Caspase-3 and -7 mediate apoptosis of human Chang's conjunctival cells induced by enterovirus 70

Abstract

Enterovirus 70 (EV70) is the major etiological agent of acute hemorrhagic conjunctivitis (AHC). EV70 m.o.i.- (multiplicity of infection) and time-dependently induced apoptosis in human Chang's conjunctival (HCC) cells. UV- or heat-inactivated EV70 did not induce apoptosis. EV70-induced apoptosis was inhibited by cycloheximide and methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK), but not actinomycin D and guanidine.HCl (although guanidine.HCl inhibited the apoptosis induced by EV70 infection at 0.5 PFU/cell for 18 h). EV70 infection induced activation of caspase-3 and -7 and degradation of the constitutively activated caspase-6. EV70-induced apoptotic DNA ladders and activated caspase-3 and -7, correlated with virus release. Caspase inhibitor IX (Z-VD-FMK) inhibited EV70-induced apoptosis and virus release, but not intracellular viral production. The results suggest that infectious virus and the syntheses of viral proteins especially EV70 proteases, but not viral genome RNA, are required for caspase-3 and -7-mediated EV70-induced apoptosis, and that apoptosis through cell lysis promotes EV70 release from HCC cells.

Multiple Caspases Mediate Acute Renal Cell Apoptosis Induced by Bacterial Cell Wall Components

“…25 Briefly, 4% p-formaldehyde-fixed RK 13 cells on glass slides (Lab-Tek ® chamber slide systems; Nunc Nalgene International, Rochester, NY, USA) were reacted with primary antibodies (1:200 dilution in PBS) overnight at 4°C. Secondary fluorescein-isothiocyanate (FITC)-tagged goat anti-rabbit IgG (Fab) (Cappel, ICN Biomedicals, Inc., Aurora, OH, USA) (200 μL/glass slide chamber of a 1:500 dilution in PBS) was applied for 6 h. Cytological changes and IFA staining were observed by a Nikon Eclipse TE300 inverted microscope with an epi-fluorescence attachment and a single band exciter for FITC (492 nm/18×).…”

“…To assay for caspase, 25 the frozen cell lysis buffer samples were thawed and centrifuged at 10,000´g for 10 min in desktop Eppendorf microcentrifuge. Ten microliters of each sample were added per replicate 96-well microtiter plate well (Starstedt) containing 100 μL of caspase assay buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS, 10 mM DTT, 100 mM NaCl, 1 mM EDTA, and 10% sucrose) and 1 μM of p-nitoanalide (pNA)-tagged caspase-1, -2, -3, -6, -8, -9, or -10 -specific substrates (1 μM) ( Table 1) as per the manufacturer's instructions (Caspase Assay Kit; Sigma).…”

“…25 Briefly, monoclonal antibody (#9668; Cell Signaling Technology, Inc.) to human caspase-3, rabbit polyclonal antibodies against human caspases-6 (#9762) and -7 (#9492; Cell Signaling Technology, Inc.), human IL-1β (NB600-1381; Novus Biological, Littleton, CO, USA) (caspase-1 cleaves IL-1β), 24 caspase-10 (ab2012), and caspase-12 (ab62484; Abcam, Cambridge, MA, USA) were diluted in PBS and incubated with transblotted proteins on nitrocellulose membranes (Pierce Biotechnology, Inc., Rockford, IL, USA) in accordance with the manufacturer's recommendations for western blot analysis. The immunoreactive bands were detected using horseradish peroxidase-tagged secondary antibodies to rabbit IgG (Fab 2 fragment; Jackson ImmununiResearch Laboratories, Inc., West Grove, PA, USA).…”

Antiapoptotic Activity of the Cardiovirus Leader Protein, a Viral “Security” Protein

“…Since then, a wealth of data has been accumulated that shows that the activation of apoptotic pathways is a widespread, though not universal, response to picornavirus infection. Thus, apoptosis-inducing capacity was reported for coxsackieviruses B3, B4, and B5 (22,54,82), enteroviruses 70 and 71 (25,27,60,88), human rhinoviruses 1B, 9, 14, and 16 (32,92,100), foot-and-mouth disease virus (53,76), avian encephalomyelitis virus (62,63), and hepatitis A virus (16,43) and was the subject of several recent reviews (15,102). The antiapoptotic activity of picornaviruses was studied predominantly by using poliovirus (3,8,13,72) and coxsackievirus B3 (21,36,85).…”

Induction of Caspase-dependant Apoptosis by Enterovirus 70 in Human Lens Epithelial SRA01/04 Cells

“…Natural and experimental EV70 infection is generally localized to the superficial conjunctival epithelial cell layer (i.e., conjunctivitis) and corneal epithelium (i.e., keratitis) [9,10] and results in AHC. Although it cannot infect and replicate in all kinds of human cells, EV70 has been found to be able to infect and replicate in a broad range of human cells including leukocyte cells (A2.01, Daudi, HL60, Jurkat, Raji, THP-1 and U937) [11] and human eye cells such as HCC cell [12] , SRA01/04 cells [13] , human retina and retina pigment epithelial cells (unpublished). Therefore, EV70 may potentially cause human diseases other than AHC and its neurological complications.…”