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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously demonstrated a correlation between wild-type p53 expression and appearance of osteoblastic-specific differentiation characteristics, as evidenced by basal osteocalcin gene expression in a mouse osteosarcoma tumor. The study reported here further explored the possibility of p53's having a distinct transcription-activating role in bone differentiation, in addition to its proposed role in G1 arrest and apoptosis. ROS17/2.3 osteoblastic osteosarcoma cells were stably transfected with a plasmid containing wild-type p53 binding sequences fused to the chloramphenicol acetyltransferase reporter gene. These cells were used to determine the transactivating role of p53 in regulation of osteocalcin gene expression. We chose two conditions under which osteocalcin expression is known to be upregulated: exposure of osteoblastic cells to differentiation-promoting medium and to vitamin D3. Exposure of the transfected cells to differentiation-promoting medium produced an increase in p53 transactivating activity correlating with the appearance of osteocalcin expression after about 1 wk. Vitamin D3 treatment resulted in upregulation of osteocalcin activity without a corresponding change in p53 transactivation activity or expression. In separate experiments, we tested whether changes in osteocalcin expression accompanied changes in p53 activity under conditions of downregulation of cell proliferation mediated by inhibition of DNA synthesis. Hydroxyurea treatment was used to inhibit DNA synthesis and produce growth arrest in osteoblastic cells. Inhibition of osteoblast cell proliferation was associated with a fourfold increase in p53 transactivating activity and a transient increase in osteocalcin steady-state expression. These results demonstrated a close relationship between p53 and osteocalcin and suggested a regulatory role for wild-type p53 in the control of basal osteocalcin gene expression in osteoblasts.
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PMID:p53 transactivity during in vitro osteoblast differentiation in a rat osteosarcoma cell line. 1036 15

p73 is a recently cloned tumor suppressor gene that is highly homologous to p53, and the products of both possess similar functions in inhibiting cell growth and inducing apoptosis. Interestingly, the COOH-terminal region of p53 displays no significant homology with that of p73. Moreover, p73 has an additional segment at its COOH terminus. Recently, we have found two mutations of p73 with amino acid substitution (P405R and P425L) in primary neuroblastomas. Because the region (amino acid residues 382-491) contains a glutamine- and proline-rich domain, we hypothesized that it has a transactivation function, and the mutations found in tumors result in loss of function. To test it, we used the yeast GAL4 DNA-binding fusion system. Yeast transformants expressing a GAL4-p73(1-112) or a GAL4-p73alpha(380-513) fusion protein were grown in SD medium lacking histidine and tryptophan and exhibited a significant induction of beta-galactosidase activity. Transient transfection experiments revealed that both of fusion proteins could induce the chloramphenicol acetyltransferase activity in mammalian cells, indicating that the COOH-terminal as well as NH2-terminal regions of p73 had significantly high levels of transactivation activity. Furthermore, the former activity was severely impaired in two naturally occurring mutant forms found in neuroblastomas. These suggest that, unlike p53, p73 has two domains with transactivation function, one in the NH2-terminal region and the other in the COOH-terminal region. Loss of function mutation in the latter might be involved in tumorigenesis and/or tumor progression.
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PMID:Identification of a transactivation activity in the COOH-terminal region of p73 which is impaired in the naturally occurring mutants found in human neuroblastomas. 1038 37

Stimulation of transfected HepG2 cells (TFG2) with the alpha(1)-adrenergic agonist phenylephrine (PE) significantly activated p21(waf1/cip1) gene expression without affecting p53 gene expression. Northern blotting and reporter assay demonstrated that this induction was due to PE stimulation of p21(waf1/cip1) mRNA stability. To further define the underlying mechanism, we prepared a chloramphenicol acetyltransferase (CAT)-p21(waf1/cip1) 3'-untranslated region (3'-UTR) hybrid construct by inserting the 3'-UTR of p21(waf1/cip1) mRNA just downstream from the CAT coding sequence and transfected it into TFG2 cells. PE treatment enhanced the activity of this construct by 6-fold. Deletion analyses indicated that an AU-rich element (AURE) located between 553 to 625 within the p21(waf1/cip1) 3'-UTR was required for this induction. RNA gel shift assays demonstrated that this AURE bound an RNA-binding protein. This protein has been purified 5000-fold from PE-treated TFG2 cells by heparin-Sepharose and RNA affinity chromatography. SDS-polyacrylamide gel electrophoresis, UV cross-linking, and Northwestern analyses indicated the molecular mass of this protein as 24 and 52 kDa. Finally, PE treatment markedly enhanced this RNA-protein binding by a p42/44 mitogen-activated protein kinase-dependent mechanism. These data suggest that the AURE located between 553 and 625 within the p21(waf1/cip1) mRNA 3'-UTR, which binds an RNA-binding protein, is responsible for PE-induced p21(waf1/cip1) mRNA stability.
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PMID:Alpha(1) adrenergic agonist induction of p21(waf1/cip1) mRNA stability in transfected HepG2 cells correlates with the increased binding of an AU-rich element binding factor. 1076 10

