Development of a mammalian Tet-on expression cell line: glucosylceramide synthase regulates TNF-alpha-induced apoptosis.

Abstract

AbstractTumor necrosis factor-α (TNF-α) is one of the most pleiotropic of cytokines, acting as a host defense factor in myriad immunological and inflammatory responses and antitumor activity (1–3). The cytotoxic effects of TNF-α are primarily mediated through TNF-R1 and the receptor-associated proteins, TNF-R1-associated death domain protein (TRADD) and Fas-associated death domain (FADD/MORT1) (3–5). Ceramide generation and caspase activation represent potential regulation points of apoptotic signaling by TNF-α (6,7). Increased ceramide formation via sphingomyelinase represents an early event in the apoptotic cascade of TNF-α (6,8,9). In MCF-7 breast cancer cells, ceramide is one of the essential molecules in TNF-α-induced apoptosis (10–12). Increased ceramide generation induced by the P-glycoprotein blocker PSC 833, restores TNF-α-induced apoptosis in KG1a leukemia cells (13), whereas loss of ceramide production is a cause of cellular resistance to apoptosis induced by TNF-α (14). Glucosylceramide synthase (GCS), converting ceramide into glucosylceramide (15), exerts strong influence over cellular ceramide levels and is thus a major modulator of programmed cell death (16). Introducing the GCS gene into TNF-α-sensitive MCF-7 cells greatly diminishes cellular response to TNF-α cytotoxicity (12).KeywordsTransfection SolutionHost Defense FactorMammalian TransfectionRinse CellCellular Ceramide LevelThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.