Many cytotoxic drugs used in cancer chemotherapy inhibit DNA replication and induce programmed cell death (apoptosis) in tumor cells. It is unknown to what extent the inhibition of DNA replication per se is responsible for the induction of cell death by anticancer drugs. Cell death in cycling cells can be induced by treatment with known inhibitors of DNA replication, such as aphidicolin, a mycotoxin which inhibits elongation of replication by DNA polymerases alpha and delta, or mimosine, a plant amino acid which prevents initiation of replication in mammalian cells. The main goal of this application is to identify genes and genetic pathways that are involved in the induction of cell death by inhibitors of DNA replication. These genes will be identified through expression selection of genetic suppressor elements (GSE), short cDNA fragments that inhibit gene expression by encoding dominantly acting peptides or inhibitory antisense RNA. A normalized library of GSEs in a retroviral vector will be transduced into HeLa cells, and GSEs which protect cells from aphidicolin- or mimosine-induced cell death will be isolated. These GSEs will be characterized for their efficacy, effects on cell cycle progression, ability to protect cells against different DNA replication inhibitors and cell-type specificity. Full-length cDNAs for human genes corresponding to the selected GSEs will be isolated, and their structure and expression patterns in the cell cycle will be characterized. The isolated GSEs will also be tested for their effects on cellular response to replication-inhibiting chemotherapeutic drugs, to determine the role of replication-based mechanisms of cytotoxicity in their antitumor effect.
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