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Clinical Cancer Research Vol. 9, 2327-2334, June 2003
© 2003 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

DNA Mismatch Repair (MMR) Mediates 6-Thioguanine Genotoxicity by Introducing Single-strand Breaks to Signal a G2-M Arrest in MMR-proficient RKO Cells1

Tao Yan, Suzanne E. Berry, Anand B. Desai and Timothy J. Kinsella2

Department of Radiation Oncology, Ireland Comprehensive Cancer Center, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4942

Purpose: 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 G2-M arrest leading to cell death.

Experimental Design: 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 µM 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 G2-M arrest followed by cell death after 6-TG exposure, compared with the vector controls. The MMR-mediated, 6-TG-induced G2-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 G2-M arrest.

Conclusions: Our data suggest that MMR mediates 6-TG-induced G2-M arrest by introducing SSBs to signal a persistent G2-M arrest leading to enhanced cell death.




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