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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

While steroid response is generally restricted by the availability of steroid receptors, the theoretical limits of the response are not known. We have constructed a series of cell lines that stably express the estrogen receptor (ER) at levels up to 5,000,000 ERs per cell and employed these cells to explore the limits of the estrogen response. Several reporter genes with estrogen response elements upstream of the herpes thymidine kinase promoter showed hyperbolic saturation kinetics with increasing ER. Maximum response was 10 times that seen in cell lines with receptor titers comparable to physiological levels. Half-maximal responses required 500,000 receptors per cell, and cells with 5,000,000 ERs showed greater than 90% maximum induction. Estradiol dose-response studies indicated that the receptors are limiting below 500,000 ERs per cell, but at higher ER titers there are spare receptors. In contrast to most reporters, the widely used reporter pA2-CAT, which has 200 base pairs of Xenopus vitellogenin DNA between the response element and the promoter, showed squelching at ER levels beyond 500,000 per cell. Cell lines that expressed ER above this level activated pA2-CAT with a distorted hormone dependence, where saturating ligand concentrations were inhibitory. All reporters displayed squelching when the ER was provided by transient transfection at a level that we judge is 20,000,000 per cell by extrapolation from the behavior of stable cell lines. These findings suggest that saturation of the cellular capacity to mediate an estrogen response and ER-dependent squelching occur at receptor titers well above those encountered in nature. If current models of steroid hormone action are correct, the findings also imply that estrogen response elements are occupied to very small extents under normal conditions.
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PMID:The limits of the cellular capacity to mediate an estrogen response. 156 62

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

We have previously shown that the intracellular content of c-fos mRNA is rapidly induced (within 1 to 3 hours) in ovariectomized rat or mouse uteri following administration of estradiol. This induction is sensitive to actinomycin D but not to protein synthesis inhibitor puromycin, indicating an effect of estradiol at the transcriptional level, possibly mediated by the estrogen receptor. We have used transient transfection assays with defined regions of the mouse c-fos gene ligated to a reporter plasmid expressing chloramphenicol acetyl transferase to study regulation of this gene by estrogens. These recombinants were transfected in two different estrogen-responsive cell lines, GH4 and MCF-7, and stimulated with estradiol. A two- to five-fold induction of chloramphenicol acetyl transferase activity was observed with a construct containing the intact c-fos promoter and 351 bases of 5'-flanking sequence (-351/+44). A similar induction by estrogen is observed with the endogenous c-fos gene in the two cell lines as determined by RNA blot analysis. Estrogen induction is lost when a construct containing -135/+44 region of the c-fos gene is transfected. Plasmid containing the consensus estrogen response element GGTCAnnnTGACC derived from vitellogenin gene is induced 10- to 50-fold in both estrogen-responsive cell lines. Under identical conditions, the oligonucleotide containing the perfect palindrome GGTCTnnnAGACC, present around the -209 region of the c-fos gene, is completely silent when transfected under the control of thymidine kinase promoter. Additional transfection analysis with a number of c-fos promoter constructs has narrowed the estrogen response region to within the -278 to -135 region upstream of the c-fos promoter.
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PMID:Presence of an estradiol response region in the mouse c-fos oncogene. 180 51

Tamoxifen, nafoxidine, and clomiphene (1 x 10(-5) M) cause 5- to 15-fold increases in transient expression of plasmids transfected into rat somatomammotrophic pituitary tumor cell lines. To be effective, the antiestrogen must be present during the calcium phosphate transfection though it does not enhance the nuclear uptake or stability of transfected plasmid. The effect occurs with mammalian (rat growth hormone, mouse metallothionein I) or viral (thymidine kinase, Rous sarcoma virus) promoters and is inhibited by prior exposure of cells to high concentrations of estradiol but not glucocorticoid, progesterone or testosterone. Cis-tamoxifen, a conformation with much lower affinity for the estrogen receptor, has only one-fifth the effect of tamoxifen. Neither estradiol nor diethylstilbestrol have similar effects. Tamoxifen also increases endogenous rat growth hormone mRNA in these pituitary tumor cell lines. Transient expression in a number of other cell lines (JEG-3, COS-7, PC-12) is unaffected by tamoxifen suggesting the effect may be cell-type specific though MCF-7 cells are slightly responsive. The mechanism for the potent stimulation of gene transcription by these agents is not apparent but may be relevant to the mechanism of action of these agents as estrogen antagonists in vivo.
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PMID:Antiestrogens stimulate expression of transiently transfected and endogenous genes in rat pituitary tumor cell lines. 181 97

