western blotting to detect the BRCA1 protein or the FLAG epitope (see below). Western blotting was carried out as described before (23Fan S. Yuan R. Ma Y.X. Meng Q. Goldberg I.D. Rosen E.M. Oncogene. 2001; 20: 8215-8235Crossref PubMed Scopus (66) Google Scholar, 24Ma Y.X. Tomita Y. Fan S. Wu K. Tong Y. Zhao Z. Song L.N. Goldberg I.D. Rosen E.M. Oncogene. 2005; 24: 1831-1846Crossref PubMed Scopus (47) Google Scholar). The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulose membranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution). As a control, nonprecipitated lysates (50 μg of cell protein) were blotted on the same gels. The blotted protein bands were visualized using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences), with colored markers (BioRad) as size standards. In a separate experiment, cells were transfected overnight with wild-type or mutant FLAG-E6 or FLAG-E7, post-incubated for 24 h to allow gene expression, and Western-blotted (50 μg of total cell protein per lane) to detect BRCA1 (C-20 antibody), E6 protein (anti-HPV16/18 E6, Abcam Ltd.), FLAG-E7 (M2, Sigma), p53 (polyclonal antibody 240, Santa Cruz Biotechnology) (which detects both wild-type and mutant p53), RB1 (C-15 antibody, Santa Cruz Biotechnology), and α-actin (I-19 antibody, Santa Cruz Biotechnology) (control for loading and transfer). GST bead assays were performed essentially as described earlier (12Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Oncogene. 2001; 20: 77-87Crossref PubMed Scopus (228) Google Scholar, 14Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Cancer Res. 2002; 62: 141-151PubMed Google Scholar, 24Ma Y.X. Tomita Y. Fan S. Wu K. Tong Y. Zhao Z. Song L.N. Goldberg I.D. Rosen E.M. Oncogene. 2005; 24: 1831-1846Crossref PubMed Scopus (47) Google Scholar). In vitro translated (IVT) proteins were prepared by in vitro transcription and translation, using the T7 promoter of the pcDNA3 vector or the T3 promoter of the pCMV-Tag2B vector. The proteins were labeled using [35S]methionine (Amersham Biosciences) or Transend™ tRNA (Promega Corp., Madison, WI), and in vitro transcription-translation was carried out using the TnT-coupled rabbit reticulocyte lysate system (Promega), according to the manufacturer's instructions. The GST fusion proteins were generated from cDNAs cloned into the pGEX2T vector, expressed in Escherichia coli, and purified by affinity chromatography using glutathione-Sepharose (Amersham Biosciences). The source or construction of the expression vectors for various GST-BRCA1, GST-E7, and GST-E6 proteins is described above. Labeled IVT proteins were incubated with GST protein (negative control) or GST fusion proteins for 4 h at 4°C, recovered using GSH-agarose beads, eluted in boiling sample buffer, and analyzed by SDS-PAGE autoradiography. In all GST capture assays, the IVT input lanes show 10% of the protein quantity used in the GST capture assays. All experiments included a lane corresponding to capture by beads coated with GST alone, as a negative control. The GST fusion proteins were visualized by Western blotting, using anti-GST mouse monoclonal antibody 27-4577-01 (Amersham Biosciences, 1:5000). Additional details relevant to specific experiments are provided in the text or figure legends. The BRCA1 and corresponding control (scrambled sequence) siRNAs have been described earlier (29Xiong J. Fan S. Meng Q. Schramm L. Wang C. Bouzahza B. Zhou J. Zafonte B. Goldberg I.D. Haddad B.R. Pestell R.G. Rosen E.M. Mol. Cell. Biol. 2003; 23: 8668-8690Crossref PubMed Scopus (77) Google Scholar). All siRNAs were chemically synthesized by Dharmacon, Inc. The sequences used to synthesize the siRNAs were as follows: BRCA1 siRNA, 5′-AATGCCAAAGTAGCTAATGTA-3′, and control siRNA, 5′-CGATAGATACACAGATTGAAT-3′. For siRNA treatments, subconfluent proliferating cells were transfected with 50 nm of siRNA using the siPORT Amine transfection reagent (Ambion). The maximal reduction of protein levels