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 demonstrate that PCR amplification of human genomic DNA can be used for the detection of loss of heterozygosity (LOH) in tumor samples. A 250-bp fragment containing codon 72 of the human p53 gene was amplified, ThaI digested and electrophoresed. Tumor LOH is detectable both by ethidium bromide staining and autoradiography, despite 25% contamination with normal DNA. This technique provides a fast and reproducible alternative to conventional Southern blotting and has minimal sample requirements.
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PMID:Detection of loss of heterozygosity in tumor DNA samples by PCR. 193 Oct 11

DNA from archival Papanicolaou stained smears was successfully amplified using the polymerase chain reaction (PCR) to see if it could be used for retrospective genome studies such as detection of the presence of human papilloma virus (HPV) and changes in p53 gene expression. DNA was isolated and purified by treatment with proteinase K, phenol/chloroform, and isoamyl alcohol. Segments of the human beta actin and TGF beta 1 gene were amplified by PCR. Of all stains used in the preparation of Papanicolaou smears, only eosin was detectable as a greenish band in ethidium bromide treated DNA gels under ultraviolet illumination.
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PMID:PCR amplification of DNA from stained cytological smears. 768 6

A rapid (< 2.5 hrs) method for single-strand conformation polymorphism (SSCP) analysis of PCR products that allows the use of ethidium bromide staining is described. PCR products ranging in size from 117 to 256 bp were evaluated for point mutations and polymorphisms by 'cold SSCP' in commercially available pre-cast polyacrylamide mini-gels. Several electrophoretic parameters (running temperature, buffers, denaturants, DNA concentration, and gel polyacrylamide concentration) were found to influence the degree of strand separation and appeared to be PCR fragment specific. Use of the 'cold' SSCP technique and the mini-gel format allowed us to readily optimize the electrophoretic conditions for each PCR fragment. This greatly increased our ability to detect polymorphisms compared to conventional, radioisotope-labeled 'hot' SSCP, typically run under two standard temperature conditions. Excellent results have been obtained in resolving mutant PCR fragments from human p53 exons 5 through 8, human HLA-DQA, human K-ras exons 1 and 2, and rat K-ras exon 3. Polymorphisms could be detected when mutant DNA comprised as little as 3% of the total gene copies in a PCR mixture. Compared to standard 'hot' SSCP, this novel non-isotopic method has additional advantages of dramatically increased speed, precise temperature control, reproducibility, and easily and inexpensively obtainable reagents and equipment. This new method also lacks the safety and hazardous waste management concerns associated with radioactive methods.
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PMID:'Cold SSCP': a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses. 836 79

Temperature gradient gel electrophoresis (TGGE) and related methods are widely employed to detect mutations in DNA fragments. DNA melting map calculations and GC clamps have been used to enhance the detection of mutations. While generally successful, these methods have not always revealed base changes within a DNA fragment. Previous work suggested that mutations are detected if they are in a DNA's first melting domain, and the melting domain is well separated from final strand dissociation. Two criteria from the DNA melting theory were established to determine when both of these conditions are met. The criteria involve calculating the derivative melting curve as well as the melting map of a DNA sequence. The approach was applied to the cDNA sequence of the human p53 gene. Mutations in the p53 gene are common in human cancers and are generally located in four 'hot spot' regions. Calculations indicated that three DNA fragments are needed to detect base substitutions in the four hot-spot regions. Predicted melting behavior was experimentally tested with eight single base substitutions distributed among the four hot-spot regions. All mutations tested behaved as predicted and were detected by vertical TGGE. Heteroduplex DNAs formed by melting and reannealing various ratios of wild type and mutant DNA fragments were also examined. Results indicated that point mutations can be detected by ethidium bromide staining from samples containing 10% mutant and 90% wild-type sequences.
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PMID:Selecting DNA fragments for mutation detection by temperature gradient gel electrophoresis: application to the p53 gene cDNA. 837 44

