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

Abstract

The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) 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 unselected cell lines which were used to determine if the four fluorescent compounds were sensitive enough to detect the low MXR levels found in drug-sensitive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was found in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and low but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both selected and unselected cell lines was found to correlate well with MXR levels as determined by Northern blotting, r(2)=0.89 and r(2)=0.70 respectively. In contrast, rhodamine and LysoTracker were not able to reliably detect MXR. Cytotoxicity assays performed on two of the four unselected cell lines confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibition of MXR-associated ATPase activity at 1 microM FTC. Short term selections of 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 functional assay. These studies show that flow cytometric measurement of FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and unselected cell lines.

Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2)

“…The enhanced cytotoxicity of topotecan by FTC may be related to the known lowlevel expression of BCRP in parental MCF-7 cells, but a second, FTC-sensitive, mechanism of drug resistance cannot be completely discounted (Rabindran et al, 2000). Flow cytometric measurement of the inhibition of mitoxantrone or prazosin efflux by FTC correlates with levels of BCRP mRNA by Northern analysis, and as such, constitutes a sensitive and specific functional assay for BCRP (de Bruin et al, 1999;Robey et al, 2001a).…”

Functional characterization of the ABCG2 5′ non-coding exon variants: Stem cell specificity, translation efficiency and the influence of drug selection

“…The HEK293 cell line was purchased from ATCC, whereas the MCF-7 cell line, as well as its mitoxantrone (Mx) [28] and flavopiridol (Flv) [6] resistant sublines were kind gifts from Susan Bates, NIH, Bethesda, USA. HEK293 and MCF-7 cells, as well as drug resistant derivatives of the latter one were maintained in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and cells were cultured at 37°C in 5% CO 2 atmosphere.…”

“…We examined the MCF-7 human breast adenocarcinoma cell line and its Mx or Flv resistant sublines. MCF-7 is used in several studies and shows a medium level of ABCG2 expression [31], whereas the resistant sublines were established by drug selection [6,28]. We used a TaqMan ® assay designed for the 3' mRNA region that detects the entire pool of 5' UTR variants (denoted as "total" mRNA level).…”

Impact of BCRP/MXR, MRP1 and MDR1/P‐Glycoprotein on thermoresistant variants of atypical and classical multidrug resistant cancer cells

“…Thus, cells of all sublines were split and incubated with the BCRP/MXR-specific inhibitor fumitremorgin C (FTC; synthesized at the National Institutes of Health and kindly provided by L. Greenberger, 10 M), [45][46][47] with the MRP1-specific inhibitor MK-571 (free sample from Merck, 752 Whitehouse Station, NJ; 100 M), 48,49 with the PGP-specific inhibitor verapamil (Sigma, 40 M) 50,51 and with the combination of verapamil and MK-571, verapamil and FTC and MK-571 and FTC for 30 min at 37°C or 39.4°C, respectively. Thereafter, the ABC transporter substrates mitoxantrone (Lederle, Wolfratshausen, Germany, 20 M), or rhodamine 123 (Sigma, 13 M) were added for another hour at 37°C or 39.4°C.…”

“…These results are in agreement with those reporting the inhibition of FTC-caused BCRP/MXR in other human carcinoma cell lines as determined by mitoxantrone accumulation. [45][46][47] Effects of the PGP-specific inhibitor verapamil have been described in numerous reports. 50,51 In the highly PGP-overexpressing daunorubicin-selected cells, which we analyzed here, verapamil caused elevated drug accumulations, as observed in both, the thermosensitive as well as the thermoresistant variants.…”