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)

Expression of prostate-specific antigen (PA) mRNA was tested at various time periods after incubation of the human prostate tumor cell line LNCaP with the synthetic androgen R1881. Androgen-stimulated expression was observed within 6 h after addition of R1881 to the cells. Run-on experiments with nuclei isolated from LNCaP cells showed that expression of the PA gene could be regulated by R1881 on the level of transcription. DNase I footprints of the promoter region of the PA gene (-320 to +12) with nuclear protein extracts from LNCaP cells showed at least four protected regions. The protected areas include the TATA-box, a GC-box sequence, and a sequence AGAACAgcaAGTGCT at position -170 to -156, which closely resembles the reverse complement of the consensus sequence GGTACAnnnTGTTCT for binding of the glucocorticoid receptor and the progesterone receptor. Fragments of the PA promoter region were cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression plasmid into COS cells in a transient expression assay. CAT activity of COS cells grown in the presence of 1 nM R1881 was compared to untreated controls. A 110-fold induction of CAT activity was found if a -1600 to +12 PA promoter fragment was used in the construct. By further deletion mapping of the PA promoter a minimal region (-320 to -155) was identified as being essential for androgen-regulated gene expression. Mutation of the sequence AGAACAgcaAGTGCT (at -170 to -156) to AAAAAAgcaAGTGCT almost completely abolished androgen inducibility of the reporter gene constructs. One or more copies of the sequence AGAACAgcaAGTGCT cloned in front of a thymidine kinase promoter-CAT reporter gene confers androgen regulation to the reporter gene. These findings provide strong evidence for transcription regulation of the PA gene by androgens via the sequence AGAACAgcaAGTGCT. Interestingly, in addition to the AGAACAgcaAGTGCT element, an upstream region (-539 to -320) is needed for optimal androgen inducibility of the PA promoter.
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PMID:The promoter of the prostate-specific antigen gene contains a functional androgen responsive element. 172 87

Transcription of the chicken ovalbumin gene is induced both in vivo and in vitro by four classes of steroid hormones. Recent experiments identified a steroid-dependent regulatory element (SDRE) in the 5'-flanking region of the ovalbumin gene between -900 and -521. To characterize the regulatory properties of the SDRE more precisely, additional mutations were created in this region, and fusion genes prepared by linking the ovalbumin 5'-flanking region and promoter to the chloramphenicol acetyltransferase structural gene. When the ovalbumin-chloramphenicol acetyltransferase fusion genes were transfected into steroid-responsive primary oviduct cells, mutants lacking sequences between -900 and -732 were no longer responsive to estrogen, corticosterone, progesterone, or dihydrotestosterone. The SDRE did not confer steroid-dependent expression on the heterologous thymidine kinase promoter by itself but did in conjunction with the negative regulatory element identified between -350 and -100. This suggests that the two elements act as a single functional entity and that the SDRE is not behaving as a typical steroid response element. Gel shift analyses revealed that two SDRE.protein complexes were formed when nuclear protein extracts were derived from estrogen-treated chicken oviduct but that only one complex was formed with extracts from estrogen-withdrawn oviduct or from other tissues. Neither an estrogen response element oligomer nor a glucocorticoid/progesterone response element oligomer competed for either of the DNA.protein complexes. Partially purified progesterone receptor also did not bind to the SDRE. These data indicate that induction of the ovalbumin gene by steroid hormones requires complex interactions involving both the SDRE and the negative regulatory element.
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PMID:The steroid-dependent regulatory element in the ovalbumin gene does not function as a typical steroid response element. 233 44

To define the recognition sequence of the glucocorticoid receptor and its relationship with that of the progesterone receptor, oligonucleotides derived from the glucocorticoid response element of the tyrosine aminotransferase gene were tested upstream of a heterologous promoter for their capacity to mediate effects of these two steroids. We show that a 15-base-pair sequence with partial symmetry is sufficient to confer glucocorticoid inducibility on the promoter of the herpes simplex virus thymidine kinase gene. The same 15-base-pair sequence mediates induction by progesterone. Point mutations in the recognition sequence affect inducibility by glucocorticoids and progesterone similarly. Together with the strong conservation of the sequence of the DNA-binding domain of the two receptors, these data suggest that both proteins recognize a sequence that is similar, if not the same.
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PMID:A DNA sequence of 15 base pairs is sufficient to mediate both glucocorticoid and progesterone induction of gene expression. 289 Nov 34

