Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line

Selection for in vitro drug resistance can result in a complex phenotype wi th more than one mechanism of resistance emerging concurrently or sequentia lly. We examined emerging mechanisms of drug resistance during selection wi th mitoxantrone in the human myeloma cell line 8226, A novel transport mech anism appeared early in the selection process that was associated with a 10 -fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction i n intracellular drug concentration was ATP-dependent and ouabain-insensitiv e. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent az a-anthrapyrazole BBR 3390, The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BB R 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug -resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing age nt, fumitremorgin C, In contrast, fumitremorgin C had no effect on resistan ce to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cel l line, Increasing the degree of resistance to mitoxantrone in the 8226 cel l line from 10 to 37 times (8226/MR20) did not further reduce the intracell ular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topo isomerase expression and activity was not observed in the low-level drug-re sistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxant rone resistance is due to the presence of a novel, energy-dependent drug ef flux pump similar to P-glycoprotein and multidrug resistance-associated pro tein. Reversal of resistance by blocking drug efflux with fumitremorgin C s hould allow for functional analysis of this novel transporter in cancer cel l lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytop lasmic drug distribution, and alterations in topoisomerase II activity.