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• W2022176772 abstract "The breast cancer predisposition genes,BRCA1 and BRCA2, are responsible for the vast majority of hereditary breast cancer. Although BRCA2functions to help the cell repair double-stranded DNA breaks, the function of BRCA1 remains enigmatic. Here, we develop a human genetic system to study the role of BRCA1 in oxidative DNA damage. We show that human cancer cells containing mutated BRCA1 are hypersensitive to ionizing radiation. This hypersensitivity can be reversed by the expression of forms ofBRCA1 that are not growth suppressing. Reversal of hypersensitivity requires the ring finger of BRCA1, its transactivation domain, and its BRCT domain. Lastly, we show that unlike BRCA2, BRCA1 does not function in the repair of double-stranded DNA breaks. Instead, it functions in transcription-coupled DNA repair (TCR). TCR ability correlated with radioresistance as cells containing BRCA1 showed both increased TCR and radioresistance, whereas cells withoutBRCA1 showed decreased TCR and radiosensitivity. These findings give physiologic significance to the interaction ofBRCA1 with the basal transcription machinery. The breast cancer predisposition genes,BRCA1 and BRCA2, are responsible for the vast majority of hereditary breast cancer. Although BRCA2functions to help the cell repair double-stranded DNA breaks, the function of BRCA1 remains enigmatic. Here, we develop a human genetic system to study the role of BRCA1 in oxidative DNA damage. We show that human cancer cells containing mutated BRCA1 are hypersensitive to ionizing radiation. This hypersensitivity can be reversed by the expression of forms ofBRCA1 that are not growth suppressing. Reversal of hypersensitivity requires the ring finger of BRCA1, its transactivation domain, and its BRCT domain. Lastly, we show that unlike BRCA2, BRCA1 does not function in the repair of double-stranded DNA breaks. Instead, it functions in transcription-coupled DNA repair (TCR). TCR ability correlated with radioresistance as cells containing BRCA1 showed both increased TCR and radioresistance, whereas cells withoutBRCA1 showed decreased TCR and radiosensitivity. These findings give physiologic significance to the interaction ofBRCA1 with the basal transcription machinery. BRCA1 and BRCA2, the breast cancer 1 and 2 genes, are responsible for over 90% of hereditary breast cancers (1Futreal P.A. Liu Q. Shattuck-Eidens D. Cochran C. Harshmann K. Tavtigian S. Bennett L.M. Haugen-Strano A. Swensen J. Miki Y. et al.Science. 1994; 266: 120-122Crossref PubMed Scopus (1137) Google Scholar, 2Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshmann K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W. et al.Science. 1994; 266: 66-71Crossref PubMed Scopus (5289) Google Scholar, 3Wooster R. Bignell G. Lancaster J. Swift S. Seal S. Mangion J. Collins N. Gregory S. Gumbs C. Micklem G. Nature. 1995; 378: 789-792Crossref PubMed Scopus (2943) Google Scholar, 4Struewing J.P. Hartge P. Wacholder S. Baker S.M. Berlin M. McAdams M. Timmerman M.M. Brody L.C. Tucker M.A. N. Engl. J. Med. 1997; 336: 1401-1408Crossref PubMed Scopus (1981) Google Scholar). Although BRCA2 has been shown to affect the repair of double-stranded DNA breaks (Refs. 5Sharan S.K. Morimatsu M. Albrecht U. Lim D.-S. Regel E. Dinh C. Sands A. Eichele G. Hasty P. Bradley A. Nature. 1997; 386: 804-810Crossref PubMed Scopus (916) Google Scholar, 6Connor F. Bertwistle D. Mee P.J. Ross G.M. Swift S. Grigorieva E. Tybulewicz V.L. Ashworth A. Nat. Genet. 1997; 17: 423-430Crossref PubMed Scopus (363) Google Scholar, 7Abbott D.W. Freeman M.L. Holt J.T. J. Natl. Cancer Inst. 1998; 90: 978-985Crossref PubMed Scopus (182) Google Scholar, 8Patel K.J. Vu V.P. Lee H. Corcoran A. Thistlethwaite F.C. Evans M.J. Colledge W.H. Friedman L.S. Ponder B.A. Venkitaraman A.R. Mol. Cell. 1998; 1: 347-357Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar; reviewed in Ref. 9Kinzler K.W. Vogelstein B. Nature. 1997; 386: 761-762Crossref PubMed Scopus (1008) Google Scholar), a clear consensus has not been reached on the function of BRCA1. Unfortunately, BRCA1 −/− mice die at day 6.5 to day 8.5 of embryonic gestation because of lack of proliferation of the mouse blastocyst (10Gowen L.C. Johnson B.L. Latour A.M. Sulik K.K. Koller B.H. Nat. Genet. 