A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2)

The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxant rone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FT C) in order to develop functional assays for the half-transporter, MXR/BCRP /ABCP1. A measure of FTC-inhibitable efflux was generated for each compound in a series of MXR-overexpressing drug-selected cell lines and in ten unse lected cell lines which were used to determine if the four fluorescent comp ounds were sensitive enough to detect the low MXR levels found in drug-sens itive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was f ound in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and lo w but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both s elected and unselected cell lines was found to correlate well with MXR leve ls as determined by Northern blotting, r(2) = 0.89 and r(2) = 0.70 respecti vely. In contrast, rhodamine and LysoTracker were not able to reliably dete ct MXR. Cytotoxicity assays performed on two of the four unselected cell li nes confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibit ion of MXR-associated ATPase activity at 1 muM FTC. Short term selections o f the SF295, KM12, NCI-H460 and A549 cell lines in mitoxantrone resulted in a small but measurable increase in MXR by both Northern blot and functiona l assay. These studies show that flow cytometric measurement of FTC-inhibit able mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and un selected cell lines. (C) 2001 Elsevier Science B.V. All rights reserved.