SECONDARY COMBINED RESISTANCE TO THE MULTIDRUG-RESISTANCE-REVERSING ACTIVITY OF CYCLOSPORINE-A IN THE CELL-LINE F4-6RADR-CSA

Multidrug-resistant tumor cells can be resensitized by combined applic ation of the selecting cytostatic drug and a chemosensitizer, such as cyclosporin A (CsA) or a calcium channel blocker. Since clinical trial s on the circumvention of multidrug resistance (MDR) with chemosensiti zers report disparate results, we investigated whether tumor cells of the MDR phenotype can develop additional resistance to the cytostatic chemosensitizer combination. Thus, the Adriamycin(ADR)-selected, P-gly coprotein-positive MDR Friend leukemia cell line F4-6RADR was exposed to stepwise increased concentrations of CsA at a constant level of 0.0 5 mu g/ml ADR. The initial CsA concentration (plus 0.05 mu g/ml ADR) t o inhibit cell growth of F4-6RADR cells by 50% (IC50) was 0.04 mu g/ml . By continuous incubation for more than 6 months, the IC50 for CsA (a t constant ADR) was elevated to 3.6 mu g/ml (90-fold), thus generating the variant F4-6RADR-CsA. The F4-6RADR-CsA cells were cross-resistant for cyclosporin H (CsH), a non-immunosuppressive derivative of CsA. A s shown by immunocytochemistry as well as by the polymerase chain reac tion and by Western blotting including densitometry, P-glycoprotein wa s preserved in the F4-6RADR-CsA variant and was expressed at a 4-fold higher level than in F4-6RADR cells. Sodium dodecyl sulfate/polyacryla mide gel electrophoresis analysis could detect no new proteins in F4-6 RADR-CsA as compared to F4-6RADR. Interestingly, resistance of F4-6RAD R-CsA cells remained reversible for the calcium antagonists verapamil and dihydropyridine B859-35 (dexniguldipine-HCl), indicating that CsA and these compounds interfere with the P glycoprotein function by diff erent pharmacodynamic mechanisms. Transport studies with [C-14]ADR, pe rformed in the presence and absence of chemosensitizers, confirmed the good correlation of P-glycoprotein function with the pattern of resis tance found in proliferation assays. Cellular accumulation of [H-3]cyc losporin was reduced to 71% of that of the F4-6 controls in F4-6RADR-C sA cells, but remained at the level of controls in F4-6RADR cells. Res ults indicate that increased amounts of the P-glycoprotein - besides o ther, perhaps more important mechanisms that are as yet unknown partia lly mediate CsA resistance in F4-6RADR-CsA cells. We have designated t his new form of resistance ''secondary combined resistance'' (SCR). Th e results suggest that at least some clinical cases of insensitivity t o chemosensitizers or of relapse after reversing therapy could be expl ained by SCR, and that resensitizing treatment of tumor patients shoul d be based on the consideration of several chemosensitizers of differe nt pharmacodynamics.