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)

The coding region of the herpes simplex type 1 virus thymidine kinase gene was coupled to the promoter of the bovine thyroglobulin gene and introduced into the genome of mice. The viral thymidine kinase (HSV1-TK) was expressed mainly in the thyroid glands and testis. Upon treatment of transgenic females with the antiherpetic agent Ganciclovir the thyroid regressed, while the parathyroid gland was unaffected. The number of thyroid follicle cells was greatly reduced after 3 days, and they were completely absent after 7 days of treatment. After 14 days, the levels of circulating T4 and T3 were below the limits of detection, total soluble protein recovered from the thyroid and parathyroid glands together was 10% of the control value, and the level of thyroid HSV1-TK was more than 100-fold lower than that in transgenic controls. Levels of circulating PTH and calcitonin remained normal. At the time of treatment the mice were adults. Thus, the thyroid follicle cells were selectively ablated after normal development with a functional thyroid gland. When treatment with Ganciclovir was terminated after 14 days, no circulating T4 or T3 or other indications of thyroid regeneration were detected for a subsequent period of 90 days. During this time the mice gained weight more slowly than controls, at a rate consistent with the suppression of GH synthesis by thyroid deficiency. The production of mouse major urinary protein (MUP) ceased in the treated mice and was completely restored by the administration of T4. MUP production was not restored by GH, demonstrating that the expression of the Mup genes requires T4 in addition to GH.
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PMID:Specific ablation of thyroid follicle cells in adult transgenic mice. 165 24

We reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase reporter gene (tk) was expressed in the testes of transgenic mice when coupled to the promoter of a liver-specific mouse major urinary protein (MUP) gene. Here we show that HSV-1 tk is also expressed in the testis when coupled to a MUP pseudogene promoter, to a truncated MUP promoter that is not active in the liver, and to the promoter of the bovine thyroglobulin gene. Furthermore, HSV-1 tk itself was expressed in the testis, although its normal expression had been disabled by removing an upstream regulator of transcription. In every case, the same multiple transcripts were observed, with their 5' ends located downstream of the normal HSV-1 tk translation initiation codon. We conclude that the transcription of HSV-1 tk in the testis is directed by a cryptic TATA box-independent promoter located in the coding region of the gene. The longest HSV-1 thymidine kinase (TK) polypeptides synthesized in the testis were shorter than full-length TK and probably result from translational initiation at Met46 and Met60, the second and third ATG codons of the tk reading frame. Male mice of most transgenic lines were sterile, and the severity of the lesion in spermatogenesis was directly related to the level of TK expression. In the most highly expressing lines, sperm counts were low and morphologically defective sperm were common. In other sterile lines, TK was expressed at a lower level and sperm counts were normal but sperm motility was greatly reduced. Lines with the lowest levels of HSV-1 TK expression were fertile. HSV-1 TK was expressed in germ line cells, mainly in the haploid spermatids. However, low-level HSV-1 TK activity was found in the testis before the first germ cells entered meiosis, showing that if expression is confined to the germ cells, it also occurs in spermatogonia.
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PMID:The herpes simplex virus type 1 thymidine kinase is expressed in the testes of transgenic mice under the control of a cryptic promoter. 171 6

Previously we demonstrated that lines of transgenic mice carrying the herpes simplex type 1 virus thymidine kinase (HSV1-tk) reporter gene are male-sterile. Ectopic transcription of the HSV1-tk reporter in the testis was initiated downstream of the normal translation initiation codon and truncated proteins consistent with translational initiation at the second and third ATG codons were synthesized. Here we describe the effects on fertility 1) of converting the second and third ATG codons of the HSV1-tk reporter to CTG codons and 2) of utilizing the HSV type 2 thymidine kinase (HSV2-tk) reporter gene, in which the second ATG codon is located downstream of the ATP-binding pocket of the enzyme. Both reporters were coupled to the bovine thyroglobulin promoter (bTG-tk1 alpha and bTG-tk2 transgenes). The level of ectopic expression of these transgenes in the testis, relative to expression in the thyroid, was one to two orders of magnitude less than that of bTG-tk1. Sixty percent of male founders carrying the bTG-tk1 alpha and bTG-tk2 transgenes were fertile but did not transmit the transgene. In contrast, most males from subsequent generations were fertile and transmitted the transgenes at the expected frequency. This difference between founder males and male descendants is also observed with certain constructs in which the HSV1-tk reporter is coupled to other promoters. We attribute the effect to mosaicism among male founders, leading to competition between transgenic and nontransgenic spermatozoa and/or spermatogenic precursor cells and resulting in a lack of fertilization by transgenic sperm that would successfully fertilize eggs in the absence of competition.
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PMID:Different transmission rates of herpesvirus thymidine kinase reporter transgenes from founder male parents and male parents of subsequent generations. 757 10

