971. Novel Ribozyme-Based Strategy for Cancer Chemoprevention

Abstract

Top of pageAbstract Recent advances in understanding the molecular mechanisms of carcinogen-induced mutagenesis indicate that most mutations are dependent on the activity of newly-described translesion DNA polymerases, which have reduced base-pairing requirements and are able to replicate past DNA damage with potentially mutagenic consequences. In human cells in culture, reducing the level of one such polymerase, REV1, with a gene-specific ribozyme greatly reduces carcinogen-induced mutagenesis. To examine the hypothesis that reducing the mutagenic load will reduce the incidence of cancer, we are developing strategies to reduce REV1 in vivo in mouse models of carcinogenesis. We constructed vectors that express a ribozyme targeting mouse REV1 in a region homologous to the human mRNA used in our published studies. In order to improve the efficiency of gene-specific knockdown, DNA coding for the ribozyme was cloned into expression cassettes designed to target the transcripts either to the nuclear or to the cytoplasmic compartment. Using quantitative real-time PCR for REV1 mRNA with GAPDH as an internal control, we consistently found that the greatest suppression occurred when both compartments were targeted. When both compartments were targeted, REV1 expression was consistently suppressed by 80-90%. UV-induced mutant frequency at the HPRT locus in these cells was also reduced up to 80%, but UV-induced cytotoxicity was not affected. We plan to deliver these plasmids to the skin of excision repair deficient hairless xpa knockout mice, which develop squamous cell skin cancer with 100% penetrance after low doses of UV. Delivery of these expression cassettes to carcinogen-exposed tissue presents a novel chemopreventive strategy to reduce the frequency of mutations and the incidence of cancer.