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)

The multidrug resistance (MDR1) gene encodes a P-glycoprotein, which catalyzes the energy-dependent efflux of anticancer agents. Various environmental stresses including heat shock can induce the expression of endogenous MDR1 genes. In order to study the regulatory mechanisms of MDR1 gene expression, we have established human cancer KB cell lines which could stably integrate bacterial chloramphenicol acetyltransferase (CAT) gene driven by various lengths of the MDR1 promoter. Kst-6 has an integrated plasmid, pMDRCAT1, containing the human MDR1 promoter of -2 kilobases. The MDR1 gene promoter contains a typical heat shock element (HSE) motif located -152 bp to -178 bp from the initiation site. Heat shock at 45 degrees C for 90 min significantly induced CAT activity in Kst-6 cells. Northern blot analysis showed a 4-5 fold increase in CAT mRNA levels in Kst-6 cells. Deletion analysis of the MDR1 promoter demonstrated that the induction of CAT activity was observed in Kxh-14 cells containing a HSE-deleted MDR1 promoter construct, pMDRCAT7. However, further deletion analysis showed that heat shock could not induce CAT activity in Khp-1 cells containing -76 approximately +121 base sequence of the promoter, suggesting that a new heat shock responsible element was located at between -136 and -76. Gel shift assay showed that the heat shock factor (HSF) could bind to the HSE motif located at -152 bp to -178 bp in the MDR1 promoter. We also found that one distinct DNA-protein complex formed specifically within the MDR1 promoter region -99 to -66 was not significantly increased, but relatively more stabilized under mild denaturing condition in the nuclear extract of heat-shocked cells. In our present assay system, activation of the MDR1 promoter in response to heat shock appears to be mediated through both a new heat shock responsive element and MDR1 specific transcription factor.
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PMID:Activation of human multidrug resistance-1 gene promoter in response to heat shock stress. 135 36

Recent studies have suggested that a labile transcriptional repressor protein is important in the regulation of pgp mRNA expression. However, cycloheximide (CHX) the protein synthesis inhibitor used, can increase mRNAs by either stabilizing the mRNA transcript or directly activating gene transcription. To determine whether CHX posttranscriptionally increased pgp mRNA, we compared the effect of CHX, which inhibits protein synthesis by stabilizing polysomes, with puromycin (PURO), which inhibits protein synthesis by polysome destabilization. In rat hepatocytes, CHX induced pgp2 mRNA, and the increase was proportional to the degree of protein synthesis inhibition. In contrast, despite almost complete inhibition of protein synthesis, PURO did not induce pgp2 mRNA. Further studies demonstrated that PURO pretreatment could block pgp2 mRNA induction by CHX. Likewise, in cultures of primary human hepatocytes CHX, but not PURO, induced MDR1 mRNA. A polymerase chain reaction assay was developed to assess whether CHX treatment altered the length of the 3'-untranslated region (UTR) of pgp2. CHX treatment time dependently increased the length of the pgp2 3'-UTR. To determine whether CHX acts as a transcriptional agonist, we performed nuclear run-off analysis and found no increase in pgp2 gene transcription compared to untreated control. Further, transcription studies were performed by transiently transfecting HepG2 cells with plasmids containing 5' segments of human MDR1 fused with the reporter chloramphenicol acetyltransferase (CAT). These plasmids were not transcriptionally activated by CHX. In summary, our results cast doubt on the existence of a labile transcriptional repressor protein for pgp. Furthermore, these are the first studies to demonstrate that polysomal destabilization by PURO can block CHX induction of pgp.
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PMID:Induction of P-glycoprotein mRNA by protein synthesis inhibition is not controlled by a transcriptional repressor protein in rat and human liver cells. 759 4

The effect of human wild type and mutant p53 proteins on the human multidrug resistance (MDR1) promoter was studied in a p53-negative human cell line. Transient expression of MDR1 promoter-chloramphenicol acetyltransferase reporter gene constructs (MDRCAT) cotransfected with p53 expression vectors was analyzed in H358 lung carcinoma cells. Cotransfection with a wild type p53 expression vector stimulated MDRCAT activity, while cotransfection with mutant p53 expression vectors altered at amino acid positions 181, 252, 258, or 273 failed to stimulate expression. Wild type p53 stimulation of MDRCAT activity was time dependent with maximal expression occurring 24-30 h following transfection and correlating with high p53 protein levels. MDR1 promoter deletion analysis suggested that the sequences involved in wild type p53 stimulation of MDRCAT activity were contained within the region from -39 to +53 relative to the start of transcription at +1. This region contains no TATA or p53 consensus binding sequence but does contain an initiator sequence. Wild type p53 stimulation of MDRCAT expression also occurred in parental and doxorubicin-resistant SW620 colon and parental 2780 ovarian cancer cell lines, indicating that wild type p53-mediated simulation of the MDR1 promoter is not restricted to a single cell line.
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PMID:Wild type p53 stimulates expression from the human multidrug resistance promoter in a p53-negative cell line. 782 27