The mammalian paired box (Pax) genes encode a family of transcription factors involved in embryogenesis. The murine and human Pax8 genes are expressed in developing and adult thyroid as well as in the developing secretory system and at the lower level in adult kidney. In the secretory system expression is localized to the induced, extensively differentiating parts that undergo a transition from mesenchyme to epithelium. The human PAX8 gene generates at least five different alternatively spliced transcripts encoding different PAX8 isoforms. These isoforms differ in their carboxy-terminal regions downstream of the paired domain that has been shown previously to be responsible for the DNA binding. The PAX8a isoform contains a 63 amino-acid serine-rich region that is absent in the isoform PAX8b whereas PAX8c reveals a novel 99-amino-acid proline-rich region. This proline-rich region arises due to an unusual reading-frame shift in the PAX8 transcript. RNAse protection and RT(reverse transcription)-PCR analysis show the expression of all three PAX8 transcripts in human thyroid, kidney and five Wilms' tumors. Band-shift assay indicates a greatly reduced binding affinity of the isoform PAX8c to a DNA sequence from the promoter of the thyroperoxidase gene compared to the binding of PAX8a and PAX8b to this sequence. Deletion analysis of murine PAX8a indicates that its activating domain residues at the carboxy terminus of the protein which is shared by isoforms PAX8a and PAX8b. In accordance with these data PAX8a and PAX8b activate transcription from a thyroglobulin promoter as well as from a cotransfected synthetic PAX8-specific promoter/chlorampericol acetyltransferase (CAT) reporter containing a Pax8-binding oligonucleotide in front of the basal herpes simplex virus thymidine kinase (HSV-TK) promoter (P11/12-TK-CAT). However if the basal HSV-TK promoter of this reporter is substituted by a minimal adenovirus E1b TATA element, PAX8a and PAX8b fail to activate transcription. Of the three chimaeric forms containing the GAL4 DNA-binding domain at the amino-terminal end fused to the corresponding carboxy-terminal regions of the PAX8 isoforms beginning immediately downstream of the paired domain only a GAL4-PAX8b fusion significantly activates transcription from a cotransfected GAL4-specific upstream-activating-sequence (UAS)-TK-CAT reporter. Substitution of the basal HSV-TK promoter in this reporter by the minimal E1b TATA element does not affect this activation. These results indicate that the PAX8 isoforms display different functional properties and may also function differently in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Distinct functional properties of three human paired-box-protein, PAX8, isoforms generated by alternative splicing in thyroid, kidney and Wilms' tumors. 773 92

The herpes simplex type 1 virus thymidine kinase (HSV1-TK) reporter gene was coupled to a bovine thyroglobulin promoter (TG-tk construct). Within the thyroid glands of transgenic mice expression was confined to thyroid follicle cells. Infusion of Ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine) to 8 to 12 week transgenic females led to the complete loss of thyroid HSV1-TK activity (at 3 to 4 days) and thyroid follicles (between 7 and 14 days). During the first 5 days of treatment a single reciprocal oscillation in circulating thyroxine (T4) and TSH levels occurred. By 14 days the circulating tri-iodothyronine (T3) and T4 levels of all treated animals were below the detection limits of the assays, while TSH levels were elevated ten-fold and continued to increase thereafter. During 14 days of treatment the thyroids regressed, protein content fell by 80-90% and the C cells, normally dispersed within the central region of each gland, came together in aggregates. Pituitary GH levels in females rose and fell back to normal within 14 days and between 14 and 28 days fell to a level comparable with that of GH-deficient lit/lit mice. The levels of hepatic GH receptor mRNA and the predominant 6.6 kb T3 receptor mRNA were unaffected by thyrocyte ablation. Thyrocyte ablation had no effect on the level of prolactin (Prl) receptor mRNA in females, but increased Prl receptor mRNA levels in males and eliminated group 1 major urinary protein (MUP).mRNA in females. T4 replacement reversed the effects of thyrocyte ablation on MUP mRNA in females and on Prl receptor mRNA in males.2+ Despite the many physiological changes induced by thyrocyte ablation, ablated mice have been maintained for up to 1 year without thyroid hormone supplementation. T4-deficient females were normally fertile and carried pups to term. Although transgenic males expressed HSV1-TK ectopically in spermatids and spermatozoa at levels similar to thyrocyte levels, a rate of Ganciclovir infusion which successfully ablated the thyrocytes did not affect the testis. As an alternative to infusion by minipump, thyrocyte ablation could be achieved by 6 twice-daily injections of Ganciclovir, at a level of 112 micrograms Ganciclovir/g body weight per day, and fetuses in utero could be thyrocyte ablated by administering 50 or 15 micrograms/g body weight per day to pregnant females between days 14 and 18 of gestation. These data demonstrate the potential value of transgenic thyrocyte ablation in the study of the effects of thyroid hormone deprivation.
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PMID:Consequences of thyroid hormone deficiency induced by the specific ablation of thyroid follicle cells in adult transgenic mice. 796 9

