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J Biol Chem, Vol. 275, Issue 9, 6246-6251, March 3, 2000

Retroviral Transfection of Madin-Darby Canine Kidney Cells with Human MDR1 Results in a Major Increase in Globotriaosylceramide and 10⁵- to 10⁶-Fold Increased Cell Sensitivity to Verocytotoxin
ROLE OF P-GLYCOPROTEIN IN GLYCOLIPID SYNTHESIS^*

Prateek Lala, Shinya Ito^§¶, and Clifford A. Lingwood^¶**

From the Departments of  Laboratory Medicine & Pathobiology, ^§ Pediatrics, and  Biochemistry, University of Toronto and the ^¶ Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada

Retroviral infection of the Madin-Darby canine kidney (MDCK) renal cell line with human MDR1 cDNA, encoding the P-glycoprotein (P-gp) multidrug resistance efflux pump, induces a major accumulation of the glycosphingolipid (GSL), globotriaosylceramide (Gal1-4Gal1-4glucosylceramide-Gb₃), the receptor for the E. coli-derived verotoxin (VT), to effect a ~million-fold increase in cell sensitivity to VT. The shorter chain fatty acid isoforms of Gb₃ (primarily C16 and C18) are elevated and VT is internalized to the endoplasmic reticulum/nuclear envelope as we have reported for other hypersensitive cell lines. P-gp (but not MRP) inhibitors, e.g. ketoconazole or cyclosporin A (CsA) prevented the increased Gb₃ and VT sensitivity, concomitant with increased vinblastine sensitivity. Gb₃ synthase was not significantly elevated in MDR1-MDCK cells and was not affected by CsA. In MDR1-MDCK cells, synthesis of fluorescent N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-aminocaproyl (NBD)-lactosylceramide (LacCer) and NBD-Gb₃ via NBD-glucosylceramide (GlcCer) from exogenous NBD-C⁶-ceramide, was prevented by CsA. We therefore propose that P-gp can mediate GlcCer translocation across the bilayer, from the cytosolic face of the Golgi to the lumen, to provide increased substrate for the lumenal synthesis of LacCer and subsequently Gb₃. These results provide a molecular mechanism for the observed increased sensitivity of multidrug-resistant tumors to VT and emphasize the potential of verotoxin as an antineoplastic. Two strains (I and II) of MDCK cells, which differ in their glycolipid profile, have been described. The original MDR1-MDCK parental cell was not specified, but the MDR1-MDCK GSL phenotype and glycolipid synthase activities indicate MDCK-I cells. However, the partial drug resistance of MDCK-I cells precludes their being the parental cell. We speculate that the retroviral transfection per se, or the subsequent selection for drug resistance, selected a subpopulation of MDCK-I cells in the parental MDCK-II cell culture and that drug resistance in MDR1-MDCK cells is thus a result of both MDR1 expression and a second, previously unrecognized, component, likely the high level of GlcCer synthesis in these cells.

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