Methylator-induced, Mismatch Repair-dependent G₂Arrest Is Activated through Chk1 and Chk2

Abstract

2 /M arrest and how MMR mechanistically participates in this process are unknown. Here, we show that MNNG exposure results in activation of the cell cycle checkpoint kinases ATM, Chk1, and Chk2, each of which has been implicated in the triggering of the G 2 /M checkpoint response. We document that MNNG induces a robust, dose-dependent G 2 arrest in MMR and ATM-proficient cells, whereas this response is abrogated in MMR-deficient cells and attenuated in ATM-deficient cells treated with moderate doses of MNNG. Pharmacological and RNA interference approaches indicated that Chk1 and Chk2 are both required components for normal MNNG-induced G 2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMRdependent manner and was compromised in cells lacking ATM. Finally, both Chk1 and Chk2 interact with the MMR protein MSH2, and this interaction is enhanced after MNNG exposure, supporting the notion that the MMR system functions as a molecular scaffold at the sites of DNA damage that facilitates activation of these kinases. INTRODUCTIONCells are continually exposed to numerous forces and toxins capable of damaging DNA. To ensure maintenance of genome stability, cells have evolved a complex set of mechanisms to appropriately respond to genotoxic damage. Such responses include genome surveillance and DNA repair, activation of cell cycle checkpoints, and apoptosis. Tumor initiation and progression are directly linked to genomic instability and often correlate with loss of gene(s) involved in genome damage response (Hartwell and Kastan, 1994;Loeb et al., 2003). Paradoxically, many cancer treatment regimens induce DNA damage and exert their therapeutic effects through activation of growth arrest or apoptotic responses. Thus, elucidating the mechanisms and molecules that govern DNA damage response is key to understanding the molecular basis of both tumor formation and the therapeutic effects of many anticancer drugs.The nitrosourea N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG) is a well characterized monofunctional DNA alkylating agent. The cytotoxic and mutagenic potential of MNNG is chiefly attributable to its ability to alkylate (methylate) the O 6 -position of guanine, resulting in formation of O 6 -methylguanine (O 6 MeG) adducts (Goldmacher et al., 1986;Karran and Bignami, 1992). O 6 MeG forces O 6 MeG-T mispairing after DNA replication due to blocking of a hydrogen bonding position involved in complementary base pairing. This mutagenic lesion is primarily repaired via direct demethylation by the DNA repair protein methylguanine-DNA methyltransferase (MGMT) (Lindahl et al., 1982). Moreover, loss of MGMT activity renders cells extremely sensitive to MNNG and like alkylators (Kalamegham et al., 1988), underscoring the role that O 6 MeG lesions play in triggering response to this drug. O 6 MeG lesions are also recognized and repaired by the mismatch repair (MMR) system (Griffin et al., 1994;Duckett et al., 1996).In response to MNNG and other methylators, cells undergo a robust G...

Epigenetic Silencing of the Tumor Suppressor Cystatin M Occurs during Breast Cancer Progression

“…The medium was subsequently concentrated using iCON concentrators (Pierce, Rockford, IL) and protein concentration was assayed by BCA (Pierce). SDS-PAGE and immunoblotting was done using established protocols (36). Biotinylated goat anti-human cystatin E/M antibody was purchased from R&D Systems (Minneapolis, MN).…”

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…A number of interactions between MMR proteins and DNA damage signalling proteins have been reported including interactions between MSH2 and ATR, Chk1 and Chk2 (Wang and Qin, 2003;Yoshioka et al, 2006); MLH1 and ATM (Adamson et al, 2005;Brown et al, 2003); PMS2 and p73 (Shimodaira et al, 2003); the three MutL homologues, MLH1, PMS2, and PMS1 with a large number of proteins involved in cell cycle/signalling/apoptosis functions (Cannavo et al, 2007). The challenge is to determine the functional significance of these interactions and the biological context in which they operate.…”

Reduced ATR or Chk1 Expression Leads to Chromosome Instability and Chemosensitization of Mismatch Repair–deficient Colorectal Cancer Cells

“…This requires hMutS and hMutL complexes and may involve direct interactions between hMLH1 and ATM, as one signaling complex, and hMSH2 and Chk2 as another (Brown et al, 2003). In response to certain DNA damaging adducts, such as O 6 -methylguanine, hMSH2 has been found to engage in a direct signaling complex with ATR, indicating that MMR can act as damage sensors for ATR-dependent checkpoint signaling (Wang and Qin, 2003;Yamane et al, 2004;Adamson et al, 2005;Yoshioka et al, 2006). Additionally, it has been shown that hMLH1 is necessary for p53-mediated induction of apoptosis in response to alkylating and oxygen radicalinducing agents (Luo et al, 2004).…”