1996; 12: 191-194Crossref PubMed Scopus (395) Google Scholar, 11Hakem R. de la Pomba J.L. Sirard C. Mo R. Woo M. Hakem A. Wakeham A. Potter J. Reitmair A. Billia F. et al.Cell. 1996; 85: 1009-1023Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 12Liu C.Y. Flesken-Nikitin A. Li S. Zeng Y. Lee W.-H. Genes Dev. 1996; 10: 1835-1843Crossref PubMed Scopus (274) Google Scholar). Despite this embryonic lethality, work in the mouse systems has resulted in two suggestive findings. First, whenBRCA1 −/− mice are mated with p53−/− mice to generate BRCA1 −/−p53−/− mice, these double-knock out mice show reduced embryonic lethality (13Hakem R. de la Pomba J.L. Elia A. Potter J. Mak T.W. Nat. Genet. 1997; 16: 298-302Crossref PubMed Scopus (227) Google Scholar, 14Ludwig T. Chapman D.L. Papaioannou V.E. Efstratiadis A. Genes Dev. 1997; 11: 1226-1241Crossref PubMed Scopus (461) Google Scholar), suggesting that BRCA1 and p53 may lie on a common functional pathway. The second finding is that cells from BRCA1 −/− mice have a defect in transcription-coupled DNA repair (TCR) 1The abbreviations used are: TCR, transcription-coupled DNA repair; Gy, gray; F, forward linker; R, reverse linker; wt, wild type.1The abbreviations used are: TCR, transcription-coupled DNA repair; Gy, gray; F, forward linker; R, reverse linker; wt, wild type. (15Gowen L.C. Avrutskaya A.V. Latour A.M. Koller B.H. Leadon S.A. Science. 1998; 281: 1009-1012Crossref PubMed Scopus (452) Google Scholar), implying thatBRCA1 may be involved in DNA repair and/or the stress response of the cell. Despite these suggestive findings in the mouse system, there are such large differences in mouse and human BRCA1 biology that it is unclear whether the DNA repair function of mouse BRCA1 is applicable to human BRCA1. Mouse BRCA1 is only 57% homologous to human BRCA1 (10Gowen L.C. Johnson B.L. Latour A.M. Sulik K.K. Koller B.H. Nat. Genet. 1996; 12: 191-194Crossref PubMed Scopus (395) Google Scholar, 11Hakem R. de la Pomba J.L. Sirard C. Mo R. Woo M. Hakem A. Wakeham A. Potter J. Reitmair A. Billia F. et al.Cell. 1996; 85: 1009-1023Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 12Liu C.Y. Flesken-Nikitin A. Li S. Zeng Y. Lee W.-H. Genes Dev. 1996; 10: 1835-1843Crossref PubMed Scopus (274) Google Scholar), andBRCA1 appears to function differently in the two systems. Although BRCA1 has been shown to be required for cellular proliferation during mouse development, BRCA1 has been shown to be a powerful growth suppressor in both yeast and human systems (16Thompson M.E. Jensen R.A. Obermiller P.S. Page D.L. Holt J.T. Nat. Genet. 1995; 9: 444-450Crossref PubMed Scopus (543) Google Scholar, 17Holt J.T. Thompson M.E. Szabo C. Robinson-Benion C. Arteaga C.L. King M.C. Jensen R.A. Nat. Genet. 1996; 12: 298-302Crossref PubMed Scopus (380) Google Scholar, 18Humphrey J.S. Salim A. Erdos M.R. Collins F.S. Brody L.C. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5820-5825Crossref PubMed Scopus (98) Google Scholar, 19Somasundaram K. Zhang H. Zeng Y.-X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar, 20Burke T.F. Cocke K.S. Lemke S.J. Angleton E. Becker G.W. Beckmann R.P. Oncogene. 1998; 16: 1031-1040Crossref PubMed Scopus (13) Google Scholar). Although there are reports of living humans who are homozygous for BRCA1 mutations (21Boyd M. Harris F. McFarlane R. Davidson H.R. Black D.M. Nature. 1995; 375: 541-542Crossref PubMed Scopus (83) Google Scholar), mice carrying homozygous BRCA1 mutations die early in gestation. Lastly, DNA repair in a mouse cell is not necessarily indicative of repair in a human cell (22Namba M. Nishitani K. Kimoto T. Jpn. J. Exp. Med. 1977; 47: 263-269PubMed Google Scholar, 23Yagi T. Mutat. Res. 1982; 96: 89-98Crossref PubMed Scopus (33) Google Scholar, 24Walton D.G. Acton A.B. Stich H.F. Mutat. Res. 1984; 129: 129-136Crossref PubMed Scopus (34) Google Scholar). Mouse and human cells show differences in the amount of damage sustained per given DNA-damaging dose, in the kinetics of DNA repair, and in cellular survival at a given dose of a DNA-damaging agent (22Namba M. Nishitani K. Kimoto T. Jpn. J. Exp. Med. 1977; 47: 263-269PubMed Google Scholar, 23Yagi T. Mutat. Res. 1982; 96: 89-98Crossref PubMed Scopus (33) Google Scholar, 24Walton D.G. Acton A.B. Stich H.F. Mutat. Res. 1984; 129: 129-136Crossref PubMed Scopus (34) Google Scholar). Other attempts to determine the function of BRCA1 have centered on finding functional domains of BRCA1.BRCA1 contains a Zn2+ finger in its N terminus (2Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshmann K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W. et al.Science. 