Altered topoisomerase IIalpha and multidrug resistance-associated protein levels during drug selection: adaptations to increasing drug pressure.

To understand resistance to topoisomerase II inhibitors, we used four cancer cell lines (ZR-75B, MDA-MB-231, T47D, and MCF-7) and performed a single-step selection process to isolate 50 clones resistant to topoisomerase II inhibitors. Of these, 26 were isolated with VP-16 and 24 with mAMSA. Sixteen of these isolates (four from each cell line; two selected with VP-16 and two with mAMSA) were further exposed to higher drug concentrations. Characterization of the resistant sublines revealed the adaptation that occurs with increasing drug concentration during in-vitro selections. Reduced topoisomerase IIalpha mRNA level was observed in the majority of the initial isolates. This reduction was accompanied by a decrease in topoisomerase II activity. Other isolates showed increased levels of multidrug resistance-associated protein (MRP). With advancing resistance, MRP expression was increased further, concomitantly with some recovery in topoisomerase IIalpha expression and topoisomerase II activity. In these sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression. These results extend previous studies demonstrating how cellular adaptation to increasing drug pressure utilizes more than one mechanism. Reduced expression of topoisomerase IIalpha occurs early in the selection process. MRP overexpression can occur early or can help to confer high levels of resistance. In the latter case, MRP overexpression allows some recovery of topoisomerase II activity without loss of high drug resistance.