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J Biol Chem, Vol. 275, Issue 9, 6246-6251, March 3, 2000
From the Departments of 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
(Gal
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*
,
¶
**
Laboratory Medicine & Pathobiology, § Pediatrics, and
Biochemistry,
University of Toronto and the ¶ Research Institute, Hospital
for Sick Children, Toronto, Ontario M5G 1X8, Canada
1-4Gal
1-4glucosylceramide-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.
*
This work was supported by Canadian Medical Research Council
Grant MT13073.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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