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 estrogen-responsive element (ERE) present in the 5'-flanking region of the Xenopus laevis vitellogenin (vit) gene B1 has been characterized by transient expression analysis of chimeric vit-tk-CAT (chloramphenicol acetyltransferase) gene constructs transfected into the human estrogen-responsive MCF-7 cell line. The vit B1 ERE behaves like an inducible enhancer, since it is able to confer estrogen inducibility to the heterologous HSV thymidine kinase (tk) promoter in a relative position- and orientation-independent manner. In this assay, the minimal B1 ERE is 33 bp long and consists of two 13 bp imperfect palindromic elements both of which are required for the enhancer activity. A third imperfect palindromic element is present further upstream within the 5'-flanking region of the gene but is unable to confer hormone responsiveness by itself. Similarly, neither element forming the B1 ERE can alone confer estrogen inducibility to the tk promoter. However, in combinations of two, all three imperfect palindromes can act cooperatively to form a functional ERE. In contrast a single 13 bp perfect palindromic element, GGTCACTGTGACC, such as the one found upstream of the vit gene A2, is itself sufficient to act as a fully active ERE. Single point mutations within this element abolish estrogen inducibility, while a defined combination of two mutations converts this ERE into a glucocorticoid-responsive element.
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PMID:The estrogen-responsive element as an inducible enhancer: DNA sequence requirements and conversion to a glucocorticoid-responsive element. 348 Jul 98

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

We have investigated the ability of several transcriptionally inactive estrogen receptor (ER) mutants to block endogenous ER-mediated transcription in MCF-7 human breast cancer cells. In transient transfections of MCF-7 cells, two of the mutants, a frame-shifted ER (S554fs) and a point-mutated ER (L540Q), strongly inhibit the ability of endogenous wild-type ER to activate transcription of estrogen-regulated reporter plasmids. A third mutant, ER1-530, which is missing 65 residues from its carboxy-terminus, is a weaker repressor of estradiol-stimulated transcription. When an estrogen response element (ERE)-thymidine kinase-chloramphenicol acetyltransferase reporter gene is used, S554fs, L540Q, and ER1-530 suppress the transcriptional activity of endogenous MCF-7 ER by 87%, 97%, and 62%, respectively. The magnitude of dominant negative repression is promoter specific; when an ERE-pS2-chloramphenicol acetyltransferase reporter is employed, inhibition of endogenous ER activity by equivalent amounts of S554fs, L540Q, and ER1-530 ranges from 85-97%. Dose-response studies show the S554fs mutant to be the most potent of the three ER mutants as a repressor of estrogen action in these cells. In addition, elevated levels of intracellular cAMP, achieved by the addition of 3-isobutyl-1-methylxanthine plus cholera toxin to cells, fail to compromise the effectiveness of these mutants as dominant negative ERs despite the cAMP-enhanced transcriptional activity of ER. The mutants are also powerful repressors of the agonist activity of trans-hydroxytamoxifen-stimulated ER transcription. The dominant negative activity of the three mutants is lost when the A/B domain of these receptors is deleted, implying an important role for this N-terminal region of the ER in the ability of these mutants to inhibit endogenous wild-type ER activity. All in all, the data suggest that S554fs in particular is a reasonable candidate for studies designed to use a dominant negative ER to inhibit the estrogen- and tamoxifen-stimulated growth of human breast cancer cells.
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PMID:Repression of endogenous estrogen receptor activity in MCF-7 human breast cancer cells by dominant negative estrogen receptors. 762 51

