Genetic predisposition to genomic instability in cancer.

Ataxia telangiectasia and Bloom's syndrome are cancer prone genomic instability disorders for which the causative genes ATM and BLM have only recently been identified. The cellular localisation and biochemical roles of these proteins are currently being defined. ATM shares sequence homology with several lipid and protein kinases but at the outset of this work a kinase function for ATM itself was not established and the extent to which the kinase domain contributed to the cellular phenotype of A-T was unknown. In addition the role of ATM heterozygosity and genomic instability in breast cancer had been suggested but not elucidated. During the course of this work, the anti-ATM antibody (CN-12) and anti-BLM antibody (IHIC-27), were developed and affinity purified. Antibody CN-12 successfully demonstrated the presence of the 350 kDa ATM protein by immunoblotting and immunoprécipitation in HeLa cells. The protein was largely nuclear in localisation with a minor microsomal fraction but was found to be absent in AT2RO cells which contain a homozygous truncating mutation of ATM. Tumour cell lines provided by the NCI showed varying expression of ATM except for the promyelocytic leukaemia cell line HL60 in which no ATM was detected. Antibody IHIC-27 demonstrated the presence of a 180 kDa protein in HeLa cells which was absent in the Bloom's cell line GM8505. Studies using antibodies directed to the C and N-termini showed BLM instability and cell fractionation studies demonstrated that the protein was nuclear in localisation. Anti-BLM monoclonal antibodies 185a and 32e were able to identify the fusion protein to which they were raised and BLM immunoprecipitated by IHIC-27 but could not identify BLM in whole cell lysates. N either ATM nor BLM antibodies were usefu l for immunohistochemical studies with the protocols tested despite successful immunostaining of the positive control. Studies of immunoprecipitated ATM using a variety of assay \ conditions* with p53 peptides and PHAS-I as substrates did not reveal any serine / threonine protein kinase activity, although low levels of autophosphorylation were identified. H1299 cells transfected with a mutant ATM kinase domain did not exhibit a dominant negative phenotype, which curtailed further studies of the cellular function of the domain. DNA repair in peripheral blood lymphocytes from breast cancer patients and controls was assessed by the comet assay. Although the patients generally showed less DNA repair than the controls at 30 min and 120 min, the difference was not found to be statistically significant. AIMS The general purpose of this work was to elucidate the mechanisms underlying the genomic instability seen in a taxi a-telangiectasia. Bloom's syndrome and breast cancer by studying the gene products of ATM and BLM and by investigating the DNA repair capacity in breast cancer patients. The specific goals are listed below: 1) To raise and characterise polyclonal antibodies versus ATM and BLM proteins which are abnormal in the genetic instability disorders ataxiatelangiectasia and Bloom's syndrome respectively. 2) To determine the levels of expression of these proteins in normal and tumour cell lines and in the tissue from a variety of human tumours. 3) To investigate the putative protein kinase activity of ATM protein with a view to developing an ATM kinase assay for use in determining the integrity of ATM signalling in the lymphocytes of normal women and women with breast cancer. It was hoped that this would enable the development of a test to determine an individuals risk of breast cancer. 4) To investigate the role of the ATM kinase domain in the cellular phenotype of ATM using a dominant negative strategy. 5) To examine the constitutional ability of DNA repair in lymphocytes from normal women and women with breast cancer using the comet assay. The intention was to assess the importance of DNA repair defects as a cause of genomic instability in the aetiology of this disease and to identify differences between the two populations which would be useful as a test of an individuals potential risk of developing breast cancer.