Somatic mutations in the p53 tumor suppressor gene are the most common genetic alterations found in human malignancies. In the present study, we studied 36 primary human breast carcinomas, using a polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing analysis of exons 2 through 9 for the presence of p53 gene mutations. Six of 36 (17%) breast cancers contained mutations within the core domain of the p53 protein responsible for sequence-specific DNA binding (codons 102-292); all 5 missense mutations clustered between codons 240 and 291 (codons 240, 243, 250, 285, and 291), whereas one nonsense mutation occurred at codon 199. By using recombinant PCR in vitro mutagenesis, we introduced point mutations at codons 199 from Gly to stop (gly199stop), 240 from Ser to Ile (ser240Ile), 250 from Pro to Ala (pro250ala), 285 from Glu to Lys (glu285lys), and 291 from Lys to Asn (lys291asn), and all the p53 sequences were subcloned into the CMVneoBam vector under the control of the cytomegalovirus (CMV) promoter. To test whether the mutants p53 were functionally wild-type (wt) or mutant, we transfected them to p53-null Saos-2 cells with a reporter plasmid containing a p53-responsive element, and performed chloramphenicol acetyltransferase (CAT) assay. Transient CAT assay for transcriptional activation revealed that one group, including gly199stop, ser240ile, glu285lys, and lys291asn, abolished the transcriptional activity, whereas the other group, including pro250ala, retained stronger transcriptional transactivation activity than that of wt p53.
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PMID:Identification of p53 gene mutations in breast cancers and their effects on transcriptional activation function. 1090 Nov 65

The expression of the human presenilin-1 cellular gene is suppressed by the p53 protooncogene. The rapid kinetic of the down-regulation has suggested that it may result from a primary mechanism. We show here that p53 also suppresses the transcription of a presenilin-1 promoter-chloramphenicol acetyltransferase reporter synthetic gene in transient infection assays in neuroblastoma (SK-N-SH) and hepatoma (HepG2) cell lines. Only a minimum promoter including sequences from -35 to + 6 from the transcription initiation is sufficient to confer down-regulation. We have previously defined a crucial DNA element controlling 90% of the expression of the gene within the same short area, and the identification of the transcription factors involved should also provide insights into the regulation of PS1 by p53. This region contains an Ets transcription factor binding motif, and a 2-base pair alteration within the core sequence (GGAA to TTAA) of the Ets consensus also reduced transcription by more than 90%. We now show that Ets1 and Ets2 indeed transactivate a PS1 promoter-chloramphenicol acetyltransferase reporter including the (-35 to +6) fragment. Furthermore, in vitro translated Ets2 binds specifically to the -10 Ets motif in electrophoretic mobility shift assays. Therefore, Ets1/2 factors bind specifically to the -10 Ets element and activate PS1 transcription. We also show that the coactivator p300 enhances the activation by Ets1 and Ets2 as well as the repression by p53. p300 is known to interact with p53 as well as with Ets1 and Ets2. We show that p53 does not bind directly to the PS1 promoter. Hence the repression of PS1 transcription by p53 is likely to be mediated through protein-protein interactions.
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PMID:Regulation of transcription of the human presenilin-1 gene by ets transcription factors and the p53 protooncogene. 1094 70

Under ICH guidelines, short-term carcinogenicity assays such as the Tg.AC assay are allowed alternatives for one species in the 2-year rodent bioassay. The Tg.AC transgenic mouse, which carries the v-Ha-ras oncogene under control of the zeta-globin promoter, develops skin papillomas in response to dermal application of carcinogens and tumor promoters. The appropriate specificity of the Tg.AC model for testing pharmaceuticals has not been systematically evaluated. The selection of candidate test compounds among noncarcinogenic pharmaceuticals would be aided by a high-throughput in vitro prescreen correlative of activity in the in vivo Tg.AC assay. Here we describe the development of a prescreen based on correct response to 24 compounds tested previously in Tg.AC mice. The in vitro prescreens, chosen to reflect molecular pathways possibly involved in Tg.AC papilloma formation, consisted of a zeta-globin promoter-luciferase construct stably expressed in K562 cells (Zeta-Luc) and three of the stress-response element-chloramphenicol acetyltransferase (CAT) fusion constructs stably expressed in HepG2 cells that are part of the CAT-Tox (L)iver assay. The stress response elements chosen were the c-fos promoter, the gadd153 promoter, and p53 response element repeats. Of the four assays, the gadd153-CAT assay showed the strongest concordance with activity in the Tg.AC assay, correctly classifying 78% of Tg.AC positive and 83% of Tg.AC negative compounds. The correlation was further improved by adding the Zeta-Luc assay as a second-stage screen. These cell-based assays will be used in a novel approach to selecting candidate compounds that challenge the specificity of the Tg.AC assay toward pharmaceuticals.
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PMID:Evaluation of in vitro reporter gene induction assays for use in a rapid prescreen for compound selection to test specificity in the Tg.AC mouse short-term carcinogenicity assay. 1096 10

