ENHANCED REPAIR OF A CISPLATIN-DAMAGED REPORTER CHLORAMPHENICOL-O-ACETYLTRANSFERASE GENE AND ALTERED ACTIVITIES OF DNA-POLYMERASES ALPHA AND BETA, AND DNA-LIGASE IN CELLS OF A HUMAN-MALIGNANT GLIOMA FOLLOWING IN-VIVO CISPLATIN THERAPY

Current evidence suggest an important role for increased repair of drug-induced DNA damage as one of the major mechanisms involved in tumor cell resistance to cis-DDP. In this study, we examined the DNA repair capacity and the activities of three DNA repair related proteins, namely, DNA polymerases alpha and beta, and total DNA ligase in cells of a malignant oligodendroglioma obtained from a patient before therapy and compared it with those of a specimen of the tumor acquired after the patient had failed cis-DDP therapy. DNA repair capacity was quantitated as the extent of reactivation of the chloramphenicol-O-acetyltransferase (CAT) gene in a eukaryotic expression vector that had been damaged and inactivated by prior treatment with cis-DDP and then transfected into the tumor cells. The extent of DNA-platinum adduct formation in the expression vector was determined by flameless atomic absorption spectrometry. The level of cis-DDP resistance of cells of the two tumors was determined with the capillary tumor stem cell assay. We observed a 2.8-fold increased capacity to repair Pt-DNA adducts and reactivate the CAT gene in cells of the tumor obtained after cis-DDP therapy, compared to cells of the untreated tumor. This was associated with increases of 9.4-foId and a 2.3-fold, respectively, in DNA polymerase beta and total DNA ligase activities in cells of the treated tumor. At 5 mu M cis-DDP, there was a 5.9-fold increase in the in vitro cis-DDP resistance of post-therapy tumor cells relative to cells of the untreated tumor. No significant difference in DNA polymerase cu activity was observed between the two tumors. These data suggest that the enhanced ability to repair cis-DDP induced DNA damage, mediated, in part, by increased tumor DNA polymerase beta and DNA ligase activities, plays an important role in the in vivo acquisition of cis-DDP resistance in human malignant gliomas, and that these proteins and/or their encoding genes may represent critical targets for strategies to overcome such resistance clinically. (C) 1994 Wiley-Liss, Inc.