The long-range goal of this project is to elucidate the molecular mechanisms underlying the preferential killing of cancer cells by therapeutic agents acting on DNA topoisomerases. Specifically, three types of questions will be addressed: (1) How do these anticancer drugs interact with their targets? (2) Upon binding of the drug to their targets, what are the subsequent cellular events leading eventually to cell death? (3) How is the expression of the gene TOP2 encoding human DNA topoisomerase II regulated by cell growth and differentiation? What is the relation between the regulation of this key enzyme, which participates in a number of vital processes including DNA replication and transcription, and the genetic programming of normal and malignant cells? Parallel approaches will be undertaken using yeast strains permeable to the anticancer drugs, and human and yeast genes TOP1 and TOP2 encoding respectively DNA toposomerases I and II of the two organisms. The methods of molecular genetics will be used to identify mutations in the TOP genes of yeast and human leading to drug- resistance or hypersensitivity, using yeast and yeast expressing the human genes in these studies. Cellular events leading to cell killing by the anti- cancer drugs will be studied initially using the yeast model. The effects of the drugs on recombination and repair will be examined, and second site suppressors or hypersensitive mutants will be identified to determine the participants of the pertinant cellular processes. The regulation of human TOP2 gene will be studied in mammalian cells transfected with various clones of the resected or mutagenized human gene. The effects of mitogen stimulation of quiescent cell on the expression of the gene in various constructs will be examined. The effects of expressing the gene constitutively in stably transformed cells will also be investigated.
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