Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells.
A mitoxantrone-resistant human MCF-7 breast cancer subline (MCF/MX) which is approximately 4000-fold resistant to mitoxantrone was isolated by serial passage of the parental wild-type MCF-7 cells (MCF/WT) in stepwise increasing concentrations of drug. MCF/MX cells were also approximately 10-fold cross-resistant to doxorubicin and etoposide but were not cross-resistant to vinblastine. Intracellular accumulation of radiolabeled mitoxantrone was markedly reduced in MCF/MX cells relative to that in the drug-sensitive MCF/WT cells. This decrease in intracellular drug accumulation into MCF/MX cells was associated with enhanced drug efflux, which was reversed when cells were incubated in the presence of sodium azide and 2, 4-dinitrophenol, suggesting an energy-dependent process. Incubation of MCF/MX cells with verapamil did not affect either the accumulation of mitoxantrone or the level of resistance in these cells. Furthermore, RNase protection and Western blot analyses failed to detect the expression of the mdr1 RNA or P-glycoprotein, a drug efflux pump known to be associated with the development of multidrug resistance in vitro. However, a polyclonal antibody directed against a synthetic peptide corresponding to the putative ATP binding domain of P-glycoprotein reacted with two (M(r) 42,000 and 85,000) membrane proteins from MCF/MX cells which were not found in MCF/WT. Functional assays and Western blot analysis for topoisomerase II revealed no differences in topoisomerase II activity or protein levels in MCF/MX cells. Thus, resistance in this cell line is apparently associated with enhanced drug efflux involving a pathway distinct from the mdr1-encoded multidrug transporter P-glycoprotein.
Nakagawa
M
M
Medicine Branch, National Cancer Institute, Bethesda, Maryland 20892.
Schneider
E
E
Dixon
K H
KH
Horton
J
J
Kelley
K
K
Morrow
C
C
Cowan
K H
KH
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism; D000906:Antibodies; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D003429:Cross Reactions; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance; D005047:Etoposide / Q000494:pharmacology; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D008942:Mitoxantrone / Q000493:pharmacokinetics* / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000906:Antibodies; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D005047:Etoposide; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D008942:Mitoxantrone; D014700:Verapamil; D004250:DNA Topoisomerases, Type II