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.

Summary Repression of mitochondrial respiration represents an evolutionarily ancient cellular adaptation to hypoxia and profoundly influences cell survival and function; however, the underlying molecular mechanisms are incompletely understood. Primarily utilizing pulmonary arterial endothelial cells as a representative hypoxic cell type, we identify the iron-sulfur cluster assembly proteins (ISCU1/2) as direct targets for repression by the hypoxia-induced microRNA-210 (miR-210). ISCU1/2 facilitate the assembly of iron-sulfur clusters, prosthetic groups that are critical for electron transport and mitochondrial oxidation-reduction reactions. Under in vivo conditions of up-regulating miR-210 and repressing ISCU1/2, the integrity of iron-sulfur clusters is disrupted. In turn, by repressing ISCU1/2 during hypoxia, miR-210 decreases the activity of prototypical iron-sulfur proteins controlling mitochondrial metabolism, including Complex I and aconitase. Consequently, miR-210 represses mitochondrial respiration and associated downstream functions. These results identify important mechanistic connections among microRNA, iron-sulfur cluster biology, hypoxia, and mitochondrial function, with broad implications for cellular metabolism and adaptation to cellular stress.