DEVELOPMENT OF AN INTRINSIC P-GLYCOPROTEIN-MEDIATED DOXORUBICIN RESISTANCE IN QUIESCENT CELL-LAYERS OF LARGE, MULTICELLULAR PROSTATE TUMOR SPHEROIDS

Growing multicellular prostate tumor spheroids develop quiescent cell subpopulations in central regions with features of intrinsic multicell -mediated drug resistance. Doxorubicin (dox) uptake was significantly reduced in large spheroids (diameter 400 +/- 70 mu m), which consist p redominantly of quiescent cells, as compared to small spheroids (diame ter 100 +/- 50 mu m), which consist entirely of proliferating cells, A fter removal of dox from the incubation medium, dox fluorescence decli ned more efficiently in large spheroids, which led to a decreased dox toxicity as revealed by colony-forming assays. Verapamil significantly increased dox retention in large spheroids and, consequently, augment ed dox toxicity. At a depth 80 mu m from the spheroid periphery, a sig nificantly decreased dox fluorescence was observed in the deep, quiesc ent cell layers of large spheroids. The P-glycoprotein-mediated multid rug resistance (MDR)-reversing agents verapamil, cyclosporin A, quinid ine, sodium orthovanadate and tamoxifen significantly increased dox fl uorescence at this depth, whereas genistein, indomethacin, probenecid and brefeldin A, which reverse multidrug-resistance-associated protein (MRP) function, exerted no effect, Anti-P-glycoprotein immunohistoche mistry of multicellular tumor spheroids revealed an increase of P-glyc oprotein expression in large speroids as compared to small spheroids, which was most prominent in the Ki-67-negative, quiescent cell layers 60 to 100 mu m distant from the periphery of the spheroid, indicating that the MDR phenotype is related to cell quiescence, This was corrobo rated by whole-cell patch-clamp experiments, where the C219 antibody, which is directed against the ATP-binding site of P-glycoprotein, sign ificantly inhibited P-glycoprotein-associated, volume-activated chlori de currents in quiescent, but not proliferating cells from multicellul ar tumor spheroids. (C) 1998 Wiley-Liss, Inc.