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)

Both neu gene overexpression and loss of estrogen receptor (ER) expression have been found to correlate with a poor prognosis in human breast cancer. Studies of breast tumor specimens have suggested that these two factors are not independent, leading us to hypothesize that there is a causal relationship between loss of ER and overexpression of neu. In this report, we confirm that ER can negatively regulate the expression of the neu gene protein product, p185neu, in two ER positive but not an ER negative breast cancer cell line(s). We have produced sublines which stably express human ER from a previously ER negative human breast cancer cell line. We demonstrate that the expression of ER in these cell lines is sufficient to confer the ability to respond to estradiol by down-regulating neu expression at both the protein and RNA levels. Utilizing neu promoter-chloramphenicol acetyltransferase constructs in transient cotransfection assays, we have also shown that this regulation occurs at the transcriptional level and requires the presence of both ER and estradiol. Furthermore, utilizing promoter deletion constructs, we provide evidence that a 140-base pair region of the neu promoter is required for this transcriptional regulation. When placed in a heterologous promoter, this 140-base pair region allows transcriptional repression by estradiol stimulated ER; thus, it represents an estrogen responsive region within the neu promoter. Finally, we have used gel mobility shift analysis to demonstrate an alteration in the nuclear factor(s) binding to this promoter region in estradiol stimulated versus estradiol deprived breast cancer cells. This study provides the first evidence that the inverse clinical correlation between neu and ER expression may be due to transcriptional repression of neu by estradiol stimulated ER.
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PMID:Transcriptional repression of the neu protooncogene by estrogen stimulated estrogen receptor. 135 36

We describe an assay employing the competitive binding of estrogen receptor (ER) with basal transcription factors on a constitutive promoter (cytomegalovirus-hormone response element[s]-chloramphenicol acetyltransferase [CMV-(HRE)n-CAT, containing a hormone response element(s) between the TATA box and the start site of transcription]) to examine the DNA-binding ability of the human ER in whole cells. We used this promoter interference assay to examine the DNA binding of ER in cell lines containing high and low levels of endogenous ER, as well as in CHO cells expressing wild-type and mutant ERs from cotransfected expression vectors. The ER is capable of binding to the promoter interference constructs in the absence of added ligand, and estrogen (estradiol) or antiestrogen (trans-hydroxytamoxifen or ICI 164,384) enhances or stabilizes this interaction. The binding of unoccupied ER to reporter gene activation plasmids results in ligand-independent transactivation, presumably due to the TAF-1 function of the receptor. DNA binding of ER in the absence of ligand is observed in cells containing endogenous ER, or expressed ER, and occurs in cells with high or low receptor contents. Although estrogen- and antiestrogen-occupied ER complexes bind to DNA and reduce the template promoter activity, the extent of suppression achieved by ICI-bound ERs is consistently less than that achieved with the other ligands, presumably caused by the fact that ICI rapidly reduces the level of ER in most of the cells examined. However, the ICI-ER complexes that remain are in sufficient quantity to bind to gene activation reporter constructs, and in these cells, ICI still behaves as a pure antagonist of gene transcription and does not activate reporter genes. Hence, obstruction of ER DNA binding or reduction of ER in target cells may contribute to, but cannot fully explain, the pure antagonist character of the antiestrogen ICI 164,384. In addition, DNA binding by the ER alone is clearly not sufficient for ensuring full activation of transcription and argues for an intermediate in the receptor activation of promoters.
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PMID:Examination of the DNA-binding ability of estrogen receptor in whole cells: implications for hormone-independent transactivation and the actions of antiestrogens. 140 42

