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)

Multidrug resistance in mammalian cells is often associated with the overproduction of a membrane glycoprotein, P-glycoprotein, that is encoded by mdr genes. Multidrug resistance cell lines selected with either vinblastine, colchicine, or taxol from the drug-sensitive murine macrophage-like cell line J774.2 overexpress the mdr1a and/or mdr1b genes, and overproduce P-glycoprotein. To elucidate the mechanisms of mdr1b gene expression, the mdr1b 5'-flanking sequences have been isolated from a normal mouse liver genomic library and analyzed by gel shift and DNase I footprinting assays. These analyses have demonstrated three nuclear protein binding sites, from -82 to -59 (site 1), from -123 to -101 (site 2), and from -272 to -249 (site 3), which interact with proteins present in nuclear extracts from both sensitive and resistant cells. Although site 1 contains a partially conserved AP-2 consensus sequence, our results indicate that the nuclear protein binding to site 1 is not AP-2 protein. The sequence of site 2 is conserved in the murine mdr1a, human mdr1, and hamster pgp1 promoters. Such conservation suggests that this sequence may have an important role in mdr gene expression. The use of a transient chloramphenicol acetyltransferase expression vector containing the basal promoter for herpes simplex virus thymidine kinase (tkCAT) and either site 1 or site 2 or both revealed that the sequences of sites 1 and 2 enhanced tkCAT activity. DNase I footprinting analyses demonstrated that site 3 is recognized by human AP-1 protein, indicating that the nuclear protein binding to this site is an AP-1-like protein. These observations suggest that mdr1b gene expression is mediated by preexisting transcription factors present in sensitive and resistant cells.
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PMID:Three distinct nuclear protein binding sites in the promoter of the murine multidrug resistance mdr1b gene. 809 13

P-glycoprotein, the product of the multidrug resistance (mdr) gene family, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents and is also expressed normally in a variety of mammalian tissues. In rodents during pregnancy, there is a dramatic overproduction of the mdr1b form of P-glycoprotein at the lumenal surface of the secretory epithelium of the gravid uterus. An expression vector, mdr1b-CAT, was constructed by fusion of this promoter region to a reporter gene, the bacterial chloramphenicol acetyltransferase (CAT) gene. R5020, a progesterone agonist, increased approximately 3-fold the expression of mdr1b-CAT when transfected into T47D cells, a cell line that constitutively expresses the progesterone receptor. A far greater response to R5020 was observed when the cells were co-transfected with an expression vector for the A form of the progesterone receptor, but not the B form. A series of 5'-deleted clones of the mdr1b-CAT construct indicated that the region of responsiveness was located in the first untranslated exon of the gene. Furthermore, sequences from the first exon were able to confer responsiveness to the non-responsive thymidine kinase-CAT vector. This study demonstrates that progesterone specifically regulates the activity of the mdr1b promoter and that this response is directed solely by the A form of the progesterone receptor.
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PMID:Progesterone regulates the murine multidrug resistance mdr1b gene. 809 15

The human mdr1 gene encodes a putative drug efflux pump (P-glycoprotein) whose overexpression is associated with the development of multidrug resistance (MDR). The promoter and 5'-flanking DNA of this gene were isolated from a human genomic DNA library and used to prepare a series of chloramphenicol acetyltransferase (CAT) fusion vectors under the transcriptional control of the mdr1 promoter (mdrCAT vectors). Transient transfection of these mdrCAT vectors produced CAT activities similar to those produced by transfection of CAT vectors containing viral promoters. The regulation of mdr1 expression was examined in two MDR tumor cell lines selected for resistance to doxorubicin and their corresponding parental cell lines. Although nuclear run-on analysis indicates that the expression of the mdr1 gene in these two MDR cell lines is regulated by transcriptional mechanisms, mdrCAT expression was not significantly increased in either of these lines relative to parental cells. Thus, the sequences involved in the transcriptional regulation in these cells are apparently not included in the constructs studied (-4741 to +286). Analyses of a series of deletion constructs show that the basal mdr1 promoter activity is encoded by sequences that span a region adjacent to the transcription start site (-134 to +286) and that sequences 3' to the start of mdr1 transcription are necessary for proper initiation of transcription in vivo. Structural and functional studies indicate that an initiator (Inr) sequence surrounding the major transcription start site governs accurate initiation of mdr1 transcription.
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PMID:Identification of 5' and 3' sequences involved in the regulation of transcription of the human mdr1 gene in vivo. 809 20

