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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using a plasmid substrate which integrates into the genome, we determined that the rate of homologous recombination was suppressed by p53. Human tumor cell lines, mutant or null for p53 had recombination rates 10000-times greater than primary fibroblasts. When isogenic cell pairs from tumor cells or primary fibroblasts were compared, differing only in one genetic change which inactivated p53, the recombination rate increased > 100-fold. Functional inactivation of p53 by dominant mutant p53, by large T antigen of SV40 virus, by E6 protein of human papilloma virus, or by genetic deletion led to the same result. Our results suggest that p53 suppresses spontaneous homologous recombination, and that p53 is not required for recombination to proceed. The mechanism of recombination suppression may be related to the reported association of p53 with Rad 51, but the functional consequences of this association are not yet established. It is suggested that suppression of homologous recombination is the means by which p53 maintains genetic stability.
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PMID:Inactivation of p53 results in high rates of homologous recombination. 915 Mar 91

The RAD17 gene product of S. Pombe is an essential component of the checkpoint control pathway which responds to both DNA damage and disruption of replication. We have identified a human cDNA that encodes a polypeptide which is structurally conserved with the S. Pombe Rad17 protein. The human gene, designated hRAD17, predicts an encoded protein of 590 amino acids and a molecular weight of 69 kD. Amino acid sequence alignment revealed that hRadl7 has 28.3% and 52.5% similarity with the S. Pombe Rad17 protein, and 21.8% identity and 45.8% similarity to the budding yeast cell cycle checkpoint protein, Rad 24. When introduced into the S. Pombe rad17 mutant, hRAD17 was able to partially revert its hydroxyurea and ionizing radiation hypersensitivity, but not its UV hypersensitivity. Permanent overexpression of the hRAD17 gene in human fibrosarcoma cells resulted in p53 activation and a significant reduction of S- and G2/M-phase cells accompanied by an accumulation of the G1-phase population, suggesting that hRAD17 may have a role in cell cycle checkpoint control. Immunostaining of HT-1080 cells transiently transfected with a hRAD17 construct confirmed the nuclear accumulation of p53, which mimics the induction caused by DNA damage. Using FISH analysis, we have mapped the hRAD17 locus to human chromosome 5q11.2.
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PMID:hRAD17, a structural homolog of the Schizosaccharomyces pombe RAD17 cell cycle checkpoint gene, stimulates p53 accumulation. 1020 30

p53 mutants in tumours have a reduced affinity for DNA and a reduced ability to induce apoptosis. We describe a mutant with the opposite phenotype, an increased affinity for some p53-binding sites and an increased ability to induce apoptosis. The apoptotic function requires transcription activation by p53. The mutant has an altered sequence specificity and selectively fails to activate MDM2 transcription. Loss of MDM2 feedback results in overexpression of the mutant, but the mutant kills better than wild-type p53 even in MDM2-null cells. Thus the apoptotic phenotype is due to a combination of decreased MDM2 feedback control and increased or unbalanced expression of other apoptosis-inducing p53 target genes. To identify these genes, DNA chips were screened using RNA from cells expressing the apoptosis-inducing mutant, 121F, and a sequence-specificity mutant with the reciprocal phenotype, 277R. Two potential new mediators of p53-dependent apoptosis were identified, Rad and PIR121, which are induced better by 121F than wild-type p53 and not induced by 277R. The 121F mutant kills untransformed MDM2-null but not wild-type mouse embryo fibroblasts and kills tumour cells irrespective of p53 status. It may thus expand the range of tumours which can be treated by p53 gene therapy.
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PMID:Increased apoptosis induction by 121F mutant p53. 1044 8

The aim of this study was to examine Bax, Bcl-2 and Bcl-XL proteins in human pancreatic cancer cell lines and to clarify the mechanism of radiation resistance. PANC-1 and AsPC-1 pancreatic cell lines were used, both having mutated p53. Radioresistant PANC-1/Rad cells and AsPC-1/Rad cells were obtained by repeated 5 Gy irradiation of PANC-1 cells and AsPC-1 cells, respectively. Radiation was found to inhibit the growth of PANC-1 cells and AsPC-1 cells. After exposure to radiation, detached cells were subjected to FITC-TUNEL staining to calcualte the ratio of apoptosis. TUNEL positive ratios increased dose-dependently in both cell lines. Western blotting showed that the basal level of the Bax/Bcl-2 ratio reflected the radiosensitivity of these cell lines, and Bax expression was obviously upregulated after irradiation in the presence of mutated p53, but Bcl-2 expression remained almost constant. Both PANC-1/Rad and AsPC-1/Rad cells had greater Bcl-XL expression than the parental cells, and the basal level of the Bax/Bcl-2 ratio was no longer predictive of radiosensitivity. Upregulated expression of Bax protein after irradiation was not related to induction of apoptosis in these cells, suggesting that overexpression of Bcl-XL and functional reconstruction of Bcl-2 family proteins are important factors in acquired radioresistance.
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PMID:Role of Bcl-2 family proteins (Bax, Bcl-2 and Bcl-X) on cellular susceptibility to radiation in pancreatic cancer cells. 1065 30

