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Differential expression of sphingolipids in MRP1 overexpressing HT29 cells
Author(s) -
Kok Jan W.,
Veldman Robert J.,
Klappe Karin,
Koning Hans,
Filipeanu Catalin M.,
Müller Michael
Publication year - 2000
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/1097-0215(20000715)87:2<172::aid-ijc3>3.0.co;2-k
Subject(s) - multiple drug resistance , colchicine , biology , efflux , p glycoprotein , microbiology and biotechnology , cell culture , sphingolipid , biochemistry , drug resistance , genetics
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. Int. J. Cancer 87:172–178, 2000. © 2000 Wiley‐Liss, Inc.