DNA mismatch repair (MMR) mediates 6-thioguanine genotoxicity by introducing single-strand breaks to signal a G2-M arrest in MMR-proficient RKO cells.
OBJECTIVE
The DNA mismatch repair (MMR) system plays an important role in mediating cell death after treatment with various types of chemotherapeutic agents, although the molecular mechanisms are not well understood. In this study, we sought to determine what signal is introduced by MMR after 6-thioguanine (6-TG) treatment to signal a G(2)-M arrest leading to cell death.


METHODS
A comparison study was carried out using an isogenic MMR(+) and MMR(-) human colorectal cancer RKO cell system, which we established for this study. Cells were exposed to 6-TG (3 micro M x 24 h) and then harvested daily for the next 3-6 days for growth inhibition assays. Cell cycle effects were determined by flow cytometry, and DNA strand breaks were measured using pulsed-field gel electrophoresis and alkaline Comet assays.


RESULTS
We first established MMR(+) RKO cell lines by transfection of human MutL homologue 1 (hMLH1) cDNA into the hMLH1-deficient (MMR(-)) RKO cell line. The ectopically expressed hMLH1 protein restored a MMR-proficient phenotype in the hMLH1(+) transfectants, showing a significantly increased and prolonged G(2)-M arrest followed by cell death after 6-TG exposure, compared with the vector controls. The MMR-mediated, 6-TG-induced G(2)-M arrest started on day 1, peaked on day 3, and persisted to day 6 after 6-TG removal. We found that DNA double-strand breaks were comparably produced in both our MMR(+) and MMR(-) cells, peaking within 1 day of 6-TG treatment. In contrast, single-strand breaks (SSBs) were more frequent and longer lived in MMR(+) cells, and the duration of SSB formation was temporally correlated with the time course of 6-TG-induced G(2)-M arrest.


CONCLUSIONS
Our data suggest that MMR mediates 6-TG-induced G(2)-M arrest by introducing SSBs to signal a persistent G(2)-M arrest leading to enhanced cell death.
Yan
Tao
T
Department of Radiation Oncology, Ireland Comprehensive Cancer Center, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4942, USA.
Berry
Suzanne E
SE
Desai
Anand B
AB
Kinsella
Timothy J
TJ
D048868:Adaptor Proteins, Signal Transducing; D020137:Base Pair Mismatch / Q000235:genetics*; D002352:Carrier Proteins; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D008938:Mitosis / Q000187:drug effects*; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D013866:Thioguanine / Q000633:toxicity*; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004277:DNA, Single-Stranded; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000070957:MutL Protein Homolog 1; D013866:Thioguanine