CERAMIDE REVERSES BREFELDIN-A (BFA) RESISTANCE IN BFA-RESISTANT CELL-LINES

We have found that C-6 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 C-6 ceramide resulted in (i) a pronounced increase in BFA cytotoxicity as measured by the inhibition of [H-3]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. C-6 ceramide was the most effective among the ceramides with different acyl chain lengths. Interestingly, dihydro-C-6 ceramide, which lacks the trans double bond in the sphingoid base, had no effect. On the other hand, C-6 ceramide did not enhance BFA sensitivity in BFA-sensitive Vero cells. The LD(50) of C-6 ceramide were similar in Vero and BER-40 cells. Fluorescence microscopic studies revealed that C-6 ceramide induced the redistribution of P-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 C-6 ceramide also restored the effect of BFA on the beta-COP distribution in BER-40 and MDCK cells. These results indicate that C-6 ceramide restores the BFA sensitivity in BFA-resistant BER-40 and MDCK cells.