Although 17 beta-estradiol (E2) replacement therapy has been shown to be effective in treating postmenopausal osteoporosis, the underlying mechanism remains unclear. The presence of low levels of functional endogenous estrogen receptor (ER) in some osteoblastic cells has been demonstrated, and the suggestion that the abundance of ER may be rate-limiting in the action of E2 on these cells has been made. To study the mechanism of ER in regard to E2-mediated effects, we stably transfected a human osteosarcoma cell line, SaOS-2, with an expression vector, pMV-7-ER, containing the human ER gene. We characterized six of the stably transfected clones. One of the stable clones, SaOS-2-ER, expressed extra copies of ER genes integrated into the genome as detected by Southern blot analysis, showed a significantly increased level of ER mRNA by RT-PCR, and contained an increased level of ER cytosolic protein as detected by an ER-specific EIA. The overexpressed ER was functional and sensitive to E2 in a dose-dependent fashion after transient transfection with a vector containing an estrogen response element (ERE) linked to a chloramphenicol acetyltransferase (CAT) reporter gene. Scatchard analysis revealed a single high-affinity binding site with a Kd similar to values obtained for the ER in MCF-7 breast cancer cells. These SaOS-2-ER cells had altered osteoblast phenotypic features including growth inhibition, decreased basal alkaline phosphatase activity, and decreased IL-6 expression and secretion. In response to E2, a greater than 2-fold increase in TGF-beta 1 mRNA was quantitatively measured in these ER-overexpressing osteoblasts. These cells may provide a sensitive and unique model for understanding the mechanism of E2 and ER in overall bone metabolism.
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PMID:Generation and characterization of a human osteosarcoma cell line stably transfected with the human estrogen receptor gene. 763 12

Since we have observed effects of growth factors and cAMP as well as estradiol (E2) on regulation of expression of some genes stimulated by the estrogen receptor (ER), we have undertaken studies to examine directly whether activators of protein kinases can modulate transcriptional activity of the ER. We find that activators of protein kinase-A [cholera toxin plus 3-isobutyl-1-methylxanthine (CT+IBMX)] and protein kinase-C [12-O-tetradecanoylphorbol-13-acetate (TPA)] markedly synergize with E2 in ER-mediated transcriptional activation. When a reporter plasmid [with a minimal promoter containing a TATA region and estrogen-responsive elements (ERE) linked to a chloramphenicol acetyltransferase (CAT) reporter gene] was transfected into MCF-7 human breast cancer cells, which contain endogenous ER, E2 evoked a dose-dependent increase in CAT activity. While treatment with protein kinase-A or -C activator alone evoked only very low CAT activity, the maximal (approximately 25-fold) CAT activity stimulated by E2 alone was increased 2- to 3-fold (to approximately 60 times the control value) upon cotreatment with either of the protein kinase activators. Interestingly, antiestrogen abolished all of the CAT activity induced by E2 and protein kinase activators. Immunoblots showed that TPA reduced ER levels to 30% of control values after 24 h, while CT+IBMX increased levels about 1.5-fold. Scatchard binding analysis revealed no change in the binding affinity of E2 to ER by these agents. Gel mobility shift competition assays with extracts prepared from cells that had been treated with E2 and protein kinase activators did not reveal any quantitative or qualitative changes in the binding of ER to the ERE in vitro. In ER-deficient Chinese hamster ovary (CHO) cells transfected with the reporter gene and varying amounts of an ER expression vector, the level of CAT activity obtained by cotreatment with E2 and CT+IMBX was 3-fold higher than that observed with E2 alone over the range of different ER amounts tested. This ER-mediated synergism was still retained in an amino-terminal A/B-domain-deleted ER mutant lacking the hormone-independent transcriptional activation function (TAF-1), but was greatly reduced in two hormone-binding domain (region E) mutants that exhibit significantly diminished ligand-dependent transcriptional activation. TPA did not show any synergistic activation with E2 in CHO cells, indicating differences in responses between cell types.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators. 768 75