Most oral contraceptives (OC) contain a progestin in combination with an estrogen, and the progestin component in OC includes one of the following 19-nortestosterone derivatives: norethynodrel; norethindrone; or norgestrel (levonorgestrel). It is well known that estrogens promote the growth of breast cancer. However, progestins have recently also been implicated in the development of breast cancer. We have compared and contrasted the ability of synthetic progestins to stimulate the proliferation of cultured human breast cancer cells and examined their possible mechanism of action. We found that some progestins used in OC were able to stimulate the growth of estrogen receptor-positive (ER+) MCF-7 and T47DA18 human breast cancer cells but not ER- MDA-MB-231, BT-20, and T47DC4 human breast cancer cells. However, two other progestins, MPA and R5020, which are not used in OC, were either not able to stimulate or only slightly stimulated growth. The potency of norethynodrel [median effective dose (EC50) = 4 x 10(-8) M] and norethindrone (EC50 = 3 x 10(-8) M) was greater than norgestrel (EC50 = 2 x 10(-7) M) in MCF-7 cells. E2 (EC50 = 8 x 10(-13) M) was an even more potent stimulator of growth. More importantly, the progestin-induced growth stimulation was blocked by the antiestrogens 4-hydroxytamoxifen and ICI 164,384 but not the antiprogestin 17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)-17 alpha-(1-propynyl)-estra-4, 9-dien-3-one (RU486). To determine whether the proliferative action of progestins was mediated through the ER, cells were transfected with a chloramphenicol acetyltransferase reporter gene containing an estrogen response element derived from vitellogenin 2A gene. The progestins which stimulated the growth of breast cancer cells also increased chloramphenicol acetyltransferase activity. The induction of chloramphenicol acetyltransferase activity was blocked by the addition of the antiestrogens 4-hydroxytamoxifen and ICI 164,384 but not the antiprogestin RU486. This study provides direct evidence that the 19-nortestosterone derivatives in OC have estrogenic properties and suggests that activation of ER, but not progesterone receptor, is the growth-stimulatory mechanism for these synthetic progestins. Our results may help to explain the conflicting evidence linking OC and breast cancer risk. A rigorous evaluation of the "total" estrogenic potential of OC might produce a better correlation with breast cancer risk.
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PMID:Estrogenic potential of progestins in oral contraceptives to stimulate human breast cancer cell proliferation. 142

The 5'-flanking region of the human lactoferrin gene was isolated from a human placental genomic library. This genomic clone contains a 16-kilobase pair (kbp) insert and produces seven fragments when digested with the SacI restriction enzyme. We sequenced one of the fragments that comprises 1294 bp of the 5'-flanking sequence, 79 bp of the first exon, and 690 bp of the first intron. A major transcription start site was mapped by primer extension. The region immediately upstream from the transcription initiation site following the first exon is abundant in G and C nucleotides. In the promoter and 5'-flanking region within a 300-bp stretch (-465 to -165) of the DNA, we found a noncanonical TATA box (ATAAA), CAAT-like sequence (CAAC) and sequences homologous to the consensus SP1 binding site, Pu.1/Sp.1 binding element (PU box), two half-palindromic estrogen response elements (EREs; GGTCA), an imperfect ERE (GGTCAAGGCGATC), and a sequence resembling the chicken ovalbumin upstream promoter transcription factor (COUP-TF) binding site (GTCTCACAGGTCA). The COUP-TF binding site and the imperfect ERE shared five nucleotides (GGTCA). With the exception of the two half-palindromic EREs, the elements with very well matched sequences were also found in the corresponding positions in the mouse lactoferrin gene. The synthetic oligonucleotide, including the 26 bp of COUP/ERE sequence, was cloned before the SV40 promoter in a chloramphenicol acetyltransferase reporter construct. These chimeric plasmids were transiently transfected into human endometrium carcinoma RL95-2 cells to assess hormone responsiveness. We found that the COUP/ERE element acted as an enhancer in response to estrogen stimulation. In vitro DNase I footprinting analysis showed binding of the estrogen receptor on the imperfect ERE. In contrast to the mouse lactoferrin COUP/ERE element, COUP-TF does not interact with this element, as demonstrated by band shift assay and site-directed mutagenesis. Therefore, the molecular mechanisms of the estrogen action that govern the lactoferrin gene expression differ between mouse and human.
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PMID:Differential molecular mechanism of the estrogen action that regulates lactoferrin gene in human and mouse. 148 Jan 83