The chicken vitellogenin II gene is transcriptionally activated by estrogens. In transient transfection experiments in human T47D cells that contain receptors for various steroids, we showed estradiol, progestin, and androgen responses of a chimeric chicken vitellogenin II construct. This construct consists of DNA sequences from -626 to -590 upstream of the start of transcription of the chicken vitellogenin gene linked to the herpes simplex virus thymidine kinase promoter driving the transcription of the bacterial chloramphenicol acetyltransferase gene. Treatment of the transfected T47D cells with a combination of estradiol and the progestin R5020 led to a superinduction of chloramphenicol acetyltransferase activity, showing a synergistic action of these two steroids. This synergism was not observed upon treatment of the transfected cells with estradiol and the androgen dihydrotestosterone. Using point mutations in the vitellogenin gene fragment, we showed in functional and in in vitro DNase I footprinting assays with a purified progesterone receptor that, for the synergistic action of estradiol and R5020 to occur, the progesterone receptor must be bound to the vitellogenin gene fragment. The progesterone receptor-binding site was localized at -610 to -590, close to the consensus sequence (-626 to -613) for estrogen receptor binding and function. We therefore demonstrate here that two different steroid hormones can be functionally synergistic through the interaction of their corresponding receptors with two different binding sites adjacent to one another.
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PMID:Estrogen and progesterone receptor-binding sites on the chicken vitellogenin II gene: synergism of steroid hormone action. 324 57

Celiptium (Ce) is an antitumor drug used in the therapy of breast carcinomas, which are to a large extent dependent on estrogens. We have studied the effect of Ce on some proteins induced by estradiol (E2) in the rat uterus. It was observed that Ce administered at the same time or before E2, was able to inhibit the induction by E2 of fetal thymidine kinase (TK-F), of creatine kinase of brain-type (CK-BB) and of progesterone receptor (PR). When Ce was given after E2, its inhibitory effects were less evident. Results seemed to indicate that Ce could bind the acceptor sites for E2 receptor and thus inhibit the activity of E2-regulated genes. This assumption was corroborated by the fact that Ce did not modify the activity of enzymes not submitted to E2 regulation.
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PMID:Inhibition by Celiptium of the fetal thymidine kinase synthesis induced by estrogens in the rat uterus. 359 39

Hypothyroidism has been believed to affect the female reproductive system, but relatively few studies have been made on the direct effects of the thyroid hormone on the uterus, and the results have been conflicting. In the present paper, thymidine kinase (TK) and progesterone receptor (PrgR) in cytosol were assayed in the rat to study the uterine response to estrogen. Furthermore, this uterine response to estrogen was compared between hypothyroid and euthyroid rats. Hypothyroidism was induced by surgical operation performed at 21 days of age. Immature rats: Estradiol (E2) was injected on day 26. No significant difference in increases of uterine wet weight, TK and PrgR was noted between hypothyroid and euthyroid rats 30 h after E2 injection. Adult rats: Ovariectomy was added at 21 days or 65 days of age. E2 was injected on day 70. No significant difference in increases of uterine wet weight, TK and PrgR was noted between hypothyroid and euthyroid rats 24 h or 48 h after E2 injection. These results suggest that hypothyroidism may have no direct effects on such uterine responses to estrogen as wet weight, TK and PrgR.
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PMID:[Uterine response to estrogen in the rat of hypothyroidism]. 712 68

Thymidine kinase (TK) is involved in DNA synthesis by the salvage pathway. In this study, thymidine kinase (TK) was determined in routinely prepared cytosols of primary tumors from 290 breast-cancer patients. Enzyme activity was measured using a radioenzymatic method optimized for detection of the fetal isoenzyme. High levels of TK (> or = 126 mU/mg protein) were positively associated with histological grade in both pre/peri-and post-menopausal patients. In pre/peri-menopausal patients, high concentrations of TK were also found more frequently in progesterone receptor (PgR)-negative tumors than in PgR-positive samples. In post-menopausal patients, high levels of TK were associated with large tumor size, estrogen receptor (ER) negativity and PgR negativity. In univariate analysis, high levels of TK were strongly associated with shorter overall survival in both pre/peri- (p = 0.001) and post-menopausal patients (p = 0.02). Pre/peri-menopausal patients whose tumors had high levels of TK also had an increased risk of relapse (p = 0.001). In multivariate analysis (including treatment protocol, patient age, lymph-node involvement, tumor size, histological grade, ER and PgR status), TK status was found to be an independent prognostic factor for recurrence-free survival in pre/peri-menopausal patients with a weight similar to that of PgR status. In post-menopausal patients, TK was the only factor selected for overall survival.
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PMID:Prognostic value of cytosolic thymidine kinase activity as a marker of proliferation in breast cancer. 770 35