Checkpoints of DNA integrity are conserved throughout evolution, as are the kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia- and Rad-related), which are related to phosphatidylinositol (PI) 3-kinase [1] [2] [3]. The ATM gene is not essential, but mutations lead to ataxia telangiectasia (AT), a pleiotropic disorder characterised by radiation sensitivity and cellular checkpoint defects in response to ionising radiation [4] [5] [6]. The ATR gene has not been associated with human syndromes and, structurally, is more closely related to the canonical yeast checkpoint genes rad3(Sp) and MEC1(Sc) [7] [8]. ATR has been implicated in the response to ultraviolet (UV) radiation and blocks to DNA synthesis [8] [9] [10] [11], and may phosphorylate p53 [12] [13], suggesting that ATM and ATR may have similar and, perhaps, complementary roles in cell-cycle control after DNA damage. Here, we report that targeted inactivation of ATR in mice by disruption of the kinase domain leads to early embryonic lethality before embryonic day 8.5 (E8.5). Heterozygous mice were fertile and had no aberrant phenotype, despite a lower ATR mRNA level. No increase was observed in the sensitivity of ATR(+/-) embryonic stem (ES) cells to a variety of DNA-damaging agents. Attempts to target the remaining wild-type ATR allele in heterozygous ATR(+/-) ES cells failed, supporting the idea that loss of both alleles of the ATR gene, even at the ES-cell level, is lethal. Thus, in contrast to the closely related checkpoint gene ATM, ATR has an essential function in early mammalian development.
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PMID:Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice. 1080 16

The oral use of chewing tobacco has greatly increased in recent years, and this usage is associated with cancers of the mouth, lip, nasal cavities, esophagus and gut. Oral cancer accounts for 3% of all cancers in U.S.A. and is the seventh most common cancer. Previous studies in our laboratory have demonstrated the protective abilities of a novel IH636 grape seed proanthocyanidin extract (GSPE) against reactive oxygen species both in vitro and in vivo models, and provided significantly better protection as compared to vitamins C, E and beta-carotene. In the recent past, we have demonstrated smokeless tobacco (STE)-induced oxidative stress, apoptotic cell death in a primary culture of normal human oral keratinocytes (NHOK), and have compared the protective abilities of vitamins C and E, singly and in combination, and GSPE in this pathobiology [Free Rad. Biol. Med., 26, 992-1000 (1999)]. In the present study, we have assessed the protective role of vitamins C and E, and GSPE against STE-induced modulation of intracellular oxidized states in NHOK cells as demonstrated by laser scanning confocal microscopy. Approximately 11%, 26%, 28% and 50% protection were observed following incubation with vitamin C, vitamin E, a combination of vitamins C plus E, and GSPE, respectively. DNA fragmentation was assessed as an index of oxidative DNA damage and similar results were observed. Furthermore, the cellular viability and functional roles of Bcl-2, p53 and c-myc genes were assessed in STE-induced oxidative stress in NHOK cells. NHOK cells were treated with STE (0-200 micrograms/ml) for 24 h and changes in the expression of Bcl-2, p53 and c-myc genes were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), and the protective effect of GSPE was assessed. Approximately a 2.0-fold increase in p53 gene expression was observed following incubation of the oral keratinocytes with 100 micrograms/ml of STE, beyond which the expression of p53 decreased, confirming increased apoptotic cell death with a higher concentration of STE as reported earlier. GSPE significantly modulated STE-induced changes in p53. The expression of antiapoptotic Bcl-2 gene decreased with STE treatment and the expression of Bcl-2 gene increased significantly following preincubation with GSPE. No significant change in the expression of transcription factor c-myc gene responsible for cell cycle growth was observed following incubation with STE and/or GSPE. Thus, c-myc may not be involved in STE-induced cytotoxicity towards NHOK cells. These results suggest that antioxidant protection of STE-induced cellular injury is associated with alterations in Bcl-2 and p53 expression.
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PMID:Protective effects of antioxidants against smokeless tobacco-induced oxidative stress and modulation of Bcl-2 and p53 genes in human oral keratinocytes. 1169 99

ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase required for survival after DNA damage. A critical role for ATR has been hypothesized to be the regulation of p53 and other cell cycle checkpoints. ATR has been shown to phosphorylate p53 at Ser(15), and this damage-induced phosphorylation is diminished by expression of a catalytically inactive (ATR-kd) mutant. p53 function could not be examined directly in prior studies of ATR, however, because p53 was mutant or because cells expressed the SV40 large T antigen that blocks p53 function. To test the interactions of ATR and p53 directly we generated human U2OS cell lines inducible for either wild-type or kinase-dead ATR that also have an intact p53 pathway. Indeed, ATR-kd expression sensitized these cells to DNA damage and caused a transient decrease in damage-induced serine 15 phosphorylation of p53. However, we found that the effects of ATR-kd expression do not result in blocking the response of p53 to DNA damage. Specifically, prior ATR-kd expression had no effect on DNA damage-induced p53 protein up-regulation, p53-DNA binding, p21 mRNA up-regulation, or G(1) arrest. Instead of promoting survival via p53 regulation, we found that ATR protects cells by delaying the generation of mitotic phosphoproteins and inhibiting premature chromatin condensation after DNA damage or hydroxyurea. Although p53 inhibition (by E6 or MDM2 expression) had little effect on premature chromatin condensation, when combined with ATR-kd expression there was a marked loss of the replication checkpoint. We conclude that ATR and p53 can function independently but that loss of both leads to synergistic disruption of the replication checkpoint.
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PMID:ATR is not required for p53 activation but synergizes with p53 in the replication checkpoint. 1171 32

In response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM-/- fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEF(p53-/-). However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21(CIP1) induction, resulting in partial release of the G(2)/M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis.
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PMID:ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53. 1182 15

The Rad51 gene is the mammalian homologue of the bacterial RecA gene and catalyses homologous recombination in mammalian cells. In some cell types Rad51 has been shown to interact with p53, leading to inhibition of Rad51 activity. Here, we show a two- to four-fold increase in gene-targeting frequency at the HPRT locus using murine ES clones preengineered to overexpress Rad51, and a twofold increase in targeting frequency when a Rad51 expression cassette was cointroduced to wild-type ES cells with the targeting construct. In addition to its effect on homologous recombination, we show that Rad51 may down-regulate illegitimate recombination. We investigated the dependence of these phenomena upon p53 and found no evidence that the Rad 51-mediated increase is affected by the functional status of p53, a conclusion supported by the observed cytoplasmic localisation of p53 in ES cells following electroporation. Furthermore, in the absence of additional Rad51, p53-deficient ES cells do not have elevated rates of homologous recombination with extrachromosomal DNA. These findings demonstrate that Rad51 levels modify both homologous and illegitimate recombination, but that these phenomena are independent of p53 status.
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PMID:Elevated expression of exogenous Rad51 leads to identical increases in gene-targeting frequency in murine embryonic stem (ES) cells with both functional and dysfunctional p53 genes. 1274 58

Long-term exposure (72 h) to hedamycin, a monofunctional DNA alkylator of the pluramycin class of antitumor antibiotics, decreased growth of mammalian cells by 50% at subnanomolar concentrations. Short-term treatment (4 h) rapidly reduced DNA synthesis by 50% also at subnanomolar concentrations, but substantially higher levels were needed to block RNA synthesis while protein synthesis even at very high hedamycin concentrations remained unaffected. Hedamycin treatment at concentrations below its growth IC(50) induced only a transient and temporary accumulation of cells in G(2). Somewhat higher concentrations resulted in substantial S-phase arrest, and at increasing concentrations, complete cell cycle arrest in G(1) was observed without the appearance of a sub-G(1) cell population. Neither inhibition of cell growth nor cell cycle arrest appeared to be dependent on ataxia and Rad-related kinase expression. DNA damage checkpoint proteins including p53, chk1, and chk2 were differentially activated by hedamycin depending on the concentration and duration of treatment. The level of downstream cell cycle regulators such as cdc25A, E2F1, cyclin E, and p21 were also altered under conditions that induced cell cycle arrest, but atypically, p21 overexpression was observed only in S-phase-arrested cells. Apoptotic indicators were only observed at moderate hedamycin concentrations associated with S-phase arrest, while increasing concentrations, when cells were arrested in G(1), resulted in a reduction of these signals. Taken together, the responses of cells to hedamycin are distinct with regard to its effect on cell cycle but also in the unusual concentration-dependent manner of activation of DNA damage and cell cycle checkpoint proteins as well as the induction of apoptotic-associated events.
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PMID:DNA damage responses triggered by a highly cytotoxic monofunctional DNA alkylator, hedamycin, a pluramycin antitumor antibiotic. 1514 Oct 15


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