Phosphatidylcholine and phosphatidylethanolamine behave as substrates of the human MDR1 P-glycoprotein

Irene Bosch, Kyriaki Dunussi-Joannopoulos, Rui Lian Wu, Stephen T. Furlong, James Croop

Research output: Contribution to journalArticle

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Abstract

The multidrug resistant cell line CEM/VBL300 and the parental CEM T- lymphoblastic cell line from which it was derived were used to study the accumulation of fluorescent phospholipid analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). The fluorescent analogs NBD-PC, NBD-PE, and NBD-PS and [3H]PC were delivered in liposomes prepared by ethanol injection. Fluorescence microscopy demonstrated decreased accumulation of the NBD-PC analog in the multidrug resistant cell line compared to the parental cell line. Verapamil enhanced NBD-PC accumulation in the resistant cells. Similar results were obtained with insect cells expressing high levels of recombinant human MDR1. Elimination of NBD fluorescence on the outer leaflet of the plasma membrane with dithionite permitted quantification of the internal cellular fluorescence by FACS analysis. The drag resistant CEM/VBL300 cells accumulated approximately 10% the amount of NBD-PE and 20% the amount of NBD-PC compared to CEM drug sensitive cells. No difference in internal accumulation of NBD-PS was found between the drug resistant and drug sensitive cell lines. The internal accumulation of NBD-PE and NBD-PC was enhanced by the MDR reversal agents verapamil, cyclosporin A, and SDZ PSC 833 in the CEM/VBL300 cells but not in the CEM cells. The increased accumulation was dose dependent, and the relative potency of the reversal agents paralleled their ability to circumvent multidrug resistance. In addition, the monoclonal antibody UIC2 directed against the P-glycoprotein produced similar results. The evidence presented here suggests that PC and PE but not PS behave as substrates for human MDR1 P-glycoprotein.

Original languageEnglish (US)
Pages (from-to)5685-5694
Number of pages10
JournalBiochemistry
Volume36
Issue number19
DOIs
StatePublished - May 13 1997
Externally publishedYes

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P-Glycoprotein
Phosphatidylcholines
Phosphatidylserines
Cells
Cell Line
Substrates
Verapamil
Fluorescence
Pharmaceutical Preparations
Dithionite
Fluorescence microscopy
Multiple Drug Resistance
Cell membranes
Fluorescence Microscopy
Liposomes
Cyclosporine
Drag
Insects
phosphatidylethanolamine
Phospholipids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphatidylcholine and phosphatidylethanolamine behave as substrates of the human MDR1 P-glycoprotein. / Bosch, Irene; Dunussi-Joannopoulos, Kyriaki; Wu, Rui Lian; Furlong, Stephen T.; Croop, James.

In: Biochemistry, Vol. 36, No. 19, 13.05.1997, p. 5685-5694.

Research output: Contribution to journalArticle

Bosch, Irene ; Dunussi-Joannopoulos, Kyriaki ; Wu, Rui Lian ; Furlong, Stephen T. ; Croop, James. / Phosphatidylcholine and phosphatidylethanolamine behave as substrates of the human MDR1 P-glycoprotein. In: Biochemistry. 1997 ; Vol. 36, No. 19. pp. 5685-5694.
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abstract = "The multidrug resistant cell line CEM/VBL300 and the parental CEM T- lymphoblastic cell line from which it was derived were used to study the accumulation of fluorescent phospholipid analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). The fluorescent analogs NBD-PC, NBD-PE, and NBD-PS and [3H]PC were delivered in liposomes prepared by ethanol injection. Fluorescence microscopy demonstrated decreased accumulation of the NBD-PC analog in the multidrug resistant cell line compared to the parental cell line. Verapamil enhanced NBD-PC accumulation in the resistant cells. Similar results were obtained with insect cells expressing high levels of recombinant human MDR1. Elimination of NBD fluorescence on the outer leaflet of the plasma membrane with dithionite permitted quantification of the internal cellular fluorescence by FACS analysis. The drag resistant CEM/VBL300 cells accumulated approximately 10{\%} the amount of NBD-PE and 20{\%} the amount of NBD-PC compared to CEM drug sensitive cells. No difference in internal accumulation of NBD-PS was found between the drug resistant and drug sensitive cell lines. The internal accumulation of NBD-PE and NBD-PC was enhanced by the MDR reversal agents verapamil, cyclosporin A, and SDZ PSC 833 in the CEM/VBL300 cells but not in the CEM cells. The increased accumulation was dose dependent, and the relative potency of the reversal agents paralleled their ability to circumvent multidrug resistance. In addition, the monoclonal antibody UIC2 directed against the P-glycoprotein produced similar results. The evidence presented here suggests that PC and PE but not PS behave as substrates for human MDR1 P-glycoprotein.",
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AB - The multidrug resistant cell line CEM/VBL300 and the parental CEM T- lymphoblastic cell line from which it was derived were used to study the accumulation of fluorescent phospholipid analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). The fluorescent analogs NBD-PC, NBD-PE, and NBD-PS and [3H]PC were delivered in liposomes prepared by ethanol injection. Fluorescence microscopy demonstrated decreased accumulation of the NBD-PC analog in the multidrug resistant cell line compared to the parental cell line. Verapamil enhanced NBD-PC accumulation in the resistant cells. Similar results were obtained with insect cells expressing high levels of recombinant human MDR1. Elimination of NBD fluorescence on the outer leaflet of the plasma membrane with dithionite permitted quantification of the internal cellular fluorescence by FACS analysis. The drag resistant CEM/VBL300 cells accumulated approximately 10% the amount of NBD-PE and 20% the amount of NBD-PC compared to CEM drug sensitive cells. No difference in internal accumulation of NBD-PS was found between the drug resistant and drug sensitive cell lines. The internal accumulation of NBD-PE and NBD-PC was enhanced by the MDR reversal agents verapamil, cyclosporin A, and SDZ PSC 833 in the CEM/VBL300 cells but not in the CEM cells. The increased accumulation was dose dependent, and the relative potency of the reversal agents paralleled their ability to circumvent multidrug resistance. In addition, the monoclonal antibody UIC2 directed against the P-glycoprotein produced similar results. The evidence presented here suggests that PC and PE but not PS behave as substrates for human MDR1 P-glycoprotein.

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