Transcriptional repression mediated by the p53 tumour suppressor.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
S Benchimol
Publications and source records attributed to S Benchimol.
Structure and functionality of a designed p53 dimer.
P53 is a homotetrameric tumor suppressor protein involved in transcriptional control of genes that regulate cell proliferation and death. In order to probe the role that oligomerization plays in this capacity, we have previously designed and characterized a series of p53 proteins with altered oligomeric states through hydrophilc substitution of residues Met340 or Leu344 in the normally tetrameric oligomerization domain. Although such mutations have little effect on the overall secondary structural content of the oligomerization domain, both solubility and the resistance to thermal denaturation are substantially reduced relative to that of the wild-type domain. Here, we report the design and characterization of a double-mutant p53 with alterations of residues at positions Met340 and Leu344. The double-mutations Met340Glu/Leu344Lys and Met340Gln/Leu344Arg resulted in distinct dimeric forms of the protein. Furthermore, we have verified by NMR structure determination that the double-mutant Met340Gln/Leu344Arg is essentially a "half-tetramer". Analysis of the in vivo activities of full-length p53 oligomeric mutants reveals that while cell-cycle arrest requires tetrameric p53, transcriptional transactivation activity of monomers and dimers retain roughly background and half of the wild-type activity, respectively.
Binding Sites↗
Abnormalities of the ARF-p53 pathway in oral squamous cell carcinoma.
Oral squamous cell carcinoma (OSCC) is associated with heavy smoking and drinking, but the molecular pathway of tumorigenesis is not understood. Inactivation of the p53 tumor suppressor gene is likely to play an important role since p53 mutation is frequently found. The p14ARF tumor suppressor gene is functionally linked to p53, because it is activated by oncogenes and causes p53-dependent growth arrest and apoptosis. The relationship between p14ARF and p53 inactivation has not been described for OSCC. We studied 25 cases of OSCC to determine if there is an inverse correlation between p53 mutation and p14ARF inactivation by homozygous deletion or mutation. p53 mutation was found in 16 of 25 cases (64%), including nine missense and seven truncating mutations. While all cases with missense mutations showed abnormal accumulation of p53 protein, there were also five carcinomas which showed increased p53 staining in the absence of mutation. p14ARF deletion or mutation was found in eight cases (32%), six of which also demonstrated p53 mutation. Our findings indicate that OSCC often involves loss of both p14ARF and p53 function and suggest that inactivation of these two tumor suppressor genes are not functionally equivalent during tumorigenesis.
Adult↗
Regulation of PTEN transcription by p53.
PTEN tumor suppressor is frequently mutated in human cancers and is a negative regulator of PI3'K/PKB/Akt-dependent cellular survival. Investigation of the human genomic PTEN locus revealed a p53 binding element directly upstream of the PTEN gene. Deletion and mutation analyses showed that this element is necessary for inducible transactivation of PTEN by p53. A p53-independent element controlling constitutive expression of PTEN was also identified. In contrast to p53 mutant cell lines, induction of p53 in primary and tumor cell lines with wild-type p53 increased PTEN mRNA levels. PTEN was required for p53-mediated apoptosis in immortalized mouse embryonic fibroblasts. Our results reveal a unique role for p53 in regulation of cellular survival and an interesting connection in tumor suppressor signaling.
Animals↗
p53-dependent pathways of apoptosis.
Explore the source record for details and available documents.
Animals↗
Post-translational modification of p53 protein in response to ionizing radiation analyzed by mass spectrometry.
The p53 tumor suppressor protein promotes cell cycle arrest or apoptosis in response to DNA damage and other forms of stress. p53 protein functions as a transcription factor by binding to specific DNA sequences and regulating the transcription of target genes. This activity of p53 is reported to be regulated by phosphorylation and acetylation occuring at various sites on the molecule. Here, we have used a direct and non-radioactive approach involving mass spectrometric analysis of p53 protein to identify sites that are covalently modified in vivo, either constitutively or in response to ionizing radiation. Following partial purification by immuno-affinity chromatography and enzymatic in-gel digestion, the resulting p53 peptides were analyzed by MALDI-TOF and nanoelectrospray mass spectrometry. Mass spectrometry analyses identified four sites at the N terminus that were phosphorylated in response to irradiation, a single constitutive phosphorylation site at serine 315 and several acetylation sites.
