DETECTION OF DNA-DAMAGE IN TRANSCRIPTIONALLY ACTIVE GENES BY RT-PCR AND ASSESSMENT OF REPAIR OF CISPLATIN-INDUCED DAMAGE IN THE GLUTATHIONE-S-TRANSFERASE-PI GENE IN HUMAN GLIOBLASTOMA CELLS

Cisplatin is an anticancer agent frequently used as an alternative to the nitrosoureas in brain tumor chemotherapy. We describe the use of a technique of quantitative reverse transcription-polymerase chain reac tion (RT-PCR) to examine the damage induced in the glutathione S-trans ferase (GST)-pi gene by cisplatin and the subsequent repair of this da mage in cells of the MGR3 human glioblastoma multiforme cell line. The relationship between cisplatin dose and the extent of damage in the G ST-pi gene was determined over cisplatin concentrations (0-10 mu M) wi thin the clinically achievable range. Total RNA was purified from cont rol and cisplatin-treated cells, and both the full-length GST-pi cDNA and a control 200-bp beta-actin cDNA were amplified by RT-PCR. The cDN A reaction products were electrophoresed, Southern hybridized, and qua ntitated densitometrically. A decrease in GST-pi mRNA representing dam age to the GST-pi gene was observed with increasing cisplatin concentr ations, up to a maximum of 75% at 10 mu M cisplatin. Repair of the GST -pi gene in cells treated with cisplatin, assessed as recovery of tran scriptional activity of the gene, was shown to occur even after 48 hr following drug removal. A potent RNA polymerase II inhibitor, alpha-am anitin, was used to show that the GST-pi mRNA quantitated in this RT-P CR assay resulted from de novo RNA transcription of the GST-pi gene wi th little contribution from preexisting GST-pi transcripts. The result s demonstrate that the GST pi gene, which is actively transcribed and often overexpressed in human glioma cells, is a target for cisplatin, but that the damage to the gene is efficiently repaired in these cells . The RT-PCR assay has the potential for use in the detection of DNA d amage induced by genotoxic agents in other actively transcribed genes and for assessing the repair of gene-specific DNA lesions in cells. (C ) 1997 Academic Press, Inc.