Human tumors frequently develop resistance to many of the widely used chemotherapeutic agents. Many different mechanisms have been proposed for the molecular basis of this resistance. Studies on drug resistance in tumor cells have often focused on the mechanism of action of the drug, and looked for resistance due to altered drug concentration in the cell, different models of drug inactivation or altered damage repair. As such, other pathways that may be involved with the cellular cytotoxic response to individual drugs have been overlooked. We propose to use the cellular slime mold Dictyostelium discoideum in an unbiased approach to identify novel molecular targets that can be modulated to increase sensitivity of tumor cells to chemotherapeutic drugs. The genes and pathways of Dictyostelium are highly conserved with those of humans, and molecular genetic methods are well developed for this organism. Our preliminary system on cisplatin resistance resulted in the identification of 6 genes. Significantly, none of these had been previously associated with cisplatin, and each represents a potential new target for therapy. The goal of the present study is to demonstrate the general utility of this system and to show that it can be applied to the understanding of resistance to other drugs. We have focused on four classes of DNA damaging drugs that damage DNA by different mechanisms. These include: both intra- and inter-strand crosslinkers, monoalkylators, and oxygen radicals. We will 1) create a comprehensive Dictyostelium insertional mutant library, 2) isolate mutants resistant to drugs of each of the four classes and identify the cognate genes, and 3) test the mutants for cross-resistance to the other drugs. These studies will identify new mechanisms for drug resistance and new targets for chemotherapeutic intervention which can subsequently be validated in human cells.
