The ultimate goal of this project continues to be: to overcome tumor resistance to the anticancer drug bleomycin (BLM) and to broaden the spectrum of human cancers that can be treated with BLM. The overall objective is to investigate the role of the BLM-inactivating enzyme, BLM hydrolase, in the resistance of human tumors to BLM and to develop strategies to overcome this resistance. This will be accomplished by a) cloning and sequencing human tumor BLM hydrolase cDNA, transfecting this cDNA into BLM-sensitive tumor cells and determining their responsiveness to BLM b) introducing a neutralizing BLM hydrolase antibody to BLM- resistant cells and determining their responsiveness to BLM, c) investigating the mechanism by which some human tumors metabolize higher levels of BLM, determining the levels of BLM hydrolase activity, its gene expression, protein amount and turnover rate in human tumors, and relating these parameters to the antitumor activity of BLM in these tumors, d) determining the metabolic pathways involved in the complex BLM metabolism observed in human tumors, sensitizing these tumors using inhibitors of BLM metabolism, and e) isolating and characterizing the various BLM metabolites formed in human tumors. Cloning of the full length BLM hydrolase cDNA will be carried out using a partial length BLM hydrolase cDNA insert that we have recently isolated from a Burkitt's lymphoma cDNA library. This probe will also be used to determine gene expression levels of BLM hydrolase in human tumor cells. Neutralization of intracellular BLM hydrolase will be carried out by electroporation of BLM-resistant human tumor cells with affinity-purified antibody that we have recently prepared against a 14 amino acid BLM hydrolase peptide. This neutralizing antibody will also be used to measure tumor content of BLM hydrolase. BLM hydrolase activity will be measured by high performance liquid chromatography. The in vivo antitumor studies will be carried out using human tumor cell lines with various degrees of BLM- resistance in the nude mouse xenograft model. Human tumor cell lines, including those transfected with BLM hydrolase cDNA, and human tumor fragments from fresh biopsies, will be implanted subcutaneously into athymic nude mice and examined for their responsiveness to BLM and for their BLM hydrolase content, activity and expression levels. These studies will enhance our understanding of the mechanism by which BLM hydrolase levels influence the responsiveness of human tumors to BLM treatment and will allow us to develop strategies to overcome BLM resistance.