Multidrug resistance genes (mdr) that encode P-glycoproteins (P-gp) are transcriptionally regulated in normal tissues and in some multidrug-resistant (MDR) cells. Several lines of evidence suggest that regulation of P-gp overexpression at the transcriptional level is also important in human tumors. In murine MDR cells, mdr1a and/or mdr1b genes are overexpressed and P-gp isoforms are overproduced. To identify the mdr1a promoter regions that are required for transcription, the promoter has been linked to the chloramphenicol acetyltransferase (CAT) gene in transient expression vectors. 5'-Deletions of the promoter sequences have demonstrated that the region between -155 to +89 bp is crucial for basal activity of the mdr1a gene. DNase I footprinting, methylation interference, and gel retardation assays identified two nuclear protein binding sites within these sequences. One of the nuclear protein binding sites contains an 11-bp DNA sequence that interacts with nuclear protein(s) and is conserved in the promoters of the murine mdr1a and mdr1b, hamster pgp1, and human MDR1 genes. The conserved SP1 site (5'-GGGCGGG-3') that is present further downstream was shown to interact with its nuclear factor. These observations suggest that at least part of mdr gene transcriptional regulation is mediated by conserved mdr cis-regulatory elements and common nuclear factors.
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PMID:Identification of two nuclear protein binding sites and their role in the regulation of the murine multidrug resistance mdr1a promoter. 791 38

Revealing the regulatory mechanisms involved in P-glycoprotein expression is important to our understanding of multidrug resistance (MDR) in tumor cells. The MDR1 gene encoding P-glycoprotein contained a promoter sequence (-157 to -125) that was found to be homologous with other mdr gene promoters and that specifically interacted with a nuclear protein. The nuclear protein was identified, using a HeLa lambda gt11 cDNA expression library, to be the transcriptional regulator nuclear factor for interleukin-6 (NF-IL6), a member of the C/EBP family of transcription factors that bound an NF-IL-6-like consensus element 5'-TTTCGCAGT-3'. Furthermore, a glutathione S-transferase fusion protein (10.1-glutathione S-transferase) containing the partial NF-IL6 cDNA was also found to specifically interact with the MDR1 promoter sequence. Co-transfection of an NF-IL6 expression vector with a chloramphenicol acetyltransferase reporter gene driven by 1018 base pairs of MDR1 5'-flanking sequences demonstrated that NF-IL6 trans-activated the MDR1 promoter. This trans-activation was significantly reduced when the NF-IL6 element in the reporter gene construct was deleted or mutated. Identification of NF-IL6 as an important transcriptional regulator and the implications of its potential role in MDR1 gene induction in response to a variety of stimuli are discussed.
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PMID:NF-IL6, a member of the C/EBP family of transcription factors, binds and trans-activates the human MDR1 gene promoter. 796 62

NF-R2 is a DNA-binding protein that interacts with the MDR1 gene proximal promoter sequence. We previously reported that NF-R2 binds within the promoter's -126 and -102 regions, which contain the ATTCAGTCA motif. In the present study, we have purified NF-R2 from the nuclear extract of K562/ADM cells, a multidrug-resistant cell line derived from human myelogenous leukemia K562 cells, using sequential chromatography on Sephacryl S-300, DEAE-Sepharose, heparin-Sepharose and a DNA affinity column consisting of a repetitive synthetic ATTCAGTCA motif coupled to Sepharose. NF-R2 runs as a single protein of 75 kDa on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). CAT (chloramphenicol acetyltransferase) expression assay and gel mobility shift competition assay with mutated promoters revealed that the ATTCAGTCA motif is a positive regulatory element of MDR1 gene and that the motif is important for NF-R2 binding. These results suggest that NF-R2 may be involved in the positive regulation of the MDR1 gene transcription.
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PMID:Purification and characterization of NF-R2 that regulates the expression of the human multidrug resistance (MDR1) gene. 809 26

The human MDR1 gene can be induced in response to various environmental stimuli. To examine whether such stress-induced activation of the MDR1 gene can be modulated by protein kinase, we employed a stable human cancer KB cell line which contained the bacterial chloramphenicol acetyltransferase (CAT) gene directed by the MDR1 gene promoter. H-7, a protein kinase C inhibitor, at more than 40 microM inhibited activation of the MDR1 promoter that was induced by ethylmethane sulfonate, 5-fluorouracil or UV irradiation. DNA binding activity of nuclear factors recognizing the MDR1 promoter was augmented in KB cells treated with UV, but decreased in cells treated concomitantly with H-7. Okadaic acid alone was able to induce the promoter activation, and this induction was dependent on specific promoter sequences. Okadaic acid also enhanced the DNA binding activity of nuclear factors recognizing the MDR1 promoter. The phosphorylation of transacting factors may modulate the MDR1 gene promoter activity.
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PMID:Involvement of protein kinase in environmental stress-induced activation of human multidrug resistance 1 (MDR1) gene promoter. 810 Jul 81

To investigate multidrug-resistance gene (MDR1) promoter efficacy and drug inducibility in cells with different multidrug-resistance phenotypes, multidrug-resistant HCT15 and drug-sensitive KM12 human colon carcinoma cell lines were transfected with constructs incorporating the chloramphenicol acetyltransferase (CAT) reporter gene, driven by wild-type and point-mutated MDR1 promoter regions. The basal CAT expression level in HCT15 cells was markedly elevated compared to KM12 cells. CAT induction by vincristine was dose-dependent over a broad concentration range (40-500 ng/ml) in both lines. The induction levels were related to the cells' MDR phenotype, with the multidrug-resistant HCT15 cells showing the greater effect. In both cell types, basal and drug-induced CAT expression were significantly enhanced by the point-mutated promoter regions. The findings support the possible exploitation of the MDR1 promoter for construction of drug-inducible and MDR-cell-targeted expression vectors for use in gene therapy.
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PMID:Vincristine induction of mutant and wild-type human multidrug-resistance promoters is cell-type-specific and dose-dependent. 860 50

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