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.

To address the role of a plausible protease cascade in daunorubicin-triggered apoptosis, we evaluated the effect of cell-permeant protease inhibitors on its signal transduction pathway. Treatment of U937 and HL-60 cells with 0.5-1 microM of the chemotherapeutic drug daunorubicin induced a greater than 30% activation of neutral sphingomyelinase activity within 4-10 min with concomitant sphingomyelin hydrolysis and ceramide generation. DNA fragmentation and the classical morphological features of apoptosis were observed within 4-6 h. Pretreatment of cells with the serine protease inhibitors N-tosyl-L-phenylalanyl chloromethyl ketone (20 microM) or dichloroisocoumarin (20 microM) for 30 min inhibited daunorubicin-induced neutral sphingomyelinase activation, sphingomyelin hydrolysis, ceramide generation, and apoptosis. Other cell-permeant protease inhibitors such as pepstatin, leupeptin, and antipain had no such effect. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. Daunorubicin-induced NF-kappaB activation was inhibited by dichloroisocoumarin but not by N-tosyl-L-phenylalanyl chloromethyl ketone, suggesting that this transcription factor can be activated independently of ceramide and is not directly implicated in the apoptotic pathway. These results suggest that inhibitors of serine proteases can act upstream of ceramide in drug-triggered apoptosis and that neutral sphingomyelinase activation is either directly or indirectly serine protease dependent.

The purpose of this study was to characterize mitoxantronetreated with chemotherapy alone remains low. In first-line induced cytotoxicity in KG1a and TF-1, two P-glycoprotein therapy, the 'gold standard' induction regimen consists of the expressing AML cell lines which display early differentiation administration of the anthracycline daunorubicin (DNR) in phenotypes, compared to more mature HL-60 and U937 cells.association with cytosine-arabinoside (Ara-C). While the KG1a and TF-1 cells were found to be 30-40-fold more resistant DNR/Ara-C combination induces a relatively high rate of to mitoxantrone than HL-60 and U937 cells. Uptake and efflux of mitoxantrone were similar for all cell lines. Moreover, a complete remission, the high incidence of early relapse indi- less recognized by the P-gp 9 and therefore may be more active Keywords: mitoxantrone; chemoresistance; apoptosis; leukemia than DNR towards immature AML cells.Mitoxantrone, an anthracenedione derivative, is a potent antitumor agent with proven activity against a number of Introduction human neoplasias, including AML. [10][11][12] The cytotoxic action Acute myeloid leukemia (AML) is a neoplastic disorder of mitoxantrone is believed to be associated with its ability to characterized by the clonal expansion of leukemic myeloid stabilize a covalent DNA-topoisomerase II reaction product, cells. In spite of apparent uniformity, AML cells consist of a the so-called cleavable complex (for review see Ref. 10). More heterogeneous cellular population. A small fraction of leurecently, it has been demonstrated that mitoxantrone induces kemic progenitor cells (CFU-L) proliferate and differentiate programmed cell death (apoptosis) in certain leukemia cells 13 into terminal division blast cells which comprise the vast although it remains unclear whether mitoxantrone-induced majority of leukemic cells observed in the blood and marrow apoptosis is a direct consequence of the mitoxantrone-topoof AML patients. 1 Similar to normal hemopoiesis, the leuisomerase II interaction. kemic myeloid progenitor compartment appears to be heteroTo the best of our knowledge, no study has been performed geneous with the less mature progenitors providing the selfevaluating the cytotoxic effects of mitoxantrone on AML cells renewal activity of the AML clone. 2 The phenotypic identifiderived from distinct stages of differentiation. Therefore, we cation of the most primitive AML cells has been lacking until have evaluated drug transport, drug-topoisomerase II interacrecent studies showed that leukemia initiating cells (SL-IC) tions, and cytotoxicity of mitoxantrone in two AML cell lines which can engraft SCID mice, and which can therefore be which express the early differentiation phenotype (KG1a and considered as leukemic stem cells, display the early CD34 + TF-1) compared to two AML cell lines which display a more CD33 − CD38 − phenotype. 3 Thus, the elimination of this early mature phenotype (HL-60 and U937). Our results show that AML cell subpopulation by chemotherapy is probably the ...