In adult mice of the transgenic strain TG66.19, in which expression of herpes simplex type 1 virus thymidine kinase (HSVI-TK) is driven in thyrocytes from the thyroglobulin promoter, the drug Ganciclovir causes the death (ablation) of thyrocytes. Ablation occurred in the absence of thyrocyte proliferation or nuclear DNA synthesis, but was accompanied by transient expression of proliferating cell nuclear antigen and the dying thyrocytes exhibited the ultrastructural features of apoptosis. Control experiments show that the apoptosis is a result of the production of Ganciclovir phosphates in thyrocytes that express HSV1-TK. However, cell death was not dependent upon the presence of a functional copy of the oncosuppressor gene p53. We conclude that the apoptosis is probably not mediated by induction of DNA damage and occurs via a pathway that is independent of p53. The fact that Ganciclovir phosphate can kill cells by a p53-independent apoptotic pathway is encouraging in relation to tumour ablation by methods based on transfection with HSV1-tk genes and administration of Ganciclovir.
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PMID:Ganciclovir-induced ablation non-proliferating thyrocytes expressing herpesvirus thymidine kinase occurs by p53-independent apoptosis. 870 May 54

To develop gene therapy targeting thyroid carcinoma, the recombinant retrovirus (LNTGTK) carrying herpes simplex virus thymidine kinase (HSV-TK) gene under the control of thyroglobulin (TG) promoter was constructed and its efficacy was investigated in 3 thyroid cell lines; a differentiated normal rat thyroid cell line (FRTL5), malignant rat thyroid carcinoma cells derived from FRTL5 (FRTC) and a human anaplastic thyroid carcinoma cell line (FRO). TG mRNA was detected by Northern blot analysis in FRTL5 cells and by RT-PCR in FRTC cells when cultured with 2 U/L TSH and its expression levels were decreased by TSH withdrawal. However, either methods revealed no TG expression in FRO cells. In vitro cytotoxic assays demonstrated TG expression status-dependent cell killing by transduction of LNTGTK followed by ganciclovir (GCV) treatment. Thus, LNTGTK transduction increased the GCV sensitivity approximately 13,000- and approximately 160-folds in the presence of TSH and approximately 4- and approximately 27-folds in the absence of TSH in FRTL5 and FRTC cells, respectively. In contrast, there was no difference in the GCV cytotoxicity between parental and transduced FRO cells. Significant growth inhibition, but not complete eradication, of transduced FRTC cells was observed in in vivo subcutaneous tumor models of nude mice. These results demonstrate that retrovirus-mediated transduction of HSV-TK gene under the control of the TG promoter confers the GCV sensitivity selectively to TG-expressing thyroid cells. This system may therefore be feasible for gene therapy targeting TG-expressing thyroid carcinomas.
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PMID:Retrovirus-mediated suicide gene/prodrug therapy targeting thyroid carcinoma using a thyroid-specific promoter. 972 55

