Staurosporine and anti-Fas (CD95) induce caspase activation in the HT-22 murine hippocampalk cell line Lucy M. Elphick, 1John E. Eriksson, George E.N. Kass and Nick J. Toms. School of Biomedical and Life Sciences, University of Surrey, Guildford, GU2 7XH and 1Turku Centre for Biotechnology, POB 123, FIN-20521 Turku, Finland.

Print abstract Search PubMed for: Elphick LM Eriksson JE Kass GEN Toms NJ

Emerging data suggest that the Fas/CD95 receptor signalling pathway may play an important role in inducing delayed neuronal cell death following acute ischaemic CNS insults (Jin et al., 2001). In the present study, we have employed the murine hippocampal HT-22 cell line to examine the molecular mechanisms of Fas-induced cell death.

Twenty four hours after seeding (4,000 cells/cm²⁾ in DMEM / 2% FCS medium, cells were exposed to either mouse 200 ng ml^-1 anti-Fas (Jo-2, Pharmingen) / 1 µg ml^-1 cycloheximide (anti-Fas/CHX) or 1 µM staurosporine. Cell viability was then determined via monitoring lactate dehydrogenase (LDH) release (Roche). All caspase inhibitors were added 30 min prior to toxin addition. Caspase-3 activity was examined by both flow cytometry and confocal microscopy using an anti-active caspase-3 phycoerythrin-conjugated antibody (1:10, Pharmingen). Cytochrome c translocation was examined via confocal microscopy using 0.05% (w/v) saponin-permeabilised cells with mouse anti-cytochrome c (1:100, Pharmingen) / rabbit anti-mouse-FITC (1:100, DAKO) antibodies. Chromatin condensation was examined via Hoechst 33258 (2µg ml^-1) fluorescence. Cell viability data are expressed as mean (± s.e.m.) percentage of total available cellular LDH released (with total LDH content determined by Triton X-100 (0.1% v/v)). All statistical analyses were performed via one-way ANOVA (Tukey-Kramer post test).

After 48 hrs, anti-Fas/CHX (63.6% ± 2.2%, n=8, P<0.001) and staurosporine (72.4% ± 9.2%, n=5, P<0.001) induced significant cell death; an effect significantly reduced by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-FMK, 100µM) (anti-Fas/CHX + z-VAD-FMK = 47.5% ± 2.5%, n=6, P<0.001; staurosporine + z-VAD-FMK = 26.6% ± 6.5%, n=5, P<0.01). After 24 hrs, the caspase-3 selective inhibitor acetyl Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO, 50µM) inhibited both anti-Fas/CHX (50.8% ± 3.6%, n=3, P<0.01) and staurosporine (40.8% ± 16.2%, n=3, P<0.001)-induced cell death. Quinoline-Val-Asp-CH2-O-Ph (Q-VD-OPH, 50µM) also afforded significant protection versus anti-Fas/CHX induced cell death (24.4% ± 1.8%, n=3, P<0.001). Over 48 hours, significant caspase-3 activation accompanied both anti-Fas/CHX (30hrs, 43.4% ± 1.9%, n=3, P<0.001) and staurosporine (24hrs, 75.0% ± 5.6%, n=3, P<0.001) insults. Confocal microscopy confirmed caspase-3 activation and cytosolic cytochrome c translocation in both anti-Fas/CHX and staurosporine cytotoxic paradigms. After 24 hrs, chromatin condensation (morphological indicator of apoptosis) occurred in both sets of treatments.

These data show that HT-22 cells are susceptible to Fas-induced apoptosis, which is accompanied with activation of caspase-3 and cytochrome c release. Therefore, HT-22 cells represent a useful cell system in which to examine neuronal ischaemic apoptosis.

Jin, K. et al., (2001), J. Cereb. Blood Flow & Metab. 21, 1411-1421.

We wish to thank Dr P. Maher (The Scripps Research Institute, La Jolla, California) for donating the HT-22 cell line.