Glycosylation of ceramide potentiates cellular resistance to tumor necrosis factor-alpha-induced apoptosis

Ceramide, as a second messenger, initiates one of the major signal transduc tion pathways in tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and pr oduces glucosylceramide. By introduction of the GCS gene, cytotoxic resista nce to TNF-alpha has been conferred in human breast cancer cells. MCF-7/GCS -transfected cells expressed 4.1-fold higher levels of GCS activity and exh ibited a 15-fold (P < 0.0005) greater EC50 for TNF-alpha compared with the parental MCF-7 cell line. DNA fragmentation and DNA synthesis studies showe d that TNF-alpha had little influence on the induction of apoptosis or on g rowth arrest in MCF-7/GCS cells, compared to MCF-7 cells. These studies rev eal that TNF-alpha resistance in MCF-7/GCS cells is closely related to cera mide hyperglycosylation, a hallmark of this transfected cell line, and resi stance was not aligned with changes in TNF receptor 1 expression. This work demonstrates that GCS, which catalyzes ceramide glycosylation, potentiates cytotoxic resistance to TNF-alpha. (C) 1998 Academic Press.