The dependence of some cell types on serum factors for growth may represent a powerful, but poorly studied, model for antimitogenic pathways. In this study, we examine ceramide as a candidate intracellular mediator of serum factor dependence. In Molt-4 leukemia cells, serum withdrawal caused a significant arrest in cell cycle progression (80% of cells in G0/G1), accompanied by a modest apoptotic cell death (12%). Serum deprivation of these cells resulted in significant sphingomyelin hydrolysis (72%; corresponding to hydrolysis of 47 pmol/nmol phosphate), which was accompanied by a profound and progressive elevation (up to 10-15-fold) in endogenous levels of ceramide. Withdrawal of serum caused the activation of a distinct, particulate, and magnesium-dependent sphingomyelinase. The addition of exogenous C6-ceramide induced a dramatic arrest in the G0/G1 phase of the cell cycle comparable to the effects observed with serum withdrawal, albeit occurring much sooner. Unlike serum withdrawal, however, the addition of C6-ceramide resulted in more pronounced apoptosis. Because of the previously noted ability of exogenously added phorbol esters to inhibit ceramide-mediated apoptosis, we investigated the hypothesis that endogenous activation of the diacylglycerol/protein kinase C pathway may modulate the response to serum withdrawal. Indeed, serum withdrawal resulted in 3-4-fold elevation in endogenous diacylglycerol levels. The addition of exogenous diacylglycerols resulted in selective attenuation of ceramide's effects on apoptosis but not on cell cycle arrest. Thus, the combination of ceramide and diacylglycerol recapitulated the complex effects of serum withdrawal on cell cycle arrest and apoptosis. These studies identify a novel role for ceramide in cell cycle regulation, and they may provide the first evidence for an intracellular signal transduction pathway in mammalian cells mediating cell cycle arrest. These studies also underscore the importance of lipid second messengers and the significance of the interplay between glycerolipid-derived and sphingolipid-derived lipid mediators.

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

Triggering of the Fas/APO-1 cell-surface receptor induces apoptosis through an uncharacterized chain of events. Exposure of Fas-sensitive cells to an agonist monoclonal antibody induced cell death and a 200-300% elevation in endogenous levels of the sphingolipid ceramide, a proposed intracellular mediator of apoptosis. In contrast, similar treatment of Fas-resistant cells caused insignificant changes in ceramide levels. Because resistant cell lines expressed the Fas antigen, these results indicate that these cells have a defect in the proximal signaling events leading to ceramide generation. Exposure of the resistant cell lines to a synthetic analog of ceramide induced apoptosis, thus bypassing Fas resistance and indicating that the signaling pathways downstream of ceramide were intact. Furthermore, activation of protein kinase C with the diacylglycerol analog phorbol 12-myristate 13-acetate significantly reduced Fas-induced cytotoxicity, suggesting opposing roles for ceramide and protein kinase C in regulation of apoptosis. These results provide evidence for ceramide as a necessary and sufficient lipid mediator of Fas-mediated apoptosis and suggest this process may be modulated via activation of additional signal-transduction pathways.The Fas (APO-1, CD95) cell-surface antigen belongs to the nerve growth factor/tumor necrosis factor receptor family of type I membrane proteins and mediates apoptosis (physiological cell death) after ligand binding or crosslinking with agonist antibodies (1-4). The physiologic significance of this cell death is demonstrated by the lymphoproliferation and generalized lymphproliferative disease mouse models of autoimmune disease in which pathogenesis results from the absence of Fas function due to either mutations in the Fas gene (i.e., lpr) or its ligand (i.e., gld) (5-7). In lprcg mice, a point mutation within the coding region for the cytoplasmic domain results in the replacement of Ile-225 with asparagine abrogating signal transduction. Although this domain does not contain any known enzymatic activity (e.g., kinase, phosphatase), the carboxylterminal 15 amino acids have been shown to modulate its apoptotic signal (8). Additional insight into Fas function has been gained from the existence of significant homology with the intracellular domain of tumor necrosis factor receptor type I (TNFR) (3, 9). The death signal conveyed by human TNFR can be disrupted by converting Leu-351, the amino acid corresponding to mouse Fas Ile-225, to alanine or asparagine, which further suggests similar mechanisms for Fas and TNFR (9).Recently, the sphingolipid ceramide has been implicated as an early intermediary in tumor necrosis factor a (TNF-a)-induced differentiation and apoptosis (10-12). In response to TNF-a treatment, ceramide is generated rapidly by the reversible hydrolysis of sphingomyelin via activation of a "sphingomyelin cycle." Free ceramide subsequently activates a serine/ threonine phosphoprotein phosphatase 2A (13) and/or a serine/threonine protein kinase (14). Although the ...