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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Real-time quantitative polymerase chain reaction (QPCR) using the Roche LightCycler was used to verify the expression of asparagine synthetase (ASNS) identified by microarray analysis as a target of p53 transrepression and mutant p53 transactivation. A p53-null cell line derived from lung carcinoma, H1299, was infected with recombinant adenovirus expressing wild-type (WT) p53, mutant p53-D281G, or beta-galactosidase as a control. After 24 h of infection, RNA was harvested and used for microarray analysis. ASNS was one of several genes whose expression was down-regulated by WT p53 and up-regulated in the presence of mutant p53. Expression levels of ASNS were measured relative to an exogenously applied quality-control nucleic acid template. Real-time PCR product accumulation was monitored using the intercalating dye, SYBR Green I, which exhibits a higher fluorescence upon binding of double-stranded DNA. Relative gene expression was calculated using conditions at the early stages of PCR, when amplification was logarithmic and, thus, could be correlated to initial copy number of gene transcripts. ASNS was found to be down-regulated in the presence of WT p53 and up-regulated by mutant p53.
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PMID:Real-time polymerase chain reaction quantitation of relative expression of genes modulated by p53 using SYBR Green I. 1282 26

Mutations of the tumor suppressor gene p53 are common in bladder cancer. To determine whether p53 gene transfer would lead to decreased viability of bladder cancer cells, we studied the effect of p53 gene transfer in human bladder cancer cell lines with either mutant or wild-type p53. Bladder cancer cell lines 5637 and J82 (which express only mutant p53) and 253J-BV (which expresses wild-type p53) were transduced with vectors containing the beta-galactosidase gene (Ad5-lacZ), wild-type human p53 gene (Ad5CMV-p53), or no foreign gene (DL312 or Ad5-polyA). X-gal staining of cells exposed to Ad5-lacZ showed that the adenoviral vector was capable of transducing each of the cell lines. Increases in p53, p21(waf1/cip1) and bax protein were demonstrated following exposure to Ad5CMV-p53, and there was a dose-dependent increase in the number of apoptotic cells. Cell viability was decreased in all three cell lines, although J82 was less sensitive than either 5637 or 253J-BV. To determine whether cisplatin increases sensitivity of J82 cells to Ad5CMV-p53, we performed median effect analysis for cisplatin combined with Ad5CMV-p53 or DL312. The combination index for cisplatin plus Ad5CMV-p53 revealed synergy, whereas cisplatin and DL312 were only additive. These results suggest that forced p53 gene expression is cytotoxic to human bladder cancer cells with either p53 mutant or wild-type background, and that combination with cisplatin is a potential method for overcoming resistance.
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PMID:Adenoviral p53 gene transfer in human bladder cancer cell lines: cytotoxicity and synergy with cisplatin. 1469 72

The poor prognosis for patients with metastatic osteosarcoma (OS) indicates that new therapeutic options should be explored. Studies with adenoviral-mediated p53 gene transfer have been conducted in many cancer types including cervical, ovarian, prostatic and head and neck tumors. However, limited work has been carried out with pediatric cancers, including OS. Using three viral constructs containing cDNA for wild-type p53, mutant p53 (Cys135Ser) and lacZ, we studied the effect of adenoviral-mediated gene therapy in four OS cell lines: Saos-2 (p53-/-), HOS (R156P), KHOS/NP (R156P) and MNNG (R156P, F270L). We demonstrated that the virus efficiently enters the cells using the beta-galactosidase assay. Using the MTT assay, we have shown a dose-dependent decrease in cell viability 72 h post-treatment that occurs with Ad-wtp53 but not with Ad-mutp53. We have also shown that treatment with Ad-wtp53 significantly increases sensitivity of the cell lines to cisplatin and doxorubicin, chemotherapeutic agents commonly used in the treatment of OS. Our results indicate that restoration of wt p53 function in OS cells provides a basis for novel approaches to treatment of this disease.
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PMID:Adenovirus-mediated p53 gene therapy in osteosarcoma cell lines: sensitization to cisplatin and doxorubicin. 1621 Oct 88

