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 concentrations of the anti-mitotic, microtubule-disrupting agent colchicine. This HT29(col) cell line displayed a 25-fold increase in colchicine resistance and exhibited cross-resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT-PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29(col) cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5-carboxyfluorescein, an increased efflux activity of HT29(col) cells was measured, as compared to the wild-type HT29 G(+) cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5-carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29(col) cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P-glycoprotein-specific substrate. Colchicine-induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis-related signal transduction processes, was much more abundant in HT29(col) cells, which is indicative of a stress response.
Kok
J W
JW
Department of Physiological Chemistry, Groningen Institute of Drug Studies, University of Groningen, Groningen, The Netherlands. j.w.kok@med.rug.nl
Veldman
R J
RJ
Klappe
K
K
Koning
H
H
Filipeanu
C M
CM
Müller
M
M
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000096:biosynthesis; D003078:Colchicine / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D005047:Etoposide / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D005699:Galactosylceramides / Q000096:biosynthesis; D005963:Glucosylceramides / Q000096:biosynthesis; D019073:HT29 Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects; D008938:Mitosis / Q000187:drug effects; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D017239:Paclitaxel / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D015398:Signal Transduction; D013107:Sphingolipids / Q000096:biosynthesis*; D014750:Vincristine / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D002518:Ceramides; D003078:Colchicine; D004317:Doxorubicin; D005047:Etoposide; D005452:Fluoresceins; D005699:Galactosylceramides; D005963:Glucosylceramides; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D017239:Paclitaxel; D011422:Propionates; D011804:Quinolines; D013107:Sphingolipids; D014750:Vincristine; C059141:verlukast; C045132:4-carboxyfluorescein