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A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2)
R W Robey1, Y Honjo, A van de Laar
1Developmental Therapeutics Department, Medicine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 12N226, 9000 Rockville Pike, Bethesda, MD 20892, USA.
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.
Insights
Functional assays using fumitremorgin C (FTC) and fluorescent compounds like mitoxantrone and prazosin effectively measure MXR/BCRP/ABCP1 half-transporter activity. This method reliably detects MXR levels in various cell lines.
Area of Science:
- Pharmacology
- Molecular Biology
- Cell Biology
Background:
- The multidrug resistance-associated protein (MXR), also known as Breast Cancer Resistance Protein (BCRP) or ABCP1, is a half-transporter implicated in drug resistance.
- Developing sensitive assays to measure MXR function is crucial for understanding its role in cellular drug transport and resistance.
Purpose of the Study:
- To develop and validate functional assays for measuring MXR/BCRP/ABCP1 activity.
- To assess the sensitivity and specificity of fluorescent compounds and an antagonist in detecting MXR levels in various cell lines.
Main Methods:
- Utilized fluorescent compounds (rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, BODIPY-prazosin) and fumitremorgin C (FTC), a specific MXR antagonist.
- Generated measures of FTC-inhibitable efflux in MXR-overexpressing and unselected cell lines.
- Correlated efflux measurements with MXR mRNA levels determined by Northern analysis and MXR-associated ATPase activity.
Main Results:
- FTC-inhibitable efflux of mitoxantrone and prazosin was detected in specific cell lines (SF295, KM12, NCI-H460, A549) and correlated well with MXR levels.
- Rhodamine 123 and LysoTracker Green were not reliable for MXR detection.
- FTC demonstrated specific inhibition of MXR, with IC50 of 1 microM for ATPase activity.
Conclusions:
- Flow cytometry measuring FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific assay for MXR function.
- This assay can detect MXR activity in both drug-selected and unselected cell lines, including those with low MXR expression.