Cisplatin (cis-diamminedichloroplatinum (II)) is a metal coordination complex that kills cells by mechanisms initiated by its interaction with DNA. Using biochemical and genetic approaches, the goal of this project is to continue our work probing the how this compound kills cells. The practical value of the work stems from the use of this compound as an anticancer drug with organotropic specificity for the testis, and to a lesser extent, ovary. While study of the anticancer activity of cisplatin is not our goal, any basic mechanisms we uncover could be of value in the design of new selective toxins. In previous work we discovered that the architecture of cisplatin-DNA adducts is unusual in that it attracts certain cellular proteins. We have postulated and in some cases demonstrated that there are deleterious effects of these interactions. First, the binding of cellular proteins to adducts shields the adducts from DNA repair enzymes; the adducts persist and have an enhanced likelihood of killing the cells. Second, some adduct binding proteins are transcription factors. If the transcription factor becomes associated with an adduct, and not its promoter, gene expression is adversely affected. Third, adducts attract mismatch repair proteins and possibly disrupt their role in genetic recombination. The proposed work is divided into three areas. Having established that adducts act as molecular decoys for transcription factors in vitro, we plan experiments to test whether a transcription factor, specifically hUBF (which regulates rRNA synthesis), is hijacked in vivo.
Our second aim i s to continue recent work that has shown that mutations in specific recombination genes can result in enhanced sensitivity to cisplatin by as much as 10^4 fold over an isogenic wild type cell. Our proposal is to continue to uncover the mechanism by which recombination affects cellular responses to cisplatin and, in addition, determine by using genetic techniques if the mismatch repair pathway interacts with recombination pathways in affecting cellular survival. Finally, we plan biochemical experiments to complement the aforementioned genetic studies on recombination and mismatch repair. It is hypothesized that cisplatin adducts will be substrates for purified recombination enzymes and that mismatch repair proteins, which control many aspects of recombination, may interfere with the orderly progress of events that would otherwise allow tolerance of the adducts. Taken together these studies will provide mechanistic detail on the toxic effects of an important DNA damaging agent.
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