The p53 tumor suppressor protein functions as an activator and also as a repressor of gene transcription. Currently, the mechanism of transcriptional repression by p53 remains poorly understood. To help clarify this mechanism, we carried out studies designed to identify the minimal repression domain that inhibits p53 transcriptional activities. We found only eight amino acids (339) of the COOH-terminal domain (termed P53MRD) that possess activities of repression. The exact location of this minimal domain is on the E6-binding region, and it lacks the ability of tetramerization. P53MRD is able to repress the transcription of p53 while not affecting VP16. The mutants (amino acids M340P and F341D) of native p53 also lost transcriptional repression of the thymidine kinase chloramphenicol acetyltransferase promoter. These results suggest that this eight-amino acid element is required for the repression of p53.
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PMID:p53 amino acids 339-346 represent the minimal p53 repression domain. 1100

Cyclopentenone prostaglandins inhibit virus replication in several DNA and RNA virus models. In this report we investigated the effect of prostaglandin A1 (PGA1) on HIV-1 transcription in human CD4+ Jurkat T lymphocyte cells. A dramatic reduction of HIV-1 RNA levels was detected up to seven days post infection in both unstimulated and phorbol 12-mystrate 13-acetate (PMA)-stimulated cells treated with PGA1- PGA1 treatment of cells was also effective in inhibiting the transcription of a chloramphenicol acetyltransferase (CAT) reporter gene, under the control of HIV-1 LTR, in Jurkat-Tat cells. We also show that PGA1 induced the synthesis of 70-kDa heat-shock protein (HSP70) in this cell system and the induction correlated with the drug-antiviral activity. PGA1 was also found to induce the loss of the tumor suppressor p53 protein, in the "proliferative" conformation, in a time correlation with the induction of the HSP70 As the "proliferative" p53 has been involved in the positive trans-activation of the HIV-1 LTR its depletion could contribute to the inhibitory mechanisms of PGA1 on virus transcription.
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PMID:Inhibition of HIV-1 transcription by cyclopentenone prostaglandin A1 in Jurkat T lymphocytes. 1103 55

The lung resistance-related protein (LRP) was identified as the human major vault protein (MVP), and is overexpressed in various multidrug-resistant cancer cell lines and clinical samples. We characterized DNA sequences upstream to the transcription initiation site of the MVP gene in the human non-small cell lung cancer cell line SW-1573. A 1.9-kb and a shortened 0.7-kb fragment of the 5'-upstream genomic region show strong promoter activity in chloramphenicol acetyltransferase (CAT) reporter assays. The promoter is TATA-less and contains an inverted CCAAT-box and a Sp1 site located near to a p53 binding motif. An alternative 3'-splice site of intron 1 results in a splicing variant within the 5'-untranslated region of MVP mRNA.
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PMID:Cloning and initial analysis of the human multidrug resistance-related MVP/LRP gene promoter. 1107 64

Virus-like particles (VLPs) composed of recombinant capsid protein L1 and L2 of human papillomavirus type 16 were conjugated with polylysine (PL) and gene transfer was performed using VLP-PL conjugates to allow the expression of targeted gene. When HeLa cells were incubated with VLP-PL conjugate coupled with plasmid cytomegalovirus beta-galactosidase (pCMVbeta-gal), about 10% of cells were transfected and demonstrated beta-galactosidase activity. Hence chloramphenicol acetyltransferase activity was also expressed significantly in VLP-PL-plasmid simian virus 2 chloramphenicol acetyl transferase (pSV2CAT)-transfected cells, VLP-PL conjugate was tested whether it could transfer a tumor suppressor gene, pCMVp53, to HeLa cells and the exogenously provided p53 gene complexed to VLP-PL conjugate was detected from HeLa cells by polymerase chain reaction (PCR) analysis. Interestingly, additional increase of transfection efficiency was demonstrated in the presence of poloxamer 407 when C-33A cells were transfected with VLP-PL-pCMVbeta-gal complex. The result support the notion that VLP-PL conjugate may be a promising vector to transfer genetic materials into cancer cells and poloxamer 407 can be used for enhancing the transfection efficiency of VLP-PL conjugate.
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PMID:Improvement of gene transfer to cervical cancer cell lines using non-viral agents. 1112 65


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