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)

Human papillomavirus (HPV) infection, in particular that by HPV type 16, is positively associated with cervical/genital cancer. In contrast, human adeno-associated virus (AAV) infection is negatively associated with these same cancers. AAV has also been found to inhibit the oncogenic properties of a variety of DNA viruses, including bovine papillomavirus type 1, a relative of HPV-16. Taken together, these findings suggested the possibility that AAV and HPV-16 might interact, with AAV inhibiting HPV-16's oncogenic phenotype. Here this hypothesis is addressed using tissue culture assays measuring HPV-16-directed phenotypes. It is found that the cotransfection of AAV type 2 Rep78-positive plasmids resulted in the inhibition of HPV-16 sequence containing plasmids in oncogenic transformation/focus formation, G418-resistant colony formation, and chloramphenicol acetyltransferase expression assays. These data are consistent with the hypothesis that AAV's negative association with cervical cancer is at least partially due to its ability to inhibit HPV-16 expression.
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PMID:Adeno-associated virus inhibits human papillomavirus type 16: a viral interaction implicated in cervical cancer. 817 38

In order to develop systems to express mammalian proteins in human skin-derived cells, we tested 6 different viral and 1 eukaryotic promoter (pCMV, pRSV, pSV, pMMTV, pPoly E, pPoly L, pHMT) for their ability to drive the expression of the chloramphenicol acetyltransferase (CAT) enzyme in different human skin-derived cells. DNA was transfected in human keratinocytes derived from normal foreskin and cervix, in the HPV-negative cervical cancer line HT-3 and in malignant melanoma cell lines (SK-Mel 23, SK-Mel 37) using a liposome-based technique or calcium precipitation. Transfection efficacy was controlled by cotransfection of a beta-galactosidase gene construct. The enzymatic activity of the CAT-gene expression was determined by incubation of the cell extract prepared from the transfected cells with 14 C-labeled chloramphenicol. The CMV-promoter was highly active in all skin- or mucosal-derived cells. In contrast to the strong CMV-promoter, the RSV-, SV-, and HMT-promoter were less active and varied in dependence of the cell type. The pattern of the promoter activity differed between benign and transformed genital keratinocytes. Only the SV-promoter showed a comparable strong basal activity, which was restricted to the SK-Mel 37 cells. In conclusion, the promoter activity has to be tested for each cell type depending on the aims of the gene expression.
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PMID:Differential promoter activity in benign and malignant human cells of skin origin. 858 24

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-dependent pathway and that their promoter activity is dependent on the presence of Pit-1
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PMID:Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1. 861 14

Growth factors coordinately regulate a variety of genes associated with pathological states including tumor invasion and metastasis. Overexpressed epidermal growth factor receptor (EGFR) on tumor cell surfaces is associated with enhanced cell attachment and migration into extracellular matrices, which promotes tumor aggressiveness. We have demonstrated that epidermal growth factor (EGF) up-regulates the cell surface adhesion molecule CD44 at both the mRNA and protein levels on mouse fibroblasts expressing full-length wild-type EGFR (NR6-WT) but not on EGFR-deficient cells (NR6-P). This increases cell attachment to hyaluronic acid. In this investigation, transcriptional regulation of CD44 by EGF was confirmed by defining an EGF-regulatory element. By employing human CD44 gene promoter-chloramphenicol acetyltransferase (CAT) constructs transfected into NR6-WT cells, EGF inducibility was observed within a 120-base pair (bp) DNA fragment located 450 bp upstream of the RNA initiation site. Differential EGF inducibility was found among different cell lines chosen, indicating a 3.2- and 1.8-fold enhancement in DU145 cells carrying exogenous wild-type EGFR and in MCF-7 cells, respectively, while minimal EGF induction was found in cervical cancer HeLa cells. Utilizing gel shift assays, a time-dependent increase of DNA-protein complex formation was found upon EGF stimulation in NR6-WT cells but not in NR6-P cells. Based upon these observations, a novel 22-bp EGF regulatory element (ERE) (5'--604CCCTCTCTCCAGCTCCTCTCCC-583-3') was isolated from the CD44 gene promoter. This ERE conferred DNA-protein binding ability in vitro, as well as the full functional recovery of EGF inducibility of CAT activity when linked to a homologous CD44 promoter or a SV40 promoter driving a CAT reporter gene. A two-base mutation of the ERE completely eliminated its binding activity as well as its EGF inducibility of CAT expression. Our studies indicate that EGF induces CD44 gene expression through an interaction between a specific ERE and putative novel transcriptional factor so as to regulate cell attachment to extracellular matrix.
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PMID:Epidermal growth factor induces CD44 gene expression through a novel regulatory element in mouse fibroblasts. 916 42

