The long-term objective of this project is to understand at the cellular and molecular levels the mechanisms responsible for the activity of the new anticancer drug tirapazamine (TPZ) towards hypoxic cells. This drug is activated at low-oxygen level to a highly reactive free radical that damages DNA, thereby killing hypoxic cells. In addition, at non toxic doses, TPZ preferentially sensitizes hypoxic cells to the anticancer drug cisplatin, and clinical trials of the drug combined with radiation and with cisplatin show very promising results. However, the mechanisms both of cytotoxicity and potentiation of cisplatin are poorly understood. We propose three Specific Aims. First, we propose to identify and characterize the nuclear reductase responsible for the metabolism of TPZ to its DNA- damaging radical. We will use both biochemical and genomics approaches, in the latter case using the powerful methodology of chip arrays with specially constructed deletion mutants of all of the open reading frames of Saccharomyces cerevisiae.
In Specific Aim 2 we will test the hypothesis that the mechanism by which TPZ produces DNA double-strand breaks (dsb's) and cytotoxicity in hypoxic cells is by poisoning the catalytic cycle of topoisomerase II (topo II). Our approaches for this will include using specific inhibitors of the topo II catalytic cycle, using various topo II mutants, using transfected cells with various levels of topo II and by measuring topo II binding to DNA in individual cells both in vitro and in tumors in vivo. In the third Specific Aim we will test the hypothesis that the potentiation of cisplatin cytotoxicity by TPZ under hypoxic conditions is the result of inhibiting topo II, thereby delaying the repair of the cisplatin-induced DNA interstrand cross-links. To test this hypothesis we will employ inhibitors of the topo II catalytic cycle as well as cells induced to express high levels of topo II following TPZ treatment
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