We have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyltransferase, linked to regions of mouse c-fos, to identify a specific estrogen response element (ERE) in this protooncogene. This element is located in the untranslated 3'-flanking region of the c-fos gene, 5 kilobases (kb) downstream from the c-fos promoter and 1.5 kb downstream of the poly(A) signal. This element confers estrogen responsiveness to chloramphenicol acetyltransferase reporters linked to both the herpes simplex virus thymidine kinase promoter and the homologous c-fos promoter. Deletion analysis localized the response element to a 200-base pair fragment which contains the element GGTCACCACAGCC that resembles the consensus ERE sequence GGTCACAGTGACC originally identified in Xenopus vitellogenin A2 gene. A synthetic 36-base pair oligodeoxynucleotide containing this c-fos sequence conferred estrogen inducibility to the thymidine kinase promoter. The corresponding sequence also induced reporter activity when present in the c-fos gene fragment 3 kb from the thymidine kinase promoter. Gel-shift experiments demonstrated that synthetic oligonucleotides containing either the consensus ERE or the c-fos element bind human estrogen receptor obtained from a yeast expression system. However, the mobility of the shifted band is faster for the fos-ERE-complex than the consensus ERE complex suggesting that the three-dimensional structure of the protein-DNA complexes is different or that other factors are differentially involved in the two reactions. When the 5'-GGTCA sequence present in the c-fos ERE is mutated to 5'-TTTCA, transcriptional activation and receptor binding activities are both lost. Mutation of the CAGCC-3' element corresponding to the second half-site of the c-fos sequence also led to the loss of receptor binding activity, suggesting that both half-sites of this element are involved in this function. The estrogen induction mediated by either the c-fos or the consensus ERE was blunted by the antiestrogen tamoxifen. Based on these studies, we believe the 3'-fos ERE sequence we have identified may be a major cis-acting element involved in the physiological regulation of the gene by estrogens in vivo.
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PMID:Identification of an estrogen response element in the 3'-flanking region of the murine c-fos protooncogene. 151 37

Recent studies have demonstrated the presence of estrogen receptor (ER) in both normal human osteoblast-like and osteoblast-like osteosarcoma cells. The number of ER in cultured osteoblastic cells is very low (200-500 sites/cell). This has complicated characterization of the biological role of estrogens in bone cells. To study the responsiveness of bone cells to estrogens, we established osteoblast-like cell lines expressing higher ER levels. ROS 17/2.8, an osteoblastic cell line, was stably transfected with the cDNA encoding for the mouse ER. After a selection period, positive clones were isolated and evaluated for the presence of ER by both Northern blot analysis and ligand binding assays. Using these techniques, we detected a significant increase in the level of both ER transcript and binding compared to that in wild-type cells. The levels of expressed ER protein were similar to those reported in normal human osteoblast-like cells in primary culture (approximately 2000 sites/cell). To test whether the exogenously inserted ER was responsive, both wild-type and ER stably transfected cells were transiently transfected with a reporter construct containing an estrogen-responsive element linked to a truncated thymidine kinase promoter and a chloramphenicol acetyltransferase (CAT) reporter gene. Exposure of the cells to increased concentrations of estradiol induced a slight increase in CAT activity in wild-type cells (approximately 1.5-fold) at maximal stimulation; however, it provoked a clear concentration-dependent increase in CAT activity in the ER stably transfected cells, with a maximal stimulation of approximately 10-fold. This event was receptor mediated, since ICI 164,384, an ER antagonist, blocked the enhancement of estradiol-induced CAT activity, and it was specific, since other steroid hormones did not stimulate CAT activity. Finally, we evaluated the ability of ER to modulate an endogenous estrogen-responsive gene by measuring the activity of the enzyme alkaline phosphatase. In addition, diethylstilbestrol, a synthetic estrogen agonist, increased the activity of both the CAT reporter gene and the endogenous alkaline phosphatase enzyme. In summary, we have established osteoblast-like cells expressing high levels of an exogenously inserted ER, which has characteristics similar to those of the endogenous ER in terms of its Kd. Finally, the exogenous ER regulates both exogenously inserted construct (VITERECAT) and endogenous properties of the cells (enzymatic activity and proliferation).
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PMID:Estrogens modulate the responsiveness of osteoblast-like cells (ROS 17/2.8) stably transfected with estrogen receptor. 157 85

