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

---

Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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)
...
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.
...
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.
...
PMID:Regulation by estrogen through the 5'-flanking region of the mouse calbindin-D28k gene. 777 78

Human progesterone target tissues contain two progesterone receptors: B-receptors (hPRB), which are 933 amino acids in length, and A-receptors (hPRA), which lack the N-terminal 164 amino acids. The two isoforms differ functionally when they are occupied by agonists or antagonists. We postulated that the unique 164-amino acid, B-upstream segment (BUS) is in part responsible for the functional differences between the two isoforms and have constructed a series of hPR expression vectors encoding BUS fused to isolated down-stream functional domains of the receptors. These include the two transactivation domains: activation function-1 (AF1), located in a 90-amino acid segment just up-stream of the DNA-binding domain (DBD) and nuclear localization signal (NLS), and AF2, located in the hormone-binding domain. BUS is a highly phosphorylated domain, and contains the serine residues responsible for the hPRB triplet protein structure. The construct containing BUS-DBD-NLS binds tightly to DNA when aided by accessory nuclear factors. In HeLa cells, BUS-DBD-NLS strongly and autonomously activates transcription of chloramphenicol acetyltransferase (CAT) from a promoter containing two progesterone response elements (PRE2-TATAtk-CAT). Transcription levels with BUS-DBD-NLS are equivalent to those seen with full-length hPRB, and are higher than those seen with hPRA. BUS specifically requires an intact hPR DBD to be transcriptionally active. DBD mutants that cannot bind DNA or whose DNA binding specificity has been switched to an estrogen response element cannot cooperate in BUS transcriptional activity. The function of BUS-DBD-NLS is promoter and cell specific. It does not transactivate a CAT reporter driven by the mouse mammary tumor virus promoter in HeLa cells and poorly transactivates PRE2-TATAtk-CAT in PR-negative T47D breast cancer cells. However, in the breast cancer cells, BUS-DBD-NLS transactivation of PRE2-TATAtk-CAT can be reconstituted by either elevating cellular levels of cAMP or linking BUS and DBD to AF1 or AF2 of hPR, each of which alone is also inactive in these cells. We conclude that hPRB contains a unique third activation function (AF3) located within BUS and requiring the functional DBD of hPR. Depending on the promoter or cell tested, AF3 can activate transcription autonomously, or it can functionally synergize with AF1 or AF2. Autonomous AF3 function may explain the unexpected transactivating actions of antiprogestin-occupied hPRB, an issue of importance in hormone-resistant breast cancers and in tissue-specific agonist-like effects of hormone antagonists.
...
PMID:A third transactivation function (AF3) of human progesterone receptors located in the unique N-terminal segment of the B-isoform. 785 52

Synthetic ligands for steroid receptors represent important drugs in the control of fertility and in the therapy of a large variety of endocrinological diseases. In the present study we describe the establishment of different biochemical and molecular biological screening methods. We developed a microtiter plate assay for the induction of the de novo synthesis of alkaline phosphatase in T47D cells as a suitable and fast system for the measurement of actions of progestogenic and antiprogestogenic compounds. We compared several progestogenic activities with relative molar binding affinities (RBA) to the progesterone receptor. The ED50 values for the induction of alkaline phosphatase are in good accordance with RBA to the progesterone receptor. Furthermore, glucocorticoid and antiglucocorticoid effects were measured in the stable transfected breast cancer cell line ZR75/-763AGP-CAT. The construct AGP-CAT contains the glucocorticoid responsible element of the rat alpha-1-acid glycoprotein (AGP) gene with the bacterial chloramphenicol acetyltransferase (CAT) gene. The rat hepatoma Reuber cell line H4-II-E with the tyrosine aminotransferase gene is a further suitable marker of glucocorticoid action and was used as a second model for glucocorticoid activity. Thus, we demonstrated in three cell systems the antiprogestogenic and antiglucocorticoid activities of the model compound mifepristone.
...
PMID:Normal and stable transfected cancer cell lines: tools for a screening of progestogenic, antiprogestogenic and antiglucocorticoid substances. 788 82

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.
...
PMID:Cloning of the rat progesterone receptor gene 5'-region and identification of two functionally distinct promoters. 814 66

MDA-MB-231 human breast cancer cells express the aryl hydrocarbon (Ah) receptor; however, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) does not induce CYP1A1 gene expression or chloramphenicol acetyltransferase (CAT) activity in cells transiently transfected with pRNH11c, and Ah-responsive plasmid derived from the 5'-flanking region of the human CYP1A1 gene. However, when MDA-MB-231 cells were treated with 10 nM TCDD and co-transfected with pRHN11c and a human estrogen receptor (hER) expression plasmid (delta hER), there was approximately a 10-fold increase in CAT activity. The restoration of Ah-responsiveness in MDA-MB-231 cells by expression of nuclear hER was highly specific since parallel studies in which plasmids that express the progesterone receptor and Jun nuclear proteins did not restore Ah-responsiveness to this cell line. Moreover, in cells transiently transfected with the pRNH11c and delta hER plasmids and 10 nM TCDD, overexpression of the Jun protein inhibited the effects of the hER on Ah-responsiveness. Plasmids that express truncated forms of the hER were also active in MDA-MB-231 cells but were not as effective as the complete hER. These studies reveal a unique function for the ER in MDA-MB-231 cells in which expression of this protein results in restoration of Ah-responsiveness.
...
PMID:Restoration of aryl hydrocarbon (Ah) responsiveness in MDA-MB-231 human breast cancer cells by transient expression of the estrogen receptor. 820 98

