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BRCA1 Activates a G₂-M Cell Cycle Checkpoint following 6-Thioguanine–Induced DNA Mismatch Damage

¹ Department of Radiation Oncology, Case Western Reserve University and ² Case Comprehensive Cancer Center/University Hospitals Case Medical Center, Cleveland, Ohio

Requests for reprints: Timothy J. Kinsella, Department of Radiation Oncology, LTR6068, University Hospitals Case Medical Center, 11100 Euclid Avenue, Cleveland, OH 44106-6068. Phone: 216-844-2530; Fax: 216-844-4799; E-mail: timothy.kinsella{at}UHhospitals.org.

Human DNA mismatch repair (MMR) is involved in the response to certain chemotherapy drugs, including 6-thioguanine (6-TG). Consistently, MMR-deficient human tumor cells show resistance to 6-TG damage as manifested by a reduced G₂-M arrest and decreased apoptosis. In this study, we investigate the role of the BRCA1 protein in modulating a 6-TG–induced MMR damage response, using an isogenic human breast cancer cell line model, including a BRCA1 mutated cell line (HCC1937) and its transfectant with a wild-type BRCA1 cDNA. The MMR proteins MSH2, MSH6, MLH1, and PMS2 are similarly detected in both cell lines. BRCA1-mutant cells are more resistant to 6-TG than BRCA1-positive cells in a clonogenic survival assay and show reduced apoptosis. Additionally, the mutated BRCA1 results in an almost complete loss of a G₂-M cell cycle checkpoint response induced by 6-TG. Transfection of single specific small interfering RNAs (siRNA) against MSH2, MLH1, ATR, and Chk1 in BRCA1-positive cells markedly reduces the BRCA1-dependent G₂-M checkpoint response. Interestingly, ATR and Chk1 siRNA transfection in BRCA1-positive cells shows similar levels of 6-TG cytotoxicity as the control transfectant, whereas MSH2 and MLH1 siRNA transfectants show 6-TG resistance as expected. DNA MMR processing, as measured by the number of 6-TG–induced DNA strand breaks using an alkaline comet assay (±z-VAD-fmk cotreatment) and by levels of iododeoxyuridine-DNA incorporation, is independent of BRCA1, suggesting the involvement of BRCA1 in the G₂-M checkpoint response to 6-TG but not in the subsequent excision processing of 6-TG mispairs by MMR. [Cancer Res 2007;67(13):6286–92]

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