Genetic predisposition to genomic instability in cancer.

Vessey, Carina Jayne (1998). Genetic predisposition to genomic instability in cancer. PhD thesis The Open University.



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 immunoprecipitation 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. Neither ATM nor BLM antibodies were useful 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.

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