Susana Gamen

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.

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.

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.

Resistance to apoptosis correlates with a highly proliferative phenotype and loss of Fas and CPP32 (caspase-3) expression in human leukemia cells

Apoptosis induced by effector cells of the immune system or by cytotoxic drugs is a main mechanism mediating the prevention or elimination of tumoral cells. For instance, the human T‐cell leukemia Jurkat is sensitive to Fas‐induced apoptosis and to activation‐induced cell death (AICD), and the promonocytic leukemia U937 is sensitive to Fas‐ and TNF‐induced apoptosis. In this work, we have analyzed the mechanisms of resistance to physiological or pharmacological apoptosis in human leukemia by generating highly proliferative (hp) sub‐lines derived from Jurkat and U937 cells. These hp sub‐lines were resistant to Fas‐ and TNF‐induced apoptosis, as well as to AICD. This was due to the complete loss of Fas and TNFR surface expression and, in the case of Jurkat‐derived sub‐lines, also of CD3, CD2 and CD59 molecules. The sub‐lines also completely lacked the expression of the apoptotic protease CPP32, present in parental cells. Moreover, these sub‐lines were no longer sensitive to doxorubicin‐induced apoptosis, which was efficiently blocked by the general caspase inhibitor Z‐VAD‐fmk in the parental cell lines. These data suggest a molecular mechanism for the development of resistance of leukemic cells to physiological and pharmacological apoptosis inducers, giving rise to highly proliferative tumoral phenotypes. These results also indicate that Fas and CPP32 could be useful prognostic markers for the progression and/or therapy outcome of human leukemias. Int. J. Cancer 75:473–481, 1998.

mtDNA‐depleted U937 cells are sensitive to TNF and Fas‐mediated cytototxicity

It has been proposed that TNF cytotoxicity is mediated by reactive oxygen intermediates generated by uncoupling of mitochondrial respiration. We have compared sensitive U937 cells and derived cell lines depleted of mtDNA for their ability to undergo TNF‐ and Fas‐induced apoptosis. Cells lacking around 98% of mtDNA were still sensitive to TNF‐induced apoptosis. U937 cells devoid of mtDNA (U937‐ϱ°) were resistant to TNF, but this was due to the loss of its 55 kDa receptor. U937‐ϱ° cells were also resistant to docosahexaenoic acid, which causes U937 cell death by lipid peroxidation. These cells were sensitive to anti‐Fas toxicity. The results indicate that TNF and Fas‐induced toxicity occurs by a mechanism mostly independent of mitochondrial free radical generation.