1994; 266: 66-71Crossref PubMed Scopus (5289) Google Scholar), a transcriptional activation domain in its C terminus (25Chapman M.S. Verma I.M. Nature. 1996; 382: 678-679Crossref PubMed Scopus (436) Google Scholar, 26Monteiro A.N.A. August A. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13595-13599Crossref PubMed Scopus (428) Google Scholar), and a BRCT domain at its far C terminus (27Bork P. Hofmann K. Bucher P. Neuwald A.F. Altschul S.F. Koonin E.V. FASEB J. 1997; 11: 68-76Crossref PubMed Scopus (661) Google Scholar, 28Callebaut I. Mornon J. FEBS Lett. 1997; 400: 25-30Crossref PubMed Scopus (485) Google Scholar). BRCA1 has been shown to interact with the basal transcriptional machinery (RNA polymerase II, TFIIH, TFIIE, and RNA helicase A) (29Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar, 30Anderson S.F. Schlegel B.P. Nakajima T. Wolpin E.S. Parvin J.D. Nat. Genet. 1998; 19: 254-256Crossref PubMed Scopus (340) Google Scholar),BRCA2 (31Chen J. Silver D.P. Walpita D. Cantor S.B. Gazdar A.F. Tomlinson G. Couch F.J. Weber B.L. Ashley T. Livingston D.M. Scully R. Mol. Cell. 1998; 2: 317-328Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar), and Rad51 (29Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar). BRCA1 also colocalizes with Rad51 to discrete nuclear foci when exposed to UV radiation (32Scully R. Anderson S.F. Chao D.M. Wei W. Ye L. Young R.A. Livingston D.M. Parvin J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5605-5610Crossref PubMed Scopus (422) Google Scholar). Although BRCA1 and BRCA2 show no homology (2Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshmann K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W. et al.Science. 1994; 266: 66-71Crossref PubMed Scopus (5289) Google Scholar, 3Wooster R. Bignell G. Lancaster J. Swift S. Seal S. Mangion J. Collins N. Gregory S. Gumbs C. Micklem G. Nature. 1995; 378: 789-792Crossref PubMed Scopus (2943) Google Scholar), mutations of these genes both cause breast cancer, and these two genes show identical expression throughout development (33Rajan J.V. Wang M. Marquis S.T,. Chodosh L.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13078-13083Crossref PubMed Scopus (204) Google Scholar). Cells that lack BRCA2 have been shown to be hypersensitive to ionizing radiation and have a decreased capacity to repair double-stranded DNA breaks (5Sharan S.K. Morimatsu M. Albrecht U. Lim D.-S. Regel E. Dinh C. Sands A. Eichele G. Hasty P. Bradley A. Nature. 1997; 386: 804-810Crossref PubMed Scopus (916) Google Scholar, 6Connor F. Bertwistle D. Mee P.J. Ross G.M. Swift S. Grigorieva E. Tybulewicz V.L. Ashworth A. Nat. Genet. 1997; 17: 423-430Crossref PubMed Scopus (363) Google Scholar, 7Abbott D.W. Freeman M.L. Holt J.T. J. Natl. Cancer Inst. 1998; 90: 978-985Crossref PubMed Scopus (182) Google Scholar, 8Patel K.J. Vu V.P. Lee H. Corcoran A. Thistlethwaite F.C. Evans M.J. Colledge W.H. Friedman L.S. Ponder B.A. Venkitaraman A.R. Mol. Cell. 1998; 1: 347-357Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). In addition, BRCA2interacts directly with Rad51 (5Sharan S.K. Morimatsu M. Albrecht U. Lim D.-S. Regel E. Dinh C. Sands A. Eichele G. Hasty P. Bradley A. Nature. 1997; 386: 804-810Crossref PubMed Scopus (916) Google Scholar, 34Chen P.L. Chen C.F. Chen Y. Xiao J. Sharp Z.D. Lee W.