Inhibition of caspase activity delays apoptosis in a transfected NS/0 myeloma cell line.

The productivity of NS/0 myeloma batch cultures is often compromised by the premature induction of apoptosis, now established to be the predominant method of cell death during culture decline. Caspase proteases have recently been shown to play a major role in the transmission of signals for apoptotic cell death. Using a specific inhibitor that targets a range of caspases (Z-VAD-fmk) we assessed whether inhibition of caspase activity could prolong the viability of NS&vbar;h=0 cells under conditions that cause apoptotic cell death in batch cultures. Z-VAD-fmk was found to significantly reduce apoptotic cell death (by approximately 50%) induced by cytotoxins and to preserve membrane integrity to a similar extent. In conditions of low serum, Z-VAD-fmk reduced certain features of apoptosis (e.g., DNA fragmentation), but only marginally improved viability. In medium-depleted batch cultures, Z-VAD-fmk afforded a delay of between 24 and 48 h in both the induction of apoptosis and loss of viability. Despite an apparent increase in viability in Z-VAD-fmk-treated NS&vbar;h=0 cultures, no improvement in productivity could be demonstrated, suggesting that at least some normal pathways for protein production are shut down upstream of caspase activation. An examination of mitochondrial membrane potential (Deltapsim) in Z-VAD-fmk-treated and untreated NS&vbar;h=0 cells revealed only a small initial difference (5%) in the levels of Deltapsim depolarization. Similar levels of mitochondrial dysfunction, despite caspase inactivity, may therefore be responsible for the comparable productivity in untreated and Z-VAD-fmk-treated cultures. Thus, this study suggests that, while a delay in cell death due to caspase inhibition may reduce problems associated with cellular disintegration, it does not permit productivity improvements in this type of culture.

McKenna SL ，Cotter TG 《BIOTECHNOLOGY AND BIOENGINEERING》

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》

Sensitivity of photoreceptor-derived cell line (661W) to baculoviral p35, Z-VAD.FMK, and Fas-associated death domain.

Tuohy G ，Millington-Ward S ，Kenna PF ，Humphries P ，Farrar GJ ... -  《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》

The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death.

Hirsch T ，Marchetti P ，Susin SA ，Dallaporta B ，Zamzami N ，Marzo I ，Geuskens M ，Kroemer G ... -  《ONCOGENE》

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》