Human papillomavirus type 16 (HPV-16) infection is positively associated with cervical cancer, whereas adeno-associated virus (AAV) infection is negatively associated with this same cancer. In earlier studies these two virus types have been shown to directly interact, with AAV inhibiting or enhancing papillomavirus functions depending upon the specific circumstances. One defined interaction between these two viruses is the ability of the AAV Rep78 major regulatory protein to inhibit gene expression of the E6 promoter of BPV-1 (bovine papillomavirus type 1) and HPV types 16 and 18. As Rep78 is a DNA binding transcription factor, we considered whether Rep78 might bind HPV-16 DNA. Here, Rep78 is demonstrated to bind a 44-base pair region (nucleotides 14-56) within the HPV-16 p97 promoter using the electrophoretic mobility shift assay. This region is important for HPV-16 because it includes functional Sp1 and E2 protein binding motifs as well as part of the origin of replication. Furthermore, two Rep78 amino acid substitution mutants, at positions 77 or 64-65, were identified that did not recognize p97 DNA. Both of these Rep78 mutants were found to be defective for inhibition of p97 promoter activity in HeLa and T-47D nuclear extracts in vitro, in a transient chloramphenicol acetyltransferase assay, as well as defective for full inhibition of HPV-16-directed focus formation. These data, taken together, strongly suggest that the Rep78-p97 promoter interaction is at least partially responsible for Rep78-mediated inhibition of HPV-16. Finally, the finding that Rep78 specifically recognizes p97 DNA is surprising because the p97 promoter region contains no GAGC motifs, the core motif for Rep78 recognition. These data suggest that the p97 promoter may represent a new prototypical DNA target type for Rep78.
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PMID:Binding of the human papillomavirus type 16 p97 promoter by the adeno-associated virus Rep78 major regulatory protein correlates with inhibition. 1053 69

To facilitate the investigations of HPV-16 late gene expression HPV-16 reporter plasmids were generated using previously described sub-genomic HPV-16 plasmids, named pBEL and pBELM, that, similar to the full viral genome, produce primarily HPV-16 early mRNAs and very little, if any, late mRNAs in cervical cancer cells. The HPV-16 late L1 gene was replaced by the chloramphenicol acetyltransferase (CAT) reporter gene, or green fluorescent protein (GFP), preceded by the poliovirus internal ribosome entry site (IRES). Results show that the reporter genes mimic the expression of L1 from these plasmids. For example, overexpression of adenovirus E4orf4 protein (E4orf4), polypyrimidine tract binding protein (PTB), arginine/serine-rich SRp30c protein (SRp30c) or alternative splicing factor/splicing factor 2 (ASF/SF2) induced an increased expression of CAT or GFP. Stable cell lines with reporter plasmids pBELCAT and pBELMCAT were also generated. An induction of CAT was observed in HPV-16 reporter cell lines in the presence of the small molecule phorbol 12-myristate 13-acetate (TPA). Further experiments identified the TPA-inducible, hnRNP A2/B1 protein as a regulator of HPV-16 late gene expression. In conclusion, the HPV-16 reporter plasmids and reporter cell lines described herein can be used to identify small molecules and cellular factors that regulate HPV-16 gene expression.
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PMID:Development and validation of a novel reporter assay for human papillomavirus type 16 late gene expression. 2248 15