How cells respond to exposed DNA ends often will determine their ultimate fate. Frequently, broken DNA ends will result in an irreversible termination of proliferation, either in the form of cell death (apoptosis) or terminal cessation of proliferation (senescence). In other cases, if the damage to the DNA is repairable, the cell pauses to correct the damaged DNA and then continues to proliferate. The goal of these studies is to investigate the molecular signal transduction pathway that recognizes DNA strand breaks and causes cells to cease proliferation, either as a consequence of exposure to environmental carcinogens or as part of the normal program of cellular ageing. One gene product that seems to be required for a proper cellular response to multiple forms of DNA damage that occur throughout the cell's proliferative cycle is the product of the gene responsible for the autosomal recessive disorder ataxia telangiectasia (AT), referred to as ATM for AT Mutated gene. The role of the ATM protein in transmitting signals that result in the inhibition of cyclin/cdk protein kinase complexes is being investigated. We have raised antisera in rabbits to three peptides whose sequences correspond to predicted sequences in the carboxyl-terminal half of the ATM protein and one peptide sequence corresponding to a region in the amino terminal region. We are characterizing these antisera as well as three rabbit polyclonal antisera raised by our collaborator Dave Hill of Oncogene Research. We have been successful in reproducibly detecting the 350 kDa ATM protein in extracts from NHF and HeLa cells that is lacking in AT fibroblasts. Studies to characterize the normal expression pattern of the ATM protein during the cell cycle, the normal subcellular localization of the ATM protein, the localization after exposure to DNA damaging agents, and the levels and post-translational modifications after exposure to DNA damaging agents are underway. The results of these studies will contribute to a better understanding of the normal molecular events regulating cellular senescence and the cell cycle checkpoint delay following DNA damage. In addition, these studies hold the potential for proving insight into mechanisms of action of certain NTP compounds, in particular those compounds that have been classified as non-genotoxic carcinogens.
