The overall objective of this project is to investigate the involvement of the bleomycin (BLM)-inactivating enzyme, BLM hydrolase, in the resistance of human tumors to the antineoplastic agent, BLM, and to develop strategies to overcome this resistance. This will be accomplished by a) determining the amount of BLM hydrolase, its activity levels and the levels of expression of the gene encoding this enzyme in BLM-resistant and sensitive human tumors and relating these parameters to the antitumor activity of BLM in these tumors, b) cloning and sequencing the gene encoding BLM hydrolase, transfecting this gene into BLM-sensitive tumor cells and determining their responsiveness to BLM, c) investigating the effects of known BLM hydrolase inhibitors on the antitumor activity of BLM in vivo, and d) evaluating the antitumor activity of several BLM analogs and determining the relationship between metabolism of the analogs by BLM hydrolase and their antitumor potency. Cloning of the BLM hydrolase gene will be carried out using a recently isolated partial length BLM hydrolase cDNA probe. Tumor content of BLM hydrolase will be determined using anti-BLM hydrolase monoclonal and polyclonal antibodies and BLM hydrolase activity will be measured by a recently developed high speed liquid chromatography method. The above studies will be carried out in vivo using humor tumor cells with various degrees of resistance to BLM. Human tumor cell lines, including those transfected with the BLM hydrolase gene 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 level. The human tumor xenografts in nude mice will also be used to evaluate the antitumor activity of several BLM analogs and the effects of known BLM hydrolase inhibitors on the antitumor potency of these analogs. These studies will enhance our understanding of the mechanism by which BLM hydrolase levels influence the responsiveness of human tumor cells to BLM treatment, and will allow us to develops strategies to overcome BLM resistance and to broaden the spectrum of human tumors that can be treated with BLM.