required a 72-h incubation with the siRNA. Prior studies established that under these conditions, none of the siRNAs caused cytotoxicity, based on cell morphology and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Where appropriate, statistical comparisons were made using the two-tailed Student's t test. Rescue (Reversal) of the BRCA1-mediated Repression of ER-α Transcriptional Activity by HPV Oncoproteins E7 and E6—We tested the ability of several different DNA tumor virus-encoded transforming oncoproteins to rescue (reverse) the BRCA1-mediated repression of ER-α activity, including HPV oncoproteins E6 and E7, the adenovirus E1A oncoprotein, and the SV40 large T protein. Schematic diagrams showing the domain structure of these proteins are provided in Fig. 1A. We utilized a standard transient transfection assay of ER-α transcriptional activity using an estrogen-responsive reporter, ERE-TK-Luc (12Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Oncogene. 2001; 20: 77-87Crossref PubMed Scopus (228) Google Scholar, 14Fan S. Ma Y.X. Wang C. Yuan R.Q. Meng Q. Wang J.A. Erdos M. Goldberg I.D. Webb P. Kushner P.J. Pestell R.G. Rosen E.M. Cancer Res. 2002; 62: 141-151PubMed Google Scholar, 24Ma Y.X. Tomita Y. Fan S. Wu K. Tong Y. Zhao Z. Song L.N. Goldberg I.D. Rosen E.M. Oncogene. 2005; 24: 1831-1846Crossref PubMed Scopus (47) Google Scholar). As illustrated in Fig. 1B, in the absence of E7 or E6, exogenous wtBRCA1 reduced the estrogen (E2)-stimulated reporter activity to less than 1% of the control value (p < 0.001, two-tailed t test) in human breast (MCF-7 and T47D) and prostate (DU-145) cancer cells, whereas the empty pcDNA3 vector had little or no effect on the reporter activity. In contrast, expression vectors encoding the E7 and E6 oncoproteins from HPV-16 (an oncogenic HPV strain) rescued the wtBRCA1-mediated repression of ER-α activity in HPV-negative human breast and prostate cancer cell lines (Fig. 1B). Besides E7 and E6, several other DNA viral oncoproteins were also able to rescue the BRCA1 inhibition of ER-α transcriptional activity. Thus, in studies of DU-145 human prostate cancer and T47D breast cancer cells, the adenovirus E1A-(243R) and SV40 large T oncogenes also rescued the BRCA1 inhibition of ER-α activity (p < 0.001) (Fig. 1, C and D, respectively). The E1A-(243R) transforming protein is the product of an alternatively spliced mRNA, the full-length form of which encodes the E1A-(289R) protein (30Hurwitz D.R. Chinnadurai G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 163-167Crossref PubMed Scopus (32) Google Scholar). E1A-(243R) differs from E1A-(289R) in that E1A-(243R) is missing a conserved region (CR3) that is present in E1A-(289R). Studies of a small series of mutant E1A-(243R) genes revealed that expression vectors encoding proteins with an N-terminal mutation or deletion (RG2, Δ38–67, RG2/928m, and Δ15–35) failed to rescue the BRCA1 repression, whereas those containing several other mutations (928m and YH47/928m) retained the ability to reverse the BRCA1-mediated repression of ER-α activity (p < 0.001) (Fig. 1C). E1A-(928m) encodes a mutant protein with a defective retinoblastoma (RB) protein binding domain, suggesting that the RB binding domain is dispensable for the rescue of BRCA1-mediated repression. Expression vectors encoding wild-type SV40 large T and a mutant with a defective RB binding domain both rescued the BRCA1 repression (p < 0.001) (Fig. 1D), again suggesting that interaction with RB family proteins (RB1, p107, and p130) is not required for rescue. For reference, Fig. 1E shows the effect of wtBRCA1 on ER-α signaling in three human cervical cancer cell lines, each of which contains an oncogenic HPV genome (HPV-16 or HPV-18) as follows: CaSki, SiHa, and HeLa. As compared with the HPV-negative breast and prostate cancer cell lines for which wtBRCA1 caused a reduction in estrogen-stimulated ER-α activity t
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