-H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5287-5292Crossref PubMed Scopus (336) Google Scholar). When coupled with the finding that BRCA1 −/− mouse cells are deficient in TCR, all these findings suggest a role for BRCA1 in the cellular response to DNA damage. To determine the role of BRCA1 in the cellular response to DNA damage, we have developed a human genetic system. HCC1937 is a human breast cancer cell line originally established from a family carrying a known cancer-causing BRCA1 mutation, 5283insC. We show that: 1) HCC1937 cells are hypersensitive to ionizing radiation; 2) replacement of critical regions of BRCA1 can reverse this radiation sensitivity; and 3) TCR correlates with radioresistance as cells containing BRCA1 show both increased TCR and radioresistance. HCC 1937 cells were grown in RPMI medium (Life Technologies, Inc.) supplemented with 5 mm glutamine, 1× antibiotic/antimycotic (Sigma), 1× ITSA (Sigma), and 5% fetal bovine serum (Summit). HCC1937 cells were transfected with 1 μg of DNA using Cellfectin (Life Technologies, Inc.). Using β-galactosidase, we determined an average transfection efficiency of 20%. XRV15B cells were a gift of Gilbert Chu (Stanford University). These were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 5 mm glutamine, and 1× antibiotic/antimycotic. V8 cells are a cell line generated by our lab from a breast cancer patient who carries an exon 11BRCA1 mutation (R1203X). These cells are heterozygous for this mutation (data not shown). MCF-7 cells (hemizygous wild-typeBRCA1), HBL100 cells and Capan-1 cells (hemizygous mutantBRCA2) were obtained from American Type Tissue Collection and grown as instructed. Two types of cell survival assays were used: the colony forming assay and the transient transfection cell survival assay. The colony forming assay was performed as described previously (36Abbott D.W. Holt J.T. J. Biol. Chem. 1997; 272: 14005-14008Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The transient transfection cell survival (34Chen P.L. Chen C.F. Chen Y. Xiao J. Sharp Z.D. Lee W.-H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5287-5292Crossref PubMed Scopus (336) Google Scholar) assay was performed as described with the following changes. Approximately 1 × 106 HCC1937 cells were transfected at 85% confluency with 1 μg of indicated plasmid and Cellfectin (Life Technologies, Inc.). 18 h later, the plates of cells were exposed to the various doses of ionizing radiation. Cells were then refed every 2–3 days. 8 days following irradiation (10 days following transfection), the cells were trypsinized, and the total cell number was determined by Coulter Counter. Relative survival refers to the cell number from a given transfection at a given dose compared with the cell number from an unirradiated plate of the same transfected cells plated on the same day and grown for the same length of time. The pMT vector was a gift from Frank Rauscher (Wistar Institute, Philadelphia, PA). This vector contains a Zn2+-inducible promoter and the neomycin resistance gene. Full-length BRCA1 was cloned into this vector via theNotI and HindIII sites. The Δ702–834 deletion was generated by digestion of full-length pMT-BRCA1 withHpaI followed by religation. Δ3–29 was constructed by digestion of pMT-BRCA1 with NotI andBclI and then ligated insertion of the following forward (F) and reverse (R) linkers: F, 5′-GGCCGCGAATTCGAAATGGATTT-3′, and R: 5′-GATCAAATCCATTTCGAATTCGC-3′. Δ3–471 was constructed by digestion with NotI and AflII and then ligated insertion of F, 5′-GGCCGCGAAATGGATAAC-3′, and R, 5′-TTAAGTTATCCATTTCGC. Δ512–1284 was constructed by digestion with EcoNI and then ligated insertion of F, 5′-ATCAGGCCTTA-3′, and R, 5′-CTAAGGCCTGA-3′. Δ922–1664 was constructed by digestion with BsrGI and then ligated insertion of F, 5′-GTACAGCTGGT-3′, and R, GTACACCAGCT-3′. Δ1293–1864 was constructed by digestion with NheI and then ligated insertion of a stop linker F, 5′-CTAGCCTCTAGATAG-3′, and R, 5′-CTAGCTATCTAGAGG-3′. Stably transfected cells were generated by transfecting 1 μg of pMT-BRCA1Δ702–834 into HCC1937 cells. Cells were then selected in 50 μg/ml G418 (Sigma) for 4 weeks. Approximately 20 individual colonies were selected and analyzed forBRCA1Δ702–834 expression by Western blotting. Briefly, total cell lysates were prepared. 