Transcription of the lactoferrin gene is stimulated by estrogen in mouse uterus. To study direct estrogen regulation of this gene at the molecular level, we cloned and analyzed the 5'-flanking region of the mouse lactoferrin gene. Sequence analysis revealed a putative estrogen-responsive element (ERE) overlapping with a chicken ovalbumin up-stream promoter (COUP) element located at position -349 to -329 from the transcription initiation site. The ERE element differed from the consensus ERE sequence by one nucleotide at the second position of the 3' half of the element (G to A); the COUP element differed by one nucleotide from the chicken COUP element. Synthetic oligonucleotide containing the mouse lactoferrin COUP/ERE element was inserted into the reporter chloramphenicol acetyltransferase vector, then transiently transfected into human endometrium carcinoma RL95-2 cells to assess hormone responsiveness. We found that the COUP/ERE element confers estrogen action to both homologous and heterologous promoters. Nuclear proteins from diethylstilbestrol-treated mouse uteri and proteins from estrogen receptor expression vector-transfected RL95-2 whole cell extract bound in vitro to COUP/ERE element specifically, as assessed by band-shift assay. By using antibodies specific to the estrogen receptor and the COUP transcription factor, we demonstrated that both proteins were present in mouse uterine tissue and interacted specifically with the COUP/ERE element, as shown by the superband shift. Competition experiments with specific ERE or COUP oligonucleotides also confirmed the interaction between lactoferrin COUP/ERE element with the estrogen receptor and the COUP transcription factor. Therefore, we named this sequence mERM, the mouse lactoferrin estrogen response module.
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PMID:Estrogen response module of the mouse lactoferrin gene contains overlapping chicken ovalbumin upstream promoter transcription factor and estrogen receptor-binding elements. 158 12

Chronic administration of estradiol inhibits transcription of the gene encoding the alpha-subunit of pituitary glycoprotein hormones. Here, we show, using transfection analyses and a filter binding assay, that 1500 basepairs of proximal 5' flanking sequence of the human alpha-subunit gene lack a functional estrogen response element when transfected into heterologous cell lines, and fail to bind estrogen receptor purified from calf uterus. Yet, this same region of the alpha-subunit gene confers estradiol responsiveness (transcriptional suppression) to the bacterial chloramphenicol acetyltransferase gene in transgenic mice. A smaller promoter fragment of the bovine alpha-subunit gene also confers responsiveness to estradiol in transgenic mice, suggesting that the same element may mediate the steroid responsiveness of both promoters. Furthermore, regulation by estradiol of the chimeric human or bovine alpha-chloramphenicol acetyltransferase genes is pituitary specific, underscoring the physiological significance of these studies. Based on these results, we conclude that estradiol regulates expression of the alpha-subunit gene in vivo through a mechanism that does not involve high affinity binding of estrogen receptor to the alpha-subunit gene. Whether this mechanism is manifest at the level of the pituitary or hypothalamus remains to be determined.
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PMID:Estradiol inhibits transcription of the human glycoprotein hormone alpha-subunit gene despite the absence of a high affinity binding site for estrogen receptor. 171 10

