Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.
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PMID:Empty capsids in column-purified recombinant adenovirus preparations. 1158 34

T47D cells represent an estrogen-responsive human ductal carcinoma cell line which expresses detectable levels of estrogen receptor (ER). We have previously shown that estradiol (E(2)) treatment of T47D cells causes an increase in the level of p53 and a concomitant phosphorylation of retinoblastoma protein (pRb). In the present study, we have analysed the expression of p53 and phosphorylation state of pRb and compared the effects of E(2) and triiodothyronine (T(3)) on these phenomena. Cells were grown in a medium containing charcoal-treated serum to deplete the levels of endogenous steroids. Upon confluency, the cells were treated with T(3) (10(-12) to 10(-7) M) for 24 h and the presence of p53 and pRb was detected by Western analysis. E(2) treatment of cells caused a 2-3-fold increase in the level of p53. Presence of T(3) in the medium caused a gradual increase in the level of p53 in a concentration-dependent manner. Under the above conditions, pRb was phosphorylated (detected as an upshift during SDS-PAGE) in the presence of E(2) and T(3). Supplementation of growth medium with T(3) (1 microM) caused an increase in the rate of proliferation of T47D cells and induced hyperphosphorylation of pRb within 4 h; this effect was maintained for up to 12 h. When ICI 164 384 (ICI) (1 microM), an ER antagonist, was combined with E(2) (1 nM) or T(3) (1 microM), effects of hormones on cell proliferation and hyperphosphorylation of pRb were blocked. Western analysis of p53 was supplemented with its cytolocalization by immuno-labeling using laser scanning confocal fluorescence microscopy, which revealed an ICI-sensitive increase in the abundance of p53 in hormone-treated cells. Steroid binding analysis revealed lack of competition by T(3) for the [(3)H]E(2) binding. These results indicate that T(3) regulates T47D cell cycle progression and proliferation raising the p53 level and causing hyperphosphorylation of pRb by a common mechanism involving ER and T(3) receptor (T(3)R)-mediated pathways.
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PMID:Estrogen-like effects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells. 1185 Aug 4

Several proteins with important roles in oncogenesis have been shown to regulate the function of the E2F-1 transcription factor, which is known to activate the expression of genes required for proliferation and apoptosis. Here we identify the MDMX oncoprotein as an E2F-1-binding factor, from a yeast-two hybrid screen using a portion of the E2F-1 protein as "bait." We demonstrate that the region within MDMX needed for the E2F-1:MDMX interaction is located in the central part of the protein, C-terminal of the p53-binding domain. The region within E2F-1 needed for this association is adjacent to the DNA binding domain. Further, when expressed in vivo or in vitro the MDMX protein migrates as two isoforms on SDS-PAGE, the faster migrating isoform having the stronger affinity for the E2F-1 proteins. It appears that this interaction reduces the ability of E2F-1 to bind DNA. Expression of MDMX along with E2F-1 and Dp-1 in Saos2 cells reduces the ability of E2F-1 to bind to its consensus DNA sequence, without altering E2F-1 protein levels. These data indicate that the MDMX protein is capable of associating with E2F-1 and negatively regulating its DNA binding ability.
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PMID:The E2F-1 transcription factor is negatively regulated by its interaction with the MDMX protein. 1253 31

E-box is one of potential cis-regulatory elements for the p53 gene. It was previously reported that USF bound to the E-box of the p53 gene. Recently, we demonstrated that an 80-kDa protein other than USF bound to the E-box and activated the transcription of the p53 gene. In the present study, the 80-kDa protein was partially purified and characterized. First, we confirmed that nuclear factors bound to the E-box in sequence-specific manner by the oligonucleotide competition assay. The binding protein to the E-box was partially purified by a sequence-specific DNA affinity chromatography. The active fraction was analyzed by SDS-PAGE and southwestern blotting assay, which showed that the 80-kDa protein was enriched. The binding activity of the 80-kDa protein was not decreased in the presence of 1.4 M urea. In addition, the binding activity was stable up to 50 degrees C. Treatment of EDTA showed that the 80-kDa protein did not require divalent cation such as Mg2+ for the maximum DNA binding activity. The competition assay with non-specific competitor, poly (dI-dC) showed that the 80-kDa protein had high affinity to its binding site. These biochemical properties provide useful insights into the 80-kDa nuclear factor binding to the p53 promoter.
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PMID:Partial purification and characterization of an 80-kDa transcription factor binding to bHLH motif in the rat p53 promoter. 1254 19