Mice that carry the wild-type herpes simplex virus type 1 (HSV1) thymidine kinase (tk) gene coupled to the bovine thyroglobulin (bTG) promoter (bTG-tk1 mice) express viral TK at a high level in the thyroid gland, and at an equally high level, ectopically, in the testis, which renders the males sterile. When the bTG promoter was coupled either to a variant of HSV1-tk (differing from the wild type in 2 nucleotides) (bTG-tk1alpha mice) or to the herpes simplex virus type 2 (HSV2) tk gene (bTG-tk2 mice) viral TK was expressed at high levels in the thyroid gland, and much lower levels in the testis, which causes a reduction in male fecundity rather than sterility. Here, we compare the expression of the three transgenes in the two tissues. Thyroids of all mice exhibited a 1.3 kb RNA initiated at or near the bTG cap site. Testes of all mice exhibited mainly 5'-end-shortened RNAs (bTG-tk1 and bTG-tk1alpha mice, approx. 1.2 kb and 0.9 kb; bTG-tk2 mice, approx. 1.2 kb) initiated from cryptic initiation sites in the HSV1-tk and HSV2-tk coding regions. Also, less abundant RNAs initiated near the bTG cap site were expressed from all three transgenes. Thyroids of bTG-tk1 and bTG-tk1alpha mice contained the full-length HSV-TK protein and a truncated variant previously shown to originate at a non-ATG start codon. Testes of these mice exhibited both proteins but relatively less of the full-length protein. We attribute the high level of viral TK in the testes of bTG-tk1 mice to the expression of a predominant protein of Mr 39000 that originates from ATG-2. Thyroid and testis of bTG-tk2 mice contained only the full-length HSV2-TK protein.
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PMID:Herpesvirus thymidine kinase transgenes that do not cause male sterility are aberrantly transcribed and translated in the testis. 976 91

Our recent study demonstrates the feasibility of the thyroglobulin (TG) promoter in transcriptionally targeted gene therapy for thyroid carcinomas expressing TG, albeit less effectively than the constitutive viral promoter. The present study was, therefore, designed to enhance the activity of the TG promoter with the Cre-loxP system. Our data demonstrate that the in vitro cytotoxic effect of herpes simplex virus thymidine kinase/ganciclovir obtained with the TG promoter and the Cre-loxP system is approximately 5-10-fold higher than that with the TG promoter alone. Enhanced tumor growth inhibition was also observed in in vivo tumor models. These data indicate the usefulness of the Cre-loxP system to enhance the activity of a tissue (or tumor)-specific promoter in transcriptionally targeted cancer gene therapy.
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PMID:Enhanced efficacy of transcriptionally targeted suicide gene/prodrug therapy for thyroid carcinoma with the Cre-loxP system. 1039 42

Normally, thyroid cancer is a disease with a good prognosis, but about 30% of the tumours dedifferentiate and may finally develop into highly malignant anaplastic thyroid carcinomas with a mean survival time of less than 8 months. Due to the loss of thyroid-specific functions associated with dedifferentiation, these tumours are inaccessible to standard therapeutic procedures such as radioiodide therapy and thyroxine-mediated thyrotrophin suppression. Medullary thyroid carcinomas are also highly aggressive. Here, therapy is limited to surgery, and no alternative is left if patients do not respond to this standard procedure. Obviously, new approaches would be desirable. Several novel approaches are currently being tested for the treatment of thyroid cancer. Many of them utilise methods of gene therapy, but follow different strategies: (1) reintroduction of the tumour suppressor p53 into a background lacking functional p53; (2) suicide gene therapy with ganciclovir and a transduced gene for herpes simplex virus thymidine kinase controlled by the thyroglobulin promoter; (3) strengthening of the antitumour immune response by expression of an adenovirus-delivered interleukin-2 (IL-2) gene; (4) induction of an immune response by DNA vaccination against the tumour marker calcitonin; (5) transduction of the thyroid sodium/iodide transporter gene to make tissues that do not accumulate iodide treatable by radioiodide therapy; (6) blocking of the expression of the oncogene c-myc by antisense oligonucleotides. While these approaches are still tested in vitro or in animal models, first results from pilot studies concerning other novel treatment modalities are available: (7) radioimmunotherapy exploits the carcinoembryonic antigen expressed on medullary thyroid carcinomas to target a radiolabelled antibody to the tumour; and (8) retinoic acid is used for a redifferentiation therapy in the case of thyroid cancer. Hopefully, one or the other of these novel strategies may probably extend after some time the current therapeutic repertoire for thyroid cancers and provide a perspective for otherwise untreatable patients.
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PMID:Innovative strategies for the treatment of thyroid cancer. 1087 26


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