A flow cytometric technique utilizing the continuous incorporation of bromodeoxyuridine (BrdU) into asynchronous cells to measure radiation-induced cell cycle delay is described. Following the incorporation of the BrdU label the cells are stained with ethidium bromide and the bis-benzimidazole Hoechst 33258. These fluorochromes have differential staining patterns. Hoechst 33258 fluoresces blue and is quenched by BrdU incorporated into cellular DNA during S phase. Ethidium bromide fluoresces red and is not quenched by BrdU. Therefore in cells that are cycling and synthesizing DNA new G1 and G2 compartments are created and this can be used to measure cell cycle delays following ionizing radiation to asynchronous cells. We have used this technique to evaluate two cell lines: a normal diploid human embryo fibroblast cell line MRC 5, which has inducible p53 and shows delays at both G1 and G2 checkpoints, and the human cervix carcinoma cell line HX 156. This cell line has been infected with human papilloma virus (HPV) 16, and therefore has inactivated p53 function and is blocked only at the G2 checkpoint. Using this method, cell cycle-dependent effects relating to the G2 block can be observed. The radiation-induced G2 block differs from that induced by drugs or heating in that cells are blocked in G2 irrespective of the phase of the cell cycle they are treated in. This method allows these different types of G2 block to be quantified.
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PMID:Application of a bromodeoxyuridine-Hoechst/ethidium bromide technique for the analysis of radiation-induced cell cycle delays in asynchronous cell populations. 860 62

Loss of cell cycle control and the inability of the cell to repair DNA at cell cycle checkpoints results in the propagation of genetic lesions which ultimately leads to cancer. To further our understanding of these pathways in pituitary tumorigenesis, we have investigated the effects of DNA damage by gamma radiation in a murine pituitary adenoma (AtT20) cell line with attention to cell cycle checkpoint responses, the induction of apoptosis, and the expression of known regulators of these processes. Irradiated cells exhibited characteristic morphologic changes of apoptosis beginning at 24 h, which included cell shrinkage, chromatin condensation, and cytoplasmic vacuolization, yet the ability to exclude trypan blue was retained for several days. DNA fragmentation could be demonstrated by ethidium bromide staining beginning at 24 h post-irradiation. By propidium iodide staining and flow cytometry, irradiated cells demonstrated G1 and G2 arrest at 24 h, followed at 48 h by a shift to a sub-G1 position of the apoptotic cell population. The G1 arrest coincided with an induction of p53 protein by Western blot analysis which peaked at 4 h post-radiation and persisted beyond 48 h. Expression of c-myc in irradiated cells was found to progressively decrease at 12, 24, and 48 h. Basal expression of the bcl-2 gene in AtT20 cells was found to be 15-fold higher than in normal mouse pituitary by RNase protection assay. Bcl-2 mRNA and protein levels, however, remained unchanged at 24 and 48 h following gamma-irradiation, suggesting that apoptosis occurs independently of bcl-2 gene expression in these cells following this stimulus, as reported in other cell types. We conclude that AtT20 cells undergo G1 and G2 arrest following DNA damage and that a significant proportion of cells then undergo apoptosis. The G1 arrest at 24 h is concurrent with a strong induction of p53 protein, while c-myc expression progressively diminishes. Bcl-2 is highly expressed in this cell line. The absence of variation in bcl-2 expression during apoptosis could be related to its high basal level in these cells.
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PMID:Molecular and cellular responses to DNA damage in a murine pituitary adenoma cell line. 879 54

DNA was extracted from unstained 5-microns sections of neutral buffered 10% formalin-fixed paraffin-embedded tissue by proteinase K digestion without detergents followed by boiling, proteinase K digestion with ionic detergents with and without phenol chloroform extraction and ethanol precipitation, sonication with proteinase K followed by boiling, or boiling alone. Serial 1:10 dilutions of the extracted DNA were subject to polymerase chain reaction (PCR) amplification of a 255-bp portion of the p53 gene. Digestion with proteinase K without ionic detergents followed by boiling (without phenol chloroform extraction) gave the best yield, enabling visualization of ethidium bromide-stained PCR product from a DNA dilution corresponding to 0.1 mm2 of tissue containing of the order of 10(3) nuclear profiles. Proteinase K digestion with detergents followed by phenol-chloroform extraction was no more effective than simple boiling. Although the success of PCR from preserved tissue will vary with the fixative and size of the amplified fragment, DNA extracted with this optimized method can be used for identification of viruses, loss of heterozygosity, and immunoglobulin gene rearrangements in paraffin-embedded tissue without radioisotopes.
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PMID:Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR. 886 37