Several studies have demonstrated that regulation of P-glycoprotein gene expression at the transcriptional level is complex and involves multiple regulatory mechanisms. To investigate the transcriptional regulation of P-glycoprotein genes, genomic DNA fragments containing the 5' end of the hamster pgp1 and pgp2 genes were isolated and characterized. The pgp1 5' flanking sequences were linked to the chloramphenicol acetyltransferase (CAT) reporter gene and a series of 5' deletions were constructed. Transient expression of these CAT constructs into Chinese hamster ovary (CHO) cells revealed that the pgp1 promoter is regulated by multiple positive and negative regulatory elements. One particular region between -489 and -255 was shown to possess silencer activity. This region contains two putative negative elements that are also present in the silencer regions of several other genes. Intron 1 sequences of the Pgp genes were also examined and shown to be highly conserved both between family members and across species. Transient expression studies revealed that intron 1 sequences possess enhancer activity. Thus, it was demonstrated that sequences upstream and downstream of the transcriptional start site are important for the regulation of P-glycoprotein gene expression.
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PMID:Structural and functional analysis of 5' flanking and intron 1 sequences of the hamster P-glycoprotein pgp1 and pgp2 genes. 810 Apr 49

The multidrug resistance 1 (MDR1) gene encodes a M(r) 170,000 membrane glycoprotein termed P-glycoprotein, which catalyzes the energy-dependent efflux of multiple anticancer agents. We investigated the activation of the MDR1 gene promoter by UV light irradiation in human cancer KB cells after both transient and stable transfection assays of the MDR1 promoter fused to the chloramphenicol acetyltransferase (CAT) gene. Following exposure to UV irradiation, CAT gene expression was about 20-fold increased. A series of promoter dissection analyses showed that two elements extending from -136 to -76 of the 5' flanking sequence and from +1 to +121 of the sequence downstream from the initiation site were required for the stress induction of MDR1 promoter activity. Gel shift assays showed that the specific DNA binding activities of the transacting protein to the MDR1 promoter were augmented in nuclear extracts from the cells treated with UV irradiation. A DNA sequence, an inverted CCAAT box, was identified that specifically bound to this protein, and mutation of this sequence abolished the binding of this protein. Two guanines in the inverted CCAAT box were found to be critical, as methylation of these guanines abrogated the binding. Nuclear run-on assay demonstrated that the transcription level was increased about 5-fold. These results suggest that the activation of the MDR1 promoter may result from transcriptional rather than posttranscriptional events. These studies will provide the basis for understanding the regulatory mechanism for appearance of the drug-resistant phenotype during cancer chemotherapy.
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PMID:Enhanced expression of the human multidrug resistance 1 gene in response to UV light irradiation. 846 53

The overexpression of P-glycoprotein is thought to be responsible for resistance to chemotherapy in some non-responsive cancers. The mechanism by which P-glycoprotein is overexpressed in human tumors is poorly understood. However, several lines of evidence suggest that the major regulatory mechanism of P-glycoprotein overexpression in human tumors is at the transcriptional level. During tumor progression one of the most commonly observed alterations is mutation of the p53 tumor-suppressor gene. It has been shown that the p53 protein plays a role in transcriptional regulation. To gain insight into the effect p53 protein may have on P-glycoprotein promoter activity, we transiently co-transfected plasmids containing the hamster pgp1 or human mdr1 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene with plasmids encoding either wild-type or mutant p53 protein into Chinese hamster ovary (CHO) cells. In this report, we show that wild-type p53 protein represses P-glycoprotein promoter activity, while mutant forms of p53 protein enhance P-glycoprotein promoter activity. Furthermore, we present data which indicate that the transcriptional regulatory effects of p53 are mediated through interactions with pgp1/mdr1 core promoter sequences. These findings have implications for our understanding of the molecular mechanism(s) by which p53 protein functions as a transcriptional regulator of gene expression. In addition, our results suggest a mechanism by which P-glycoprotein may be overexpressed in human cancers that also express mutant forms of p53 protein.
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PMID:The core promoter region of the P-glycoprotein gene is sufficient to confer differential responsiveness to wild-type and mutant p53. 850 78

