The long-term goal of this project is to understand the mechanisms that govern activation of cell cycle arrest and apoptosis in response to nitrosoureas, a class of DNA alkylating agent. While these responses are key to limiting the ability of nitrosoureas to cause cancer, little is known concerning the pathways that control growth arrest or cell death in response to these drugs. We found that the DNA damage-response kinase ATM, an essential component in triggering various checkpoint pathways is activated in response to the nitrosourea MNNG. Further, the mismatch repair system, a DNA repair mechanism suspected to play a key role in MNNG-induced responses, is required for the ATM-dependent activation of the checkpoint kinase Chk2. We hypothesize that MNNG treatment activates multiple pathways that control cell cycle arrest and apoptosis. We will test this hypothesis by addressing the following Aims: 1) Determine the pathway controlling G2 arrest following low-dose MNNG exposure. Our studies are consistent with the hypothesis that mismatch repair-dependent activation of the ATM>Chk2>cdc25C> p34cdc2 pathway controls activation of G2 arrest after low-dose (5 microM) MNNG exposure. We will test the hypothesis that Chk2 is required to activate this G2 arrest using mutant cell lines and RNAi technology. 2) Determine the pathway controlling G2 arrest following high-dose MNNG exposure. Unlike response to 5 microM MNNG, we find that G2 arrest in response to 25 microM MNNG is both ATM and mismatch repair independent. Our preliminary studies lead us to hypothesize that this high-dose arrest is controlled by activation of redundant ATR>Chk1>cdc25C>p34cdc2 and ATR>Chk2>cdc25C>p34cdc2 pathways. We will use mutant cell lines, pharmacological inhibitors and RNAi technology to test this hypothesis. 3) Determine the pathway that triggers p53-dependent apoptosis in response to MNNG. ATM, Chk2, and mismatch repair are each required for proper phosphorylation of p53 in response to genotoxic stress; therefore, we hypothesize that each of these molecules/mechanisms are required to trigger the p53-dependent apoptogenic pathway. We will test this hypothesis using a panel of normal and mutant lymphoblasts and cells of epithelial origin. Understanding the molecules, mechanisms and pathways that control cellular response to MNNG and other genotoxins is important in preventing cancer and improving our ability to design effective cancer therapeutics.
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