Glucosylceramide synthase does not attenuate the ceramide pool accumulating during apoptosis induced by CD95 or anti-cancer regimens

Ceramide (Cer) accumulating during the execution phase of apoptosis is gene rated from plasma membrane sphingomyelin (SM), which gains access to a sphi ngomyelinase due to phospholipid scrambling (Tepper, A. D., Ruurs, P., Wied mer T., Sims, P., Borst, J., and van Blitterswijk W. J. (2000) J. Cell. Bio l; 150, 155-164), To evaluate the functional significance of this Cer pool, we aimed to convert it to glucosylceramide (GlcCer), by constitutive overe xpression of glucosylceramide synthase (GCS). Jurkat cells, retrovirally tr ansduced with GCS cDNA, showed a 10-12-fold increase in GCS activity in vit ro and a 7-fold elevated basal GlcCer level in vivo. However, Cer accumulat ing during apoptosis induced by ligation of the death receptor CD95, treatm ent with the anti-cancer drug etoposide, or exposure to gamma -radiation wa s not glycosylated by GCS. Likewise, Cer liberated at the plasma membrane b y bacterial SMase was not concerted by the enzyme. Thus, GCS, located at th e Golgi, is topologically segregated from Cer produced in the plasma membra ne. In contrast, de novo synthesized Cer as well as an exogenously supplied cell-permeable Cer analog were efficiently glycosylated, apparently due to different. Cer topology and distinct physicochemical behavior of the synth etic Cer species, respectively. Exogenous cell-permeable Cer species, despi te their conversion by GCS, effectively induced apoptosis. We also observed that GCS activity is down-regulated in cells undergoing apoptosis. In conc lusion, GCS can convert de novo synthesized Cer but not SM-derived Cer, and , therefore, the ability of GCS overexpression to protect cells ii om possi ble detrimental effects of Cer accumulation is limited.