MICROFLUOROMETRIC EVALUATION OF CALCEIN ACETOXYMETHYL ESTER AS A PROBE FOR P-GLYCOPROTEIN-MEDIATED RESISTANCE - EFFECTS OF CYCLOSPORINE-A AND ITS NONIMMUNOSUPPRESSIVE ANALOG SDZ-PSC-833

A microtiter plate-based fluorometric assay for functional measurement of 170-kDa P-glycoprotein (Pgp)-mediated transport using fluorescent calcein as a probe is described. The myeloma RPMI 8226 cell line and t wo of its doxorubicin-resistant Pgp-expressing sublines, dox40 (high e xpression) and dox6 (low expression), were used as models. Nonfluoresc ent calcein acetoxymethyl ester (calcein/AM) was added to the cells an d subsequent accumulation of calcein was measured in a 96-well scannin g fluorometer after 30 min. There was an inverse relationship between Pgp expression and calcein/AM accumulation, which increased dose-depen dently in the presence of cyclosporin A (CsA) and the nonimmunosuppres sive analogue SDZ PSC 833 (PSC) in the Pgp-expressing cell lines. PSC appeared to restore uptake more effectively than CsA at low concentrat ions. Calcein accumulation was also increased in Pgp-expressing cells by the addition of the Pgp substrate vincristine and the metabolic inh ibitor potassium cyanide, KCN. No effect was observed in parental cell lines. When parental and dox40 cells were mixed, 10% of dox40 cells c ould reproducibly be detected. The results indicate that microtiter-pl ate determination of calcein accumulation is a simple and sensitive me thod for functional determination of Pgp-mediated drug transport. The method may become useful, not only for preclinical screening for novel and improved resistance modifiers, but also for determination of Pgp activity in individual clinical tumor samples. (C) 1994 Academic Press , Inc.