The epipodophyllotoxin etoposide (VP-16) is utilized extensively in cancer chemotherapy. A wide variety of experiments indicate that VP-16 stabilizes covalent adducts between DNA and the nuclear enzyme topoisomerase (topo) II. Upon removal of VP-16, the adducts are reversed, yet the cells proceed to die. These observations suggest that the topo II-DNA adducts initiate a series of event which culminate in cell death. This grant proposal is aimed at elucidating the poorly understood events between the formation of topo II-DNA adducts and the death of target cells. Preliminary results suggest that treatment of human leukemia cells with VP-16 in the presence of nucleic acid synthesis inhibitors-- particularly RNA synthesis inhibitors-- markedly diminishes the cytotoxicity of VP-16 without altering the number of topo II-DNA adducts. This observation suggests that ongoing nucleic acid synthesis is required to convert reversible topo II-mediated lesions into cytotoxic damage. It has been proposed that topo II-mediated nonhomologous recombination is involved. Consistent with this view, VP-16 has been observed to be mutagenic and possibly leukemogenic. We now propose to utilize two model systems (Saccharomyces cerevisiae bearing a temperature sensitive mutation in RNA polymerase b and mouse L cells transfected with a plasmid bearing the chloramphenicol acetyltransferase gene behind an inducible promotor) to test the hypothesis that ongoing RNA synthesis helps convert the reversible topo II-DNA adducts into cytotoxic DNA lesions and to assess the nature of this irreversible DNA damage. These experiments should provide insight into he mechanism of cytotoxicity of VP-16, into the nature of DNA damage induced by this agent, and into potential mechanisms of resistance.
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