The possibility that appropriately designed chemotherapy could act selectively against p53-defective tumor cells was explored in MCF-7 human breast cancer cells. These cells were chosen because they have normal p53 function but are representative of a tumor cell type that does not readily undergo p53-dependent apoptosis. Two sublines (MCF-7/E6 and MCF-7/mu-p53) were established in which p53 function was disrupted by transfection with either the human papillomavirus type-16 E6 gene or a dominant-negative mutant p53 gene. p53 function in MCF-7/E6 and MCF-7/mu-p53 cells was defective relative to control cells in that there were no increases in p53 or p21Waf1/Cip1 protein levels and no G1 arrest following exposure to ionizing radiation. Survival assays showed that p53 disruption sensitized MCF-7 cells to cisplatin (CDDP) but not to several other DNA-damaging agents. CDDP sensitization was not limited to MCF-7 cells since p53 disruption in human colon carcinoma RKO cells also enhanced sensitivity to CDDP. Contrary to the other DNA-damaging agents tested, CDDP-induced DNA lesions are repaired extensively by nucleotide excision, and in agreement with a defect in this process, MCF-7/E6 and MCF-7/mu-p53 cells exhibited a reduced ability to repair a CDDP-damaged chloramphenicol acetyltransferase-reporter plasmid transfected into the cells. Therefore, we attributed the increased CDDP sensitivity of MCF-7 cells with disrupted p53 to defects in G1 checkpoint control, nucleotide excision repair, or both. The G2 checkpoint inhibitor pentoxifylline exhibited synergism with CDDP in killing MCF-7/E6 cells but did not affect sensitivity of the control cells. Moreover, pentoxifylline inhibited G2 checkpoint function to a greater extent in MCF-7/E6 than in the parental cells. These results suggested that, in the absence of p53 function, cancer cells are more vulnerable to G2 checkpoint abrogators. Our results show that a combination of CDDP and pentoxifylline is capable of synergistic and preferential killing of p53-defective tumor cells that do not readily undergo apoptosis.
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PMID:Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline. 771 69

Mouse calbindin-D28k expression is regulated in vivo by estradiol in ovaries, uterus, and oviduct. To determine whether estrogen can have an effect on the transcription of the calbindin-D28k gene, the human breast cancer cells T47D were transiently transfected with a plasmid containing a 1.1 kilobase (kb) PstI/SacII fragment (-1075/+34) of the mouse calbindin gene ligated to the chloramphenicol acetyltransferase (CAT) gene and cotransfected with human estrogen receptor expression vector. T47D cells, transfected and treated with estradiol (10(-11) - 10(-7) M for 64-65 h), exhibited a dose-dependent increase in CAT activity (up to 6.2-fold). Transfection of MCF-7 breast cancer cells with the chimeric gene construct alone also resulted in an estradiol-dependent induction in CAT activity. Deletion mutant analysis demonstrated that there are two regions of the mouse calbindin-D28k promoter (between -1075/-702 and between -175/-78) that contribute to the induction by estradiol. These fragments, when linked to the thymidine kinase promoter to construct a heterologous promoter chimera, were able to convert the thymidine kinase promoter to estrogen responsiveness. In these regions there are multiple imperfect half-palindromic estrogen-responsive elements. Gel retardation assays demonstrated weak protein-DNA interactions that were competed with cold oligonucleotide containing the vitellogenin estrogen-response element. These findings indicate that the mouse calbindin-D28k promoter is capable of conferring estrogen responsiveness, which may be mediated by several imperfect half-palindromic estrogen-responsive elements, and suggest, in light of previous studies concerning 1,25-dihydroxyvitamin D regulation, multiple steroid regulation of the calbindin-D28k gene.
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PMID:Regulation by estrogen through the 5'-flanking region of the mouse calbindin-D28k gene. 777 78

