Javier Naval

Implication of Mitochondrial Hydrogen Peroxide Generation in Ceramide-induced Apoptosis

The key events implicated in ceramide-triggered apoptosis remain unknown. In this study we show that 25 μm C6-ceramide induced significant H2O2 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 H2O2 production was observed in C6-ceramide-treated U937 ρ° cells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-κB and AP-1 is mediated by mitochondrial derived reactive oxygen species. Both H2O2 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). H2O2 production was also inhibitable by ruthenium red, suggesting a role of mitochondrial calcium homeostasis alterations in ceramide-induced 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.

Differential Secretion of Fas Ligand- or APO2 Ligand/TNF-Related Apoptosis-Inducing Ligand-Carrying Microvesicles During Activation-Induced Death of Human T Cells

Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments ∼500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.

Involvement of APO2 ligand/TRAIL in activation- induced death of Jurkat and human peripheral blood T cells

The interaction of Fas with Fas ligand (FasL) mediates activation‐induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti‐APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)‐prestimulated Jurkat or human PBMC on non‐activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti‐APO2L and anti‐Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA‐activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.

Doxorubicin-induced apoptosis in human T-cell leukemia is mediated by caspase-3 activation in a Fas-independent way

It has recently been proposed that doxorubicin (DOX) can induce apoptosis in human T‐leukemia cells via the Fas/FasL system in an autocrine/paracrine way. We show here that treatment of Jurkat cells with either anti‐Fas antibodies, anthracyclin drugs or actinomycin D induces the activation of CPP32 (caspase‐3) and apoptosis. However, DOX treatment did not induce the expression of membrane FasL or the release of soluble FasL and co‐incubation with blocking anti‐Fas antibodies prevented Fas‐induced but not DOX‐induced apoptosis. All the morphological and biochemical signs of apoptosis induced by anti‐Fas or DOX can be prevented by Z‐VAD‐fmk, a general caspase inhibitor. DEVD‐cho, a specific inhibitor of CPP32‐like caspases which completely blocks Fas‐mediated apoptosis, prevented drug‐induced nuclear apoptosis but not cell death. We conclude that: (i) DOX‐induced apoptosis in human T‐leukemia/lymphoma is Fas‐independent and (ii) caspase‐3 is responsible of DOX‐induced nuclear apoptosis but other Z‐VAD‐sensitive caspases are implicated in cell death.

Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis

Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8+ T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA−/− or gzmB−/− mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, ΔΨm loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA−/− but not from gzmB−/− mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA−/− or gzmB−/− mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.

A Role of the Mitochondrial Apoptosis-Inducing Factor in Granulysin-Induced Apoptosis

Granulysin is a cytolytic molecule released by CTL via granule-mediated exocytosis. In a previous study we showed that granulysin induced apoptosis using both caspase- and ceramide-dependent and -independent pathways. In the present study we further characterize the biochemical mechanism for granulysin-induced apoptosis of tumor cells. Granulysin-induced death is significantly inhibited by Bcl-2 overexpression and is associated with a rapid (1–5 h) loss of mitochondrial membrane potential, which is not mediated by ceramide generation and is not inhibited by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide generation induced by granulysin is a slow event, only observable at longer incubation times (12 h). Apoptosis induced by exogenous natural (C18) ceramide is truly associated with mitochondrial membrane potential loss, but contrary to granulysin, this event is inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide-induced apoptosis is also completely prevented by Bcl-2 overexpression. The nuclear morphology of cells dying after granulysin treatment in the presence of caspase inhibitors suggested the involvement of mitochondrial apoptosis-inducing factor (AIF) in granulysin-induced cell death. We demonstrate using confocal microscopy that AIF is translocated from mitochondria to the nucleus during granulysin-induced apoptosis. The majority of Bcl-2 transfectants are protected from granulysin-induced cell death, mitochondrial membrane potential loss, and AIF translocation, while a small percentage are not protected. In this small percentage the typical nuclear apoptotic morphology is delayed, being of the AIF type at 5 h time, while at longer times (12 h) the normal apoptotic morphology is predominant. These and previous results support a key role for the mitochondrial pathway of apoptosis, and especially for AIF, during granulysin-induced tumoral cell death.

