Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To further investigate the role of p53 gene inactivation in gastric tumorigenesis, the mutational status of the p53 gene in primary human gastric cancer samples was examined. Reverse transcriptase polymerase chain reaction and subsequent direct sequencing of the p53 gene from gastric cancer samples revealed frequent point mutations of the p53 gene: some of these coincided with those previously identified in gastric cancer cell lines. In addition, both allelic deletion analysis using pYNZ 22 and polymerase chain reaction-restriction fragment length polymorphism analysis demonstrated an allelic deletion of the p53 gene in cancer tissue which contained a point mutation of the p53 gene in the remaining allele. Transfection of the wild-type or mutant p53 genes into gastric cancer cells showed that the wild-type but none of the mutated p53 genes suppressed the colony formation of gastric cancer cells. Furthermore, the incorporation of thymidine into DNA was reduced in cancer cells expressing the wild-type p53 gene. The glutathione S-transferase-wild type p53 fusion protein bound to simian virus 40 large T antigen in COS-1 cell lysate. None of the p53 fusion proteins containing mutations at codons 143, 175, 248, or 273 bound to simian virus 40 large T antigen. By contrast, two different mutant p53 fusion proteins containing mutations specifically observed in gastric cancer bound to simian virus 40 large T antigen. These results indicate that inactivation of the p53 gene through mutations and the allelic deletion may play an important role in gastric tumorigenesis. These mutations may cause a conformational change in the p53 protein resulting in the loss of the suppression by p53 of the growth of gastric cells, partly through disruption of the association of p53 protein with a cellular component.
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PMID:p53 gene mutations in human gastric cancer: wild-type p53 but not mutant p53 suppresses growth of human gastric cancer cells. 132 85

Leukemia in the soft-shell clam, Mya arenaria, is characterized by tumor cells which are detected initially in the hemolymph. This disease is much more common in clams inhabiting polluted waters, suggesting an environmental component to its pathogenesis. In this study, leukemia cells were identified using a murine monoclonal antibody, 1E10, which recognizes a leukemia-specific protein expressed by tumor cells. Mutant p53 protein was detected using a murine monoclonal antibody (PAb 240) which reacts with mutant p53. Using immunofluorescence, the reactivity of clam cells to the 1E10 antibody was evaluated along with mutant p53 protein reactivity. Reverse transcriptase-polymerase chain reactions followed by sequence analyses were utilized to examine clams with hemocytes reacting with the p53 antibody for possible p53 gene mutations. Mutant p53 protein was expressed by tumor cells from five animals with advanced disease (in which greater than 90% of cells reacted with 1E10). A C-->G transversion was detected at the end of exon 6 from two of the five animals that reacted with both the mutant p53 antibody and 1E10. This substitution changes the amino acid of this codon from proline to alanine. Overall, our results suggest that environmentally induced alterations in p53 can contribute to the pathogenesis of leukemia in soft-shell clams inhabiting polluted water and/or sediment.
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PMID:Detection of mutant p53 in clam leukemia cells. 916 98

The effects of the macromolecular synthesis inhibitors 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), actinomycin D, and cycloheximide on the human gastric cancer TMK-1 cell line were studied. These agents inhibited DNA, RNA, or protein synthesis efficiently and induced cell death rapidly in a wide range of concentrations. After 8 hr of exposure to these agents, the cells exhibited morphological features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, and formation of apoptotic bodies. Western blot analysis revealed that these inhibitors altered the protein levels of apoptosis-related gene products such as c-Myc, Bcl-X(S), and the mutant p53 (mp53) in TMK-1 cells markedly. The c-myc mRNA and protein levels were decreased initially and were then induced markedly to a new level after 4 hr of exposure to DRB, a RNA polymerase II inhibitor. The Bcl-X(S) levels were increased rapidly after treatment with all of these agents, whereas the levels of Bcl-X(L) and Bax remained largely unchanged. Northern blot analysis indicated that the c-myc overexpression is concomitant to DRB-induced DNA fragmentation and that the increased mp53 protein level was mainly a posttranscriptional event. Our observations suggest that the up-regulation of Bcl-X(S) may serve as an important mechanism for the apoptosis triggered by these inhibitors. This study also provides evidence for the notion that interference with the cellular survival pathway may lead to apoptosis.
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PMID:Effects of transcription and translation inhibitors on a human gastric carcinoma cell line. Potential role of Bcl-X(S) in apoptosis triggered by these inhibitors. 917 10

