UP-REGULATION OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE-ACTIVITY IN MELPHALAN-RESISTANT HUMAN MULTIPLE-MYELOMA CELLS EXPRESSING INCREASED GLUTATHIONE LEVELS

Levels of intracellular glutathione (GSH) and the GSH-related enzymes gamma-glutamylcysteine synthetase (gamma-GCS) and gamma-glutamyltransp eptidase (gamma-GT) were measured in the melphalan-resistant human mul tiple myeloma cell line 8226/LR-5 and were compared to those measured in the drug-sensitive 8226/S and doxorubicin-resistant 8226/Dox40 cell lines. Both GSH and gamma-GCS activity, the rate-limiting step in the de novo synthesis of GSH, were elevated by a factor of approximately 2 in the melphalan-resistant 8226/LR-5 cells relative to the other two lines. gamma-GT activity was not elevated significantly in the /LR-5 cells. Northern analysis with a probe specific for the large subunit o f human liver gamma-GCS identified two bands (3.2 and 4.0 kb), both of which were increased by a factor of 2-3 in the 8226/LR-5 line. Levels of gamma-GCS mRNA expression were comparable in the /S and /Dox40 cel l lines. Levels of gamma-GT mRNA were similar in the /S and /LR-5 line s but were reduced in the /Dox40 cells. These data suggest that the in creased GSH levels associated with resistance to melphalan in the 8226 /LR-5 myeloma cells is attributable to up-regulation of gamma-GCS. Thi s observation is consistent with recent demonstrations of up-regulatio n of gamma-GCS in melphalan-resistant prostate carcinoma cells and cis platinum-resistant ovarian carcinoma cells, suggesting that increased expression of gamma-GCS may be an important mediator of GSH-associated resistance mechanisms.