The mRNA for transforming growth factor beta 3 (TGF-beta 3) includes a long (1.1-kb) 5' noncoding region which exerts a potent inhibitory effect on translational efficiency. We now report that many human breast cancer cell lines (T47-D, SK-BR-3, ZR-75-1, and BT-474) express two mRNA species for TGF-beta 3: the 3.5-kb transcript previously described as the only TGF-beta 3 mRNA species in cells and a novel 2.6-kb transcript which lacks approximately 870 nucleotides from the 5' noncoding region. The 5' end of the shorter transcript was sequenced, establishing it to be a 5' truncation of the full-length TGF-beta 3 transcript. Estradiol decreased mRNA levels of both TGF-beta 3 mRNA transcripts to an equivalent degree in estrogen receptor-positive cells. In contrast, the synthetic progestin gestodene altered the relative abundance of the two transcripts, preferentially diminishing the expression of the 2.6-kb transcript. The potential for enhanced mRNA translation attributable to the shorter 5' noncoding region was evaluated by transfection of cells with chimeric plasmid constructs in which the transcription unit consisted of coding sequence for chloramphenicol acetyltransferase downstream of the 5' noncoding sequence from TGF-beta 3. The translational efficiency of chloramphenicol acetyltransferase-encoding mRNA containing the shorter 5' noncoding region of the 2.6-kb TGF-beta 3 transcript was approximately seven times greater than with the full-length 5' noncoding region of TGF-beta 3. Polysome analysis of TGF-beta 3 mRNA in SK-BR-3 cells supported the hypothesis that the 2.6-kb transcript was more actively engaged in translation.
...
PMID:Enhanced translational efficiency of a novel transforming growth factor beta 3 mRNA in human breast cancer cells. 826 30

The 5'-sequences flanking the human MUC1 gene have been analyzed for their ability to direct expression of a reporter gene (the chloramphenicol acetyltransferase gene (CAT)) in cell lines that normally express or do not express the MUC1 gene. A construct containing 2.9 kilobase pairs of MUC1 5'-flanking sequence sequence showed expression of CAT in breast and pancreatic cell lines but not in the non-epithelial cell lines HT-1080, SK23, and HTB96. Deletion analysis showed that maximum expression was obtained in ZR-75 (breast cancer line) and HPAF (pancreatic cancer line) with only 743 base pairs of 5'-flanking sequence. Sequences within 1.6 kilobase pairs of the transcriptional start site showed enhancing activity in a vector carrying an enhancerless SV40 promoter. Analysis of proximal 5'-sequences in a promoterless CAT vector carrying the SV40 enhancer showed that sequences between -60 and -150 were crucial for tissue-specific expression. An Sp1 site at -99/-90 and an E box (E-MUC1) at -84/-64 in this region were shown by mutational analysis to play a role in the regulation of transcription. Gel shift analysis with oligonucleotides and nuclear extracts of ZR-75 showed protein binding to both of these sites. Sp1 binding activity was similar in ZR-75 and HT1080 cells, whereas binding of factors to the E-MUC1 oligonucleotide revealed quantitative and qualitative differences between epithelial and non-epithelial cells.
...
PMID:Analysis of the tissue-specific promoter of the MUC1 gene. 838 9

The protein kinase A stimulator cAMP can potentiate the ability of progestins to induce the transactivation function of the human progesterone receptor (hPR). We questioned in the present study whether cAMP could functionally cooperate with the progestin antagonist RU486. In T47D human breast cancer cells, RU486 behaves as a pure antagonist with respect to induction of the progesterone-responsive mouse mammary tumor virus chloramphenicol acetyltransferase (MMTV-CAT) reporter gene. It fails to stimulate MMTV-CAT expression and completely inhibits induction by the synthetic progestin R5020. However, when RU486 is combined with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), MMTV-CAT is induced to levels approaching that stimulated by R5020 alone. Also, RU486 in the presence of 8-Br-cAMP is only partially effective in antagonizing R5020 action. The agonist activity exhibited under these conditions appears to be due to RU486 acting through hPR as evidenced by the fact that 8-Br-cAMP alone has no effect on MMTV-CAT, whereas induction by the combination of 8-Br-cAMP and RU486 is dose responsive to RU486 in a saturable manner and can be inhibited by the type I antiprogestin (prevents hPR-DNA binding) ZK98299, which does not exhibit positive functional cooperation with cAMP. Acquisition of agonist activity in the presence of 8-Br-cAMP also extends to the type II antiprogestin (permits hPR-DNA binding) ZK112993. Since RU486 is also a type II antagonist, these results suggest that detection of functional synergism between cAMP and antiprogestins may require binding of the hPR-antagonist complex to DNA. We propose that cross-talk between second messenger and steroid receptor signal transduction pathways may be one mechanism for resistance to steroid antagonists that frequently develops in breast cancer.
...
PMID:The progesterone antagonist RU486 acquires agonist activity upon stimulation of cAMP signaling pathways. 838 50


<< Previous 1 2 3 4 5 6 7 Next >>

---