Ionizing radiation has been reported to promote accelerated or premature senescence in both normal and tumour cell lines. The current studies were designed to characterize the accelerated senescence response to radiation in the breast tumour cell in terms of its dependence on functional p53 and its relationship to telomerase activity, telomere lengths, expression of human telomerase reverse transcriptase (hTERT, the catalytic subunit of telomerase) and human telomerase RNA (hTR, the RNA subunit of telomerase), as well as the induction of cytogenetic aberrations. Studies were performed in p53 wild-type MCF-7 cells, MCF-7/E6 cells with attenuated p53 function, MDA-MB231 cells with mutant p53 and MCF-7/hTERT cells with constitutive expression of hTERT. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP assay), telomere lengths by the terminal restriction fragment (TRF) assay, hTR and hTERT expression by reverse transcriptase-polymerase chain reaction (RT-PCR), senescence by beta-galactosidase staining, and apoptosis by TdT-mediated d-UTP-X nick-end labelling (TUNEL assay). Widespread and extensive expression of beta-galactosidase, a marker of cellular senescence, was evident in MCF-7 breast tumour cells following exposure to 10 Gy of ionizing radiation. Radiation did not suppress expression of either hTERT or hTR, alter telomerase activity or induce telomere shortening. Senescence arrest was also observed in irradiated MCF-7/hTERT cells, which have elongated telomeres due to the ectopic expression of the catalytic component of telomerase. In contrast to MCF-7 cells, irradiated MDA-MB231 breast tumour cells and MCF-7/E6 cells failed to senesce and instead demonstrated a delayed apoptotic cell death. Irradiation produced chromosome end associated abnormalities, including end-to-end fusions (an indicator of telomere dysfunction) in MCF-7 cells, MCF-7/hTERT cells, as well as in MCF-7/E6 cells. When cells were maintained in culture following irradiation, proliferative recovery was evident exclusively after senescence while the cell lines which responded to radiation by apoptosis continued to decline in cell number. Accelerated senescence in response to ionizing radiation is p53 dependent and associated with telomer dysfunction but is unrelated to changes in telomerase activity or telomere lengths, expression of hTERT and hTR. In the absence of functional p53, cells are unable to arrest for an extended period, resulting in apoptotic cell death while accelerated senescence in cells expressing p53 is succeeded by proliferative recovery.
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PMID:p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells. 1630 15

Gene therapy for a variety of human cancers containing the mutant p53 (mt-p53) gene has been performed by direct injection of a retroviral or adenoviral vector containing the wild-type p53 (wt-p53) gene. Because many individuals with skin squamous cell carcinoma (SCC) have been shown to carry the p53 gene mutation, these patients are candidates for p53 gene therapy. For this reason, we established ponasterone A-inducible the wild-type p53 (wt-p53) protein-expressing clones by transfecting a ponasterone-inducible vector containing the wt-p53 gene into HSC-1 cells, which harbor the mutated p53 (m/w) at codon 173 (GTG --> TTG in one allele). Upon the induction of the wt-p53 protein, severe growth suppression was observed. Based on the results of the expression patterns of the p21, p16, RB, BAX and Bcl-2 proteins, as well as on the results of senescence-associated beta-galactosidase staining, the suppression was caused by senescence-like growth arrest of the cells. Although it is generally accepted that the suppression of tumor cell growth is caused by p53-induced apoptosis, permanent G1 arrest induced by p53 is also an important part of the growth-suppression mechanism in p53 gene therapy. The present results should expand the possibilities for p53 gene therapy for human skin SCCs containing the mutant p53 gene.
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PMID:Establishment of ponasterone A-inducible the wild-type p53 protein-expressing clones from HSC-1 cells, cell growth suppression by p53 expression and the suppression mechanism. 1900 4

EBV-associated nasopharyngeal cancer (NPC) occurs with high frequency in China and is a major cause of morbidity and mortality. To explore the potential use of adenovirus-mediated tumor suppressor p53 gene therapy In NPC, we first examined the in vitro effects of p53 introduced into the NPC cell lines RPMI 2650, Fadu and Detroit 562. p21(WAF1/CIP1) induction by chemotherapy was used as a functional assay which revealed that RPMI 2650 expresses wild-type p53 whereas Fadu and Detroit 562 encode mutant p53. Infection with p53-expressing adenovirus (Ad-p53) induced apoptosis and inhibited cell growth in all three NPC cell lines, regardless of the endogenous p53 status. Adenovirus infectivity was greatest in RPMI 2650 cells, with 100% of the cells expressing beta-galactosidase following Ad-LacZ infection using an MOI of 100, as compared to 20-30% infectivity with the other NPC lines. Using RPMI 2650 cells injected into nude mice, we developed an animal model for nasopharyngeal cancer. Established tumors (0.6-0.8 cm) were injected with 5x10(9) PFU Ad-LacZ, Ad-p53 or PBS in a 100 mu l volume. We found evidence for in vivo expression of beta-galactosidase or p53 and p21 up to two weeks following Ad-LacZ or Ad-p53 virus injection respectively. Objective regression of tumor size was observed at two weeks in 4/6 Ad-p53-treated tumors, but not in Ad-LacZ or PBS-treated tumors. The results provide an animal model for human nasopharyngeal cancer, and indicate a potential use of p53 in its therapy in vivo.
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PMID:Adenovirus-mediated p53 gene therapy in nasopharyngeal cancer. 2152 3

