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-glutamyltranspeptidase (gamma-GT) were measured in the melphalan-resistant human multiple 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 of 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 cell lines. Levels of gamma-GT mRNA were similar in the /S and /LR-5 lines but were reduced in the /Dox40 cells. These data suggest that the increased GSH levels associated with resistance to melphalan in the 8226/LR-5 myeloma cells is attributable to up-regulation of gamma-GCS. This observation is consistent with recent demonstrations of up-regulation of gamma-GCS in melphalan-resistant prostate carcinoma cells and cisplatinum-resistant ovarian carcinoma cells, suggesting that increased expression of gamma-GCS may be an important mediator of GSH-associated resistance mechanisms.
Mulcahy
R T
RT
Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, University of Wisconsin-Madison 53792.
Bailey
H H
HH
Gipp
J J
JJ
D015152:Blotting, Northern; D002470:Cell Survival / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance / Q000502:physiology; D005721:Glutamate-Cysteine Ligase / Q000502:physiology*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008558:Melphalan / Q000494:pharmacology*; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000201:enzymology / Q000378:metabolism*; D012313:RNA / Q000032:analysis; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000502:physiology*; D005723:gamma-Glutamyltransferase / Q000502:physiology
D012313:RNA; D004317:Doxorubicin; D005723:gamma-Glutamyltransferase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D008558:Melphalan