P-glycoprotein, expressed in multidrug resistant cells, is not responsible for alterations in membrane fluidity or membrane potential.
Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to MDR in tumors. However, the physiological role of P-gp in normal tissues is not well understood. Previous studies on multidrug-resistant cells have suggested changes in membrane fluidity and membrane potential associated with P-gp expression, but interpretation of these studies is difficult, because most experimental cells have been selected for long periods in the presence of cytotoxic drugs and may have other host alterations. Therefore, we created two cell lines in which a transfected human MDR1 cDNA is repressed by tetracycline and induced in the absence of tetracycline. One cell line was derived from a mouse embryonic fibroblast cultured from a double (mdr1a/1b) knockout mouse, and the other was from a human HeLa cell line. Analysis of the kinetics of expression of P-gp showed that the mRNA had a half-life of approximately 4 h, and the protein had a half-life of approximately 16 h. P-gp cell surface expression (measured with monoclonal antibody MRK-16) and P-gp function (measured with a fluorescent substrate, rhodamine 123) was characterized by using fluorescence-activated cell sorting. No differences in membrane potential using the fluorescent probe oxonol or in membrane "fluidity" using fluorescent anisotropy probe or electron spin resonance probe were observed in the tet-repressible P-gp-expressing cells. In contrast, several drug-selected cells that express P-gp showed an increase in membrane fluidity and membrane potential. These results suggest that expression of P-gp per se has little effect on membrane fluidity or membrane potential, and it does not have H(+) pump activity. The changes in these parameters observed in drug-selected cells must reflect other host adaptations to drug selection.
Alemán
Claudina
C
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4254, USA.
Annereau
Jean-Philippe
JP
Liang
Xing-Jie
XJ
Cardarelli
Carol O
CO
Taylor
Barbara
B
Yin
Jun Jie
JJ
Aszalos
Adorjan
A
Gottesman
Michael M
MM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology*; D018432:Drug Resistance, Multiple / Q000502:physiology*; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D004578:Electron Spin Resonance Spectroscopy; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006207:Half-Life; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008560:Membrane Fluidity / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology*; D017494:Proton Pumps / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000502:physiology; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D009363:Neoplasm Proteins; D017494:Proton Pumps; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011993:Recombinant Fusion Proteins; D013752:Tetracycline