20 μg of total protein was electrophoresed on a 7% SDS-polyacrylamide gel. The electrophoresed protein was then transferred to polyvinylidene difluoride membrane and blotted using the indicated antibodies (1:1000 dilution). After appropriate washing and exposure to the horseradish peroxidase-conjugated secondary antibody, BRCA1Δ702–834 was visualized using ECL (Amersham Pharmacia Biotech). This assay was performed as described previously (7Abbott D.W. Freeman M.L. Holt J.T. J. Natl. Cancer Inst. 1998; 90: 978-985Crossref PubMed Scopus (182) Google Scholar). Briefly 1 × 105 cells (XRV15B, HCC1937, or HCC1937BRCA1D702–834; clone 1) were embedded into agarose plugs under isotonic conditions. The cell plugs were then irradiated at 4 °C with 10 Gy ionizing radiation and placed into complete medium at 37 °C to perform double-stranded break repair for the given amount of time. After the given amount of repair time, the cell plugs were digested overnight at 55 °C in 10 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.5% SDS, 50 mm EDTA, and 2 μg/ml proteinase K (Life Technologies, Inc.). The digested cell plugs were then embedded into a 0.7% agarose gel and subjected to pulsed field gel electrophoresis (3 V/cm, 45-s pulse time) for 72 h. The separated DNA was then transferred to a Gene-Screen filter, and Southern blotting was performed. The probe was generated by isolation of total genomic DNA from the given cell line followed by Random Prime labeling using [α-32P]ATP (Prime-It II, Stratagene). 1 × 107 HCC1937 cells or 1 × 107 BRCA1Δ702–834 (clone 1) cells for each time point were incubated in complete medium supplemented with 10 μmbromodeoxyuridine (Sigma) and 25 μCi of [3H]thymidine immediately prior to irradiation with 10 Gy. The zero time point was iced immediately. Remaining samples were incubated for the indicated times at 37 °C. Cells were then washed three times with phosphate-buffered saline, and DNA purification was performed by standard methods (proteinase K, phenol/chloroform/ethanol precipitation). The DNA was then digested with KpnI, repurified by phenol/chloroform and ethanol precipitation, and incubated in phosphate-buffered saline containing 0.1% bovine serum albumin with an antibody directed against bromodeoxyuridine (Sigma) overnight at 4 °C. Following this incubation, 10 μl of goat anti-mouse antibody (Life Technologies, Inc.) was added incubated by 4 h and then collected by microcentrifugation for 15 min. Unbound DNA diluted 1:5 with TE and used directly for Southern blotting experiments. Aliquots of both the bound and unbound DNA fractions were scintillation counted to determine the total amount of repaired DNA. Both bound and unbound DNA samples from each time point were then electrophoresed on a 1% agarose gel, blotted to nylon membrane, and probed with strand-specific probes for the human DHFRgene. To generate strand-specific probes for transcribed regions of the human DHFR genes, Bluescript II (Stratagene) was digested with HindIII and BamHI. The following oligonucleotide linker from the DHFR gene was then inserted into Bluescript II (F, 5′-AGCTTCAGAGAACTCAAGTAAGTACCTTAACATAAATTCACCACAAG-3′, and R, 5′-GATCCTTGTGGTGAATTTATGTTAAGGTACTTACTTGAGTTCTCTGA-3′ (43Mellon I. Spivak G. Hanawalt P.C. Cell. 1987; 51: 241-249Abstract Full Text PDF PubMed Scopus (1037) Google Scholar). After construction, this plasmid containing DHFR plus the linker was digested with either HindIII or BamHI and transcribed with T3 polymerase or T7 polymerase in the presence of [α-32P]GTP to generate strand-specific probes. To study the role of BRCA1 in the cellular response to DNA damage, we have utilized a human breast cancer cell line (HCC1937) that contains only a mutated BRCA1 (35Tomlinson G.E. Chen T.T. Stastny V.A. Virmani A.K. Spillman M.A. Tonk V. Blum J.L. Schneider N.R. Wistuba I.I. Shay J.W. Cancer Res. 1998; 58: 3237-3242PubMed Google Scholar). The functional allele in these cells contains a frameshift mutation that deletes the BRCT domain of BRCA1. To determine whether cells lacking functional BRCA1 are sensitive to ionizing radiation, we performed colony forming assays (36Abbott D.W. Holt J.T. J. Biol. Chem. 1997; 272: 14005-14008Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). In comparison with MCF-7 cells, V8 cells and HBL100 cells, HCC1937 cells were significantly more radiation-sensitive. HCC1937 cells were approximately 2 logs more sensitive to ionizing radiation than HBL100 cells at 6 Gy and were approximately 30 to 40 times more sensitive than either V8 or MCF-7 cells (Fig. 1). Radiation sensitivity