FasL and TNF-related apoptosis-inducing ligand (TRAIL) belong to a subgroup of the TNF superfamily which induce apoptosis by binding to their death domain containing receptors. In the present study we have utilized a panel of seven cell lines derived from human malignant gliomas to characterize molecular pathways through which FasL and TRAIL induce apoptosis in sensitive glioma cells and the mechanisms of resistance in cell lines which survive the death stimuli. Our ®ndings indicate that FADD and Caspase-8 are essential for FasL and TRAIL mediated apoptosis in glioma cells. One sensitive cell line (D270) can be protected from FasL and TRAIL induced death by anti-apoptotic Bcl-2 family members while another (D645) cannot, implying that these lines may represent glioma examples of type II and type I cells respectively. For the ®rst time we demonstrate resistance to FasL but not to TRAIL within the one glioma cell line. Furthermore, we report distinct mechanisms of resistance within dierent glioma lines, including downregulation of Caspase-8 in U373MG. Cycloheximide sensitized four of the resistant cell lines suggesting the presence of labile inhibitors. None of the known apoptosis inhibitors examined accounted for the observed resistance, suggesting novel inhibitors may exist in glioma cells. Oncogene (2001) 20, 5789 ± 5798.

Background and Purpose-AM-36 is a novel arylalkylpiperazine with combined antioxidant and Na ϩ channel blocking actions. Individually, these properties have been shown to confer neuroprotection in a variety of in vitro and in vivo animal models of stroke. Preliminary studies have shown that AM-36 is neuroprotective in vivo. The purpose of the present study was to assess the neuroprotective and behavioral outcome after delayed administration of AM-36 in an endothelin-1-induced, middle cerebral artery model of cerebral ischemia in conscious rats. Methods-Conscious male hooded Wistar rats were subjected to middle cerebral artery occlusion by perivascular microinjection of endothelin-1 via a previously implanted cannula. AM-36 (6 mg/kg IP) or vehicle was administered intraperitoneally 30, 60, or 180 minutes after middle cerebral artery occlusion. Functional outcome was determined 24, 48, and 72 hours after stroke by neurological deficit score, motor performance, and sensory hemineglect tests. Rats were killed at 72 hours, and infarct area and volume were determined by histology and computerized image analysis. Results-Endothelin-1-induced middle cerebral artery occlusion resulted in marked functional deficits and neuronal damage. AM-36 significantly reduced cortical damage when administration was delayed until 30, 60, or 180 minutes after stroke. Interestingly, neuronal damage was time-dependently reduced, with the greatest protection found when AM-36 was administered 180 minutes after stroke. Striatal damage was significantly reduced after treatment with AM-36 at 180 minutes after stroke. Functional outcome paralleled histopathology. Rota-rod performance, sensory hemineglect, and neurological deficit scores returned to preischemia levels in AM-36 -treated rats by 72 hours after stroke when administration was delayed by 180 minutes after stroke. Conclusions-AM-36 potently protects against both neuronal damage and functional deficits even when administered up to 180 minutes after induction of stroke. In fact, the greatest protection was found when administration was delayed by 180 minutes after stroke. The possible mechanisms of action of AM-36 are discussed. The present findings suggest that AM-36 may have great promise in the acute treatment of human stroke.

This study characterized the ability of a new member of the p35 family, p49, to inhibit a number of mammalian and insect caspases. p49 blocked apoptosis triggered by treatment with Fas ligand (FasL), Tumor necrosis factor-related apoptosisinducing ligand (TRAIL) or ultraviolet (UV) radiation but provided negligible protection against apoptosis induced by the chemotherapeutic drug cisplatin. The caspase cleavage site in p49 was determined, and mutation of the P1 residue of this site abolished the ability of p49 to inhibit caspases, implying that p49 inhibits caspases through an analogous suicide ± substrate mechanism to p35. Unlike p35, p49 inhibited the upstream insect caspase DRONC.

BACKGROUNDBetter treatments are required urgently for patients with malignant glioma, which currently is incurable. Death ligands, such as tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL), may offer promise for the treatment high‐grade glioma if such ligands induce apoptotic signaling in vivo in glioma cells. Caspase 8 is required for death ligand signaling, and its levels may influence the sensitivity of glioma cells to death ligands. It also may act as a tumor suppressor protein. The authors analyzed caspase 8 expression levels in ex vivo glioma specimens and explored potential mechanisms of its regulation.METHODSEleven glioblastomas, 5 anaplastic astrocytomas, and 3 low‐grade astrocytomas were studied. The levels of caspase 8, caspase 10, cellular FLICE inhibitory protein (c‐FLIP), and signal transducer and activator of transcription (STAT)‐1 were assayed using quantitative immunoblotting. Caspase 8 mRNA was measured by Northern blot analysis. The methylation status of the caspase 8 gene was determined by bisulfate modification of genomic DNA, cloning, and sequencing. Statistical analyses were performed using nonparametric (Spearman) correlations.RESULTSSome ex vivo glioma samples lacked detectable caspase 8, with many expressing barely detectable levels. No tumors expressed significant amounts of caspase 10 or c‐FLIP. A strong association was found between caspase 8 mRNA and protein levels. Neither expression of the transcription factor STAT‐1 nor caspase 8 gene methylation correlated with caspase 8 levels.CONCLUSIONSThe absence of caspase 8 protein in many resected glioma samples implied that many patients with glioma may not benefit from death ligand‐based treatments, unless caspase 8 (or caspase 10) protein expression can be elevated. Demethylating agents are unlikely to boost caspase 8 levels in glioma cells, but treatments that increase caspase 8 mRNA levels may up‐regulate expression of the protein. Cancer 2005. © 2005 American Cancer Society.

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Fifty percent of high-grade glioma patients die within a year of diagnosis and less than two percent survive five years postdiagnosis. Elucidating apoptosis signaling pathways may assist in designing better adjuvant therapies. Preliminary characterizations suggested that glioma cells may either employ mitochondrial-independent or -dependent death receptor-induced apoptotic pathways, characteristic of cells termed type I and type II, respectively. In the present study, we generated panels of clonal transfectants overexpressing various levels of Bcl-2, in two parental glioma cell lines. These cells were used to explore molecular factors determining the necessity for mitochondrial amplification of death receptor signaling. Moderate Bcl-2 expression was sufficient to render one glioma cell line (D270) resistant to apoptosis induced by Fas ligand or TRAIL, consistent with these cells being type II. However, expression of even very high levels of Bcl-2 in a second line (D645) did not affect death ligand sensitivity, indicative of a type I phenotype. D270 cells expressed much less caspase-8 protein than D645 cells. Enforced overexpression of caspase-8 (or cytoplasmic Diablo/Smac) in D270 cells overcame Bcl-2 inhibition of death ligand-induced apoptosis, converting them from type II to type I. This indicates that caspase-8 levels can influence the requirement for mitochondrial involvement in death receptor apoptotic signaling in glioma cells.

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