Multidrug resistance (MDR) is a phenomenon associated with the emergence of simultaneous cross-resistance to the cytotoxic action of a wide variety of structurally and functionally unrelated antineoplastic agents. The present study was undertaken to determine if 8226 human myeloma cells possessing the MDR phenotype had an increased ability to resist the intercalating drug doxorubicin (DOX) via glutathione-based detoxification systems. Glutathione S-tranferase (GST) was isolated by affinity chromatography, and the enzyme activity was assessed using 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates. There was no difference in overall GST activity between the sensitive and resistant cells. Using a cDNA probe (pGTSS1-2) for the human placental, anionic GST isoenzyme, no overexpression of mRNA for this isoenzyme was noted in the resistant line. When glutathione peroxidase activity (GSH-px) was assessed using either H2O2 or cumene hydroperoxide as substrate, again there was no difference in enzyme activity. Non-protein sulfhydryl (NPSH) levels were found to be elevated significantly in the resistant 8226/DOX40 subline (19.2 ± 0.1 nmol NPSH/106 cells) as compared to the drug-sensitive parental subline 8226/S (11.6 ± 1.9 nmol NPSH/106 cells) (P < 0.001). In addition, when the 8226/DOX40 cells were cultured in medium without doxorubucin, there was a consistent decline in NPSH values reaching a steady state identical to that of the 8226/S cells. However, the decrease in NPSH level was not accompanied by a change in the level of doxorubicin resistance as assessed by colony-forming assays. Depletion of glutathione by d,l-buthionine-S,R-sulfoximine had no effect on doxorubicin sensitivity in either subline. Thus, it appears that GSH-based detoxification systems are not causally involved in maintaining the MDR phenotype in 8226 human myeloma cells; rather they appear to comprise an epiphenomenon associated with the resistance selection procedure.
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