seemed to correlate with the genetic status of BRCA1. Cells with two copies of wt BRCA1 (HBL100) were more resistant than cells with one copy of wt BRCA1 (MCF-7), which were more resistant than cells without wt BRCA1 (HCC1937). Survival curves were generated on the XRV15B Ku86-deficient cell line (37Errami A. Smider V. Rathmell W.K. He D.M. Hendrickson E.A. Zdzienicka M.Z. Chu G. Mol. Cell. Biol. 1996; 16: 1519-1526Crossref PubMed Scopus (157) Google Scholar) and the BRCA2-deficient Capan-1 cell line, and these survival curves were nearly identical to that of the HCC1937 cell line (Fig. 1). Because only a handful of cell lines have been shown to be as radiation-sensitive as the XRV15B cell line (38Hendrickson E.A. Qin X.Q. Bump E.A. Schatz D.G. Oettinger M. Weaver D.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4061-4065Crossref PubMed Scopus (283) Google Scholar), this finding indicates that the sensitivity of the HCC1937 cell line is likely to be a real effect. We used a recently described method to measure cell survival after transient transfection and irradiation (34Chen P.L. Chen C.F. Chen Y. Xiao J. Sharp Z.D. Lee W.-H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5287-5292Crossref PubMed Scopus (336) Google Scholar) to determine whether reintroduction of BRCA1 into the HCC1937 cells could reverse the radiation sensitivity of these cells. Surprisingly, we were unable to demonstrate that reintroduction of full-length BRCA1 into HCC1937 reverses radiosensitivity because this assay depends on the continued growth of the cells after transfection. Thus, becauseBRCA1 is such a potent suppressor of cell growth (16Thompson M.E. Jensen R.A. Obermiller P.S. Page D.L. Holt J.T. Nat. Genet. 1995; 9: 444-450Crossref PubMed Scopus (543) Google Scholar, 17Holt J.T. Thompson M.E. Szabo C. Robinson-Benion C. Arteaga C.L. King M.C. Jensen R.A. Nat. Genet. 1996; 12: 298-302Crossref PubMed Scopus (380) Google Scholar, 18Humphrey J.S. Salim A. Erdos M.R. Collins F.S. Brody L.C. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5820-5825Crossref PubMed Scopus (98) Google Scholar, 19Somasundaram K. Zhang H. Zeng Y.-X. Houvras Y. Peng Y. Zhang H. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar,39Jensen D.E. Proctor M. Marquies S.T. Gardner H.P. Ha S.I. Chodosh L.A. Ishov A.M. Tommerup N. Vissing H. Sekido Y. et al.Oncogene. 1998; 16: 1097-1112Crossref PubMed Scopus (584) Google Scholar), transfection of BRCA1 stops cell growth and does not allow us to accurately measure cell survival following irradiation. To overcome this problem, we constructed a variety of BRCA1deletion mutants and tested them for the ability to suppress growth in the HCC 1937 cells. Each of these mutants contains an intact nuclear localization signal (40Chen C.F. Li S. Chen Y. Chen P.L. Sharp Z.D. Lee W.-H. J. Biol. Chem. 1996; 271: 32863-32868Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 41Thakur S. Zhang H.B. Peng Y. Le H. Carroll B. Ward T. Yao J. Farid L.M. Couch F.J. Wilson R.B. Weber B.L. Mol. Cell. Biol. 1997; 17: 444-452Crossref PubMed Scopus (225) Google Scholar); however, these mutants lack important functional regions of BRCA1 (Fig.2 A). Because bothBRCA1 and BRCA1Δ3–29 suppress cell growth, they cannot be scored in an assay requiring cell growth. All other mutants tested, however, destroyed the growth suppressive effect ofBRCA1 and could therefore be used in the cell growth and survival assay (Fig. 2). The HCC1937 cells contain an endogenousBRCA1 mutation that eliminates the BRCT domain but leaves the transactivation domain intact. Because the HCC1937 cells are radiation-sensitive, this indicates that the BRCT domain is necessary for radiation resistance. In this assay, a deletion mutant that destroyed the N-terminal Zn2+ finger domain plus additional N-terminal sequences (BRCA1Δ3–471) could not rescue radiation sensitivity (Fig. 2 B). Mutants that deleted both the BRCT domain and the transactivation domain (BRCA1Δ1293–1864) or the transactivation domain alone (BRCA1Δ922–1664) could not rescue radiation sensitivity (Fig. 2 B). These findings demonstrate that the Zn2+ finger, the transactivation domain and the BRCT domain are all necessary for radiation resistance. In contrast to these