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  • The EMBO Journal (2003) 22, 2245 - 2254
  • doi:10.1093/emboj/cdg216

Methylation-induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1

Petr Cejka1, Lovorka Stojic1, Nina Mojas1, Anna Marie Russell2, Karl Heinimann2, Elda Cannavó1, Massimiliano di Pietro1, Giancarlo Marra1 and Josef Jiricny1

  1. Institute of Molecular Cancer Research, University of Zürich, August Forel-Strasse 7, CH-8008 Zürich, Switzerland
  2. Research Group Human Genetics, Departments of Research and Clinical-Biological Sciences, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland

Correspondence to:

Josef Jiricny, E-mail: jiricny@imr.unizh.ch

Received 9 September 2002; Accepted 13 March 2003; Revised 6 March 2003

The mismatch repair (MMR) gene hMLH1 is mutated in approx50% of hereditary non-polyposis colon cancers and transcriptionally silenced in approx25% of sporadic tumours of the right colon. Cells lacking hMLH1 display microsatellite instability and resistance to killing by methylating agents. In an attempt to study the phenotypic effects of hMLH1 downregulation in greater detail, we designed an isogenic system, in which hMLH1 expression is regulated by doxycycline. We now report that human embryonic kidney 293T cells expressing high amounts of hMLH1 were MMR-proficient and arrested at the G2/M cell cycle checkpoint following treatment with the DNA methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), while cells not expressing hMLH1 displayed a MMR defect and failed to arrest upon MNNG treatment. Interestingly, MMR proficiency was restored even at low hMLH1 concentrations, while checkpoint activation required a full complement of hMLH1. In the MMR-proficient cells, activation of the MNNG-induced G2/M checkpoint was accompanied by phosphorylation of p53, but the cell death pathway was p53 independent, as the latter polypeptide is functionally inactivated in these cells by SV40 large T antigen.

  • Keywords:

    • cell cycle checkpoint,
    • hMLH1,
    • methylating agent,
    • mismatch repair,
    • TetOff