FLUORESCENCE METHODS TO ASSESS MULTIDRUG-RESISTANCE IN INDIVIDUAL CELLS

Purpose: Microscopic methods to measure the activity of drug extrusion systems important in multidrug resistance in individual cells were de veloped. Methods: Multidrug-resistant (MDR) and parental lines of hams ter CHO and pituitary GH(3) cells were incubated with the acetoxymethy lester (AM) forms of several fluorescent calcium-sensing dyes, fura2, indo1 and fluo3. The AM forms of these compounds are hydrolyzed by int racellular esterases and then trapped in cells, and the AM forms of th e dyes are excellent substrates for P-glycoprotein (Pgp). Results: The fluorescent free acid forms of fura2, indo1 and fluo3 did not accumul ate in MDR lines unless a chemosensitizer such as cyclosporin A, R(+)v erapamil, quinidine, or progesterone was included during loading to pr event the cells from extruding the AM forms of the dyes before they co uld be hydrolyzed. Cyclosporin A increased the fluorescence due to int racellularly trapped fura2 free acid from 8- to 20-fold and was maxima lly effective at <5 mu M. Fluorescence microscopy was employed to meas ure fura2 free acid accumulation by parental and MDR cell lines using excitation at the Ca2+-insensitive wavelength. When MDR cells were inc ubated with rhodamine 123 and fura2/AM, no fluorescence was detectable . Cellular fluorescence was dramatically increased by inclusion of cyc losporin A, quinidine, progesterone, or R(+)verapamil. There was no me asurable decline in the fura2 free acid fluorescence in 1 h while the fluorescence due to rhodamine 123 diminished rapidly in cells overexpr essing Pgp. Conclusions: These fluorescence methods detect drug-extrud ing activity in individual cells and therefore have the potential to p rovide complementary information to studies quantifying protein or mRN A levels of Pgp or other efflux pumps. In addition, they provide a rap id and quantifiable method for screening multidrug resistance reversin g agents.