Crm-A, bcl-2 and NDGA inhibit CD95L-induced apoptosis of malignant glioma cells at the level of caspase 8 processing

Abstract

Susceptibility to CD95 (Fas/APO-1)-mediated apoptosis in human glioma cells depends on CD95 expression and unknown factors that regulate signal transduction. Thus, LN-18 cells are highly sensitive to CD95 ligand (CD95L) whereas LN-229 cells require coexposure to inhibitors of RNA or protein synthesis for induction of apoptosis. Here, we report that caspase 8 and 3 activation, poly(ADP-ribose)polymerase cleavage and apoptosis are inhibited by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), or ectopic expression of crm-A or bcl-2. CD95L-induced glioma cell apoptosis does not involve ceramide generation. Apoptosis induced by exogenous ceramide resembles CD95-mediated apoptosis in that bcl-2 is protective but differs in that NDGA and crm-A have no effect and in that cycloheximide (CHX) inhibits rather than potentiates ceramide-induced cell death. We conclude that caspase 8 and caspase 3 activation, but not ceramide generation, are required for CD95 ligand-induced apoptosis of glioma cells and that bcl-2, crm-A and NDGA all act upstream of caspases to inhibit apoptosis.

Death receptor-independent cytochrome c release and caspase activation mediate thymidine kinase plus ganciclovir-mediated cytotoxicity in LN-18 and LN-229 human malignant glioma cells

“…These cells have been shown here ( Figure 3b) and previously 11 to resist death receptor-mediated apoptosis. Puro control and crm-A-transfected cells were cotransGene Therapy fected with a plasmid encoding eGFP.…”

“…Furthermore, a two-fold approach was employed to examine a possible role for death ligand/receptor interactions in TK/GCV-mediated cell death. TK/GCV cytotoxicity was examined in LN-18 and LN-229 sublines engineered to express the viral caspase inhibitor, crm-A, which inhibits death ligand-induced cell death 11 and in a LN-18 subline selected for resistance to cytotoxic cytokines including CD95L, LN-18-R. 8 As expected, cell death induced by Ad-CD95L was abrogated by crm-A ( Figure 3b). In contrast, crm-A had no effect on TK/GCVinduced cytotoxicity (Figure 3c, d).…”

“…The generation of glioma cell sublines expressing crm-A has been described. 11 Glioma cell sublines engineered to express human BCL-X L were obtained by electroporation (Biorad Gene Pulser, 250 V, 950 F; Biorad, Munich, Germany) using the pSFFV-BCL-X L plasmid, kindly provided by Dr CB Thompson (Chicago, IL, USA) or the empty neo plasmid as a control. Transgene expression was assessed by immunoblot analysis.…”

APRIL, a new member of the tumor necrosis factor family, modulates death ligand-induced apoptosis

“…Vincristine, teniposide, lomustine and cisplatin killed glioma cells irrespective of the presence of APRIL ( Figure 3D ± F). Since we have previously described caspase 3 as a key executioner in death ligand-induced apoptosis of malignant glioma cells, 15 we next examined the induction of caspase 3-like activity by CD95L in APRIL-versus control-transfected glioma cells. APRIL transgene expression resulted in significant inhibition of DEVD-amc cleavage upon treatment with CD95L, both in the absence and presence of CHX, in LN-229 and U373MG cells (Figure 4).…”

Increased expression and activation of poly(ADP-ribose) polymerase (PARP) contribute to retinal ganglion cell death following rat optic nerve transection

“…Equal amounts of protein were diluted in 66 sample buffer, boiled for 5 min and loaded onto 8% polyacrylamide gels. To test for antibody specificity, 2 ml of a rat glioma cell line (LN-18) lysate, in which PARP protein had successfully been detected using Western blot analysis, 44 were loaded onto a separate lane as positive control. Proteins were transferred onto nitrocellulose membranes, immersed in blocking solution (5% milk, 0.1% Tween 20 in PBS; 12 ± 15 h at 48C) and incubated with a polyclonal rabbit anti-PARP antibody (1 : 2500 in PBS with 1% milk, 0.1% Tween 20; 2 h at RT; Boehringer-Mannheim, Germany).…”