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

This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.
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PMID:Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene. 193 51

Although the estrogen responsiveness and estrogen receptors of Xenopus hepatocytes have been well described, oocytes of this species have not previously been shown to contain estrogen receptors (ER). Recombinant human ER (HER) was expressed in oocytes in a dose dependent fashion as measured by [35S]methionine incorporation into newly synthesized proteins. Chloramphenicol acetyl transferase (CAT) reporter plasmids, driven by a herpes simplex thymidine kinase promotor with or without a 17 base pair estrogen response element (ERE) from the vitellogenin A2 gene, were also injected into oocytes. When injected without the accompanying HER sequences, the construct containing the ERE expressed 10-fold more CAT activity, and this response was saturable as demonstrated by injecting increasing amounts of reporter plasmid. These results suggest either the activity of small amounts of a Xenopus ER (measured here by LH-20 assay), or the presence of some endogenous oocyte protein other than the ER that can interact with this ERE. When HER was co-expressed with ERECAT, CAT expression was suppressed over a wide range of HER concentrations. This unexpected repression may be due to displacement of an estrogen receptor or other endogenous oocyte regulatory protein on the ERE. HER's positive regulatory activity may require transcription factors that are lacking or insufficient in the oocyte. Alternatively the simple 17 base pair ERE may not provide DNA binding sites for such transcription factors.
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PMID:Human estrogen receptor introduced into the Xenopus oocyte represses expression from an artificial frog estrogen response element. 203 57

To examine the functional relationship between distinct cis-active elements within the distal enhancer region of the rat PRL gene, we have used deletional and mutational analysis of that region in transient transfection studies in GH3 pituitary tumor cells. Results from these studies demonstrate that the region of the PRL distal enhancer containing the Pit-1-binding sites is critical not only for enhancer activity and the response to cAMP, but also for the response to estradiol. An interaction of the estrogen receptor with factors conferring basal enhancer activity is suggested by studies with a mutant distal enhancer region in which the PRL estrogen response element was converted to a palindromic estrogen response element. To directly examine potential interactions, cotransfection studies using PRL distal enhancer reporter gene constructs and expression vectors for Pit-1 and rat estrogen receptor were performed in two heterologous cell lines. The activity of the reporter gene under the control of the PRL distal enhancer linked to either the thymidine kinase promoter or the PRL proximal promoter was not significantly altered by cotransfection with the Pit-1 expression vector in COS-1 or RAT-1 cells. Coexpression of these reporter constructs and an expression vector for estrogen receptor resulted in only a slight response to estradiol. However, when both Pit-1 and estrogen receptor were cotransfected with the distal enhancer reporter gene, a marked induction was observed in response to estradiol, and this activity was dependent upon the concentration of the Pit-1 expression vector.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene. 208 92

Estrogen and androgen receptors within the liver have been reported to modulate the hepatic regenerative response to partial hepatectomy. Moreover, cyclosporine has several untoward effects that might occur as a consequence of alterations in sex hormone activity. To evaluate these questions the following experiments were performed. Estrogen and androgen receptors in cytosol were quantitated in livers of rats treated with cyclosporine or olive oil vehicle before and after partial hepatectomy or a sham operation. Ornithine decarboxylase activity and thymidine kinase activity were assessed as indices of hepatic regeneration. Preoperative levels of estrogen receptor activity in the hepatic cytosol were significantly greater in rats treated with cyclosporine as compared to vehicle treated controls (P less than 0.01). In contrast, preoperative levels of androgen receptor activity in the cyclosporine-treated and vehicle-treated animals were similar. Following partial hepatectomy, a reduction in the activity of both sex hormone receptors in the hepatic cytosol was observed and was compatible with results described previously in normal animals. Unexpectedly the preoperative levels of ornithine decarboxylase (P less than 0.01) and thymidine kinase activity (P less than 0.01) were significantly greater in the rats treated with cyclosporine as compared to the vehicle treated controls. As expected, ornithine decarboxylase activity (at 6 hr) and thymidine kinase activity (at 24 hr) rose and peaked in response to a partial hepatectomy but were significantly greater (P less than 0.05) in the rats treated with cyclosporine as compared to the vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of cyclosporine on hepatic cytosolic estrogen and androgen receptor levels before and after partial hepatectomy. 229 96


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