Caspases are the main executioners of Fas-mediated apoptosis, irrespective of the ceramide signalling pathway

Abstract

Tumor necrosis factor a (TNF) or cytotoxic anti-Fas antibodies lead to the activation of apoptotic proteases (caspases) and to sphingomyelinase-mediated ceramide generation. Caspases and ceramide are both known to induce apoptosis on its own, but their relative contribution to Fas-and TNF-induced cell death is not well established. We report here that rapid apoptosis induced by TNF in U937 cells or anti-Fas in Jurkat cells, in the presence of cycloheximide, induced only a very low increase (520%) in the cell ceramide content. Neither treatment with inhibitors of sphingomyelinases nor incubation of cells with fumonisin B 1 , which inhibits de novo ceramide synthesis, prevented TNF and Fas-mediated apoptosis. Increasing or depleting the cell ceramide content by prolonged culture in the presence of monensin or fumonisin B1, respectively, did not prevent TNF and Fas-mediated apoptosis. Treatment of cells with sphingomyelinase inhibitors did not affect to the activation of CPP32 (caspase-3) induced by TNF or anti-Fas antibodies. Chromatin condensation and fragmentation in cells treated with anti-Fas or TNF was abrogated by peptide inhibitors of caspases, which also inhibited Fas-, but not TNF-induced cell death. These results indicate that while ceramide does not seem to act as a critical mediator of TNF and Fas-induced apoptosis, it is generated as a consequence of CPP32 activation and could contribute to the spread of the intracellular death signal.

Involvement of Sphingosine in Mitochondria-dependent Fas-induced Apoptosis of Type II Jurkat T Cells

“…Notwithstanding, there is disagreement as to both the extent of the increase in ceramide levels as well as the timing, resulting in controversy regarding its role in apoptosis (22)(23)(24)(25)(26)(27). The results presented here may reconcile many findings, as we observed a biphasic ceramide response to Fas ligation in type II Jurkat cells as follows: a rapid and transient low amplitude increase in the cellular concentration of ceramide followed by a delayed, persistent, and robust elevation.…”

“…In contrast to numerous studies demonstrating potential involvement of ceramide in Fas signaling (10 -21), others have challenged the concept of a role for ceramide (22)(23)(24)(25)(26)(27). Furthermore, it is possible that a further metabolite of ceramide may be the active component produced as a result of sphingomyelin turnover.…”

Role of caspases and apoptosis-inducing factor (AIF) in cladribine-induced apoptosis of B cell chronic lymphocytic leukemia

“…Chromatin condensation and nuclear fragmentation during apoptosis were analyzed by pphenylenediamine (PPDA) staining and fluorescence microscopy as described previously. 10 …”

“…Solubilized proteins from equal numbers of extracted cells (4 × 10 6 Trypan blue-negative cells for each lane) were resolved by SDS-12% PAGE, transferred to nitrocellulose membranes (Hybond-C extra, Amersham, Barcelona, Spain) and incubated with primary antibodies diluted in TBS-T (10 mM Tris/HCl pH 8, 0.12 M NaCl, 0.1% Tween-20, 0.05% sodium azide), containing 5% skimmed milk, as described previously. 10 Primary antibodies used were: mouse monoclonal anti-caspase-3 (0.25 g/ml, clone 19, BD Transduction Laboratories, Madrid, Spain); monoclonal anti-caspase-7 (1 g/ml, clone B94-1, BD Pharmingen, Madrid, Spain); monoclonal anti-caspase-8 (1 g/ml, UBI, clone 5F7) rabbit antiserum anti-human caspase-9 (1/6000, cat. No.…”

Cytolytic T cells induce ceramide-rich platforms in target cell membranes to initiate graft-versus-host disease

“…47 Algeciras-Schimnich et al 45 did not detect Fas-containing platforms in SKW.4, K50, or H9 lymphoid cells lines. This probably resulted from failure to protect Fc receptors while using agonistic anti-Fas Apo-1-3 antibody, resulting in their nonspecific clustering and the masking of specific Fas clustering within platforms.…”