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)

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.
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PMID:The tumor suppressor p53 is bound to RNA by a stable covalent linkage. 170 9

Different variations of the antigen retrieval technique using different retrieval solutions have been evaluated for their effectiveness in restoring the antigenicity of six intranuclear antigens, each of which is a potentially valuable prognostic indicator in formalin-fixed, paraffin-embedded tissue sections. The results of immunohistochemical staining for estrogen receptor, progesterone receptor, androgen receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen were compared following the different antigen retrieval approaches. The strongest immunostaining signal with the clearest background was obtained by microwave heating of dewaxed paraffin sections for 10 minutes in 0.05 mol/L glycine HCl (pH 3.5) or in citrate buffer solution (pH 6). Urea solution, distilled water, and lead thiocyanate solution yielded improvements with some antigens, but less consistently and less impressively than glycine HCl buffer or citrate buffer. Following antigen retrieval nuclear staining was sharply defined and could be achieved consistently in a variety of tissues after formalin fixation for as long as 7 days. The duration of fixation, however, was an important variable; generally, the longer the fixation time the more vigorous the retrieval procedure required. This study demonstrates the ability to stain a variety of intranuclear antigens, which are not readily demonstrable otherwise, in formalin-paraffin sections with a high degree of consistency and reproducibility. The availability of methods that are effective in paraffin sections may facilitate studies of the possible value of these markers as prognostic indicators for predicting the response of major tumors to different forms of therapy. This study also provided insight into the basic principles of the antigen retrieval method, which may be helpful in attempts to develop a more uniformly standardized technique applicable to many different antigen systems.
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PMID:Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques. 792 19

Primary hyperparathyroidism is a common disorder characterized by aberrant growth and function of solitary or multiple parathyroid glands. Many, if not all, parathyroid adenomas are examples of benign clonal neoplastic growth. The molecular events associated with the development of parathyroid neoplasia have not been well characterized. We examined benign and malignant parathyroid tissues for structural abnormalities of the p53 tumor suppressor gene. To screen for mutations in the p53 gene, we analyzed polymerase chain reaction-amplified DNA by denaturing gradient gel electrophoresis. DNA was isolated from 26 benign parathyroid adenomas and 3 parathyroid carcinomas, and polymerase chain reaction was used to amplify DNA fragments corresponding to the 4 evolutionarily conserved domains within exons 5, 7, and 8 of the p53 gene in which the majority of point mutations have been identified. Amplified DNA fragments were electrophoresed through polyacrylamide gels with linearly increasing gradients of the denaturants urea and formamide. After electrophoresis, the gels were examined for the presence of abnormally migrating bands, which represent DNA with altered melting points due to nucleotide sequence changes. Amplified fragments were of the expected size in DNA from 26 parathyroid adenomas and 3 parathyroid carcinomas. Denaturing gradient gel electrophoresis studies failed to disclose evidence of mutations in exons 5, 7, and 8 of the p53 gene in these neoplasms. We conclude that p53 point mutations do not appear to be a primary event responsible for neoplastic growth in parathyroid tissue.
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PMID:Absence of p53 point mutations in parathyroid adenoma and carcinoma. 828 93

Androgen ablation induces programmed death of androgen-dependent prostatic glandular cells, resulting in fragmentation of their genomic DNA and the cells themselves into apoptotic bodies. Twenty percent of prostatic glandular cells undergo programmed death per day between day 2 and 5 after castration. During this same period, < 1% of prostatic glandular cells enter the S phase of the cell cycle, documenting that > 95% of these die in G0. During the programmed death of these G0 glandular cells, a futile DNA repair process is induced secondary to the DNA fragmentation. This futile DNA repair is not required, however, since inhibition of this process by > 90% with an appropriately timed hydroxy-urea dosing regimen had no effect upon the extent of the programmed death of these cells after castration. Likewise, p53 gene expression is not required since the same degree of cell death occurred in prostates and seminal vesicles after castration of wild-type and p53-deficient mice.
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PMID:Cell proliferation, DNA repair, and p53 function are not required for programmed death of prostatic glandular cells induced by androgen ablation. 841 31

Highly purified p53 protein from different sources was able to degrade DNA with a 3'-to-5' polarity, yielding deoxynucleoside monophosphates as reaction products. This exonuclease activity was dependent on Mg2+ and inhibited by addition of 5 mM nucleoside monophosphates. This exonuclease activity is intrinsic to the wild-type p53 protein: it copurified with p53 during p53 preparation; only purified wild-type p53, but not identically purified mutant p53 proteins displayed exonuclease activity; the exonuclease activity could be reconstituted from SDS gel-purified and urea-renatured p53 protein and mapped to the core domain of the p53 molecule; and finally, purified p53 protein could be UV-cross-linked to GMP. A p53-intrinsic exonuclease activity should substantially extend our view on the role of p53 as a "guardian of the genome."
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PMID:p53 Protein exhibits 3'-to-5' exonuclease activity. 867 15

