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 revealed that the expression of P-glycoprotein/multidrug resistance genes is crucial for the development of resistance to a number of lipophilic cancer chemotherapeutic agents. To better understand the regulatory mechanisms of pgp gene expression, we isolated and characterized a DNA fragment containing the 5' portion of a Chinese hamster pgp gene. DNA sequence analysis revealed that this gene is pgp1, the hamster homologue of murine mdr3/mdr1a. This gene is expressed at a higher level in intestines than in kidney and liver, consistent with the expression pattern for the murine mdr3/mdr1a gene. The major transcription start site, determined by the S1 nuclease protection, RNase protection, and primer extension methods, lies 67 nucleotides upstream of the murine and human downstream transcription start site. A chimera containing 101 base pairs upstream from this start site and the chloramphenicol acetyltransferase (CAT) gene was able to direct CAT expression in transient transfection experiments. The AP-1 site, located at -48 base pairs, was crucial for the full pgp1 promoter activity, as demonstrated by site-directed mutagenesis of this site, enhancement of the CAT expression by cotransfection with the expression vectors encoding c-Jun/c-Fos genes, but sequestration with those containing retinoic acid receptor genes. The sequestration effect could be partially abolished when c-Jun/c-Fos genes were also included in cotransfection. An AP-1 or AP-1-like site is also present at the same location in both human and mouse mdr homologues. The involvement of AP-1 in the expression of mammalian pgp1-class genes is discussed.
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PMID:Analysis of the Chinese hamster P-glycoprotein/multidrug resistance gene pgp1 reveals that the AP-1 site is essential for full promoter activity. 166 Nov 34

The overproduction of P-glycoprotein, an integral membrane protein thought to function as a drug efflux pump, is the hallmark of the multidrug resistance phenotype. In murine multidrug resistant J774.2 cell lines, distinct mdr genes, mdr1a and mdr1b, encode unique P-glycoprotein isoforms. To examine the transcriptional regulation of the mdr1b gene, its promoter was isolated and characterized. The transcription initiation site was mapped by primer extension, and the 5'-flanking region was sequenced. Several potential regulatory elements were identified in this region. A transient expression vector was constructed by fusion of 540 base pairs of 5'-flanking sequence and part of the first untranslated exon to the chloramphenicol acetyltransferase (CAT) gene. When transfected into monkey kidney COS-1, rat pituitary GH3 or T47D human breast cells, the mdr1b 5'-flanking sequences were capable of driving CAT expression. Transient transfection studies using deletion subclones of the mdr1b-CAT construct were done to locate potential cis-acting sequences. The studies indicate the presence of cis-acting elements in the 5'-flanking region of the mdr1b gene. The implications of these findings for expression and regulation of the mdr1b gene are discussed.
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PMID:Structural and functional analysis of the mouse mdr1b gene promoter. 167 Dec 22

We have previously shown that the multidrug-resistance/P-glycoprotein gene, mdr3/mdr1a, is activated in mouse hepatocellular carcinomas (HCC). In this study, we show that in a number of HCC-derived cell lines (Hepa1c1c, Hepa1c1c-BprC1, and Hepa1-6) mdr3 is expressed at high levels. To investigate transcriptional regulation of mdr3 in these cells, we have isolated a DNA fragment containing the 5' portion of the mouse mdr3 gene and performed a functional analysis of its promoter. Transient transfection assays using various lengths of the promoter sequence to direct expression of the chloramphenicol acetyltransferase (CAT) reporter gene revealed that the sequence located -94 nucleotides upstream from mouse mdr3 transcription start site functions as a negative element in mouse hepatoma cells. A canonical AP-1 binding sequence TGA-GTCA located at -117 is at least in part responsible for the negative effect from the following observations: (i) Alteration of this AP-1 sequence by site-directed mutagenesis enhanced CAT expression. (ii) Expression of CAT reporter gene was elevated when double-stranded DNA containing the AP-1 sequence, but not mutated sequences, was used as a competitor in cotransfection experiment. (iii) Enhancement of the CAT expression was also seen in cotransfection experiments using recombinant plasmid DNA expressing the c-jun/c-fos proteins, which interact with AP-1 sequences. Interestingly, the proximal region of the hamster pgp1 promoter shares striking sequence similarity with that of the mouse mdr3 gene, including the AP-1 site, but the AP-1 site in the hamster promoter serves as a positive regulator. Although previous studies have demonstrated that positive and negative transcription factors can modulate gene expression through interactions with c-jun/c-fos, this is the first study to show that an AP-1 site functions as a negative cis-element in the regulation of gene expression.
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PMID:Structural and functional analyses of the promoter of the murine multidrug resistance gene mdr3/mdr1a reveal a negative element containing the AP-1 binding site. 168 3