The performance of agarose and polyacrylamide (PAGE) gels in quantitating polymerase chain reaction (PCR) amplified c-erbB2 and p53 gene sequences (213 and 133 base pairs, respectively) was studied by applying image analysis on photographed ethidium bromide stained gels. The 5 mm thick agarose gels were more insensitive than the 1 mm thin PAGE gels and already started to show saturation effects within a narrow concentration range. The explanation is the greater dilution of band-associated products within the volume of the gel and the inefficient penetration of exciting UV light through the gel. Such effects produced inconsistency and dramatic variations in the band ratio estimates. In the system used by us, agarose electrophoresis and also electrophoresis using 5 mm thick PAGE gels have only a limited value in quantitation of PCR products with the band density ratios, but both can be used well in qualitative work. The far thinner (1 mm) PAGE gels, which did not markedly absorb UV light, performed better in the light of band density ratio estimates. The linear or approximately linear range of concentrations was wider. The coefficient of variation of band ratio, when estimated from the same gels, after loading them with aliquots of identical DNA from several PCR amplified tubes, was in the range of 4%. The absolute values of band densities had a more remarkable variation (than 4%) in both agarose and PAGE gels. Our recommendation is that quantitation of PCR products be done with band density ratio estimates, and in thin PAGE gels.
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PMID:Basics of quantitative polymerase chain reaction: 2. Electrophoresis and quantitation of polymerase chain reaction products. 890 16

Tat (transactivator of transcription) is essential for HIV-1 replication in vivo and in vitro. Tat-(65-80), an RGD containing domain, has been shown to regulate proliferative function of a variety of cell lines, including a human adenocarcinoma cell line, A549. The exact cellular and molecular mechanisms by which these effects are mediated, remain unknown. To evaluate the hypothesis that Tat-(65-80) modulates the expression of immediate early genes (IEG) c-jun, c-myc, c-fos and the tumor suppressor gene p53, serum starved A549 cells were incubated with Tat-(65-80) or heat-inactivated Tat-(65-80) at 10 ng/ml. Total cellular RNA was isolated from the cells at various time points (0-24 hours). In each case, 5 micrograms of RNA was reverse transcribed in 20 microliters of reaction volume. Equal amounts of cDNA were subjected to polymerase chain reaction (PCR) and analyzed by electrophoresis. The photographic negatives of the ethidium bromide stained gels were quantitated by densitometric scanning and normalized to corresponding beta-actin PCR products. Treatment with Tat-(65-80) showed a twofold induction of c-jun at 0.5 h. Peak expression occurred at 60 minutes and remained above baseline at 24 hours (h). c-myc was increased at 0.5 h, reached a twofold increase at 2 h and remained above baseline at 24 h. c-fos increased seven fold at 0.5 h and declined subsequently to baseline at 8 h. p-53 gene was reduced fivefold at 0.5 h and remained downregulated thereafter. These results show that Tat-(65-80) can modulate growth related genes in human lung epithelial cells.
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PMID:An RGD containing peptide from HIV-1 Tat-(65-80) modulates protooncogene expression in human bronchoalveolar carcinoma cell line, A549. 912 88

The murine double minute 2 (MDM2) protein facilitates G1 to S phase transition by activation of E2F-1 and can enhance cell survival by suppressing wild-type p53 (wtp53) function. In this study, we examined MDM2 expression and function in multiple myeloma (MM) cells. MDM2 is strongly and constitutively expressed in MM cell lines (ARH-77, RPMI 8226, and OCI-My5) and in the cells of plasma cell leukemia (PCL) patients, but is not expressed in normal bone marrow mononuclear cells (BM MNCs). Treatment of MM cells with MDM2 antisense, but not sense, nonsense, or scrambled, oligodeoxyribonucleotides (ODNs) decreased DNA synthesis and cell viability; it also induced G1 growth arrest, as evidenced by propidium iodide (PI) staining and induction of retinoblastoma protein (pRB) to E2F-1 binding. Moreover, inhibition of MDM2 using antisense ODNs also triggered MM cell apoptosis as evidenced by acridine orange-ethidium bromide staining. We next studied the association of MDM2 with wtp53 and/or mutant p53 (mtp53), E2F-1, CDK4, and p21. MDM2 constitutively binds to E2F-1 in all MM cells, to both wtp53 and mtp53, and to p21 in tumor cells lacking p53. These data suggest that MDM2 may enhance cell-cycle progression in MM cells both by activating E2F-1 and by downregulating cell-cycle inhibitory proteins (wtp53 and p21). Overexpression of MDM2 may therefore contribute to both growth and survival of MM cells, suggesting the potential utility of treatment strategies targeting MDM2 in MM.
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PMID:MDM2 protein overexpression promotes proliferation and survival of multiple myeloma cells. 929 33


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