Exposure of neuronal P012 cells, differentiated by nerve growth factor, to tumor necrosis factor-a (TNFa) and bacterial Iipopolysaccharide (LPS) resulted in de novo synthesis of inducible nitric oxide synthase (iNOS) mRNA and protein with an increase up to 24 h. Brain NOS expression was unaffected. The induction of iNOS in differentiated P012 cells was associated with cell death characterized by features of apoptosis. The NOS inhibitors Nmonomethylarginine, aminoguanidine, and 2-amino-5,6dihydro-6-methyl-4H-1 ,3-thiazine~HCI prevented TNF-a/ LPS-induced cell death and DNA fragmentation, suggesting that the TNF-ct/LPS-induced cell death is mediated by iNOS-derived NO. This hypothesis is supported by the finding that addition of L-arginine, which serves as a precursor and limiting factor of enzyme-derived NO production, potentiated TNF-ct/LPS-induced loss of viability. Key Words: Differentiated P012 cells-Inducible nitric oxide synthase-N-Monomethylarginine-DNA fragmentation -Apoptosis. oxide synthase; bNOS and iNOS, brain NOS and inducible NOS, respectively; PBS, phosphate-buffered saline; TNF-a, tumor necrosis factor-a; TUNEL, terminal transferase dUTP nick end-labeling.

Cytokines such as transforming growth factor-beta (TGF-beta) are thought to mediate escape from immune surveillance in human malignant glioma. Here, we report that ectopic expression of the small TGF-beta-binding proteoglycan, decorin, inhibits not only TGF-beta bioactivity but also TGF-beta 1 and TGF-beta 2 mRNA transcription and TGF-beta protein synthesis by human LN-18, LN-229, T98G and rat C6 glioma cells in vitro. Ectopic expression of decorin in C6 rat glioma cells results in strong inhibition of tumor formation in vivo. Decorin-expressing C6 gliomas grow initially but regress to very small residual tumors at 12 weeks after implantation whereas all control animals die or have to be killed within 4 weeks. Decorin-expressing tumors show a four-fold increase of infiltration by activated T cells and a 1.6-fold increase in total B and T cells. Chronic steroid-mediated immunosuppression abrogates the inhibitory effects of decorin gene transfer. We conclude that decorin-induced inhibition of TGF-beta release by glioma cells significantly enhances antiglioma immune responses in vivo. Clinical evaluation of decorin gene therapy for human malignant gliomas may be warranted.

Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D‐ and cycloheximide‐sensitive caspase (ICE‐like) activity. The peptide inhibitor of caspase activity, N‐acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde, was more potent than acetyl‐Tyr‐Val‐Ala‐Asp‐chloromethyl ketone in preventing NGF withdrawal‐induced apoptosis, suggesting an important role for caspase‐3 (CPP32)‐like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N‐tert‐butyl‐α‐phenylnitrone, and the antioxidant, N‐acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL‐2 or the BCL‐2‐binding protein BAG‐1 blocked NGF withdrawal‐induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL‐2 and BAG‐1 was more protective than expression of either protein alone.

In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonylVal-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.

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