The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery.
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PMID:p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner. 937 54

Previous studies have shown that the apoptotic response of cells following DNA damage requires p53 expression. Wild-type p53 protein levels increase in response to DNA damage and its growth-suppressive action is thought to be mediated by transcriptional activation of the p21/WAF1/CIP1 gene, the product of which is a potent inhibitor of cyclin-dependent kinases. The mechanism by which elevated p53 levels lead to apoptosis is not known, but is believed to involve transcriptional activation of apoptotic genes, such as BAX. We have studied transformed human cells that constitutively express high levels of the R273H mutant p53, which has been reported to lack transcriptional activation activity. We used the inability to induce the p21/Waf1/Cip1 protein as a marker to verify the lack of transcriptional activation activity. Cells expressing the R273H mutant of p53 do not show an increase in p21/Waf1/Cip1 following irradiation with ionizing or UVB radiation. Surprisingly, these cells are very susceptible to induction of apoptosis by UVB radiation, as seen by the formation of a nucleosomal ladder and the proteolytic cleavage of poly(ADP-ribose) polymerase. This suggests that the R273 mutant p53 can function normally in apoptosis but not in transcriptional activation following DNA damage. Furthermore, an inhibitor of RNA polymerase II is a potent inducer of apoptosis in these cells, demonstrating that transcription is not required for apoptosis and suggesting that stalled RNA polymerase II complexes can initiate apoptosis. Interestingly, proteolytic cleavage of p53 occurs during apoptosis in these cells, generating a 45-kDa fragment and liberating the DNA repair helicase binding domain of p53. We propose that the peptide liberated from the carboxy terminus of p53 may contribute to its apoptotic activity, possibly through interaction with the XPB and XPD DNA helicases.
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PMID:The apoptotic and transcriptional transactivation activities of p53 can be dissociated. 949 57

Tumor cells frequently lack the p53 tumor suppressor because p53 mediates apoptosis in these cells. We report here that c-Abl, and to a greater extent a c-Abl mutant defective for DNA-binding, can provoke programmed cell death in p53-deficient tumor cells. Tyrosine kinase mutant K290R is less cytotoxic. In contrast, a C-terminal deletion mutant that lacks the RNA polymerase 11 (PolII)/actin interaction domain, fails to mediate apoptosis unless expressed to very high levels. Cytotoxicity is overcome by coexpression of the apoptosis antagonist E1B 19K protein, and partially overcome by full-length retinoblastoma protein (Rb) or the C pocket fragment of Rb (SEA) that associates with c-Abl. c-Abl is also highly toxic to Saos-2 cells that are deficient for both Rb and p53, indicating that cell death is not the result of inhibition through c-Abl of the anti-apoptotic function of Rb. Finally, p53 and c-Abl combined induce apoptosis stronger than either protein alone. Unlike c-Abl-mediated cell death, apoptosis by p53 is antagonized efficiently only by full-length Rb with intact A/B pocket but not by SEA. Mutant p53 inhibits apoptosis by p53 but not c-Abl. Thus, c-Abl with intact kinase and PolII/ actin-binding domains can affect tumor cell survival independently of Rb and p53.
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PMID:c-Abl tyrosine kinase can mediate tumor cell apoptosis independently of the Rb and p53 tumor suppressors. 970 21

