IS RHODAMINE-123 AN APPROPRIATE FLUORESCENT-PROBE TO ASSESS P-GLYCOPROTEIN MEDIATED MULTIDRUG-RESISTANCE IN VINBLASTINE-RESISTANT CHO CELLS

Cellular drug resistance, which involves several mechanisms such as P- glycoprotein (P-gp) overexpression, kinetic and metabolic quiescence, or the increase in the intracellular levels of glutathione, limits the effectiveness of cancer treatment. It has been reported that function al assessment of the cationic dye rhodamine 123 (Rho123) efflux reveal s accurately the drug-resistant phenotype. To study cellular drug resi stance, we have obtained a CHO-K1 derived cell line resistant to vinbl astine by means of multistep selection. This cell line (CHOVBR) displa ys high reactivity with a monoclonal antibody (MAb) (C219) directed ag ainst an internal domain of P-gp, and an active Rho123 efflux, as show n by parallel flow cytometric and fluorometric assays. However, under similar experimental conditions, the drug-sensitive parental cell line CHO-K1 (as well as the myeloblastic KG1 and KG1a cell lines), was als o able to pump Rho123 out. These parental CHO-K1 cells had a very low reactivity against the C219 Mab, as confirmed by Western blot analysis . Both vinblastine and verapamil inhibited Rho123 efflux in CHO-K1 cel ls, but had no effect on CHOVBR cultures. Also, deprivation of vinblas tine for one month did not affect Rho123 efflux in these cells. Our re sults suggest that the activity of P-gp appears to be essential, but n ot sufficient to confer drug resistance, and that Rho123-based functio nal assays of drug resistance should be evaluated for each cellular ex perimental model.