Amino Acid Sequence↗
Pidd, a new death-domain-containing protein, is induced by p53 and promotes apoptosis.
The p53 tumour suppressor promotes cell-cycle arrest or apoptosis in response to cellular stress, such as DNA damage and oncogenesis. This role of p53 is important for its tumour-suppression function and depends, at least in part, on its ability to bind to specific DNA sequences and activate the transcription of target genes. The pathway through which p53 promotes apoptosis is not fully understood. Here we describe a new gene regulated by p53 that encodes a predicted protein of 915 amino acids in mice (910 amino acids in humans), which we have named Pidd. The mouse Pidd cDNA contains a p53 consensus DNA binding sequence upstream of the Pidd-coding region. This sequence element bound to p53 and conferred p53-dependent inducibility on a heterologous reporter gene. Moreover, Pidd RNA was induced by ionizing radiation in a p53-dependent manner and the basal level of Pidd RNA was dependent on Trp53 status. Overexpression of Pidd inhibited cell growth in a p53-like manner by inducing apoptosis. Antisense inhibition of Pidd expression attenuated p53-mediated apoptosis. Our data suggest that Pidd is an effector of p53-dependent apoptosis.
Amino Acid Sequence↗
Alternative pathways for the extension of cellular life span: inactivation of p53/pRb and expression of telomerase.
Telomere shortening may be one of several factors that contribute to the onset of senescence in human cells. The p53 and pRb pathways are involved in the regulation of cell cycle progression from G1 into S phase and inactivation of these pathways leads to extension of life span. Short dysfunctional telomeres may be perceived as damaged DNA and may activate these pathways, leading to prolonged arrest in G1, typical of cells in senescence. Inactivation of the p53 and pRb pathways, however, does not lead to cell immortalization. Cells that overcome senescence and have an extended life span continue to lose telomeric DNA and subsequently enter a second phase of growth arrest termed 'crisis'. Forced expression of telomerase in human cells leads to the elongation of telomeres and immortalization. The development of human cancer is frequently associated with the inactivation of the pRb and p53 pathways, attesting to the importance of senescence in restricting the tumor-forming ability of human cells. Cancer cells must also maintain telomere length and, in the majority of cases, this is associated with expression of telomerase activity.
Animals↗
A translation repressor element resides in the 3' untranslated region of human p53 mRNA.
The 3' untranslated region of human p53 mRNA represses translation both in vitro and in vivo. Here, we identify a cis-acting 66-nucleotide U-rich sequence in the human p53 mRNA 3' untranslated region that mediates translational repression. Using UV cross-linking, we detect a 40 kDa protein that interacts specifically with the p53 3'UTR containing the repressor element. Enhanced translation of p53 mRNA contributes to the accumulation of p53 protein in cells exposed to gamma-radiation and could be a consequence of relieving the inhibition mediated by the repressor element.
3' Untranslated Regions↗
Phosphorylation of p53 protein in response to ionizing radiation occurs at multiple sites in both normal and DNA-PK deficient cells.
The tumour suppressor gene product, p53, is involved in mediating cellular responses to DNA damage including growth arrest and/or apoptosis. The mechanism by which p53 protein senses the presence of damaged DNA is not understood. The possibility that p53 may be post-translationally modified by enzymes that are activated in response to DNA damage including DNA-dependent protein kinase (DNA-PK), poly(ADP-ribose) polymerase and stress activated protein kinase has received considerable attention. Recent studies have indicated that DNA-PK is not required for the transactivation or apoptosis-promoting activities of p53 protein. However, the possibility that other functions of p53 may be dependent on phosphorylation by DNA-PK has not been explored. Here we describe a series of experiments that compares the expression, function and phosphorylation status of p53 protein in normal and DNA-PK-deficient scid cells. While several novel p53 phosphoforms are generated in response to DNA damage in normal cells, the same phosphoforms are observed in scid cells.
