TOPOISOMERASE II--GENE REGULATI0N AND DRUG RESISTANCE

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Topoisomerase II is an essential enzyme for such cell functions as DNA replication and chromosome segregation. Acting as a dimer, topoisomerase II passes a double stranded DNA segment through a transient double strand break in a second DNA strand to modify the topology of the DNA molecule. Two isoforms of topo II (alpha and beta) are present in mammalian cells. Topo IIbeta protein levels in the cell are known to directly correlate with cell proliferation rate and topo IIalpha expression is cell cycle dependent. Topo IIbeta levels are much more constant throughout the cell cycle and the protein is more tightly associated with the nuclear scaffold. Because of the interaction of topo II with DNA in critical cellular functions, it is both a unique and natural target for anticancer drugs that can inhibit cell growth or induce cell death. However, all too often, tumor cell develop resistance to topo Il-targeted drugs. Although the drug resistance can result from structural mutations in the topo II protein, decreased levels to topo II in the cell may more often be the primary factor contributing to the cells decreased drug sensitivity. The major objective of this application is to identify and characterize the transcription factors that regulate the expression of topoisomerase IIalpha. These studies will focus on both the normal cell cycle-dependent expression of topo IIalpha and the altered expression of topo IIalpha in drug-resistant cells. These studies will include characterization of the mechanism by which topo Il-targeted drugs and the DNA damage resulting from the drug's action effects the level of topo IIalpha in the cell. Drug induced DNA strand breaks induce the tumor suppressor gene p53, which serves as a G1 checkpoint control for DNA damage. Wild type p53 has an antiproliferative and antitumorgenetic effect resulting from specific activation or suppression of gene transcription. On the other hand, mutant p53 functions as an oncogene, promoting tumorgenesis. Our initial studies provide evidence that wild type p53 may serve as a negative controlling factor for the regulation of topo Ha expression. The topo IIalpha promoter sequence required for p53 downregulation of topo IIalpha and the transcription factors interacting with the topo IIalpha promoter element will be identified and characterized. Analysis of topo II regulation in normal and tumor cells will yield vital information for the effective use of the clinically important topo II-targeted drugs.