Release of preformed Fas ligand in soluble form is the major factor for activation‐induced death of Jurkat T cells

Interaction of Fas/APO‐1 (CD95) and its ligand (FasL) plays an important role in the activation‐induced cell death (AICD) of T lymphocytes. In the present work, the contribution of soluble FasL to AICD of the human T‐cell line Jurkat has been studied. Jurkat cells prestimulated with phytohaemagglutinin (PHA) induced the death of non‐activated Jurkat cells, and also of L1210Fas, but not that of Fas‐negative L1210 cells. Culture supernatants from prestimulated Jurkat cells were highly toxic to their non‐activated counterparts. Time–course analysis revealed that PHA‐stimulated Jurkat cells quickly release (less than 15 min) to the medium a toxic molecule following a biphasic pattern, with maximal cytotoxic activities at 1 hr and 7 hr after stimulation. The cytotoxic effect of those supernatants was prevented by the addition of a blocking anti‐Fas monoclonal antibody, suggesting that PHA‐stimulated Jurkat cells exert Fas‐based cytotoxicity mainly through the release of soluble FasL. The constitutive intracellular expression of FasL in non‐activated Jurkat cells and its release as a consequence of PHA activation were detected by immunostaining and immunoblotting using an anti‐FasL antibody. These data indicate that, at least in Jurkat cells, AICD is mainly mediated by the rapid release of preformed FasL in soluble form upon stimulation.

CPP32 inhibition prevents Fas-induced ceramide generation and apoptosis in human cells

Intracellular activation of sphingomyelinase, leading to ceramide generation, and ICE‐like proteases have been implicated in TNF and Fas‐induced apoptosis, but the links between these intracellular apoptotic mediators remain undefined. We show here that a specific peptide inhibitor of the ICE‐like protease CPP32/Yama (DEVD‐cho) blocks anti‐Fasinduced apoptosis in Jurkat and U937 cells, while having no effect on TNF‐induced apoptosis in U937 cells. This peptide also prevents ceramide accumulation induced by Fas engagement. Jurkat and U937 cells, as well as their mtDNA‐depleted derived lines (π° cells), were sensitive to ceramide toxicity, which was not prevented by ICE‐like protease inhibitors. These results, taken together, suggest that ICE‐like protease activation is a prerequisite for ceramide generation and subsequent apoptosis, at least in the case of Fas‐induced cell death.

Bcl-2 family members as molecular targets in cancer therapy

Escape from apoptosis is often a hallmark of cancer cells, and is associated to chemotherapy resistance or tumor relapse. Proteins from the Bcl-2 family are the key regulators of the intrinsic pathway of apoptosis, controlling the point-of no-return and setting the threshold to engage the death machinery in response to a chemical damage. Therefore, Bcl-2 proteins have emerged as an attractive target to develop novel anticancer drugs. Current pharmacological approaches are focused on the use of peptides, small inhibitory molecules or antisense oligonucleotides to neutralize antiapoptotic Bcl-2 proteins, lowering the threshold and facilitating apoptosis of cancer cells. We discuss here recent advances in the development of Bcl-2 targeted anticancer therapies.

Fatty acid metabolism in human lymphocytes. I. Time-course changes in fatty acid composition and membrane fluidity during blastic transformation of peripheral blood lymphocytes

The time-course changes in fatty acid composition of human T-lymphocytes during blastic transformation were analysed, as well as the variations in membrane fluidity determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), using a fluorescence-activated cell sorter. The more important changes observed, in activated relative to quiescent cells, started after 24 h and consisted in an increase in the proportion of oleic (18:1(n - 9)), docosapentaenoic (22:5(n - 3)) and docosahexaenoic (22:6(n - 3)) acids and a decrease in that of linoleic (18:2(n - 6)) and arachidonic (20:4(n - 6)) acids. This represented a relative increase of 26% for 18:1, 56% for 22:5 and 84% for 22:6 in peripheral blood mononuclear cells (PBMC) and 35%, 182% and 94%, respectively, in purified T-lymphocytes, both activated for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and 19%, respectively, for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and phosphatidylethanolamine) rather than neutral lipids. The 18:1/18:0 ratio increased greatly in major cell phospholipids. The proportion of 20:4, 22:5 and 22:6 in phosphatidylinositol was not significantly altered after 72 h of activation. The molar ratio cholesterol/phospholipids was reduced in 72-h-activated lymphocytes (0.29) compared to quiescent cells (0.5). On the other hand, the stimulation of human T-lymphocytes caused a significant decrease in the order parameter (S) of DPH, according to the observed changes in lipid composition. After 72 h in culture, the S value for quiescent and stimulated T-lymphocytes was 0.530 and 0.326, respectively. In conclusion, the blastic transformation of human T-lymphocytes is associated with changes in lipid composition which modify the physical properties of their membranes. These modifications could modulate, in turn, the activity of membrane proteins implicated in the process of blastic transformation.