Signal amplification is fundamental to the normal operation of the preovulatory LH surge and is achieved through processes such as GnRH self-priming and augmentation of stimulated LH secretion by progesterone. We have proposed a model for GnRH self-priming that requires cross-communication between a GnRH receptor-activated protein kinase A pathway and the progesterone receptor (PR) to achieve amplification of the GnRH signal. We found that a pulse of GnRH administered to gonadotrope-enriched pituitary cells cultured in medium containing charcoal-treated serum plus estradiol (E2) potentiated the LH secretory response to subsequent GnRH pulses, and this potentiation could be blocked by a PR antagonist, RU486, in the absence of progesterone. Similarly, exposure of gonadotrope-enriched cultures to forskolin augmented the response to a pulse of GnRH, and the augmentation due to cAMP elevation could be reduced by RU486 in the absence of progesterone. To directly test whether stimulation with either GnRH or a cAMP analog results in transactivation of the endogenous PR, we used rat anterior pituitary cells cultured in the presence of E2 and transfected with reporter plasmids containing progesterone-responsive elements (PRE) and either a E1b or a thymidine kinase (tk) promoter linked to the chloramphenicol acetyltransferase (CAT) gene. For pituitary cells transfected with the PRE-E1b-CAT plasmid, exposure to either progesterone, GnRH, or 8-bromo-cAMP (8BrcAMP) for 6 h resulted in an induction of CAT activity which could be suppressed by coincubation with RU486. RU486 by itself had no effect on CAT activity. Similar results were obtained when a plasmid containing a different promoter (PRE-tk-CAT) was used. For cells transfected with a construct lacking a PRE (pSV2CAT), 8BrcAMP was without effect on CAT expression. When cells were made PR-deficient by omission of E2 from the incubation medium and transfected with PRE-E1b-CAT, neither progesterone, GnRH, nor 8BrcAMP was able to induce CAT activity. In summary we found that either GnRH or 8BrcAMP is able to stimulate transcription of reporter genes linked to two different PRE-containing promoters in anterior pituitary cells that contain endogenous PR; this occurred in the absence of progesterone and was suppressed by a PR antagonist. A simple interpretation of these data is that a GnRH-triggered signaling cascade can result in progesterone-independent transactivation of the PR. We propose that, in the normal operation of the preovulatory LH surge, the pathways for GnRH self-priming and progesterone augmentation converge at the PR and that the pathways serve as physiological redundancies to ensure the LH surge.
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PMID:Activation of the progesterone receptor by the gonadotropin-releasing hormone self-priming signaling pathway. 798 48

P-glycoprotein, the product of the multidrug resistance (mdr) gene family, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents and is also expressed normally in a variety of mammalian tissues. In rodents during pregnancy, there is a dramatic overproduction of the mdr1b form of P-glycoprotein at the lumenal surface of the secretory epithelium of the gravid uterus. An expression vector, mdr1b-CAT, was constructed by fusion of this promoter region to a reporter gene, the bacterial chloramphenicol acetyltransferase (CAT) gene. R5020, a progesterone agonist, increased approximately 3-fold the expression of mdr1b-CAT when transfected into T47D cells, a cell line that constitutively expresses the progesterone receptor. A far greater response to R5020 was observed when the cells were co-transfected with an expression vector for the A form of the progesterone receptor, but not the B form. A series of 5'-deleted clones of the mdr1b-CAT construct indicated that the region of responsiveness was located in the first untranslated exon of the gene. Furthermore, sequences from the first exon were able to confer responsiveness to the non-responsive thymidine kinase-CAT vector. This study demonstrates that progesterone specifically regulates the activity of the mdr1b promoter and that this response is directed solely by the A form of the progesterone receptor.
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PMID:Progesterone regulates the murine multidrug resistance mdr1b gene. 809 15

Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.
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PMID:Restoration of estrogen-dependent progesterone receptor expression in a uterine myocyte cell line. 846 59


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