The tumor suppressor gene p53 has been implicated in the loss of neuronal viability, but the signaling events associated with p53-mediated cell death in cortical and hippocampal neurons are not understood. Previous work has shown that adenovirus-mediated delivery of the p53 gene causes cortical and hippocampal neuronal cell death with some features typical of apoptosis. In the present study we determined whether p53-initiated changes in neuronal viability were dependent on members of the Bcl-2 family of cell death regulators. Primary cultures of cortical neurons were derived from animals containing Bax (+/+ and +/-) or those deficient in Bax (-/-). Cell damage was assessed by direct cell counting and by measurements of MTT activity. Neurons containing at least one copy of the Bax gene were damaged severely by exposure to excitotoxins or by the induction of DNA damage. In contrast, Bax-deficient neurons (-/-) exhibited significant protection from both types of injury. Bax protein expression was elevated significantly by glutamate exposure, but not by camptothecin-induced DNA damage in wild-type neurons. The glutamate-induced increase in Bax protein was dependent on the presence of the p53 gene. However, increased p53 expression, using adenovirus-mediated transduction, was not sufficient by itself to elevate Bax protein levels. These results demonstrate that Bax is required for neuronal cell death in response to some forms of cytotoxic injury and further support the key role for p53 activation in response to excitotoxic and genotoxic injury.

A role for p53-related modulation of neuronal viability has been suggested by the finding that p53 expression is increased in damaged neurons in models of ischemia and epilepsy. These findings were recently extended with the demonstration that mice deficient in p53 ("knock-out" mice) exhibit almost complete protection from seizure-induced brain injury, whereas wild-type mice display significant neuronal cell loss in the hippocampus and other brain regions. Because the p53 knock-out mice used in the latter study expressed a global p53 deficiency in all cell types, it was not possible to conclude that protection was conferred by the exclusive absence of p53 in neurons. Therefore, in the present study, we determined whether p53 expression in isolated neurons is directly coupled to a loss of viability associated with excitotoxic challenge. Primary cultures of hippocampal or cortical neurons were derived from animals containing p53 (+/+, +/-) or those deficient in p53 (-/-). p53-Deficient neurons appeared identical to wild-type neurons with respect to morphology, neurofilament expression, and resting levels of intracellular calcium. Neurons containing at least one copy of p53 were severely damaged by exposure to kainic acid or glutamate. Cell damage was assessed by direct cell counting and by nuclear morphology after propidium iodide staining of DNA. In contrast, neurons deficient in p53 (-/-) exhibited little or no damage in response to excitotoxin treatment. Despite their divergent outcomes, p53 (+/+) and p53 (-/-) neurons demonstrated similar sustained elevations in intracellular calcium levels triggered by glutamate exposure. Restoring p53 expression to p53-deficient neurons, using adenovirus-mediated transduction, was sufficient to promote neuronal cell death even in the absence of excitotoxin. These results demonstrate a direct relationship between p53 expression and loss of viability in CNS neurons.

Endothelial cell death may contribute to tissue injury from ischemia. Little is known, however, about the characteristics of endothelial cell death in response to hypoxia. Using an in vitro model, we found that human umbilical vein endothelial cells were resistant to hypoxia-induced cell death with only a 2% reduction in viability at 24 h and 45% reduction in viability at 48 h. Overexpression of a mutant, IB␣, via adenoviral vector did not potentiate cell death in hypoxia, indicating that nuclear factor-B activation was not involved in cytoprotection. Cell death in hypoxia was determined to be apoptotic by 3 labeling of DNA using terminal deoxynucleotidyl transferase staining and reversibility of cell death with a caspase inhibitor. Exposure of endothelial cells to hypoxia did not alter levels of proapoptotic and antiapoptotic Bcl-2 family members Bax and Bcl-X L by immunoblot analysis. In contrast, changes in p53 protein levels correlated with the induction of apoptosis in hypoxic endothelial cells. Inhibition of the proteasome increased p53 protein levels and accelerated cell death in hypoxia. Overexpression of p53 by adenoviral transduction was sufficient to initiate apoptosis of normoxic endothelial cells. These data provide a framework for the study of factors regulating endothelial cell survival and death in hypoxia.Tissue injury from ischemia and reperfusion causes significant morbidity and mortality in cardiovascular disease. Endothelial cell (EC) 1 death may contribute to the hypoxic as well as the reperfusion components of this injury. The mechanisms of cell death in hypoxia are not known but may involve calcium influx, derangements in mitochondrial function, or purine nucleotide depletion (1, 2). Limited studies suggest a role for apoptosis induced by the tumor suppressor gene p53 in hypoxia-induced cell death. Graeber et al. (3) found that tumor cells containing wild-type p53 were more sensitive to hypoxiainduced apoptosis when compared with tumor cells lacking functional p53. Long et al. (4) showed a role for p53 in cardiomyocyte apoptosis in response to hypoxia. In contrast, Amellem et al. (5) demonstrated that hypoxia-induced apoptosis occurred independent of p53 protein level in MCF-7 cells. There are few data, however, on the relative susceptibility of EC to hypoxia-induced cell death or the molecular mechanisms involved. Because EC are invariably exposed to hypoxia in ischemic conditions, this question has important therapeutic implications for the prevention of ischemic tissue damage.We used an in vitro model to examine mechanisms of EC death during hypoxia. We found that EC underwent significant cell death with features of apoptosis only after exposure to 48 h of hypoxia. Inhibition of nuclear factor-B (NF-B) activation by adenoviral-mediated overexpression of a dominant negative IB␣ mutant did not potentiate apoptosis in hypoxic EC. There was no correlation between Bax/Bcl-X L ratios and cell death. However, there was an increase in p53 protein levels concomitant with EC death. In addition,...