Ceramide reverses brefeldin A (BFA) resistance in BFA-resistant cell lines.
We have found that C6 ceramide, a cell-permeable ceramide analog, partially restored the brefeldin A (BFA) sensitivity in a BFA-resistant mutant of Vero cells (BER-40) and in the naturally BFA-resistant Madin-Darby canine kidney (MDCK) cells. Incubation of BER-40 and MDCK cells with low concentrations of C6 ceramide resulted in (i) a pronounced increase in BFA cytotoxicity as measured by the inhibition of [3H]thymidine incorporation and the inhibition of colony formation by BFA, (ii) a significant protection by BFA against ricin cytotoxicity, and (iii) an inhibition of bulk protein secretion by BFA in BER-40 and MDCK cells. Related sphingolipids including sphingosine, sphingomyelin, and lactosylceramide and other unrelated lipid second messengers such as arachidonic acid and 1,2-diacylglycerol did not elicit the protection of BER-40 and MDCK cells against ricin cytotoxicity by BFA. C6 ceramide was the most effective among the ceramides with different acyl chain lengths. Interestingly, dihydro-C6 ceramide, which lacks the trans double bond in the sphingoid base, had no effect. On the other hand, C6 ceramide did not enhance BFA sensitivity in BFA-sensitive Vero cells. The LD50 of C6 ceramide were similar in Vero and BER-40 cells. Fluorescence microscopic studies revealed that C6 ceramide induced the redistribution of beta-COP from the Golgi membranes to a more dispersed localization in both BFA-sensitive and BFA-resistant cell lines, mimicking the effect of BFA. Suboptimal concentration of C6 ceramide also restored the effect of BFA on the beta-COP distribution in BER-40 and MDCK cells. These results indicate that C6 ceramide restores the BFA sensitivity in BFA-resistant BER-40 and MDCK cells.
Oda
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Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Chen
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CH
Wu
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HC
D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D020126:Brefeldin A; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D003517:Cyclopentanes / Q000494:pharmacology*; D004285:Dogs; D004351:Drug Resistance; D012276:Ricin / Q000494:pharmacology; D013107:Sphingolipids / Q000378:metabolism; D014709:Vero Cells
D000935:Antifungal Agents; D002518:Ceramides; D003517:Cyclopentanes; D013107:Sphingolipids; D020126:Brefeldin A; D012276:Ricin