The nature of the signaling pathway(s) which initiate drug‐triggered apoptosis remains largely unknown and is of fundamental importance in understanding cell death induced by chemotherapeutic agents. Here we show that in the leukemic cell lines U937 and HL‐60, daunorubicin, at concentrations which trigger apoptosis, stimulated two distinct cycles of sphingomyelin hydrolysis (approximately 20% decrease at 1 microM) within 4–10 min and 60–75 min with concomitant ceramide generation. We demonstrate that the increase in ceramide levels, which precedes apoptosis, is mediated by a neutral sphingomyelinase and not by ceramide synthase. Indeed, potent ceramide synthase inhibitors such as fumonisin B1 did not affect daunorubicin‐triggered sphingomyelin hydrolysis, ceramide generation or apoptosis. In conclusion, we provide evidence that daunorubicin‐triggered apoptosis is mediated by a signaling pathway which is initiated by an early sphingomyelin‐derived ceramide production.

Development of atherosclerosis is believed to involve proliferation of smooth muscle cells (SMC).

The role of cytosolic phospholipase A 2 (cPLA 2 ) in the regulation of ceramide formation was examined in a cell line (L929) responsive to the cytotoxic action of tumor necrosis factor ␣ (TNF␣). In L929 cells, the addition of TNF␣ resulted in the release of arachidonate, which was followed by a prolonged accumulation of ceramide occurring over 5-12 h and reaching 250% over base line. The formation of ceramide was accompanied by the hydrolysis of sphingomyelin and the activation of three distinct sphingomyelinases (neutral Mg 2؉ -dependent, neutral Mg 2؉ -independent, and acidic enzymes). The variant cell line C12, which lacks cPLA 2 , is resistant to the cytotoxic action of TNF␣. TNF␣ was able to activate nuclear factor B in both the wild-type L929 cells and the C12 cells. However, TNF␣ was unable to cause the release of arachidonate or the accumulation of ceramide in C12 cells. C 6 -ceramide overcame the resistance to TNF␣ and caused cell death in C12 cells to a level similar to that in L929 cells. The introduction of the cPLA 2 gene into C12 cells resulted in partial restoration of TNF␣-induced arachidonate release, ceramide accumulation, and cytotoxicity. This study suggests that cPLA 2 is a necessary component in the pathways leading to ceramide accumulation and cell death.The sphingomyelin (SM) 1 cycle, first described by Okazaki et al.(1), has gained recognition over the past few years as a key mechanism for regulating anti-mitogenic signals. Activation of this cycle through the regulation of a signal-induced sphingomyelinase (SMase) results in generation of the lipid second messenger ceramide. Ceramide then modulates a number of biological fates, including growth inhibition (1-3), differentiation (2), apoptosis (4 -6), and cell cycle arrest (7). Although recent studies have begun to catalogue inducers such as TNF␣, interleukin-1␤, nerve growth factor, and Fas that are capable of signaling through the SM cycle (see Refs. 5, 6, and 8 for reviews), the mechanisms by which these inducers stimulate SMase activity remain poorly understood.TNF␣, through interaction with either a 55-or 75-kDa TNF receptor (9, 10), impacts upon a myriad of intracellular signaling cascades, including protein phosphorylation cascades, transcription factors, and lipid messengers (11). Two classes of lipid mediators have been implicated in TNF␣ signaling, glycerophospholipid metabolites and sphingolipid metabolites (11,12), and recent evidence suggests that these two classes of lipids may interact (13). In HL-60 cells, a linear correlation was established among TNF␣ stimulation, AA generation, and SM cycle activation: TNF␣-stimulated AA liberation preceded ceramide generation, and AA reproduced the effects of TNF␣ on the SM cycle (13). Although these studies suggested that AA and/or its metabolites may be involved in activation of SMase, the physiologic role of the PLA 2 /AA pathway in regulating SMase activity has not been determined.In this study, we examined the role of PLA 2 in SMase activation in the L929 murine fibroblast cell l...