Animals↗
Analysis of genomic integrity and p53-dependent G1 checkpoint in telomerase-induced extended-life-span human fibroblasts.
Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279-282, 1998; A. G. Bodnar et al., Science 279:349-352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21(Waf1/Cip1/Sdi1). TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.
Cell Survival↗
A functional wild-type p53 gene is expressed in human acute myeloid leukemia cell lines.
Explore the source record for details and available documents.
Acute Disease↗
Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation.
Recent data suggest that aberrant function of the wild type p53 protein (WTp53) may alter cellular survival following DNA damage through cellular pathways involving apoptosis and cell-cycle checkpoints, but little is known concerning it's possible role in DNA repair. In the present study, the ionizing radiation sensitivity was determined for a series of rat embryo fibroblast (REF) cell lines transfected with an activated form of the H-ras oncogene alone, or in combination with a variety of missense-mutant p53 (MTp53) alleles. Transformed REF clones which expressed exogenous MTp53 and p21ras proteins (CLASS II clones) were generally radioresistant in culture as determined by higher values for the surviving fraction after 2 Gy (SF2 value) and the radiation dose required to reduce survival to a fraction of 0.1 (D10 value), compared either to transformed REF clones expressing p21ras protein alone (CLASS I clones), or to non-transfected REF control cell lines expressing baseline endogenous levels of p21ras and WTp53 protein. The increased radioresistance observed in the CLASS II clones (following both HDR- and LDR-irradiation), was significantly correlated with increased expression of MTp53 protein, and a decreased radiation-induced G1 arrest response. The variability observed in clonogenic radiosensitivity among REF clones was not explained by differential radiation-induced apoptosis. Using the Comet assay performed after continuous low dose-rate (LDR)-irradiation, MTp53-expressing REF clones were also found to be more proficient at the rejoining of DNA double-strand breaks (DNA-dsb), compared to WTp53-expressing REF clones. These results suggest that an enhanced DNA and cellular repair capacity may, in part, explain the increased radiation survival observed in some MTp53-expressing transformed fibroblasts and tumours.
Animals↗
Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span.
Normal somatic cells have a finite life span [1] and lose telomeric DNA, present at the ends of chromosomes, each time they divide as a function of age in vivo or in culture [2-4]. In contrast, many cancer cells and cell lines established from tumours maintain their telomere length by activation of an RNA-protein complex called telomerase, an enzyme originally discovered in Tetrahymena [5], that synthesizes telomeric repeats [6-8]. These findings have led to the formation of the 'telomere hypothesis', which proposes that critical shortening of telomeric DNA due to the end-replication problem [9] is the signal for the initiation of cellular senescence [10,11]. In yeast, the EST2 gene product, the catalytic subunit of telomerase, is essential for telomere maintenance in vivo [12-14]. The recent cloning of the cDNA encoding the catalytic subunit of human telomerase (hTERT) [15,16] makes it possible to test the telomere hypothesis. In this study, we expressed hTERT in normal human diploid fibroblasts, which lack telomerase activity, to determine whether telomerase activity could be reconstituted leading to extension of replicative life span. Our results show that retroviral-mediated expression of hTERT resulted in functional telomerase activity in normal aging human cells. Moreover, reconstitution of telomerase activity in vivo led to an increase in the length of telomeric DNA and to extension of cellular life span. These findings provide direct evidence in support of the telomere hypothesis, indicating that telomere length is one factor that can determine the replicative life span of human cells.
Animals↗
ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase.
Telomere loss has been proposed as a mechanism for counting cell divisions during aging in normal somatic cells. How such a mitotic clock initiates the intracellular signalling events that culminate in G1 cell cycle arrest and senescence to restrict the lifespan of normal human cells is not known. We investigated the possibility that critically short telomere length activates a DNA damage response pathway involving p53 and p21(WAF1) in aging cells. We show that the DNA binding and transcriptional activity of p53 protein increases with cell age in the absence of any marked increase in the level of p53 protein, and that p21(WAF1) promoter activity in senescent cells is dependent on both p53 and the transcriptional co-activator p300. Moreover, we detected increased specific activity of p53 protein in AT fibroblasts, which exhibit accelerated telomere loss and undergo premature senescence, compared with normal fibroblasts. We investigated the possibility that poly(ADP-ribose) polymerase is involved in the post-translational activation of p53 protein in aging cells. We show that p53 protein can associate with PARP and inhibition of PARP activity leads to abrogation of p21 and mdm2 expression in response to DNA damage. Moreover, inhibition of PARP activity leads to extension of cellular lifespan. In contrast, hyperoxia, an activator of PARP, is associated with accelerated telomere loss, activation of p53 and premature senescence. We propose that p53 is post-translationally activated not only in response to DNA damage but also in response to the critical shortening of telomeres that occurs during cellular aging.
