BRCA1 activates a G2-M cell cycle checkpoint following 6-thioguanine-induced DNA mismatch damage.
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 G(2)-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 G(2)-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 G(2)-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 G(2)-M checkpoint response to 6-TG but not in the subsequent excision processing of 6-TG mispairs by MMR.
Yamane
Kazuhiko
K
Department of Radiation Oncology, Case Western Reserve University and Case Comprehensive Cancer Center/University Hospitals Case Medical Center, Cleveland, Ohio 44106-6068, USA.
Schupp
Jane E
JE
Kinsella
Timothy J
TJ
D019313:BRCA1 Protein / Q000096:biosynthesis* / Q000502:physiology*; D020137:Base Pair Mismatch; D002453:Cell Cycle; D002455:Cell Division; D045744:Cell Line, Tumor; D020552:Comet Assay; D053843:DNA Mismatch Repair; D016195:G2 Phase; D006801:Humans; D018613:Microscopy, Confocal; D008938:Mitosis; D009154:Mutation; D010766:Phosphorylation; D013866:Thioguanine / Q000494:pharmacology*; D014162:Transfection
D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D013866:Thioguanine