The migrating position of green fluorescent protein (GFP)-fused polypeptide varied on an SDS/urea gel by a single amino acid change in the fused polypeptide segment. An easy detection method for a single amino acid change based on this observation was called "GFP-display." Using various target polypeptides, staphylococcal protein A (SpA), Ras, p53, and human beta3 adrenergic receptor (AR), and their mobility-shift patterns resulting from the single amino acid changes, several important properties of GFP-display were revealed as follows: (i). since the binding of dodecyl sulfate ions to acidic or hydrophilic amino acids is weaker than that to basic or hydrophobic amino acids, the ions bound weakly to the fused polypeptide segment are forced to come off by high concentrations of urea prior to the ions bound strongly, resulting in the mobility shift, (ii). the mobility shift is estimated to a certain extent using a new parameter called the "GD value" calculated from the isoelectric point, hydrophilicity, and number of fused amino acids, and (iii). the fluorescence intensity of GFP-fused polypeptide tends to increase with the average hydrophilicity of the fused polypeptide segment. GFP-display will be a helpful technique for many kinds of gene or protein studies related to amino acid substitutions such as the random mutagenesis in a gene of interest.
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PMID:General properties of GFP-display, an electrophoretic analysis for single amino acid changes in target polypeptides. 1272 7

Several studies provide evidence for the anti-carcinogenic activity of resveratrol, a phytoalexin present in grapes and berries, but the precise mechanisms involved in the modulation of prostate carcinogenesis by resveratrol remain to be elucidated. The inhibitory effects induced by resveratrol in human prostate cancer cells impact diverse cellular mechanisms associated with tumor initiation, promotion, and progression. In our earlier studies with prostate cancer cells using cDNA microarray analysis, we indicated the importance of p53-mediated molecular targets of resveratrol. The present study based on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D-SDS-PAGE) followed by mass spectrometry analysis of human prostate cells that have been treated with resveratrol clearly identifies the role of phosphoglycerate mutase B. For the first time, we report on phosphoglycerate mutase B in the resveratrol-treated prostate cancer cells LNCaP, DU145, and PC-3 at the transcription level. Our observations raise the possibility of its effect on metabolic enzymes in cancer cells without affecting the normal cells.
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PMID:Resveratrol-induced cell growth inhibition and apoptosis is associated with modulation of phosphoglycerate mutase B in human prostate cancer cells: two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry evaluation. 1558 68

Temporal changes in the expression of p53 were investigated during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukaemic (MEL) cells. Cell preparations were analysed by SDS-PAGE and western immunoblotting, which detected an immunospecific band of molecular mass 53 kDa. This analysis provided semi-quantitative information. Cell extracts were analysed further by means of ELISA techniques, using a p53-specific antibody, to provide quantitative data. In time series experiments in which cells were isolated at 15, 30 and 60 min intervals, dynamic variations in the expression of the p53 protein were detected in both the untreated and the HMBA-treated MEL cells. In all cases, the effects were complex with variations in amplitude, frequency and phasing of the rhythms. The available evidence suggests that the observed patterns, like periodic variations in other systems, are modified in rhythmic fashion with respect to period and amplitude. The results add further support for our view that it is essential to consider the dynamics when interpreting the role of p53 and cellular processes in general.
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PMID:Temporal variation in the expression of the p53 protein in proliferating and differentiating murine erythroleukaemia cells. 1689 37

