The drug WR-2721 selectively protects normal vs tumor cells against radiation and antitumor drugs and is currently in clinical trials to evaluate its use in cancer treatment. The objective of the present research is to provide a chemical and biochemical basis for understanding (1) the selective uptake of the drug in normal but not tumor tissues and (2) the highly effective protection by the drug against radiation damage. Our previous studies have indicated that low uptake by tumors is due to (1) their low alkaline phosphatase activity which inhibits conversion of WR-2721 to its thiol form (WR-1065) and (2) their low pH which slows the uptake of WR-1065. Our 1st specific aim is to complete these studies by determining whether uptake occurs by a mediated process, by nonmediated passive diffusion, or by a combination of these mechanisms, and the extent to which diffusion through tissue mass limits uptake. The results of these studies will provide a basis for understanding selective uptake and for interpreting its tissue specificity. In previous studies we established that WR-1065 is the form that protects V79-171 cells. For DNA in solution we showed that cation condensation and anion depletion occurs near DNA and provided evidence that this is a significant factor in the ability of WR-1065 to protect DNA in vitro. It seems likely that these phenomena are also important in the cell and serve to determine the effectiveness of WR-1065, a dication. In our 2nd aim we will test this hypothesis with V79-171 cells in culture by comparing the radioprotection achieved when cells are loaded to the same level with thiols of varying net charge. Our 3rd specific aim is to determine how protection by WR-1065 depends upon cell cycle status and upon oxygen tension. The 4th specific aim is to test the charge dependence of thiol radioprotection with isolated nuclei and the 5th specific aim is to extend the basis for the charge dependence seen with DNA to low oxygen tension and cell-like conditions using the more sensitive SV40 DNA model system. The 6th specific aim is to establish how protection of V79-171 cells by WR- 1065 changes for different LET radiation. The results obtained will provide a critical test of the role of radioprotector net charge in determining drug effectiveness and may thereby establish a key factor in radioprotector design. The data on WR-1065 protection of cells at varying oxygen tension, cell cycle position, and radiation type are essential to understanding tissue specificity and optimal clinical application of WR 2721.
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