FLOW CYTOMETRIC ANALYSIS OF P-GLYCOPROTEIN FUNCTION USING RHODAMINE-123

The MDR1 gene product, P-glycoprotein (P-gp), works as a transmembrane efflux pump for several cytotoxic products, representing a major caus e for cancer treatment failure. Rhodamine 123 (Rh123), a law toxic flu orescent probe commonly used to assess mitochondrial bioenergetics in living cells, has also been used to measure the efflux activity of P-g p in both normal and malignant cells. Analysis of variation in cellula r fluorescence by measuring the rates of Rh123 influx and efflux, toge ther with the effect of mdr reversing agents, allows the investigation of drug-resistant phenotypes in cancer samples. We have studied the f unctional activity of P-gp in human leukemic cell lines using flow cyt ometry, taking into consideration that variables such as Rh123 cytotox icity, culture conditions, cell membrane integrity, as well as the eff ect of specific P-gp modulators, can impair the resolution of the Rh12 3-efflux measurements, The studies show that: (1) optimal non-cytotoxi c concentrations of Rh123 which allow appropriate color compensation a re in the range of 50-200 ng/ml; (2) life-gating allows accurate measu rement on the 50% average rate of Rh123 efflux; (3) relative efficienc y of P-gp inhibitors was PSC-833 > cyclosporin A > verapamil; and (4)t he presence or absence of fetal calf serum had no effect on the bioava ilability of chemosensitizer agents, with the exception of serum-free experiments, which showed a significant decrease in P-gp activity unde r the presence of PSC-833 (P = 0.05). Hence, we recommend this experim ental strategy for clinical practice better to study the cellular drug resistance phenotype.