Differential expression of sphingolipids in MRP1 overexpressing HT29 cells

We have obtained a novel multidrug resistant cell line, derived from HT29 G (+) human colon carcinoma cells, by selection with gradually increasing con centrations of the anti-mitotic, microtubule-disrupting agent colchicine. T his HT29(col) cell line displayed a 25-fold increase in colchicine resistan ce and exhibited cross-resistance to doxorubicin, VP16, vincristine and tax ol. Immunoblotting, combined with RT-PCR showed that the multidrug resistan ce phenotype was conferred by specific overexpression of the multidrug resi stance protein 1. Confocal scanning laser microscopy revealed that multidru g resistance protein I specifically localized in the plasma membrane of HT2 9(col) cells. in a functional assay, using the fluorescent multidrug resist ance protein substrate 5-carboxyfluorescein, an increased efflux activity o f HT29(col) cells was measured, as compared to the wild-type HT29 G(+) cell s. MK571, a specific inhibitor of multidrug resistance protein 1, blocked t he 5-carboxyfluorescein efflux, but only partially reversed resistance to c olchicine, indicating that additional multidrug resistance mechanisms opera te in HT29(col) cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchi cine pressure, indicating that colchicine is not a P-glycoprotein-specific substrate. Colchicine-induced overexpression of multidrug resistance protei n 1 is accompanied by a changed sphingolipid composition, i.e., enhanced re vels of glucosylceramide and galactosylceramide. In addition, ceramide, a l ipid messenger molecule involved in apoptosis-related signal transduction p rocesses, was much more abundant in HT29(col) cells, which is indicative of a stress response. Int. J. Cancer 87:172-178, 2000. (C) 2000 Wiley-Liss, I nc.