Hypoxia is an important factor that elicits numerous physiological and pathological responses. One of the major gene expression programs triggered by hypoxia is mediated through hypoxia-responsive transcription factor hypoxia-inducible factor 1 (HIF-1). Here, we report the identification and cloning of a novel HIF-1-responsive gene, designated RTP801. Its strong up-regulation by hypoxia was detected both in vitro and in vivo in an animal model of ischemic stroke. When induced from a tetracycline-repressible promoter, RTP801 protected MCF7 and PC12 cells from hypoxia in glucose-free medium and from H 2 O 2 -triggered apoptosis via a dramatic reduction in the generation of reactive oxygen species. However, expression of RTP801 appeared toxic for nondividing neuron-like PC12 cells and increased their sensitivity to ischemic injury and oxidative stress. Liposomal delivery of RTP801 cDNA to mouse lungs also resulted in massive cell death. Thus, the biological effect of RTP801 overexpression depends on the cell context and may be either protecting or detrimental for cells under conditions of oxidative or ischemic stresses. Altogether, the data suggest a complex type of involvement of RTP801 in the pathogenesis of ischemic diseases.

cDNA microarray hybridization was used in an attempt to identify novel genes participating in cellular responses to prolonged hypoxia. One of the identified novel genes, designated Hi95 shared significant homology to a p53-regulated GADD family member PA26. In addition to its induction in response to prolonged hypoxia, the increased Hi95 transcription was observed following DNA damage or oxidative stress, but not following hyperthermia or serum starvation. Whereas induction of Hi95 by prolonged hypoxia or by oxidative stress is most likely p53-independent, its induction in response to DNA damaging treatments (g-or UV-irradiation, or doxorubicin) occurs in a p53-dependent manner. Overexpression of Hi95 fulllength cDNA was found toxic for many types of cultured cells directly leading either to their apoptotic death or to sensitization to serum starvation and DNA damaging treatments. Unexpectedly, conditional overexpression of the Hi95 cDNA in MCF7-tet-off cells resulted in their protection against cell death induced by hypoxia/glucose deprivation or H 2 O 2 . Thus, Hi95 gene seems to be involved in complex regulation of cell viability in response to different stress conditions.

The protooncogene bcl-2 inhibits neuronal apoptosis during normal brain development as well as that induced by cytotoxic drugs or growth factor deprivation. We have previously demonstrated that neurons of mice deficient in Bcl-2 are more susceptible to neurotoxins and that the dopamine (DA) level in the striatum after systemic 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) administration was significantly lower than in wild-type mice. In the present study we have used transgenic mice overexpressing human Bcl-2 under the control of neuron-specific enolase promoter (NSE-hbcl-2) to test the effects of the neurotoxins 6-hydroxydopamine (6-OHDA) and MPTP on neuronal survival in these mice. Primary cultures of neocortical neurons from normal and transgenic mice were exposed to these dopaminergic neurotoxins. Addition of 6-OHDA resulted in cell death of essentially all neurons from normal mice. In contrast, in cultures generated from heterozygous NSE-hbcl-2 transgenic mice, only 69% of the cells died while those generated from homozygous transgenic mice were highly resistant and exhibited only 34% cell death. A similar effect was observed with neurons treated with MPP ؉ . Moreover, while the striatal dopamine level after MPTP injections was reduced by 32% in the wild type, the concentration remained unchanged in the NSE-hbcl-2 heterozygous mice. In contrast levels of glutathione-related enzymes were unchanged. In conclusion, overexpression of Bcl-2 in the neurons provided protection, in a dose-dependent manner, against neurotoxins known to selectively damage dopaminergic neurons. This study provides ideas for inhibition of neuronal cell death in neurodegenerative diseases and for the development of efficient neuroprotective gene therapy.

Bcl-2 is an antiapoptotic protein located in the outer mitochondrial membrane. Cellular perturbations associated with programmed cell death may be the consequence of disrupted mitochondrial function as well as excessive production of reactive oxygen species (ROS). Numerous studies indicate that Bcl-2 is involved in opposing cell death induced by oxidative stimuli, but its mode of action is uncertain. We reexamined the role of Bcl-2 by using a loss-of-function model, BcI-2 knockout mice. Brains from Bc/-2-deficient mice had a 43% higher content of oxidized proteins and 27% lower number of cells in the cerebellum relative to wild-type mice. Incubation of cerebellar neurons from Bcl-2 +1+ brains with 0.5 mM dopamine caused 25% cell death, whereas in Bc!-2-deficient cells, it resulted in 52% death; glial cells provided protection in both cultures. Splenocytes from Bc!-2-deficient mice were also killed more effectively by dopamine as well as paraquat. Bc!-2-deficient mice did not survive intraperitoneal injection of MPTP, which caused a decrease in dopamine level in the striatum of Bc!-2

Exposure of mouse thymocytes to dopamine caused apoptosis (programmed cell death). This was manifested by cellular condensation and membrane damage shown by flow cytometry measurements and scanning electron microscopic study. Dopamine also affected thymocytic nuclei and their genomic DNA integrity. Most of the DNA molecules accumulated in a subdiploid peak in flow cytometry analysis, indicating DNA fragmentation to small particles. DNA analysis showed the typical pattern of 'DNA ladder' caused by internucleosomal DNA cleavage. X-ray microanalysis of the cellular elements of dopamine-treated cells showed elevation of sodium (Na), chloride (Cl) and calcium (Ca) peaks, accompanied by reduction in phosphate (P) concentrations. Comparison of the potassium (K) and P concentrations showed significant differences between the two major death processes: necrosis (induced by exposure to sodium azide (NaN3)) and apoptosis (induced by dopamine). High concentrations of K indicated cell viability while reductions in P and elevations in Ca levels were found to be typical of apoptotic cell death. The antioxidant dithiothreitol (DTT) suppressed dopamine-induced apoptosis in thymocytes, suggesting that its toxicity may be mediated via generation of reactive oxygen radicals. Our study suggests that under certain circumstances, dopamine and/or its metabolites, may induce a process of apoptotic cell death of the dopamine-producing cells in the substantia nigra. Increased accessibility of dopamine to the nigral cell nucleus or inability to scavenge excess free radicals generated from dopamine oxidation triggering programmed cell death, may cause the progressive nigral degeneration in Parkinson's disease.