Induction of the tumor suppressor protein p53 restricts cellular proliferation. Since actively growing cells require the ongoing synthesis of ribosomal RNA to sustain cellular biosynthesis, we studied the effect of p53 on ribosomal gene transcription by RNA polymerase I (Pol I). We have measured rDNA transcriptional activity in different cell lines which either lack or overexpress p53 and demonstrate that wild-type but not mutant p53 inhibits cellular pre-rRNA synthesis. Conversely, pre-rRNA levels are elevated both in cells which express mutant p53 and in fibroblasts from p53 knock-out mice. Transient transfection assays with a set of rDNA deletion mutants demonstrate that intergenic spacer sequences are dispensable and the minimal rDNA promoter is sufficient for p53-mediated repression of Pol I transcription. However, in a cell-free transcription system, recombinant p53 does not inhibit rDNA transcription, indicating that p53 does not directly interfere with the basal Pol I transcriptional machinery. Thus, repression of Pol I transcription by p53 may be a consequence of p53-induced growth arrest.
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PMID:p53 represses ribosomal gene transcription. 1002 89

Human U1 and U6 snRNA genes are transcribed by RNA polymerases II and III, respectively. While the p53 tumor suppressor protein is a general repressor of RNA polymerase III transcription, whether p53 regulates snRNA gene transcription by RNA polymerase II is uncertain. The data presented herein indicate that p53 is an effective repressor of snRNA gene transcription by both polymerases. Both U1 and U6 transcription in vitro is repressed by recombinant p53, and endogenous p53 occupancy at these promoters is stimulated by UV light. In response to UV light, U1 and U6 transcription is strongly repressed. Human U1 genes, but not U6 genes, contain a high-affinity p53 response element located within the core promoter region. Nonetheless, this element is not required for p53 repression and mutant p53 molecules that do not bind DNA can maintain repression, suggesting a reliance on protein interactions for p53 promoter recruitment. Recruitment may be mediated by the general transcription factors TATA-box binding protein and snRNA-activating protein complex, which interact well with p53 and function for both RNA polymerase II and III transcription.
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PMID:The p53 tumor suppressor protein represses human snRNA gene transcription by RNA polymerases II and III independently of sequence-specific DNA binding. 1579 9

Che-1 is a RNA polymerase II binding protein involved in the regulation of gene transcription and, in response to DNA damage, promotes p53 transcription. In this study, we investigated whether Che-1 regulates mutant p53 expression. We found that Che-1 is required for sustaining mutant p53 expression in several cancer cell lines, and that Che-1 depletion by siRNA induces apoptosis both in vitro and in vivo. Notably, loss of Che-1 activates DNA damage checkpoint response and induces transactivation of p73. Therefore, these findings underline the important role that Che-1 has in survival of cells expressing mutant p53.
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PMID:Che-1 promotes tumor cell survival by sustaining mutant p53 transcription and inhibiting DNA damage response activation. 2070 54

The molecular mechanisms underlying mutant p53 (mutp53) "gain-of-function" (GOF) are still insufficiently understood, but there is evidence that mutp53 is a transcriptional regulator that is recruited by specialized transcription factors. Here we analyzed the binding sites of mutp53 and the epigenetic status of mutp53-regulated genes that had been identified by global expression profiling upon depletion of endogenous mutp53 (R273H) expression in U251 glioblastoma cells. We found that mutp53 preferentially and autonomously binds to G/C-rich DNA around transcription start sites (TSS) of many genes characterized by active chromatin marks (H3K4me3) and frequently associated with transcription-competent RNA polymerase II. Mutp53-bound regions overlap predominantly with CpG islands and are enriched in G4-motifs that are prone to form G-quadruplex structures. In line, mutp53 binds and stabilizes a well-characterized G-quadruplex structure in vitro. Hence, we assume that binding of mutp53 to G/C-rich DNA regions associated with a large set of cancer-relevant genes is an initial step in their regulation by mutp53. Using GAS1 and HTR2A as model genes, we show that mutp53 affects several parameters of active transcription. Finally, we discuss a dual mode model of mutp53 GOF, which includes both stochastic and deterministic components.
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PMID:Mutant p53 is a transcriptional co-factor that binds to G-rich regulatory regions of active genes and generates transcriptional plasticity. 2289


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