Many of the antiproliferative agents used to treat cancer inhibit the elongation of nascent DNA chains. In principle, targeting the molecular events involved in initiation of DNA replication at origins of DNA replication should be at least as effective as a therapeutic strategy. This possibility is relatively unexplored, largely because of the paucity of information about how initiation occurs in mammals and the lack of drugs that specifically inhibit initiation rather than elongation. We are studying the cytotoxic and replication inhibitory effects in yeast of adozelesin, a member of the cyclopropylpyrroloindole (CPI) class of experimental antitumor agents. Our choice of yeast as a model organism is based on the large extent to which pathways that regulate origin function are conserved and the relative ease with which they can be manipulated genetically. CPI drugs are extremely cytotoxic and show potent antitumor activity in mice. Adozelesin profoundly inhibits initiation of DNA replication in mammals with little or no effect on elongation. A similar inhibitory effect on initiation is induced by adozelesin in the budding yeast S. cerevisiae. It occurs in concert with the induction of a highly conserved checkpoint, and this checkpoint requires the function of an important element of pathways that regulate origin licensing in G1 and S phase, the Origin Recognition Complex (ORC). Mutations in ORC that abrogate this checkpoint sensitize cells to the lethal effects of this drug. Surprisingly, the increased lethality occurs predominantly in G1 and appears to be related to origin licensing rather than to replication of damaged DNA in S phase. This represents a novel mechanism of cytotoxicity that could explain the antitumor properties of this and related DNA damaging agents. This mechanism might be exploited therapeutically to specifically target slowly proliferating tumor cells that are in G1 which might be refractile to more conventional S phase inhibitors. This project will extend these observations through a combined genetic and biochemical analysis of the effects of this drug on initiation of DNA replication S. cerevisiae. These experiments are designed to test the hypothesis that the cytotoxicity of this drug is related to its inhibitory effect on initiation, and that this effect is different from the effects induced by less cytotoxic DNA damaging agents.
Showing the most recent 10 out of 11 publications