Previous work from this laboratory demonstrated that 17-beta estradiol (E2) can directly stimulate the transcription rate of the rat luteinizing hormone beta (LH beta) gene and that an upstream portion of the LH beta gene between -2.0 and -0.6 kilobases could confer an E2-stimulated response to a reporter gene in transient expression assays. To localize the LH beta estrogen response element (ERE) by biological function, portions of the 5'-flanking region of the LH beta gene or synthetic oligonucleotides were inserted in expression vectors next to the herpes simplex virus thymidine kinase promoter fused to the chloramphenicol acetyltransferase gene. Constructs were transfected into GH3 cells, and transfected cells were treated for 48 h with E2. E2 stimulation of activity (2-4-fold) occurred with constructs containing the 15-base pair palindromic sequence (GGACACCATCTGTCC), found at bases -1173 to -1159 relative to the transcriptional start site in the LH beta gene. A construct containing a synthetic oligonucleotide of this putative LH beta ERE was stimulated 1.7-3-fold by E2, while a construct containing two copies of the sequence was stimulated to a slightly higher level (2.5-4.0-fold). An oligonucleotide in which the palindrome was mutated failed to confer E2 stimulation, and mutation of the palindromic region within the upstream region of the LH beta gene also eliminated the E2 response. The anti-estrogen tamoxifen could not elicit a response, nor could dehydrotestosterone or dexamethasone; however, thyroid hormone treatment resulted in a 2-2.5-fold stimulation. The 15-base pair LH beta gene palindrome was found to bind estrogen receptor (ER) complex directly by gel retardation experiments. Labeled LH beta ERE DNA formed three complexes with proteins from immature rat uterine extract. Two of these were associated with ER complexes, as determined by the comigration of [3H] estradiol bound to ER with these complexes, and by the ability of anti-ER antibody to associate with these complexes. The affinity of the LH beta ERE for ER was calculated by Scatchard analysis to be 2.2-5.0 nM, an approximately 5-10-fold lower affinity than for the ERE in the vitellogenin A2 gene region. The mutated ERE, which had no biological activity, could not compete effectively for binding to ER. ER which was heat-transformed at 30 degrees C had a similar affinity (2-5 nM) for the ERE as ER occupied with E2 (2-4 nM), while ER occupied by estrone had a lower affinity (9 nM).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of an estrogen-responsive element in the rat LH beta gene. DNA-estrogen receptor interactions and functional analysis. 189 4

Mouse lactoferrin is expressed in a variety of tissues under different types of control. To understand how molecular mechanisms govern the mode of lactoferrin expression, we isolated and characterized the 5'-flanking region of the lactoferrin gene. Several clones containing lactoferrin gene fragments were isolated from a mouse (129/J) genomic library including clone lambda J14, which contains a 7.5-kilobase pair 5'-flanking sequence. Sequence analysis of the region flanking the transcription initiation site revealed the following: a TATA-like sequence, two CAAT boxes, three GC boxes including one within the first intron, an AP2 site, seven PU boxes, an AC-rich region, a B1 sequence, and an estrogen-responsive element consensus sequence over-lapping with a chicken ovalbumin upstream promoter-binding element. Footprinting analysis demonstrated that several regions, including the putative estrogen-responsive element region, in the 5'-flanking sequence were protected from DNase I digestion. Promoter fragments were cloned into a chloramphenicol acetyltransferase receptor plasmid to study functional activity. The mouse lactoferrin gene promoter was active in human endometrium carcinoma RL 95-2 cells and in rat glioma C6 cells. Multiple upstream elements modulated the basal transcriptional promoter activity. The transcription level directed by this minimal promoter was controlled by both positive (between -1739 and -922) and negative (between -2644 and -1739, and between -589 and -291) regulatory sequences. A tissue-specific regulatory sequence was critical for the establishment of lactoferrin expression in human endometrium carcinoma cells, but not in rat glioma cells located between -1739 and -922. Reporter plasmid 0.6 mL14-CAT, containing the estrogen-responsive element sequence, was estrogen-responsive in the presence of estrogen receptor in human endometrium carcinoma RL 95-2 cells.
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PMID:Characterization of estrogen-responsive mouse lactoferrin promoter. 193 12


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