Alleles↗
Mutator phenotype in Msh2-deficient murine embryonic fibroblasts.
Embryonic fibroblast cell lines were established from mice deficient, heterozygous, or proficient for Msh2, one of the three known DNA mismatch repair genes involved in hereditary nonpolyposis colon cancer (HNPCC). Cell lines were established by transfection of primary mouse embryo fibroblasts with E7 and Ras oncogenes or mutant p53. Spontaneously immortalized cells derived from the primary cultures were also studied. To determine whether these cells developed a mutator phenotype similar to that found in colon cancer cells deficient in mismatch repair, we measured mutation rates, microsatellite instability, and sensitivities to a range of DNA-damaging agents. The mutator phenotype detected in the E7 and Ras or mutant p53-immortalized Msh2-/- mouse cells was similar to that found in human mismatch repair-deficient colorectal carcinoma cell lines. Mutation rates to ouabain resistance were increased 8-12-fold relative to lines from Msh2+/+ mice, and microsatellite instability was detectable in 12-18% of subclones derived from the Msh2-/- line but was undetectable in subclones developed from the Msh2+/+ line. Furthermore, E7 and Ras or spontaneously immortalized Msh2-/- cells were significantly more resistant to the cytotoxic effects of 6-thioguanine relative to Msh2+/+ cells. In contrast, these lines showed various responses to UV light and cis-platinum, suggesting that mismatch repair deficiency was not the sole determinant for sensitivity to these DNA-damaging agents. Particular attention was paid to the properties of cells heterozygous for the Msh2 mutant gene, which would mimic the situation of an HNPCC carrier. However, our studies failed to reveal any properties of these cells that might provide a growth advantage or predispose them for the acquisition of further mutations. This observation is consistent with the model that inactivation of the wild-type Msh2 allele is a critical step for tumorigenesis in HNPCC patients.
Animals↗
Participation of the human p53 3'UTR in translational repression and activation following gamma-irradiation.
p53 protein levels have been shown to increase in a number of cells after treatment with genotoxic agents through a post-transcriptional mechanism. In gamma-irradiated human cells, the accumulation of p53 protein is accompanied by an increase in the association of p53 mRNA with large polysomes without any change in the level of p53 mRNA. This redistribution of p53 mRNA on polysomes in response to irradiation is consistent with enhanced translational activity of p53 mRNA. We demonstrate that a region of the p53 3'-untranslated region (3'UTR) inhibits translation of a chimeric reporter mRNA in vivo. Induced elevation of reporter activity after gamma-irradiation was seen in cells expressing chimeric reporter-p53 3'UTR transcripts. These data taken together demonstrate translational control of p53 gene expression after gamma-irradiation and denote a previously unsuspected and novel role for the p53 3'UTR in controlling translation.
Gamma Rays↗
Ultrasonic biomicroscopy of viable, dead and apoptotic cells.
Ultrasonic imaging is frequently used in medical diagnosis to differentiate normal and tumour tissues. Here we investigate if distinct types of cell death can be discriminated through the use of ultrasound biomicroscopy. By using a well-controlled system in vitro, we demonstrate that this imaging modality can be used to differentiate living cells, dead cells and cells that have died by programmed cell death or apoptosis. The results indicate a greater than twofold ultrasound backscatter signal from apoptotic cells in comparison to viable cells, whereas heat-killed cells exhibit an intermediate level of ultrasound backscatter. The results have potential implications in the study of disease-related biological processes involving apoptosis.
Antineoplastic Agents↗