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)

We have examined the effects of reversibly and irreversibly binding estrogenic and antiestrogenic ligands for the estrogen receptor on pS2 RNA accumulation in MCF-7 human breast cancer cells and on pS2-chloramphenicol acetyl transferase (CAT) fusion gene expression in transfected MCF-7 cells. In MCF-7 cells grown in the absence of estrogens, the reversibly binding estrogen, estradiol, and the affinity labeling estrogen, ketononestrol aziridine, KNA, evoked a 13-fold increase in pS2 RNA level. The reversibly binding antiestrogen trans-hydroxytamoxifen and the affinity labeling antiestrogens tamoxifen aziridine or desmethylnafoxidine aziridine behaved as partial agonists/antagonists. In thymidine kinase-chloramphenicol acetyltransferase (tk-CAT) fusion genes containing a 1000 base pair fragment of the pS2 5'-flanking region encompassing the estrogen responsive element of the gene [pS2 (-1100/-90) tk-CAT], estradiol and ketononestrol aziridine evoked a marked stimulation of CAT activity and, in transfected cells grown in both the presence or absence of the weak estrogen phenol red, the antiestrogens behaved as partial agonists/antagonists. This pS2 5'-flanking region displayed both estrogen-dependent and estrogen-independent enhancer activity as monitored by stimulation of CAT activity. Hormonal regulation of the transfected pS2 fusion gene was similar to that observed in the native pS2 gene of MCF-7 cells; however, antiestrogens, while still partial agonists-antagonists, were relatively more agonistic on the transfected fusion gene than on the native gene. One antiestrogen (ICI 164,384) that behaved as a pure estrogen antagonist on the native gene was a partial agonist-antagonist of pS2 gene expression in the plasmid. This study illustrates that the hormonal regulation of the pS2 gene, as characterized by the agonist-antagonist balance of estrogens and antiestrogens, is influenced by the DNA context of the pS2 estrogen responsive element. Also, the fact that estrogens and antiestrogens that form covalent bonds with the estrogen receptor modulate activity of the native pS2 gene and the pS2-tk-CAT fusion gene in a manner similar to that of their reversibly binding counterparts suggests that it may be possible to use these irreversibly binding ligands to follow the interaction of hormone-receptor complexes with regions regulating estrogenic stimulation of the pS2 gene.
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PMID:Regulation of pS2 gene expression by affinity labeling and reversibly binding estrogens and antiestrogens: comparison of effects on the native gene and on pS2-chloramphenicol acetyltransferase fusion genes transfected into MCF-7 human breast cancer cells. 246 Jul 49

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

The role of transforming growth factor-beta1 (TGFbeta1) in the regulation of estrogen receptor (ER) expression in MCF-7 cells was investigated. After treatment of the cells with 100 pM TGFbeta1, ER protein declined by about 30% at 6 h from a concentration of 413.5 fmol/mg protein in control cells to 289.5 fmol/mg protein in treated cells. The concentration of receptor remained suppressed for 24 h. Scatchard analysis demonstrated that the decrease in ER protein corresponded to a decrease in estradiol-binding sites, with no effect on the binding affinity of the ER. The dissociation constant of the estradiol-ER complex was 0.117 nM in TGFbeta1-treated cells compared to 0.155 nM in control cells. Treatment with TGFbeta1 did not influence the half-life of the ER. In TGFbeta1-treated cells, as well as in control cells, the half-life of the receptor was approximately 4 h. In contrast to the effect on ER concentration, TGFbeta1 treatment resulted in a greater decrease in the steady state level of ER messenger RNA (approximately 75%) at 6 h. By 24 h, a small recovery in the amount of messenger RNA was observed. Transcription run-on experiments demonstrated a decrease of approximately 70% in the level of ER gene transcription at 3 h. Transient transfections using an ER promoter-chloramphenicol acetyltransferase construct demonstrated that after TGFbeta1 treatment, chloramphenicol acetyltransferase activity decreased by 50%, suggesting that TGFbeta1 inhibition of the ER gene transcription is mediated through the ER promoter. Although treatment with TGFbeta1 decreased the ER concentration, the growth factor had no effect on the activity of ER, as measured by its effects on estradiol induction of progesterone receptor and pS2, suggesting that TGFbeta1 does not inhibit proliferation of MCF-7 cells by blocking ER activity.
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PMID:The role of transforming growth factor-beta in the regulation of estrogen receptor expression in the MCF-7 breast cancer cell line. 907 8

