Decrease of P-glycoprotein activity in K562/ADR cells by MbetaCD and filipin and lack of effect induced by cholesterol oxidase indicate that this transporter is not located in rafts.
The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl-beta-cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, "light," "heavy," and "extra heavy" fractions we show that no P-gp is found in the "light" fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.
Reungpatthanaphong
Paiboon
P
Laboratoire de Physicochimie Biomoléculaire et Cellulaire (LPBC/CSSB UMR 7033), Université Paris Nord, 74 rue Marcel Cachin, 93017, Bobigny, France.
Marbeuf-Gueye
Carole
C
Le Moyec
Laurence
L
Salerno
Milena
M
Garnier-Suillerot
Arlette
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D018943:Anthracyclines / Q000378:metabolism; D015153:Blotting, Western; D002463:Cell Membrane Permeability / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism*; D002789:Cholesterol Oxidase / Q000378:metabolism* / Q000494:pharmacology; D004317:Doxorubicin / Q000378:metabolism; D018432:Drug Resistance, Multiple / Q000502:physiology*; D005372:Filipin / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D021381:Protein Transport / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D049128:Sphingolipid Activator Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D047392:beta-Cyclodextrins / Q000378:metabolism* / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018943:Anthracyclines; D049128:Sphingolipid Activator Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D004317:Doxorubicin; D005372:Filipin; D002784:Cholesterol; D002789:Cholesterol Oxidase