Identification of cis-regulatory sequences is a first step in analyzing the regulation of the human multidrug-resistant 1 (MDR1) gene which encodes the 170-kilodalton membrane P-glycoprotein in normal tissues and tumor cells. We have studied several overlapping genomic clones containing the 5'-flanking region of the gene. These clones span about 30 kilobases (kb) of contiguous DNA containing 10 kb of the gene and 20 kb of the 5'-flanking sequence. The nucleotide sequence of the first exon and the 2 kb preceding the exon were determined. DNA sequences containing the 5'-flanking regions were linked to the chloramphenicol acetyltransferase (CAT) gene. For transient CAT assay, we have employed six cell lines, including human cancer KB, vincristine-resistant VJ-300 derived from KB, mouse adrenal tumor Y-1, African green monkey kidney CV-1, mouse fibroblast NIH3T3, and human adrenal carcinoma SW-13 cells. Promoter activity was very weak regardless of the length of the promoter region in mouse adrenal tumor Y-1 and monkey kidney CV-1 cells, in which endogenous P-glycoprotein was expressed. Introduction of a 700-base genomic DNA fragment from a site located at 10 kb far upstream of the initiation site increased the transcription of the CAT gene in Y-1, CV-1, and SW-13 cells. However, no significant increase in the CAT activity could be observed in NIH3T3, KB, and VJ-300 cells. This fragment markedly augmented the expression of the CAT gene regardless of orientation or position, and it acted in a cell type-specific manner even with heterogenous promoters. Our present study suggests that the 700-base pair fragment may carry a tissue-specific transcriptional enhancer that is active in at least some adrenal and kidney-derived cell lines.
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PMID:Tissue-specific enhancer of the human multidrug-resistance (MDR1) gene. 197 33

Intrinsic and acquired multidrug resistance is an important problem in cancer therapy. Multidrug resistance results from overexpression of the MDR 1 gene, which encodes a drug-efflux pump called P-glycoprotein. We have isolated a 1-kilobase genomic fragment containing the major transcription initiation sites for the human MDR 1 gene. Ribonuclease protection experiments using this fragment indicate that normal human adrenal, colon, and liver cells, the human hepatoma cell line HepG2, and vinblastine-selected human KB multidrug-resistant cells initiate transcription of the MDR 1 gene at the same site within this fragment. The 0.43-kilobase region upstream from the major transcription initiation site linked to the chloramphenicol acetyltransferase gene showed promoter activity in CV-1 monkey kidney cells and in human KB cells. The putative promoter region has a consensus CAAT box and two GC box-like sequences, but no TATA sequence. This identification and isolation of promoter sequences for the MDR 1 gene will permit studies on how expression of this gene is regulated in normal human tissues and cancers.
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PMID:Isolation and sequence of the promoter region of the human multidrug-resistance (P-glycoprotein) gene. 289 92

