Peter M. Chumakov

The antioxidant function of the p53 tumor suppressor.

It is widely accepted that the p53 tumor suppressor restricts abnormal cells by induction of growth arrest or by triggering apoptosis. Here we show that, in addition, p53 protects the genome from oxidation by reactive oxygen species (ROS), a major cause of DNA damage and genetic instability. In the absence of severe stresses, relatively low levels of p53 are sufficient for upregulation of several genes with antioxidant products, which is associated with a decrease in intracellular ROS. Downregulation of p53 results in excessive oxidation of DNA, increased mutation rate and karyotype instability, which are prevented by incubation with the antioxidant N-acetylcysteine (NAC). Dietary supplementation with NAC prevented frequent lymphomas characteristic of Trp53-knockout mice, and slowed the growth of lung cancer xenografts deficient in p53. Our results provide a new paradigm for a nonrestrictive tumor suppressor function of p53 and highlight the potential importance of antioxidants in the prophylaxis and treatment of cancer.

The candidate tumour suppressor p33ING1 cooperates with p53 in cell growth control

The candidate tumour-suppressor gene ING1 has been identified by using the genetic suppressor element (GSE) methodology. ING1 encodes a nuclear protein, p33ING1, overexpression of which inhibits growth of different cell lines. The properties of p33ING1suggest its involvement in the negative regulation of cell proliferation and in the control of cellular ageing, anchorage dependence and apoptosis. These cellular functions depend largely on the activity of p53, a tumour-suppressor gene that determines the cellular response to various types of stress. Here we report that the biological effects of ING1 and p53 are interrelated and require the activity of both genes: neither of the two genes can, on its own, cause growth inhibition when the other one is suppressed. Furthermore, activation of transcription from the p21/WAF1 promoter, a key mechanism of p53-mediated growth control, depends on the expression of ING1. A physical association between p33ING1and p53 proteins has been detected by immunoprecipitation. These results indicate that p33ING1is a component of the p53 signalling pathway that cooperates with p53 in the negative regulation of cell proliferation by modulating p53-dependent transcriptional activation.

Identification of a novel stress-responsive gene Hi95 involved in regulation of cell viability.

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 (γ- or UV-irradiation, or doxorubicin) occurs in a p53-dependent manner. Overexpression of Hi95 full-length 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 H2O2. Thus, Hi95 gene seems to be involved in complex regulation of cell viability in response to different stress conditions.

Homeostatic functions of the p53 tumor suppressor: Regulation of energy metabolism and antioxidant defense

The p53 tumor suppressor plays pivotal role in the organism by supervising strict compliance of individual cells to needs of the whole organisms. It has been widely accepted that p53 acts in response to stresses and abnormalities in cell physiology by mobilizing the repair processes or by removing the diseased cells through initiating the cell death programs. Recent studies, however, indicate that even under normal physiological conditions certain activities of p53 participate in homeostatic regulation of metabolic processes and that these activities are important for prevention of cancer. These novel functions of p53 help to align metabolic processes with the proliferation and energy status, to maintain optimal mode of glucose metabolism and to boost the energy efficient mitochondrial respiration in response to ATP deficiency. Additional activities of p53 in non-stressed cells tune up the antioxidant defense mechanisms reducing the probability of mutations caused by DNA oxidation under conditions of daily stresses. The deficiency in the p53-mediated regulation of glycolysis and mitochondrial respiration greatly accounts for the deficient respiration of the predominance of aerobic glycolysis in cancer cells (the Warburg effect), while the deficiency in the p53-modulated antioxidant defense mechanisms contributes to mutagenesis and additionally boosts the carcinogenesis process.

Small-molecule RETRA suppresses mutant p53-bearing cancer cells through a p73-dependent salvage pathway

Identification of unique features of cancer cells is important for defining specific and efficient therapeutic targets. Mutant p53 is present in nearly half of all cancer cases, forming a promising target for pharmacological reactivation. In addition to being defective for the tumor-suppressor function, mutant p53 contributes to malignancy by blocking a p53 family member p73. Here, we describe a small-molecule RETRA that activates a set of p53-regulated genes and specifically suppresses mutant p53-bearing tumor cells in vitro and in mouse xenografts. Although the effect is strictly limited to the cells expressing mutant p53, it is abrogated by inhibition with RNAi to p73. Treatment of mutant p53-expressing cancer cells with RETRA results in a substantial increase in the expression level of p73, and a release of p73 from the blocking complex with mutant p53, which produces tumor-suppressor effects similar to the functional reactivation of p53. RETRA is active against tumor cells expressing a variety of p53 mutants and does not affect normal cells. The results validate the mutant p53–p73 complex as a promising and highly specific potential target for cancer therapy.

p53 Pathway in Renal Cell Carcinoma Is Repressed by a Dominant Mechanism

Renal cell carcinoma (RCC) rarely acquires mutations in p53 tumor suppressor gene, suggesting that p53 signaling in this tumor type might be repressed by some other mechanism. In fact, all four RCC-derived cell lines we tested maintained wild-type p53 but were not capable of transactivating p53-responsive reporters and endogenous p53-responsive genes. p53 protein in RCC showed normal response to genotoxic stress, including accumulation, nuclear translocation, and activation of specific DNA binding. Functional and expression analysis of Mdm2, MdmX, and Arf showed lack of involvement of these p53 regulators in the observed defect of p53 function in RCC. However, activation of p53-mediated transactivation could be achieved by extremely high levels of p53 attained by lentivirus vector-driven transduction, suggesting the involvement of a dominant inhibitor in repression of p53-dependent transactivation in RCC. Consistently, p53 inactivation prevailed in the hybrids of RCC cells with the cells possessing fully functional p53. Remarkably, cells of normal kidney epithelium also caused partial p53 repression in cell fusion experiments, suggesting that RCC-specific p53 repression may be based on an unknown dominant mechanism also acting in normal kidney tissue.