We investigated whether the glucocorticoid-mediated mechanisms controlling P-glycoprotein (pgp2 or mdr1b) are similar in normal hepatocytes compared with the H35 hepatoma cell line. In primary rat hepatocytes, dexamethasone (DEX) caused a dose- and time-dependent decrease in the amount of the pgp2 mRNA, which correlated with functional pgp2 expression (intracellular accumulation of [3H]vincristine). The suppression of pgp2 mRNA was specific for glucocorticoids because a representative estrogen and progestin were without effect, and DEX suppression of pgp2 mRNA could be reversed by cotreatment with an anti-glucocorticoid. DEX suppression of pgp2 mRNA appears to be posttranscriptional because following actinomycin D inhibition of new RNA synthesis, the pgp2 transcript disappeared at a faster rate in DEX treated versus untreated hepatocytes. Moreover, transcriptional activity of chloramphenicol acetyltransferase plasmids containing the pgp2 promoter in primary rat hepatocytes was unaffected by DEX treatment. Thus, suppression of pgp2 mRNA by glucocorticoids in primary hepatocytes is due to a decrease in pgp2 mRNA stability. In contrast, in the H35 hepatoma cell line, DEX dose dependently increased pgp protein and pgp2 mRNA, effects which parallel transcriptional activation of the pgp2 promoter. Activation of the pgp2 promoter was specific for glucocorticoids since a representative estrogen had no significant effect on transcription of the pgp2 promoter and RU486 blocked DEX activation of pgp2 transcription. Transcriptional activation of the pgp2 promoter was not due to a global up-regulation of basal transcription factors because DEX treatment did not activate either a herpes simplex virus thymidine kinase promoter or the SV40 early gene promoter. Further studies with a panel of pgp2 5' sequence deletion plasmids revealed that the minimal promoter (-66 bp) was not activated by DEX. In contrast, inclusion of sequences up to -177 bp restored DEX-dependent transcriptional activation. These are the first studies to demonstrate that glucocorticoids regulate pgp2 by different mechanisms in normal rat hepatocytes compared to the H35 hepatoma cell line.
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PMID:Divergent regulation of the class II P-glycoprotein gene in primary cultures of hepatocytes versus H35 hepatoma by glucocorticoids. 884 10

The human multidrug resistance 1 (MDR1) gene encoding P-glycoprotein is often overexpressed in various human tumors after chemotherapy. During treatment with various chemotherapeutic agents, the MDR1 gene is activated at the transcriptional level and/or amplified, resulting in overexpression. Our previous studies demonstrated that an inverted CCAAT box (Y-box) might be a critical cis-regulatory element regulating UV or drug-induced MDR1 gene expression. We have now established various cell lines from human head and neck cancer KB cells which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene driven by various MDR1 promoter deletion constructs. Transient transfection of antisense YB-1 expression constructs resulted in a decrease of both YB-1 protein levels and DNA binding activity to the inverted CCAAT box, as determined by Western blot and gel mobility shift assays. The limited expression and binding activity due to expression of antisense YB-1 constructs were also observed when cells were treated with UV. CAT activity of constructs containing the Y-box was enhanced after treatment with UV irradiation as well as genotoxic agents such as cisplatin and etoposide. Moreover, this activation was reduced by 50-80% by transfection of antisense YB-1 expression constructs. In contrast, transfection of antisense YB-1 expression constructs had no effect on CAT activity driven by MDR1 promoter constructs not containing the Y-box. These data indicate that YB-1 is directly involved in MDR1 gene activation in response to genotoxic stress.
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PMID:Direct involvement of the Y-box binding protein YB-1 in genotoxic stress-induced activation of the human multidrug resistance 1 gene. 949 11


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