The present study evaluates the phenotypic and genotypic changes that take place during early oncogenesis. The submandibular glands of male rats were injected with a 0.5% solution of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone. Gland samples were taken at 0, 7, 30 and 150 days post-injection and submitted to histological, biochemical, immunocytochemical and PCR evaluation. Histopathological analysis was performed on hematoxylin-eosin stained slides. Total protein content was assessed by Lowry's method and the protein profile was analyzed by 12% SDS-PAGE. Bcl-2 was demonstrated by silver-enhanced gold immunolabeling. p53 immunolabeling was performed using the streptavidin-biotin system. All the treated animals developed carcinoma-like lesions at 30 and 150 days. Total protein concentration rose significantly (p < 0.05) above control values at 7, 30 and 150 days. The treated glands exhibited positive immunolabeling for p53 in the nuclei of neoplastic cells at 30 and 150 days. Treated glands also showed positive cytoplasmic immunolabeling for Bcl-2, exhibiting statistically significant differences between 7, 30 and 150 days (p = 0.0015), and with controls (p < 0.0001). No p53 mutations were observed whereas a point mutation, C-to-A, of the Bcl-2 gene was detected at 7, 30 and 150 days by PCR amplification. This mutation led to a single aminoacid change (thre --> asn) in the protein molecule. Our results suggest that the early histopathological changes correspond to quantitative and qualitative protein changes. The histopathological, biochemical, immunocytochemical and genetic alterations observed during the course of experimental carcinogenesis in the submandibular gland of the rat could constitute reproducible indices of malignant transformation applicable to human oncogenesis, given the high degree of homology between the oncogenes of mice, rats and human beings.
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PMID:Early phenotypic and genotypic alterations in submandibular gland oncogenesis in rats. 1712 Nov 94

STK11 (serine/threonine kinase 11 ), a multi-functional protein reported recently, possibly participates in a broad range of cellular processes, including regulation of cell cycle, p53-mediated apoptosis, ras-induced cell transformation and cell polarization. An efficient expression of functional STK11 in Escherichia coli will promote the study on its structure and function. Inducible prokaryotic expression vector pET-Nus-STK11 (with Nus fusion tag) was constructed with pET-44a( + ) and the cDNA of STK11 gene cloned in our lab. pET-Nus-STK11 was then expressed in both BL21 (DE3) and Rosetta-gami (DE3)pLysS on the induction of IPTG. SDS-PAGE and Western blot indicated that recombinant Nus-STK11 obtained in BL21(DE3) was in the form of inclusion body, whereas that from Rosetta-gami (DE3)pLysS was mainly in soluble fraction, and accounted for 8.9% and 16.7% of the total protein, respectively. After purification and refolding, the obtained recombinant protein was carried into SMMC-7721 cells by Chariot to observe its influence on cell growth and cell cycle. Nus-STK 1 from BL21(DE3) was proved to be lack of any tumor-suppression activity, while a growth inhibitory ratio of 47.05% on SMMC-7721 cell was observed, and cell cycle progression of SMMC-7721 cells was also arrested from G0/G1 to S phase, with the Nus-STK11 from Rosetta-gami (DE3) pLysS, indicating that the above recombinant fusion protein from Rosetta-gami (DE3)pLysS had significant biological activity. This is the first report on functional recombinant STK11 protein expressed in Escherichia coli.
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PMID:[Expression of functional human STK11 protein in Escherichia coli]. 1743 29

The p53 tumor suppressor protein is a biologically very important molecule. In addition to its central relevance in cancer, its function as an inducer of cell cycle checkpoint and apoptosis may be important in a number of cellular stress responses. However, studies of p53 interactions with other proteins have been hampered in large part by the low abundance of normal p53 in cells. Moreover, the detection of p53 in immune complexes is complicated by the presence of comigrating immunoglobulin chains in SDS-polyacrylamide gels. The method described herein, which utilizes protein A-horseradish peroxidase conjugates, in combination with chemiluminescent detection methods, allows ready sensitive detection of p53 protein in immune complexes with little interference by comigrating immunoglobulin chains. Using this method, pseudo-mutant form as one of confusing conformations of p53 mutant protein was able to be identified with the criteria as high basal level of p53 expression and immunodetection with PAb1620 in human cells.
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PMID:Sensitive immunodetection of pseudo-mutant conformation of p53 protein in human cells using immune complex with protein A conjugates. 1756 9


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