Multidrug-resistant cancer cells contain two populations of P-glycoprotein with differently stimulated P-gp ATPase activities: evidence from atomic force microscopy and biochemical analysis.
Considerable interest exists about the localization of P-gp (P-glycoprotein) in DRMs (detergent-resistant membranes) of multidrug resistant cancer cells, in particular concerning the potential modulating role of the closely related lipids and proteins on P-gp activity. Our observation of the opposite effect of verapamil on P-gp ATPase activity from DRM and solubilized-membrane fractions of CEM-resistant leukaemia cells, and results from Langmuir experiments on membrane monolayers from resistant CEM cells, strongly suggest that two functional populations of P-gp exist. The first is located in DRM regions: it displays its optimal P-gp ATPase activity, which is almost completely inhibited by orthovanadate and activated by verapamil. The second is located elsewhere in the membrane; it displays a lower P-gp ATPase activity that is less sensitive to orthovanadate and is inhibited by verapamil. A 40% cholesterol depletion of DRM caused the loss of 52% of the P-gp ATPase activity. Cholesterol repletion allowed recovery of the initial P-gp ATPase activity. In contrast, in the solubilized-membrane-containing fractions, cholesterol depletion and repletion had no effect on the P-gp ATPase activity whereas up to 100% saturation with cholesterol induced a 58% increased P-gp ATPase activity, while no significant modification was observed for the DRM-enriched fraction. DRMs were analysed by atomic force microscopy: 40-60% cholesterol depletion was necessary to remove P-gp from DRMs. In conclusion, P-gp in DRMs appears to contain closely surrounding cholesterol that can stimulate P-gp ATPase activity to its optimal value, whereas cholesterol in the second population seems deprived of this function.
Barakat
Stéphane
S
Thérapie transcriptionnelle des cellules cancéreuses, IBCP-UMR 5086 CNRS UCBL, 7 Passage du Vercors, 69367 Lyon cedex 07, France.
Gayet
Landry
L
Dayan
Guila
G
Labialle
Stéphane
S
Lazar
Adina
A
Oleinikov
Vladimir
V
Coleman
Anthony W
AW
Baggetto
Loris G
LG
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000502:physiology*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000502:physiology; D003902:Detergents; D004305:Dose-Response Relationship, Drug; D018432:Drug Resistance, Multiple / Q000502:physiology; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D006801:Humans; D018625:Microscopy, Atomic Force; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D014638:Vanadates / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D003902:Detergents; D014638:Vanadates; D002784:Cholesterol; D014700:Verapamil; D000251:Adenosine Triphosphatases