The goal of these studies is to investigate the molecular signal transduction pathways that recognize 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 (senescence). 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 gene mutated in ataxia telangiectasia ATM (AT Mutated). 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 various peptides whose sequences correspond to amino acid sequences of the ATM protein. Antibodies have been affinity purified using the ATM peptide that ellicited the antiserum found to show the best specificity in recognizing the ATM protein. We are also characterizing rabbit polyclonal antisera and a mouse monoclonal antiserum raised by our collaborator Dave Hill, Oncogene Research. We have been successful in reproducibly detecting the 350 kDa ATM protein in extracts from NHF and HeLa cells, a protein that is lacking in extracts from fibroblasts from AT patients. The ATM protein is a nuclear protein whose levels of expression do not vary over the cell cycle or in response to DNA damaging agents. Studies to characterize ATM association with other proteins and associated protein kinase activity, under normal growth conditions and following exposure to DNA damaging agents or during the process of cellular senescence, are underway.. The results of these studies will contribute to a better understanding of the normal molecular events regulating cellular senescence and cell cycle checkpoint delay.