findings, two deletion mutants (BRCA1Δ702–834 andBRCA1Δ512–1284) were able to restore radiation resistance (Fig. 2 B). These two mutants can eliminate the role ofBRCA1 as a growth suppressor but can reverse radiation sensitivity, showing that these functions can be separated. Because BRCA1 interacts with both Rad51 and BRCA2(29Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar, 31Chen J. Silver D.P. Walpita D. Cantor S.B. Gazdar A.F. Tomlinson G. Couch F.J. Weber B.L. Ashley T. Livingston D.M. Scully R. Mol. Cell. 1998; 2: 317-328Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar), two double-stranded DNA repair proteins, and because mouse cells lacking BRCA1 are deficient in TCR (15Gowen L.C. Avrutskaya A.V. Latour A.M. Koller B.H. Leadon S.A. Science. 1998; 281: 1009-1012Crossref PubMed Scopus (452) Google Scholar), we were interested in determining both the double-stranded break repair capacity and the TCR capacity of the HCC1937 cells. To study the repair capacity of HCC1937 cells lacking BRCA1 and those containing a form of BRCA1 that reverses radiation sensitivity (BRCA1Δ702–834), stably transfected cell lines were generated. HCC1937 cells were transfected withBRCA1Δ702–834 on a vector also containing the neomycin resistance gene. After 4 weeks of selection in G418, approximately 20 colonies were selected and propagated. Of these 20 colonies, two colonies expressed high levels of BRCA1Δ702–834 as shown by Western blotting (clones 1 and 2; Fig. 3,A and B). The two antibodies used for Western blotting were Ab-D (42Ruffner H. Verma I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7138-7143Crossref PubMed Scopus (179) Google Scholar) and I-20. These antibodies recognize the far C terminus of BRCA1. HCC1937 cells contain a mutant form of BRCA1 that lacks this epitope. BRCA1Δ702–834 restores this epitope; therefore, the two clones generated show expression of BRCA1Δ702–834 by Western blotting (Fig. 3 A, upper blot shows Ab-D, and bottom blot shows I-20). Because zinc is required in HCC cell medium, addition of Zn2+ gave little induction (Fig. 3 A). These two clones were then tested for radiation sensitivity in the colony forming assay. HCC1937 cells that contain only empty vector are hypersensitive to ionizing radiation, whereas those expressing BRCA1Δ702–834 are approximately 2 logs more radiation-resistant at the 6 Gy dose (Fig. 3, B andC). Thus by both transient and stable transfectionBRCA1Δ702–834 reverses the radiation sensitivity of the HCC1937 cells. The ability to generate HCC1937 cells containing stably transfectedBRCA1Δ702–834 allowed us to compare the DNA repair capabilities of parental HCC1937 cells and HCC1937 cells containingBRCA1Δ702–834. Pulsed field gel electrophoresis was then performed with Southern blotting using total genomic DNA as a probe. Under the electrophoresis conditions chosen, damaged DNA runs as a discrete band below the undamaged or repaired DNA (7Abbott D.W. Freeman M.L. Holt J.T. J. Natl. Cancer Inst. 1998; 90: 978-985Crossref PubMed Scopus (182) Google Scholar). In this experiment, HCC1937 cells repaired most damaged DNA within 2 h (Fig. 3 D, upper panel). HCC1937BRCA1Δ702–834 cells also repaired most of the damaged DNA within 2 h (Fig. 3 D, bottom panel). These cells showed slightly faster repair than parental HCC1937 cells, but the difference is too small to account for the 2 log difference in radiation sensitivity between these two cell lines. We next analyze TCR in HCC cells, with and without BRCA1gene replacement. Mouse cells containing defective BRCA1have been shown to be deficient in TCR (15Gowen L.C. Avrutskaya A.V. Latour A.M. Koller B.H. Leadon S.A. Science. 