The effect of the tumor suppressor p53 on urothelial carcinoma cells was studied by transfecting six cell lines containing different mutations in the p53 gene with an expression construct for the wild-type protein. In all cell lines, the number of cell clones resistant to a neomycin analogue was strongly diminished when pCMVhup53 was cotransfected with the resistance plasmid pRSVneo as compared to cotransfection with either a plasmid vector, a p53 deletion and a mutant p53 expression vector. Cytochemical analysis showed that cells cotransfected with pCMVhup53 and an expression plasmid for beta-galactosidase disappeared during the second day after transfection. Thus, reexpression of wildtype p53 efficiently and rapidly kills urothelial carcinoma cells, independent of the different mutations in p53 they contain.
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PMID:Rapid killing of urothelial carcinoma-cells by wild-type p53. 2155 84

We have developed human adenovirus 5 (Ad5) vectors expressing the wild type human p53 protein or a mutant p53 form under the control of the human cytomegalovirus immediate early gene promoter. Human cells infected with these vectors expressed high levels of p53, accumulating 20-40 fold more protein than found in normal human fibroblasts. The ability of the vectors to affect proliferation of cells in culture was assessed by measuring cell DNA synthesis and colony forming ability after infection with viruses. When the p53 deficient ovarian carcinoma cell line, SKOV-3, was infected with Adp53wt expressing the wild type (wt) p53 protein, a significant inhibition of cellular DNA synthesis was observed, relative to cells infected with Adp53m expressing mutant p53, or a control virus, AdLacZ, expressing bacterial beta-galactosidase. Inhibition was dependent on multiplicity of infection, with no significant effect below 5 pfu/cell, and maximal effect between 25 and 100 PFU/cell which resulted in approximately 95% inhibition of SKOV-3 cell DNA synthesis relative to mock infected cells. Infection of normal human fibroblasts with Adp53wt also inhibited DNA synthesis but to a significantly lesser degree. SKOV-3 cell survival, assayed by ability to form colonies, was reduced at least 10 fold after infection with Adp53wt compared to colony forming ability of cells after infection with either AdLacZ or Adp53m. The results of these studies indicate that p53 expressed by Ad vectors can inhibit proliferation in culture of p53 negative cells by at least 95%, and suggest that such vectors might similarly inhibit the proliferation of tumor cells in vivo.
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PMID:Inhibition of cell-proliferation by an adenovirus vector expressing the human wild type-p53 protein. 2157 32

Head and neck cancer (HNSCC) are one of the most common solid malignancies of the world, being responsible for over 350,000 deaths every year. Much of the complications in managing and treating HNSCC advent from the complex genetic and epigenetic landscape of the disease. Emerging information has shown promising results in targeting BRD4, an epigenetic regulator bromodomain that functions as a scaffold for transcription factors at promoters and super-enhancers. Here we show that by disrupting the interaction between BRD4 and histones using the bromodomain inhibitor JQ1, HNSCC cells undergo cell growth arrest followed by cellular senescence. Mechanistically, JQ1 negatively impacted the phosphorylation levels of SIRT1 along with the acetylation levels of mutant p53 (active). In vivo administration of JQ1 resulted in disruption of HNSCC growth along with the activation of cellular senescence, observed by the accumulation of DNA double-strand breaks, p16^ink4, accumulation of senescence-associated beta-galactosidase, and loss of phosphorylated Sirt1^ser47. Furthermore, we also demonstrate that JQ1 was efficient in reducing the population of cancer stem cells from HNSCC xenografts.
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PMID:Interference with the bromodomain epigenome readers drives p21 expression and tumor senescence. 3126 75


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