Implication of caspases during maedi–visna virus-induced apoptosis

Abstract

Maedi-visna virus (MVV) causes encephalitis, pneumonia and arthritis in sheep.In vitro, MVV infection and replication lead to strong cytopathic effects characterized by syncytia formation and subsequent cellular lysis. It was demonstrated previously that MVV infection in vitro induces cell death of sheep choroid plexus cells (SCPC) by a mechanism that can be associated with apoptotic cell death. Here, the relative implication of several caspases during acute infection with MVV is investigated by employing diverse in vitro and in situ strategies. It was demonstrated using specific pairs of caspase substrates and inhibitors that, during in vitro infection of SCPC by MVV, the two major pathways of caspase activation (i.e. intrinsic and extrinsic pathways) were stimulated : significant caspase-9 and -8 activities, as well as caspase-3 activity, were detected. To study the role of caspases during MVV infection in vitro, specific, cell-permeable, caspase inhibitors were used. First, these results showed that both z-DEVD-FMK (a potent inhibitor of caspase-3-like activities) and z-VAD-FMK (a broad spectrum caspase inhibitor) inhibit caspase-9, -8 and -3 activities. Second, both irreversible caspase inhibitors, z-DEVD-FMK and z-VAD-FMK, delayed MVV-induced cellular lysis as well as virus growth. Third, during SCPC in vitro infection by MVV, cells were positively stained with FITC-VAD-FMK, a probe that specifically stains cells containing active caspases. In conclusion, these data suggest that MVV infection in vitro induces SCPC cell death by a mechanism that is strongly dependent on active caspases.

Interactions of fluorochrome‐labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

“…While there is evidence that FLICA binds to proteins of molecular weight [18][19][20][21][22][23], which is in concordance with the molecular weight of large subunits of caspases (4 -6), there are certain puzzling observations that cannot be easily explained by the initially proposed mechanism of the binding, i.e., through interactions with the caspase active center only. Thus, if caspase active centers were the only binding sites for these reagents, one would expect a significant level of protection of these sites by the unlabeled caspase inhibitors (z-VAD-FMK, z-DEVD-FMK) via competitive binding.…”

“…These reagents were designed as affinity ligands to react covalently with the reactive enzymatic center of activated caspases. During the past two years several articles have been published (19,(21)(22)(23), including the papers authored by us (18,24,25), in which these reagents have been used to probe for caspase activation. Based on a similar principle, other probes were developed to detect activation of intracellular serine proteases (26).…”

Induction of antiproliferative effect by diosgenin through activation of p53, release of apoptosis-inducing factor (AIF) and modulation of caspase-3 activity in different human cancer cells

“…Fluorescence was measured with a microplate reader (Fluorolite 1000, Dynatech Laboratories, Dynex Technologies, Labsystems) using 360 nm excitation and 460 nm emission filters. Raw data [Relative Units of Fluorescence (RUF)] corresponded to the concentrations of AMC released [20].…”

Cinétique et caractérisation du phénomène “apoptosis-like” induit au cours de la congélation des spermatozoïdes bovins

“…Fluoromethylketone couple au FITC (FITC-VAD-FMK) (Promega, Charbonniere les Bains, France) a 6te utilis~ 10 pM final pour etudier I'activation des caspases [3,11]. Le Yo-Pro-t (Molecular Probes) a ete utilise a 100 nM final pour etudier les variations de permeabilit(~ membranaire [5,10].…”