1998; 281: 1009-1012Crossref PubMed Scopus (452) Google Scholar). HCC1937 cells showed a marked inability to undergo TCR (Fig.4 A, upper panels). Although the nontranscribed strand shows the expected result (little repair of the nontranscribed strand), the transcribed strand also shows little repair (compare bound and free fractions at both 2 and 4 h of repair). In contrast, cells containing BRCA1Δ702–834 were able to undergo TCR. Within 2 h after exposure, a much greater proportion of DHFR is present in the bound fraction (Fig.4 A, lower panels). Cells containingBRCA1Δ702–834 undergo TCR approximately 15 times faster than cells lacking functional BRCA1 (Fig.4 B). We have shown that cells containing a mutant BRCA1 that is known to cause cancer in human patients are hypersensitive to ionizing radiation. This radiation sensitivity can be reversed by forms of BRCA1 that do not suppress growth but maintain the Zn2+ finger, the transcription activating domain, and the BRCT domain intact. Although BRCA1 does not affect double-stranded break repair, cells lacking BRCA1 are deficient in their ability to perform TCR. These findings suggest that the interaction of BRCA1 with the transcription machinery (RNA pol II, TFIIE, TFIIF, TFIIH, and RNA helicase A) are physiologically important. This work also suggests functional differences between BRCA1 and BRCA2. WhereasBRCA1 predominantly affects TCR, cells lackingBRCA2 have been shown to be deficient in double-stranded break repair (5Sharan S.K. Morimatsu M. Albrecht U. Lim D.-S. Regel E. Dinh C. Sands A. Eichele G. Hasty P. Bradley A. Nature. 1997; 386: 804-810Crossref PubMed Scopus (916) Google Scholar, 6Connor F. Bertwistle D. Mee P.J. Ross G.M. Swift S. Grigorieva E. Tybulewicz V.L. Ashworth A. Nat. Genet. 1997; 17: 423-430Crossref PubMed Scopus (363) Google Scholar, 7Abbott D.W. Freeman M.L. Holt J.T. J. Natl. Cancer Inst. 1998; 90: 978-985Crossref PubMed Scopus (182) Google Scholar, 8Patel K.J. Vu V.P. Lee H. Corcoran A. Thistlethwaite F.C. Evans M.J. Colledge W.H. Friedman L.S. Ponder B.A. Venkitaraman A.R. Mol. Cell. 1998; 1: 347-357Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Further work can help to determine whether these differing DNA repair defects lead to the higher cancer penetrance seen in BRCA1 carriers, the higher incidence of male breast cancer seen in BRCA2 carriers, and the different histology seen in BRCA1 or BRCA2-defective tumors. We thank Gilbert Chu (Stanford University) for providing XRV15B cells, David Livingston (Dana Farber Cancer Institute), Ralph Scully (Dana Farber Cancer Institute), Michael Kastan (St. Jude's Children's Hospital), David Chen (Los Alamos National Laboratory), and Philip Browning (Vanderbilt University) for critical comments on the manuscript, and Evelyn Okediji for technical assistance." @default.
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• W2022176772 title "BRCA1 Expression Restores Radiation Resistance in BRCA1-defective Cancer Cells through Enhancement of Transcription-coupled DNA Repair" @default.
• W2022176772 cites W1487538033 @default.
• W2022176772 cites W1611268078 @default.
• W2022176772 cites W1768635289 @default.
• W2022176772 cites W1972710550 @default.
• W2022176772 cites W1973904343 @default.
• W2022176772 cites W1977035912 @default.
• W2022176772 cites W1985123116 @default.
• W2022176772 cites W2000437208 @default.
• W2022176772 cites W2002555966 @default.
• W2022176772 cites W2011275324 @default.
• W2022176772 cites W2012300945 @default.
• W2022176772 cites W2013743651 @default.
• W2022176772 cites W2019205168 @default.
• W2022176772 cites W2021851245 @default.
• W2022176772 cites W2024342001 @default.
• W2022176772 cites W2025370752 @default.
• W2022176772 cites W2030093162 @default.
• W2022176772 cites W2033423858 @default.
• W2022176772 cites W2036546668 @default.
• W2022176772 cites W2037191150 @default.
• W2022176772 cites W2038275315 @default.
• W2022176772 cites W2043560010 @default.
• W2022176772 cites W2044713924 @default.
• W2022176772 cites W2047460928 @default.
• W2022176772 cites W2048867718 @default.
• W2022176772 cites W2053003311 @default.
• W2022176772 cites W2059901912 @default.
• W2022176772 cites W2065777930 @default.
• W2022176772 cites W2068176376 @default.
• W2022176772 cites W2075417812 @default.
• W2022176772 cites W2075958559 @default.
• W2022176772 cites W2082411289 @default.
• W2022176772 cites W2091350790 @default.
• W2022176772 cites W2109465247 @default.
• W2022176772 cites W2144031018 @default.
• W2022176772 cites W2153926167 @default.
• W2022176772 cites W2155675893 @default.
• W2022176772 cites W2165579893 @default.
• W2022176772 cites W2168307556 @default.
• W2022176772 cites W2317974253 @default.
• W2022176772 cites W2328105863 @default.
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