Retroviral transfection of Madin-Darby canine kidney cells with human MDR1results in a major increase in globotriaosylceramide and 10(5)- to 10(6)-fold increased cell sensitivity to verocytotoxin - Role of P-glycoprotein inglycolipid synthesis

Retroviral infection of the Madin-Darby canine kidney (MDCK) renal cell lin e with human MDR1 cDNA, encoding the P-glycoprotein (P-gp) multidrug resist ance efflux pump, induces a major accumulation of the glycosphingolipid (GS L), globotriaosylceramide (Gal alpha 1-4Gal beta 1-4glucosylceramide-Gb(3)) , the receptor for the E. coli-derived verotoxin (VT), to effect a similar to million-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 inte rnalized to the endoplasmic reticulum/nuclear envelope as we have reported for other hypersensitive cell lines. P-gp (but not MRP) inhibitors, e.g. ke toconazole or cyclosporin A (CsA) prevented the increased Gb, and VT sensit ivity, concomitant with increased vinblastine sensitivity Gb, 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-d iazole)]-aminocaproyl (NBD)-lactosylceramide (LacCer) and NBD-Gb(3) via NBD -glucosylceramide (GlcCer) from exogenous NBD-CG-ceramide, was prevented by CsA. me therefore propose that P-gp can mediate GlcCer translocation acros s the bilayer, from the cytosolic face of the Golgi to the lumen, to provid e increased substrate for the lumenal synthesis of LacCer and subsequently Gb(3). These results provide a molecular mechanism for the observed increas ed sensitivity of multidrug-resistant tumors to VT and emphasize the potent ial of verotoxin as an antineoplastic, Two strains (I and II) of MDCK cells , which differ in their glycolipid profile, have been described. The origin al MDR1-MDCK parental cell was not specified, but the MDR1-MDCK GSL phenoty pe and glycolipid synthase activities indicate MDCK-I cells. However, the p artial drug resistance of MDCK-I cells precludes their being the parental c ell. We speculate that the retroviral transfection per se, or the subsequen t selection for drug resistance, selected a subpopulation of MDCK-I cells i n the parental MDCK-H cell culture and that drug resistance in MDR1-MDCK ce lls is thus a result of both MDR1 expression and a second, previously unrec ognized, component, likely the high level of GlcCer synthesis in these cell s.