In order to isolate experimentally induced p53 mutations, a yeast expression vector harbouring a human wild-type p53 cDNA was treated in vitro with the antineoplastic drug chloroethyl-cyclohexyl-nitroso-urea (CCNU) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutations were identified in 32 out of 39 plasmids rescued from independent ade- transformants. Ninety-two percent of CCNU induced mutations were GC-targeted single base pair substitutions, and GC > AT transitions represented 73% of all single base pair substitutions. In 70% of the cases the mutated G was preceded 5' by a purine. The distribution of the mutations along the p53 cDNA was not random: positions 734 and 785 appeared as CCNU mutational hotspots (n=3, P<0.0003) and CCNU induced only GC > AT transitions at those positions. The features of these CCNU-induced mutations are consistent with the hypothesis that O6-alkylguanine is the major causative lesion. One third of the CCNU-induced mutants were absent from a huge collection of 4496 p53 mutations in human tumours and cell lines, thus demonstrating that CCNU has a mutational spectrum which is uniquely different from that of naturally selected mutations. This strategy allows direct comparison of observed natural mutation spectra with experimentally induced mutation spectra and opens the way to a more rigorous approach in the field of molecular epidemiology.
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PMID:Determining mutational fingerprints at the human p53 locus with a yeast functional assay: a new tool for molecular epidemiology. 917 91

We report a protocol which can analyze DNA by the dideoxy method. First, we prepared DNA from paraffin specimen of colon cancer and normal tissue by the method using proteinase and phenol. Polymerase chain reaction (PCR) was performed as follows. The primers used were oligonucleotides corresponding to the sequence of exon 5 on p53. An initial denaturing step was carried out at 94 degrees C for 2 min. Products were amplified for 40 cycles at 94 degrees C for 1 min, 60 degrees C for 1 min, and 72 degrees C for 1 min. Specific PCR products derived from p53 gene were purified. Protocol for the PCR-sequencing reaction: The reaction mixture was divided into four 4 microliters fraction. Each fraction was mixed with 2 microliters of NTP solution including non-RI dideoxynucleotides (TOYOBO). PCR was carried out as follows: an initial denaturing step at 94 degrees C for 1 min, then 30 cycles at 94 degrees C for 1 min, 60 degrees C for 1 min, and 72 degrees C for 1 min. Prior to loading in a denaturing 8% polyacrylamide-6M Urea gel, the samples were heated to 94 degrees C for 2 min then quickly chilled in ice-water. Electrophoresis was carried out at 1000V for 3hr and transcribed to a nylon membrane. The ladders of DNA were obtained by Non-RI Detection Kit (TOYOBO). We determined the sequence of 167 nucleotides. Results indicated that the point mutations in DNA could be easily detected.
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PMID:[Detection of nucleotide mutation by direct sequencing method using non-radio isotopic marker]. 925 14

The INK4a tumor suppressor locus encodes p16INK4a, an inhibitor of cyclin D-dependent kinases, and p19ARF, an alternative reading frame protein that also blocks cell proliferation. Surprisingly, mice lacking p19ARF but expressing functional p16INK4a develop tumors early in life. Their embryo fibroblasts (MEFs) do not senesce and are transformed by oncogenic Ha-ras alone. Conversion of ARF+/+ or ARF+/- MEF strains to continuously proliferating cell lines involves loss of either p19ARF or p53. p53-mediated checkpoint control is unperturbed in ARF-null fibroblast strains, whereas p53-negative cell lines are resistant to p19ARF-induced growth arrest. Therefore, INK4a encodes growth inhibitory proteins that act upstream of the retinoblastoma protein and p53. Mutations and deletions targeting this locus in cancer cells are unlikely to be functionally equivalent.
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PMID:Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. 939 58

Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn2+ ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal = 4.18 kJ)/mol at 25 degrees C and 9.8 kcal/mol at 10 degrees C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal/mol, respectively. Under certain denaturing conditions, the wild-type domain forms an aggregate that is relatively highly fluorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this fluorescence under milder denaturation conditions.
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PMID:Thermodynamic stability of wild-type and mutant p53 core domain. 940 13

Genetic instability is a typical feature of tumor cells. This evidence has stimulated the development of rapid methods for detection of gene mutations. A new, improved protocol for denaturing gradient gel electrophoresis (DGGE), to screen for point mutations in genomic DNA, is reported: double gradient (DG) DGGE. In this technique, to the primary, denaturing gradient (typically 30-80% or 40-80% urea/formamide) a secondary gradient, colinear with the first, is superimposed: a porosity gradient (typically 6.5-12% polyacrylamide). The secondary gradient acts by recompacting smeared and diffuse bands of heteroduplexes, which are often indistinguishable from background fluorescence, and by augmenting the resolution between closely spaced homoduplex zones. This allows proper densitometric quantitation of the ratio of the two homoduplex bands. The reliability of this technique has been documented by detection of a number of mutations in exons 6 and 8 of the p53 gene which had escaped revelation by single-strand conformational polymorphism (SSCP) analysis. Additionally, the precise assessment of ratio of the doublet of homoduplex bands has allowed quantitation of the extent of p53 mutation in a mixed cell population extracted from a tumor specimen.
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PMID:Detection of p53 point mutations by double-gradient, denaturing gradient gel electrophoresis. 950 31


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