ALTERED GLUTATHIONE METABOLISM IN OXALIPLATIN RESISTANT OVARIAN-CARCINOMA CELLS

Elevation of glutathione (GSH) is commonly observed in cellular resist ance to a number of anticancer agents. Most frequently reported change in GSH metabolism that is associated with the elevated GSH levels is increased mRNA expression and activity of gamma-glutamyl cysteine synt hetase (gamma GCS), the first enzyme of the GSH biosynthetic pathway. We have isolated sublines of the A2780 ovarian carcinoma cell line (C1 0 and C25) that are 8- and 12-fold resistant to oxaliplatin by repeate dly exposing the cells to increasing concentrations of the platinum ag ent. The GSH levels in C10 and C25 cell sublines are 3.1- and 3.8-fold higher than the parent A2780 cell line. The mRNA levels and activitie s for gamma GCS and that for gamma-glutamyl transpeptidase (gamma GT), the GSH salvage pathway enzyme, were measured in these cells. The mRN A for gamma GT and gamma GCS were measured by RT-PCR, with quantitatio n of the PCR product by HPLC; mRNA levels are expressed as ratios to B -actin mRNA, used as an endogenous standard. GSH and gamma GCS activit y were measured by HPLC assays and gamma GT activity by a colorimetric assay. The increase in GSH in C10 and C25 was associated with an elev ation in gamma GT mRNA (2.5- and 8-fold) and gamma GT activity (2.7- a nd 2.8-fold). No changes were observed in gamma GCS mRNA levels or act ivity. The data indicate that alterations in GSH metabolism leading to elevations in cellular GSH in A2780 ovarian carcinoma cells selected for low levels of resistance to oxaliplatin are mediated by gamma GT, the 'salvage' pathway, rather than an increase in GSH biosynthesis.