To examine some of the molecular mechanisms controlling transcription of the rat progesterone receptor (PR) gene, we have cloned and sequenced the 5'-region of the gene. Northern blot analyses with a series of probes identified two regions where distinct subsets of the multiple PR gene transcripts initiated, suggesting the presence of two promoters in the gene. Promoter activities for two gene fragments encompassing these regions, -131/+65 (P; distal) and +461/+675 (P'; proximal), were demonstrated in transient transfection experiments using reporter constructs containing the gene fragments linked individually upstream of the chloramphenicol acetyltransferase (CAT) gene. Cotransfection of P-CAT or P'-CAT constructs containing two upstream GAL4 binding sites into primary cultures of rat uterine cells with a vector expressing a GAL4 DNA binding domain-VP16 activating region fusion protein resulted in a 10-fold increase in CAT activity relative to cells transfected with either reporter and a vector expressing only the GAL4 DNA binding domain. The estrogen inducibility of the promoter-CAT constructs was assessed by transfection into MCF-7 breast cancer cells, which contain high levels of estrogen receptor (ER). P'-CAT, but not P-CAT, was induced by estradiol (E2; 8-fold). In primary rat uterine cells, which contain lower levels of ER, P'-CAT required the addition of one upstream consensus estrogen response element (ERE) to be estrogen inducible, whereas P-CAT required the addition of two EREs. Point and deletion mutants of the proximal promoter region in the P'-CAT reporter, screened in MCF-7 cells, were used to identify a 20-base pair fragment (+617/+636) that retained the promoter activity and 50% of the estrogen inducibility of P'. This fragment contained an ERE-like sequence conserved in 8 of 10 positions relative to the consensus ERE. Two copies of this sequence conferred estrogen inducibility (4-fold) when placed upstream of the distal promoter in P-CAT. To examine ER-dependent stimulation of the two PR gene promoters by cAMP, P-CAT and P'-CAT reporter constructs containing two upstream consensus EREs were cotransfected into ER-negative 3T3 cells with an ER expression vector. Induction by E2 was greater than 50-fold for both constructs. Treatment of the cells with agents that increase intracellular cAMP levels, namely cholera toxin plus isobutyl methylxanthine, resulted in CAT activity that was 8% and 51% of the E2-stimulated activity for the P and P' constructs, respectively.
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PMID:Cloning of the rat progesterone receptor gene 5'-region and identification of two functionally distinct promoters. 814 66

Cathepsin D is an estrogen (17 beta-estradiol, E2)-inducible lysosomal protease. A putative estrogen receptor (ER)-Sp1-like sequence (GGGCGG(n)23ACGGG) has been identified in the non-coding strand of the cathepsin D promoter (-199 to -165), and electromobility shift assays of nuclear extracts from MCF-7 and HeLa cells confirm that both the ER and Sp1 protein bind to 32P-labeled ER/Sp1 oligo. For example, nuclear extracts from MCF-7 cells bind to the 32P-labeled ER/Sp1 oligo; however, ER/Sp1 binding can be decreased by selective competition with excess unlabeled estrogen responsive element and Sp1 oligos, immunodepletion with ER or Sp1 antibodies, and by treating cells with ICI 164,384, an antiestrogen which inhibits formation of ER homodimer. Moreover, E2-induced chloramphenicol acetyltransferase (CAT) activity in MCF-7 cells cotransfected with a human estrogen receptor expression plasmid and a plasmid containing an ER/Sp1 sequence cloned upstream to a thymidine kinase promoter and a CAT reporter. In cotreatment studies, ICI 164,384 inhibited E2-induced CAT activity. In contrast, E2 did not induce CAT activity in MCF-7 cells transfected with plasmids containing mutations in the ER or Sp1 segments of the ER/Sp1 oligo, thus confirming that both cognate binding sites are required for estrogen responsiveness.
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PMID:Estrogen receptor-Sp1 complexes mediate estrogen-induced cathepsin D gene expression in MCF-7 human breast cancer cells. 819 46

The present studies have examined the sequences responsible for regulating transcription of the human DF3 breast carcinoma-associated antigen (MUC1) gene. A region 1656 base pairs upstream to the DF3 transcription initiation site was fused to the chloramphenicol acetyltransferase gene. Transient expression assays using a series of deleted constructs demonstrated that the region from position -618 contains the regulatory sequences necessary for DF3 transcription in human MCF-7 breast cancer cells. Further analysis with internal deletion vectors and heterologous promoter constructs indicated the involvement of cis-acting elements in the fragment extending from positions -598 to -485. By gel retardation and DNA footprinting, we have identified a protein in MCF-7 cells that recognizes sequences between positions -505 and -485. The results of Southwestern studies demonstrate that this protein has an apparent molecular mass of 45 kDa. Taken together, these results suggest that DF3 gene transcription is regulated by a previously undescribed transacting factor.
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PMID:Characterization of cis-acting elements regulating transcription of the human DF3 breast carcinoma-associated antigen (MUC1) gene. 841 33


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