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)

Human tumor suppressor protein p53 is a 393-amino acid phosphoprotein that enhances transcription in response to DNA damage from several genes that regulate cell cycle progression. The tetrameric state of p53 is critical to wild-type function; the p53 tetramerization element is located in the C-terminal region of the protein. This region is phosphorylated at several evolutionarily conserved serines, suggesting that phosphorylation may be an important regulator of p53 function. In order to determine the effect of phosphorylation on tetramer formation, we synthesized phosphopeptides corresponding to p53(Ser303-Asp393) with phosphate incorporated at Ser315, Ser378, or Ser392, and at both Ser315 and Ser392. Equilibrium ultracentrifugation analysis showed that phosphorylation at Ser392 increased the association constant for tetramer formation nearly ten-fold. By itself, phosphorylation at Ser315 or Ser378 had little effect on tetramer formation, but Ser315 largely reversed the effect of phosphorylation at Ser392. Analysis by calorimetry suggests that phosphorylation may influence subunit affinity by an enthalpy driven process.
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PMID:Effect of phosphorylation on tetramerization of the tumor suppressor protein p53. 924 43

Tumor suppressor protein p53 is a tetrameric phosphoprotein that activates transcription from several cell cycle regulating genes in response to DNA damage. Tetramer formation is critical to p53's ability to activate transcription; however, posttranslational modifications and protein stabilization also contribute to p53's ability to activate transcription. To determine if phosphorylation affects tetramer formation, we synthesized phosphopeptides corresponding to residues 303-393 of human p53, which includes the domain responsible for tetramer formation. Phosphate was chemically incorporated at Ser315, Ser378, or Ser392 and also at both Ser315 and Ser392. Equilibrium ultracentrifugal analyses showed that phosphorylation at Ser392 increased the association constant for reversible tetramer formation nearly 10-fold. Phosphorylation of either Ser315 or Ser378 had little effect on tetramer formation, but phosphorylation of Ser315 largely reversed the effect of phosphorylation at Ser392. Analyses by calorimetry demonstrated that phosphorylation may influence subunit affinity (and, in turn, DNA binding) by an enthalpy-driven process, possibly between the C-terminal residues and the region immediately adjacent to Ser315. The Kd for the tetramer-monomer transition of the unphosphorylated p53 C-terminal domain was determined to be approximately 1-10 microM. Thus, in normal, undamaged cells p53 may be largely monomeric. Enhancement of tetramer formation through phosphorylation of Ser392, coupled with a DNA-damage-induced increase in its nuclear concentration, could provide a switch that activates p53 as a transcription factor in response to DNA damage.
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PMID:Phosphorylation of serine 392 stabilizes the tetramer formation of tumor suppressor protein p53. 925 8

Female sterility resulting from oocyte destruction is an unfortunate, and in many cases inevitable, consequence of chemotherapy. We show that unfertilized mouse oocytes exposed to therapeutic levels of the antitumor drug, doxorubicin (DXR), undergo apoptosis; however, fertilized oocytes do not initiate apoptosis, but enter cell-cycle arrest, when treated with DXR. Apoptosis induced by DXR in oocytes is blocked by sphingosine-1-phosphate, an inhibitor of ceramide-promoted cell death. Oocytes from Bax-deficient, but not p53-null, female mice display complete resistance to DXR-induced apoptosis in vivo and in vitro. Pretreatment of oocytes with a specific peptide inhibitor of caspases also abrogates the apoptotic response to DXR. These findings indicate that oocyte destruction caused by chemotherapy can be prevented by manipulation of apoptosis-associated signaling pathways.
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PMID:Apoptosis-associated signaling pathways are required for chemotherapy-mediated female germ cell destruction. 954 66

CDC25 phosphatases activate cyclin-dependent kinases by removing inhibitory phosphate groups on the molecules and positively regulate the cell cycle progression. The expression of CDC25A, B and C was examined in gastric carcinoma cell lines and gastric carcinoma tissues by northern blotting and immunohistochemistry. The gastric carcinoma cell lines expressed CDC25A, B and C mRNA at various levels. The expression levels of CDC25B were generally higher than those of CDC25A and C. Of the 40 gastric carcinomas, 70% of the tumors expressed CDC25B mRNA at higher levels than the corresponding normal mucosas, while 38% overexpressed CDC25A mRNA. The CDC25C expression was at very low or undetectable levels. No obvious correlation was detected between the expression of CDC25B and p53 gene mutations. Immunohistochemically, CDC25-positive tumor cells were detected in 43 (78%) of 55 gastric carcinoma cases, of which 27 (49%) were strongly positive. Strong expression of CDC25B protein was associated with advanced stage and deep invasion. Furthermore, the incidence of strong expression was significantly higher in carcinomas with nodal metastasis than in those without metastasis. These findings suggest that overexpression of CDC25B may favor development and progression and may be an indicator of malignant behavior of gastric carcinomas.
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PMID:Overexpression of cyclin-dependent kinase-activating CDC25B phosphatase in human gastric carcinomas. 941 55

The ability of endonuclease VII (endo VII) to cleave at mispairings in double-stranded DNA has recently been used for enzymatic mutation detection (EMD) [R. Youil, B.W. Kemper, R.G.H. Cotton, Proc. Natl. Acad. Sci. USA 92 (1995) 87-91]. The method is based on mapping cleavages in heteroduplex DNAs obtained from mutant and wildtype sequences. Despite the capability of endo VII to cleave at all possible mispairings, relative cleavage efficiencies vary considerably for individual mismatches and may escape detection if located in an unfavorable sequence surrounding. We report here improved reaction conditions which can increase the selectivity of the enzyme for mismatches up to 500-fold, as demonstrated with a mutation in a 247 nt long fragment from exon 7 of human gene p53. The new conditions involve replacement of Tris/HCl buffer by phosphate buffer and change from pH 8.0 to 6.5. Various concentrations of phosphate ions should be tried in the assay to meet individual requirements of the substrate.
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PMID:Enzymatic mutation detection. Phosphate ions increase incision efficiency of endonuclease VII at a variety of damage sites in DNA. 969 88

Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.
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PMID:A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53. 973 74

Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were taken in trifluoroethanol-water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.
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PMID:Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides. 975 64

Mitoguazone (methylglyoxal bisguanylhydrazone, methyl-GAG or MGBG) is a synthetic polycarbonyl derivative with activity in patients with Hodgkin's and non-Hodgkin's lymphoma, head and neck cancer, prostate cancer, and esophageal cancer. Mitoguazone has also recently been documented to have activity in patients with AIDS-related lymphoma. Among anticancer drugs, mitoguazone has a unique mechanism of action via interference with the polyamine biosynthetic pathway. Polyamines stabilize DNA structure by non-covalent cross-bridging between phosphate groups on opposite strands. In addition, mitoguazone causes uncoupling of oxidative phosphorylation. In this study, the ability of mitoguazone to induce apoptosis by inhibiting the polyamine pathway was assessed in three Burkitt's lymphoma cell lines (Raji, Ramos and Daudi) and one prostate carcinoma cell line (MPC 3). Additional evaluations were performed in two human breast cancer cell lines (MCF7 with wild-type p53 and VM4K with mutated p53) to determine whether the p53 tumor suppressor gene was required for efficient apoptosis induction. The present study demonstrated that mitoguazone induces apoptosis in all the different human cancer cell lines tested in a concentration- and time-dependent way, and triggers a p53-independent programmed cell death in the human breast cancer MCF7 cell line.
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PMID:Mitoguazone induces apoptosis via a p53-independent mechanism. 977 8

Malignant tumors contain a significant fraction of microregions that are chronically or transiently hypoxic. The experimental evidence showing that hypoxia may have a profound impact on malignant progression and on responsiveness to therapy is growing. In fact hypoxia, like other genotoxic and non-genotoxic stresses, has been shown to increase the p53 protein level, and subsequently activate target genes like p21/waf-1 which interact with cell cycle machinery or participate in apoptosis. Apoptosis is a genetically encoded program of cell death that can be activated under physiological conditions like hypoxia, and may be an important safeguard against tumour development. One of the first common manifestations of the apoptotic process, irrespective of the cell type, is the disruption of mitochondrial membrane function, including a dissipation of the delta psi m and/or a modification on the mitochondrial release of protease activators. These modifications are linked to specific patterns of bioenergetic parameters i.e. respiratory flux, mitochondrial redox potential and phosphate potential. We have studied gluconeogenesis and glycolysis pathways in intact hepatocytes isolated from fasted rats submitted to 24 h of hypoxic in vivo exposure. We have shown that hypoxia resulted in an inhibition of the gluconeogenesis pathway due to a decrease in phosphoenolpyruvate carboxykinase (PEPCK) activity and mRNA synthesis in rat hepatocytes. In conclusion, the disruption of mitochondrial membrane function in response to different oxygen content such as periarterial or perivenous PO2 led to the inhibition of gluconeogenesis and apoptosis in hypoxic cells.
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PMID:Cellular physiology and molecular events in hypoxia-induced apoptosis. 989 49

The mechanisms of action of the anticancer agent perillyl alcohol (POH), presently in Phase II clinical trials, were investigated in advanced rat mammary carcinomas. Gross and ultrastructural morphology of POH-mediated tumor regression indicated that apoptosis accounted for the marked reduction in the epithelial compartment. Characterization of cell growth and death indices revealed that apoptosis was induced within 48 h of chemotherapy, before the induction of cytostasis. RNA expression studies, based on a multiplexed-nuclease protection assay, demonstrated that cell cycle- and apoptosis-related genes were differentially expressed within 48 h of POH treatment; p21(Cip1/WAF1), bax, bad, and annexin I were induced; cyclin E and cyclin-dependent kinase 2 were repressed; and bcl-2 and p53 were unchanged. Next, a potential role for transforming growth factor beta (TGF-beta) signaling in POH-mediated carcinoma regression was explored. RNA expression studies, again based on a multiplexed-nuclease protection assay, showed that TGF-beta-related genes were induced and temporally regulated during POH treatment: (a) c-jun and c-fos were transiently induced within 12 h of chemotherapy; (b) TGF-beta1 was induced within 24 h of chemotherapy; (c) the mannose 6-phosphate/insulin-like growth factor II receptor and the TGF-beta type I and II receptors were induced within 48 h of chemotherapy; and (d) smad3 was induced during active carcinoma regression. In situ protein expression studies, based on fluorescence-immunohistochemistry in concert with confocal microscopy, confirmed up-regulation and demonstrated colocalization of TGF-beta1, the mannose 6-phosphate/insulin-like growth factor II receptor, the TGF-beta type I and II receptors, and Smad2/Smad3 in epithelial cells. Nuclear localization of Smad2/Smad3 indicated that the TGF-beta signaling pathway was activated in regressing carcinomas. Subpopulations of Smad2/Smad3-positive and apoptotic nuclei colocalized, indicating a role for Smads in apoptosis. Thus, Smads may serve as a potential biomarker for anticancer activity. Importantly, none of the POH-mediated anticancer activities were observed in normal mammary gland.
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PMID:Activation of the transforming growth factor beta signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol. 1021 1


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