ALTERED MRP IS ASSOCIATED WITH MULTIDRUG-RESISTANCE AND REDUCED DRUG ACCUMULATION IN HUMAN SW-1573 CELLS

We have analysed the contribution of several parameters, e.g. drug acc umulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a la rge set of drug-resistant variants of the human non-small-cell lung ca ncer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explai ned by reduced drug accumulation and was associated with a decrease ra ther than an increase in the low MDR1 mRNA level. To test whether a de crease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it d id not result in resistance. Tn all resistant clones we found an alter ed form of the multidrug resistance-associated protein (MRP), migratin g slightly slower during SDS-polyacrylamide gel electrophoresis than M RP in parental cells. This altered MRP was also present in non-P-gp MD R somatic cell hybrids of the SW-1573 cells, demonstrating a clear lin kage with the MDR phenotype. Treatment of crude cellular membrane frac tions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translation al modification. There was no detectable difference in sialic acid con tent. In most but not all doxorubicin-selected clones, this MDR phenot ype was accompanied by a reduction in topo II alpha mRNA level. No red uction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low lev els of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resis tance mechanisms, such as reduced topo II alpha mRNA in case of doxoru bicin selection.