TY - JOUR

T1 - A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2)

AU - Robey, R W

AU - Honjo, Y

AU - van de Laar, A

AU - Miyake, K

AU - Regis, J T

AU - Litman, Thomas

AU - Bates, S E

PY - 2001/6/6

Y1 - 2001/6/6

N2 - 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.

AB - 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.

KW - ATP-Binding Cassette Transporters

KW - Adenosine Triphosphatases

KW - Boron Compounds

KW - Breast Neoplasms

KW - Cell Survival

KW - Colonic Neoplasms

KW - Drug Resistance, Multiple

KW - Female

KW - Fluorescent Dyes

KW - Gene Expression Regulation, Neoplastic

KW - Humans

KW - Intracellular Membranes

KW - Kinetics

KW - Microsomes

KW - Mitoxantrone

KW - Neoplasm Proteins

KW - P-Glycoprotein

KW - Polymerase Chain Reaction

KW - Prazosin

KW - RNA, Messenger

KW - Transcription, Genetic

KW - Tumor Cells, Cultured

KW - Verapamil

M3 - Journal article

C2 - 11406094

VL - 1512

SP - 171

EP - 182

JO - B B A - General Subjects

JF - B B A - General Subjects

SN - 0304-4165

IS - 2

ER -