Cisplatin (cDDP) is a bifunctional DNA binding antineoplastic agent with a major role in clinical cancer. The kinetics of cDDP binding and removal suggest that DNA repair is involved in its cytotoxic effects. Resistance to cDDP in human cancer cells is multifactorial, and enhanced DNA repair may be one such mechanism. Novobiocin (NB), an inhibitor of DNA repair and of DNA topoisomerase II (top II) (not necessarily by the same mechanism), enhances cDDP cytotoxicity in a cDDP resistant human carcinoma line by increasing cDDP DNA interstrand crosslinks (ISC). We propose to investigate the role of DNA repair in drug resistance to cDDP in human cancer cells. The SCC 25 (S) and SCC 25/CP (R) (a stable 30-fold drug resistant mutant) cell lines will be utilized. The component types of cDDP adducts to genomic DNA will be assayed: total cDDP by flameless atomic absorption spectroscopy, ISC by alkaline elution and intrastrand crosslinks by Elisa employing monoclonal antibodies. Total genomic repair may be an insensitive indicator since active genes may be preferentially repaired. ISC repair in an actively transcribed gene (Dihydrofolate reductase DHFR) will be compared to an inactive (B-globin) gene in S and R cells. Comparisons of ISC formation and removal between active and inactive genes and between cell lines will be made by Southern transfer, and hybridization analysis to cDNA probes. Tolerance of DNA-CDDP adducts may be as important to resistance as removal; therefore mRNA transcription from a functional gene will be assayed and compared with DNA repair data obtained from the same gene. Transcription of the DHFR gene after cDDP exposure will be measured by Northern or dot blotting in the S and R cell lines. The effects of NB and other top Il active agents to enhance the sensitivity of R cells should elucidate the role of top II inhibition on cDDP adduct formation and repair in active and inactive genes and on levels of transcription. In the aggregate these studies will lead to a better understanding of the role of DNA repair pathways in the cellular.response to cDDP and mechanisms of cDDP resistance. The molecular consequences of inhibition of top Il on the repair of cDDP-DNA adducts may provide insight into the mechanism of cDDP resistance. Modulation of DNA repair after alkylating agent/cDDP therapy may offer a novel approach to cancer therapy and in overcoming alkylating agent resistance in the clinic.
| Eder Jr, J P; Chan, V T; Ng, S W et al. (1995) DNA topoisomerase II alpha expression is associated with alkylating agent resistance. Cancer Res 55:6109-16 |
| Eder Jr, J P; Chan, V T; Niemierko, E et al. (1993) Conditional expression of wild-type topoisomerase II complements a mutant enzyme in mammalian cells. J Biol Chem 268:13844-9 |
