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

Abstract

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␣1-4Gal␤1-4glucosylceramide-Gb 3 ), the receptor for the E. coli-derived verotoxin (VT), to effect a ϳmillion-fold increase in cell sensitivity to VT. The shorter chain fatty acid isoforms of Gb 3 (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 Gb 3 and VT sensitivity, concomitant with increased vinblastine sensitivity. Gb 3 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-Gb 3 via NBD-glucosylceramide (GlcCer) from exogenous NBD-C 6 -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 Gb 3 . 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.

The fate and function of glycosphingolipid glucosylceramide

“…Raggers et al, unpublished data). These assays should be of help in determining whether or not natural GlcCer is translocated across the plasma membrane and whether indeed similar activities exist in intracellular membranes (Lala et al 2000) (figure 3). …”

Gene expression and amplification in breast carcinoma cells with intrinsic and acquired doxorubicin resistance

“…Finally the overexpression of GM2A (ganglioside activator protein) is of relevance. This protein is involved with the breakdown of glycosphingolipids, and there is an association between P-glycoprotein overexpression and glycosphingolipid accumulation (Lala et al, 2000). Therefore overexpression of a gene involved with the breakdown of sphingolipids in DOX resistant cells is not surprising, though previously unreported.…”

Anoikis-resistant MDCK cells carrying susceptibilities to TNF-α and verotoxin that are suitable for influenza virus cultivation

“…Verotoxin, also called Shiga toxin (Stx), binds to the specific cell surface receptor globotriaosylceramide (Gala1-4Galb1-4glucosylceramide, Gb3). Increased sensitivities to VT have been observed in many multidrug-resistant ovarian carcinoma and astrocytoma cells that are correlated with the increased synthesis of Gb3 (Lala et al 2000). Wild type MDCK is resistant to VT because it synthesizes Gb5 instead of Gb3, and it becomes sensitive only after treatment with short-chain fatty acids or with tumor promoter 12-O-tetradecanoylphorbol 13-acetate (Sandvig et al1991).…”