The inactivation of the p53 gene in a large proportion of human cancers has inspired an intense search for the encoded protein's physiological and biological properties. Expression of p53 induces either a stable growth arrest or programmed cell death (apoptosis). In human colorectal cancers, the growth arrest is dependent on the transcriptional induction of the protein p21WAF1/CIP1 , but the mechanisms underlying the development of p53-dependent apoptosis are largely unknown. As the most well documented biochemical property of p53 is its ability to activate transcription of genes, we examined in detail the transcripts induced by p53 expression before the onset of apoptosis. Of 7,202 transcripts identified, only 14 (0.19%) were found to be markedly increased in p53-expressing cells compared with control cells. Strikingly, many of these genes were predicted to encode proteins that could generate or respond to oxidative stress, including one that is implicated in apoptosis in plant meristems. These observations stimulated additional biochemical and pharmacological experiments suggesting that p53 results in apoptosis through a three-step process: (1) the transcriptional induction of redox-related genes; (2) the formation of reactive oxygen species; and (3) the oxidative degradation of mitochondrial components, culminating in cell death.

NIH 3T3 fibroblasts stably transformed with a constitutively active isoform of p21(Ras), H-RasV12 (v-H-Ras or EJ-Ras), produced large amounts of the reactive oxygen species superoxide (.O2-). .O2- production was suppressed by the expression of dominant negative isoforms of Ras or Rac1, as well as by treatment with a farnesyltransferase inhibitor or with diphenylene iodonium, a flavoprotein inhibitor. The mitogenic activity of cells expressing H-RasV12 was inhibited by treatment with the chemical antioxidant N-acetyl-L-cysteine. Mitogen-activated protein kinase (MAPK) activity was decreased and c-Jun N-terminal kinase (JNK) was not activated in H-RasV12-transformed cells. Thus, H-RasV12-induced transformation can lead to the production of .O2- through one or more pathways involving a flavoprotein and Rac1. The implication of a reactive oxygen species, probably .O2-, as a mediator of Ras-induced cell cycle progression independent of MAPK and JNK suggests a possible mechanism for the effects of antioxidants against Ras-induced cellular transformation.

Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide ( O-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and°2-formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no -was seen. With cells in L-Arg-free medium, we observed *°2 formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined.°2 formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Argdepleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating°O2 and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving°2 /NO-mediated cellular injury.Nitric oxide (NO), a gaseous free radical, regulates vascular tone, platelet aggregation, leukocyte adhesion, synaptic transmission and cytostatic/cytotoxic actions of macrophages (1, 2). NO arises from the guanidino group of L-Arg in an NADPHdependent reaction catalyzed by a family of NO synthases (NOSs) (3). Three distinct isoforms of NOS, derived from separate genes, are neuronal NOS (nNOS, type I), inducible NOS (type II), and endothelial NOS (type III) (4,5). The three isoforms are similar in structure and function, utilizing L-Arg, oxygen, and NADPH as substrates and requiring FAD, FMN, calmodulin, and tetrahydrobiopterin as cofactors (6). NOS is a cytochrome P450 reductase-like hemoprotein containing FAD,-FMN, NADPH, calmodulin, and ferroprotoporphyrin IX (heme) binding sites (7). The catalytic mechanism of NOS involves flavin-mediated electron transport from NADPH to the terminal heme, where oxygen is bound and incorporated into NO and citrulline (8,9).Besides synthesizing NO, purified porcine nNOS generates hydrogen peroxide (H202) at low concentrations of L-Arg (10, 11). Purified rat nNOS produces superoxide (°O2) in an NADPH and Ca2+/calmodulin-dependent manner (12). Ques-tions not yet answered include (i) whether NOS-mediated°2 generation occurs in intact cells, (ii) how cytosolic NOS is regulated to produce either NO or°, and (iii) whether in intact cells NO and O2 generated by NOS combine to form the cytotoxic oxidant peroxynitrite (ONOO-). Furthermore, the biological significance of the°O2 generated by NOS has not been established. In the present study, we directly measure and characterize the formation of both NO and°2J in neuronal constitutive NOS-transfected human kidney 293 cells by using electron paramagnetic resonance (EPR) spin-trapping techniques. The effects of intracellular L-Arg depleti...