Functional characterization of Jurkat T cells rescued from CD95/Fas-induced apoptosis through the inhibition of caspases.

The caspases are known to play a pivotal role in the triggering and execution of apoptosis in virtually all cell types. Because inappropriate apoptosis is a prominent feature of many human diseases, the caspases are attractive targets for therapeutic intervention. In the present study we investigated whether Jurkat T lymphocytes rescued from Fas-induced cell death through the inhibition of caspases are functional. Here we show that the pan-caspase, tripeptide inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Ome) fluoromethylketone (z-VAD-FMK), inhibited the activation of caspase-2, -3, -7, and -8, and subsequently apoptosis in Jurkat T lymphocytes induced by agonistic anti-Fas. The apoptotic signals induced by the cross-linking of the Fas antigen have a relatively long half-life, as z-VAD-FMK had to be continuously present in the culture medium for 72 h after Fas stimulation in order to maintain cell survival. After 72 h, the z-VAD-FMK-rescued cells proliferate normally and responded to activation induced cell death after phytohaemaglutinin treatment, and readily undergo apoptosis when restimulated with agonistic Fas antibodies. Taken together, our results demonstrate that Jurkat T cells rescued from Fas-mediated cell death through the inhibition of caspases are functional.

Ko SC ，Johnson VL ，Chow SC 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

Caspase-1 is not involved in CD95/Fas-induced apoptosis in Jurkat T cells.

It is now well established that the caspases, a family of cysteine proteases, play a key role in apoptosis. Although overexpressing each of the caspases in cells triggered apoptosis, the precise role and contribution of individual caspases are still unclear. Caspase-1, the first caspase discovered, was initially implicated in mammalian apoptosis because of its similarity to the gene product ced-3. Using whole cells as well as an in vitro system to study apoptosis, the role of caspase-1 in Fas-mediated apoptosis in Jurkat T cells was examined in greater detail. Using various peptide-based caspase inhibitors, our results showed that N-acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone efficiently blocked Fas-mediated apoptosis in Jurkat T cells, whereas N-acetyl-Tyr-Val-Ala-Asp aldehyde, which is more specific for caspase-1, had little effect. Cell lysates derived from anti-Fas-stimulated cells, which readily induced apoptotic nuclei morphology and DNA fragmentation in isolated thymocyte nuclei, had no caspase-1 activity using proIL-1beta as a substrate. Time-course studies showed no caspase-1 activity during the activation of apoptosis in Jurkat cells by agonistic Fas antibodies. Furthermore, no pro-caspase-1 protein nor activated form of the protein was detected in normal or apoptotic Jurkat cells. In contrast, both caspase-2 and caspase-3 were readily detected as proenzymes in control cells and their activated forms were detected in apoptotic cells. Incubation of recombinant active caspase-1 with control cell lysates did not activate the apoptotic cascade as shown by the lack of detectable apoptotic nuclei promoting activity using isolated nuclei as substrate. However, under similar conditions proIL-1beta was readily processed into the mature cytokine, indicating that the recombinant caspase-1 remained active in the presence of control cell lysates. Taken together our results demonstrate that caspase-1 is not required for the induction of apoptosis in Jurkat T cells mediated by the Fas antigen.

Chow SC ，Slee EA ，MacFarlane M ，Cohen GM ... -  《EXPERIMENTAL CELL RESEARCH》

Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes.

Déas O ，Dumont C ，MacFarlane M ，Rouleau M ，Hebib C ，Harper F ，Hirsch F ，Charpentier B ，Cohen GM ，Senik A ... -  《JOURNAL OF IMMUNOLOGY》

Doxorubicin treatment activates a Z-VAD-sensitive caspase, which causes deltapsim loss, caspase-9 activity, and apoptosis in Jurkat cells.

Gamen S ，Anel A ，Pérez-Galán P ，Lasierra P ，Johnson D ，Piñeiro A ，Naval J ... -  《EXPERIMENTAL CELL RESEARCH》

Inhibition of caspase or FADD function blocks proliferation but not MAP kinase-activation and interleukin-2-production during primary stimulation of T cells.

Mack A ，Häcker G 《EUROPEAN JOURNAL OF IMMUNOLOGY》