Pyrimidine biosynthesis links mitochondrial respiration to the p53 pathway

While many functions of the p53 tumor suppressor affect mitochondrial processes, the role of altered mitochondrial physiology in a modulation of p53 response remains unclear. As mitochondrial respiration is affected in many pathologic conditions such as hypoxia and intoxications, the impaired electron transport chain could emit additional p53-inducing signals and thereby contribute to tissue damage. Here we show that a shutdown of mitochondrial respiration per se does not trigger p53 response, because inhibitors acting in the proximal and distal segments of the respiratory chain do not activate p53. However, strong p53 response is induced specifically after an inhibition of the mitochondrial cytochrome bc1 (the electron transport chain complex III). The p53 response is triggered by the deficiency in pyrimidines that is developed due to a suppression of the functionally coupled mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). In epithelial carcinoma cells the activation of p53 in response to mitochondrial electron transport chain complex III inhibitors does not require phosphorylation of p53 at Serine 15 or up-regulation of p14ARF. Instead, our data suggest a contribution of NQO1 and NQO2 in stabilization of p53 in the nuclei. The results establish the deficiency in pyrimidine biosynthesis as the cause of p53 response in the cells with impaired mitochondrial respiration.

Disruption of actin microfilaments by cytochalasin D leads to activation of p53.

Activation of p53 plays a central role in the cells response to various stress signals. We investigated whether p53 is activated upon disruption of actin microfilaments, caused by cytochalasin D (CD). We show that treatment with CD leads to accumulation of p53 in the cells and activation of p53‐dependent transcription. Treatment with CD led to arrest of G1‐to‐S transition in cells retaining wild‐type p53, while cells with inactivated p53 showed partial rescue from it. CD also induces apoptosis in p53+/+, but not in p53−/− cells. The obtained data suggest that disruption of the actin microfilaments activates p53‐dependent pathways.

Transcription of mammalian messenger RNAs by a nuclear RNA polymerase of mitochondrial origin

Transcription of eukaryotic genes is performed by three nuclear RNA polymerases, of which RNA polymerase II is thought to be solely responsible for the synthesis of messenger RNAs. Here we show that transcription of some mRNAs in humans and rodents is mediated by a previously unknown single-polypeptide nuclear RNA polymerase (spRNAP-IV). spRNAP-IV is expressed from an alternative transcript of the mitochondrial RNA polymerase gene (POLRMT). The spRNAP-IV lacks 262 amino-terminal amino acids of mitochondrial RNA polymerase, including the mitochondrial-targeting signal, and localizes to the nucleus. Transcription by spRNAP-IV is resistant to the RNA polymease II inhibitor α-amanitin but is sensitive to short interfering RNA specific for the POLRMT gene. The promoters for spRNAP-IV differ substantially from those used by RNA polymerase II, do not respond to transcriptional enhancers and contain a common functional sequence motif.

Chromosome changes caused by alterations of p53 expression

It has been proposed that p53 tumor-suppressor plays a key role in maintaining genome integrity in mammalian cells. We analyzed karyotype alterations in human and murine cell sublines expressing various exogenous human mutant (His175, Trp248, His273) or wild-type (wt) p53 cDNAs. In human pseudodiploid LIM1215 cells that contain two endogenous wt-p53 gene alleles, p53 mutants caused both an increase in the frequency of chromosome breaks and an emergence of hyperdiploid cells. Murine T12-/- and 10(1) fibroblasts lacking endogenous p53 expression have very unstable karyotypes and show a strong tendency to increase their ploidy levels during growth in culture. Transduction of a wt-p53 construct into p53-deficient cells inhibited an accumulation of highly polyploid cell variants. Transduction of mutant p53 did not show such an effect. Modification of endogenous and exogenous p53 expression by caffeine, which interferes with normal induction of p53 in response to DNA damage, showed no correlation between the induction of chromosome breaks and heteroploidy. We conclude that the caffeine- or mutant p53-induced increase in the frequency of chromosomal breaks in dividing LIM1215 cells is assonated with inactivation of wt-p53 function(s) responsible for control of G1 checkpoint and/or DNA repair, while numerical chromosome changes in these cells may be a result of elimination or modification of a separate p53 function, or due to gain-of-function activities of p53 mutants. p53 modifications may therefore cause chromosome instability by different pathways: (1) through changes in the system(s) preventing proliferation of cells with genomic alterations; and (2) by increasing the probability of events, such as chromosome non-disjunction and/or endoreduplication that can lead to chromosome gains.