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 cause for cancer treatment failure. Rhodamine 123 (Rh123), a low toxic fluorescent probe commonly used to assess mitochondrial bioenergetics in living cells, has also been used to measure the efflux activity of P-gp in both normal and malignant cells. Analysis of variation in cellular fluorescence by measuring the rates of Rh123 influx and efflux, together with the effect of mdr reversing agents, allows the investigation of drug-resistant phenotypes in cancer samples. We have studied the functional activity of P-gp in human leukemic cell lines using flow cytometry, taking into consideration that variables such as Rh123 cytotoxicity, culture conditions, cell membrane integrity, as well as the effect of specific P-gp modulators, can impair the resolution of the Rh123-efflux measurements. The studies show that: (1)optimal non-cytotoxic concentrations of Rh123 which allow appropriate color compensation are in the range of 50-200 ng/ml; (2) life-gating allows accurate measurement on the 50% average rate of Rh123 efflux; (3) relative efficiency of P-gp inhibitors was PSC-833 > cyclosporin A > verapamil; and (4) the presence or absence of fetal calf serum had no effect on the bioavailability of chemosensitizer agents, with the exception of serum-free experiments, which showed a significant decrease in P-gp activity under the presence of PSC-833 (P = 0.05). Hence, we recommend this experimental strategy for clinical practice better to study the cellular drug resistance phenotype.
Pétriz
J
J
Departament de Criobiologia i Terapia Cellular, Institut de Recerca Oncologica, Hospital Duran i Reynals, Barcelona, Spain.
García-López
J
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology*; D018432:Drug Resistance, Multiple; D005434:Flow Cytometry; D006801:Humans; D020112:Rhodamine 123; D012235:Rhodamines / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012235:Rhodamines; D020112:Rhodamine 123