methylnitronitrosoguanidine and 7-hydroxystaurosporine

SN1 DNA methylating agents such as the nitrosourea N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) elicit a G2/M checkpoint response via a mismatch repair (MMR) system-dependent mechanism; however, the exact nature of the mechanism governing MNNG-induced G2/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 G2/M checkpoint response. We document that MNNG induces a robust, dose-dependent G2 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 G2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMR-dependent 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.

Topics: Base Pair Mismatch; Cell Division; Cell Nucleus; Cells, Cultured; Checkpoint Kinase 1; Checkpoint Kinase 2; DNA Damage; DNA Methylation; DNA Repair; DNA-Binding Proteins; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; G2 Phase; Humans; Immunoblotting; Immunoprecipitation; Methylnitronitrosoguanidine; MutS Homolog 2 Protein; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; RNA Interference; RNA, Small Interfering; Staurosporine; Subcellular Fractions; Time Factors

S(N)1-type alkylating agents represent an important class of chemotherapeutics, but the molecular mechanisms underlying their cytotoxicity are unknown. Thus, although these substances modify predominantly purine nitrogen atoms, their toxicity appears to result from the processing of O(6)-methylguanine ((6Me)G)-containing mispairs by the mismatch repair (MMR) system, because cells with defective MMR are highly resistant to killing by these agents. In an attempt to understand the role of the MMR system in the molecular transactions underlying the toxicity of alkylating agents, we studied the response of human MMR-proficient and MMR-deficient cells to low concentrations of the prototypic methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We now show that MNNG treatment induced a cell cycle arrest that was absolutely dependent on functional MMR. Unusually, the cells arrested only in the second G(2) phase after treatment. Downstream targets of both ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases were modified, but only the ablation of ATR, or the inhibition of CHK1, attenuated the arrest. The checkpoint activation was accompanied by the formation of nuclear foci containing the signaling and repair proteins ATR, the S(*)/T(*)Q substrate, gamma-H2AX, and replication protein A (RPA). The persistence of these foci implied that they may represent sites of irreparable damage.

Topics: Ataxia Telangiectasia Mutated Proteins; Base Pair Mismatch; Caffeine; Cell Cycle; Cell Cycle Proteins; Cell Line; Checkpoint Kinase 1; DNA; DNA Damage; DNA Repair; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; G2 Phase; Histones; Humans; Methylation; Methylnitronitrosoguanidine; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Replication Protein A; Signal Transduction; Staurosporine; Tumor Suppressor Proteins