To determine whether arsenite has estrogen-like activities, the effects of this compound on estrogen receptor-alpha (ERalpha) and other estrogen-regulated genes were measured in the human breast cancer cell line MCF-7. Treatment of cells with 1 microM arsenite resulted in a 60% decrease in the amount of ERalpha and in a parallel decrease of 40% in ERalpha messenger RNA. Progesterone receptor concentration increased 22-fold after arsenite treatment. pS2 messenger RNA also increased 2. 1-fold after treatment. The induction of progesterone receptor and pS2 was blocked by the antiestrogen ICI-182,780. In transient cotransfection experiments of wild-type ERalpha and an estrogen response element-reporter construct, arsenite stimulated chloramphenicol acetyltransferase (CAT) activity. In growth assays, arsenite significantly stimulated the proliferation of MCF-7 cells compared with cells grown in estrogen-depleted medium. Addition of an antiestrogen blocked growth stimulation by arsenite. In binding assays, arsenite blocked the binding of estradiol to ERalpha (Ki = 5 +/- 0.5 nM; n = 3), suggesting that the compound interacts with the hormone-binding domain of the receptor. To determine whether interaction of arsenite with the hormone-binding domain results in receptor activation, COS-1 cells were transiently cotransfected with the chimeric receptors GAL-ER, which contains the hormone-binding domain of ERalpha and the DNA-binding domain of the transcription factor GAL4, and a GAL4-responsive CAT reporter gene. Treatment of cells with estradiol or arsenite resulted in a 4-fold increase in CAT activity. The effects of arsenite on the chimeric receptor were blocked by the antiestrogen, suggesting that arsenite activates ERalpha through an interaction with the hormone-binding domain of the receptor. Transfection assays with ERalpha mutants identified C381, C447, H524, and N532 as interaction sites of arsenite with the hormone-binding domain.
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PMID:Effects of arsenite on estrogen receptor-alpha expression and activity in MCF-7 breast cancer cells. 1101 13

The ability of metals to activate estrogen receptor-alpha (ERalpha) was measured in the human breast cancer cell line, MCF-7. Similar to estradiol, treatment of cells with the divalent metals copper, cobalt, nickel, lead, mercury, tin, and chromium or with the metal anion vanadate stimulated cell proliferation; by d 6, there was a 2- to 5-fold increase in cell number. The metals also decreased the concentration of ERalpha protein and mRNA by 40-60% and induced expression of the estrogen-regulated genes progesterone receptor and pS2 by1.6- to 4-fold. Furthermore, there was a 2- to 4-fold increase in chloramphenicol acetyltransferase activity after treatment with the metals in COS-1 cells transiently cotransfected with the wild-type receptor and an estrogen-responsive chloramphenicol acetyltransferase reporter gene. The ability of the metals to alter gene expression was blocked by an antiestrogen, suggesting that the activity of these compounds is mediated by ERalpha. In binding assays the metals blocked the binding of estradiol to the receptor without altering the apparent binding affinity of the hormone (K(d) = 10(-10) M). Scatchard analysis employing either recombinant ERalpha or extracts from MCF-7 cells demonstrated that (57)Co and (63)Ni bind to ERalpha with equilibrium dissociation constants of 3 and 9.5 x 10(-9) and 2 and 7 x 10(-9) M, respectively. The ability of the metals to activate a chimeric receptor containing the hormone-binding domain of ERalpha suggests that their effects are mediated through the hormone-binding domain. Mutational analysis identified amino acids C381, C447, E523, H524, N532, and D538 as potential interaction sites, suggesting that divalent metals and metal anions activate ERalpha through the formation of a complex within the hormone-binding domain of the receptor.
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PMID:Estrogen-like activity of metals in MCF-7 breast cancer cells. 1274 4