The key events implicated in ceramide-triggered apoptosis remain unknown. In this study we show that 25 M C6-ceramide induced significant H 2 O 2 production within 60 min, which increased up to 180 min in human myeloid leukemia U937 cells. Inactive analogue dihydro-C6-ceramide had no effect. Furthermore, no H 2 O 2 production was observed in C6-ceramide-treated U937 °c ells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-B and AP-1 is mediated by mitochondrial derived reactive oxygen species. Both H 2 O 2 production, transcription factor activation as well as apoptosis could be inhibited by rotenone and thenoyltrifluoroacetone (specific mitochondrial complexes I and II inhibitors) and antioxidants, N-acetylcysteine and pyrrolidine dithiocarbamate. These effects could be potentiated by antimycin A (specific complex III mitochondrial inhibitor). H 2 O 2 production was also inhibitable by ruthenium red, suggesting a role of mitochondrial calcium homeostasis alterations in ceramideinduced oxidative stress. Finally, C6-ceramide had no influence on mitochondrial membrane potential within the first 6 h. Altogether, our study points to reactive oxygen species, generated at the ubiquinone site of the mitochondrial respiratory chain, as an early major mediator in ceramide-induced apoptosis.

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.

The anthracycline daunorubicin is widely used in the treatment of acute nonlymphocytic leukemia. The drug has, of course, been the object of intense basic research, as well as preclinical and clinical study. As reviewed in this article, evidence stemming from this research clearly demonstrates that cell response to daunorubicin is highly regulated by multiple signaling events, including a sphingomyelinase-initiated sphingomyelin-ceramide pathway, mitogen-activated kinase and stress-activated protein/c-Jun Nterminal kinase activation, transcription factors such as nuclear factor B, as well as the Fas/Fas-ligand system. These pathways are themselves influenced by a number of lipid products (diacylglycerol, sphingosine-1 phosphate, and glucosyl ceramide), reactive oxygen species, oncogenes (such as the tumor suppressor gene p53), protein kinases (protein kinase C and phosphoinositide-3 kinase), and external stimuli (hematopoietic growth factors and the extracellular matrix). In light of the complexity and diversity of these observations, a comprehensive review has been attempted toward the understanding of their individual implication (and regulation) in daunorubicin-induced signaling. IntroductionThe anthracycline daunorubicin (DNR) is one of the major antitumor agents widely used in the treatment of acute myeloid leukemias (AMLs). Cytotoxicity mediated by DNR is generally thought to be the result of drug-induced damage to DNA. This damage is mediated by quinone-generated redox activity, intercalation-induced distortion of the double helix, or stabilization of the cleavable complex formed between DNA and topoisomerase II. 1 However, how and why such events should bring about cell death remains unclear, especially when one considers that DNA interaction may not be a prerequisite for anthracycline cytotoxicity. [2][3][4][5] Hence, the exploration and understanding of the process of apoptosis has forced a reconsideration of the mechanisms whereby myeloid leukemia cells respond to DNR. In this context, we have shown that, within a narrow concentration range (0.2-1 M), DNR can trigger apoptosis in the monocytic U937 or the myelocytic HL-60 AML cells but not in the immature (CD 34 ϩ ) KG1a, KG1, or HEL cells, 6 the latter being more resistant to DNR than the former. 7 These results suggested that DNR triggers apoptotic signals in drug-sensitive AML cells and that inhibition of these signals may contribute to drug resistance and, for example, to the inherent resistance of immature AML cells. For this reason, others and we have investigated the mechanism by which DNR activates apoptosis in DNR-sensitive AML cells. Implication of sphingomyelin metabolism in DNR-induced apoptosis DNR activates the sphingomyelin cycle in sensitive leukemic cellsIn the early 1990s, a number of studies provided growing evidence that sphingomyelin breakdown products (ie, ceramide [CER] and phosphorylcholine) could play an important role in mediating the action of cytokines, including tumor necrosis factor ␣ (TNF␣) and interferon. For example,...