8-Cl-cAMP, a site-selective analogue of cAMP, decreased mdr-1 expression in multidrug-resistant human breast cancer cells. A sixfold reduction of mdr-1 mRNA expression by 8-Cl-cAMP began within 8 h of treatment and was associated with a decrease in the synthesis of P-glycoprotein and with an increase in vinblastine accumulation. A reduction in mdr-1 expression after 8-Cl-cAMP treatment was also observed in multidrug-resistant human ovarian cancer cell lines. 8-Cl-cAMP is known to change the ratio between the two regulatory subunits, RI and RII, of protein kinase A (PKA). We observed that RI alpha decreased within 24 h of 8-Cl-cAMP treatment, that RII beta increased after as few as 3 h of treatment, and that PKA catalytic activity remained unchanged during 48 h of 8-Cl-cAMP treatment. The results are consistent with the hypothesis that mdr-1 expression is regulated in part by changes in PKA isoenzyme levels. Although 8-Cl-cAMP has been used to differentiate cells in other model systems, the only differentiating effect that could be detected after 8-Cl-cAMP treatment in the MCF-7TH cells was an increase in cytokeratin expression. Evidence that the reduction of mdr-1 mRNA occurred at the level of gene transcription was obtained by measuring chloramphenicol acetyltransferase (CAT) mRNA in MCF-7TH cells transfected with an mdr-1 promoter-CAT construct prior to 8-Cl-cAMP treatment. Thus, 8-Cl-cAMP is able to downregulate mdr-1 expression and suggests a new approach to reversal of drug resistance in human breast cancer.
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PMID:Downregulation of mdr-1 expression by 8-Cl-cAMP in multidrug resistant MCF-7 human breast cancer cells. 754 90

The human MDR3 (or MDR2) P-glycoprotein is probably involved in the transport of phospholipids from liver hepatocytes into bile (Smit et al. (1993) Cell 75, 451-462). In accordance with this function, MDR3 is highly expressed in human liver, but lower mRNA levels were also found in adrenal, heart, muscle and cells of the B-cell compartment. We have cloned and analyzed the MDR3 promoter region. It is GC-rich, and contains neither a TATA nor a CAAT box, but it does contain multiple putative SP1 binding sites, features also found in so-called housekeeping genes. RNase protection and primer extension analyses indicate that the MDR3 gene has multiple transcription start sites in a GC-rich region with considerable homology to the putative mouse mdr2 promoter. A 3 kb genomic fragment containing the MDR3 start sites directs transcription of a chloramphenicol acetyltransferase (CAT) reporter gene upon transient transfection in the human hepatoma cell line HepG2. This transcription is orientation dependent, and stimulated by a SV40 enhancer, indicating that the 3 kb insert contains the core promoter elements of the MDR3 gene. The promoter region contains several consensus sequences where known or putative liver-specific (C/EBP, HNF5) or lymphoid specific (Pu.1, ets-1) transcription factors may bind.
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PMID:Characterization of the promoter region of the human MDR3 P-glycoprotein gene. 789 60

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

Non-P-glycoprotein multidrug resistance of HL60/ADR cells appears to be related to overexpression of the MRP gene. Recent studies suggest that this gene may play an important role in a new form of cell resistance to certain chemotherapeutic agents. To examine mechanisms regulating transcriptional activity of this gene, a 2.2-kilobase 5'-flanking sequence of MRP has been isolated from a genomic library prepared from HL60/ADR cells. The 2.2-kilobase DNA fragment linked to the chloramphenicol acetyltransferase (CAT) gene in a reporter plasmid was found to be capable of driving expression of this gene in transient transfection experiments. This DNA containing promoter activity has been sequenced in its entirety and found to contain multiple putative regulatory sites. A series of deletion mutants linked to the CAT reporter gene was used to examine functional domains of the 2.2-kilobase sequence. The results suggest that promoter activity is contained in nucleotides -91 to +103 in a GC-rich region of the MRP genome. Promoter activity contained within this sequence, however, is modulated by both positive and negative regulatory elements. Certain of the regulatory sites contain consensus sequences for positive and negative regulatory elements which have been found in the promoter regions of other genes. Primer extension analysis indicates the presence of multiple major transcriptional start sites from the MRP promoter. Sequence analysis of MRP genomic and complementary DNAs has defined the exon/intron boundaries and the organization of a portion of the 5'-end region of the MRP genome. The results of these studies thus provide new insight in site-specific domains which may function in the regulation of MRP gene expression.
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PMID:Cloning and sequence analysis of the promoter region of the MRP gene of HL60 cells isolated for resistance to adriamycin. 804


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