Genes, variant genes and pseudogenes of the human tRNA(Val) gene family. Expression and pre-tRNA maturation in vitro.

Nine different members of the human tRNA(Val) gene family have been cloned and characterized. Only four of the genes code for one of the known tRNA(Val) isoacceptors. The remaining five genes carry mutations, which in two cases even affect the normal three-dimensional tRNA structure. Each of the genes is transcribed by polymerase III in a HeLa cell nuclear extract, but their transcription efficiencies differ by up to an order of magnitude. Conserved sequences immediately flanking the structural genes that could serve as extragenic control elements were not detected. However, short sequences in the 5' flanking region of two genes show striking similarity with sequences upstream from two Drosophila melanogaster tRNA(Val) genes. Each of the human tRNA(Val) genes has multiple, i.e. two to four, transcription initiation sites. In most cases, transcription termination is caused by oligo(T) sequences downstream from the structural genes. However, the signal sequences ATCTT and CTTCTT also serve as effective polymerase III transcription terminators. The precursors derived from the four tRNA(Val) genes coding for known isoacceptors and those derived from two mutant genes are processed first at their 3' and subsequently at their 5' ends to yield mature tRNAs. The precursor derived from a third mutant gene is incompletely maturated at its 3' end, presumably as a consequence of base-pairing between 5' and 3' flanking sequences. Finally, precursors encoded by the genes that carry mutations affecting the tRNA tertiary structure are completely resistant to 5' and 3' processing.

An interdisciplinary approach to breast and PCA. International Symposium: Breast and PCA: Genes, Hormones and the Environment, Garmisch-Partenkirchen, Germany, 22-24 April 1999.

Breast and prostate cancer (PCA) are among the leading cancer forms in the developed world. The 'International Symposium: Breast and PCA: Genes, Hormones and the Environment', and interdisciplinary meeting organized by K. Badenhoop (University Hospital, Frankfurt, Germany), J. Köhrle (University Hospital, Würzburg, Germany) and W-D. Schleuning (Schering AG, Berlin, Germany), was intended to promote the exchange of new concepts and emerging paradigms between basic science and clinical research, and to establish interactions with researchers in industry in this field.

Retinoids stimulate type I iodothyronine 5'-deiodinase activity in human follicular thyroid carcinoma cell lines.

Iodothyronine 5'-deiodinase isoenzymes generate the thyroid hormone 3,3',5-triiodothyronine from the prohormone L-T4. Basal and retinoic acid (RA)-induced type I 5'-deiodinase (5'DI) activities were studied in human thyroid carcinoma cell lines. In the follicular thyroid carcinoma line FTC-133, nanomolar concentrations of 9-cis, 13-cis-, and all-trans-RA induced 5'DI activity. Kinetics with all-trans-RA revealed 5'DI stimulation after 1 day and a maximal effect after 3 days. Increased abundance of the p27 5'DI subunit was demonstrated after RA treatment by N-bromoacetyl-[125I]T4 affinity labeling. Actinomycin-D and cycloheximide blocked RA-mediated induction. RA stimulated 5'DI activity to a lesser extent in FTC-238 cells, whereas neither basal 5'DI activity nor stimulation by RA was found in anaplastic thyroid carcinoma, human lung, or leukemia cell lines. Steady state messenger ribonucleic acid levels of RA receptor-alpha and -beta were increased after incubation of FTC-133 cells with all-trans-RA. The high 5'DI activity of differentiated rat thyroid FRTL-5 cells was not further induced by RA. Butyrate did not alter 5'DI, but increased the activity of the differentiation marker alkaline phosphatase in FTC-133 and FTC-238 cells. T4 and T3 had no effect on basal or RA-stimulated 5'DI activity. These data suggest that expression and retinoid induction of 5'DI may serve as a sensitive and functional differentiation parameter of follicular thyroid carcinoma cells.

Regulation of CD97 protein in thyroid carcinoma.

CD97 is a dimeric glycoprotein belonging to the secretin receptor superfamily and is abundantly expressed in cells of hematopoietic origin. The aim of this study was to analyze the expression of the CD97 protein in thyroid carcinomas and the role of all-trans-retinoic acid (RA) in the regulation of CD97 protein in monolayer culture of the human follicular thyroid carcinoma cell line FTC-133. In normal thyroid tissue, no immunoreactivity of CD97 could be found, whereas in differentiated thyroid carcinomas, CD97 expression was either lacking or low. Undifferentiated anaplastic thyroid carcinomas revealed high CD97 expression. The expression of CD97 protein seems to be correlated to the postoperative histopathological classification staging. Approximately 50% of FTC-133 cells expressed the CD97 protein under basal culture conditions. No differences were found in the number of CD97-positive cells after TSH, forskolin, and insulin treatment compared to control values. Epidermal growth factor treatment led to an increase in CD97 immunostaining (up to 90%), whereas phorbol 12-myristate 13-acetate slightly decreased the immunoreactivity of CD97 (from 50% to 30%). Under basal conditions, RA treatment for 72 h led to a decrease in total cell number by 33% and in CD97-positive cells from 50% to 30%. TSH, forskolin, phorbol 12-myristate 13-acetate, and insulin showed no effect after 72-h pretreatment with RA, whereas epidermal growth factor treatment led to a slight increase in the number of the CD97-positive cells (from 30% to 40%) compared to the control value. These data suggest that CD97 expression may play an important role in the dedifferentiation of thyroid tumors, and RA might interfere with this process in thyroid carcinoma by suppressing the dedifferentiation marker CD97.

The human tRNAVal gene family: organization, nucleotide sequences and homologous transcription of three single-copy genes.

At least 13 independent tRNAVal gene loci were detected in the human genome. Three of these genes were isolated and shown to occur only once in the haploid genome. No further functional tRNA genes are located on the isolated clones. Two tRNAVal genes encode the known major and minor tRNAVal isoacceptors, the third may be a pseudogene because a corresponding tRNAVal is not yet known. Comparison of extragenic sequences did not reveal significant homologies, indicating the separation of these genes early in vertebrate evolution. An Alu-type repeat was found in two of the clones within several hundred bp distance from the tDNA. All three genes are transcriptionally active in a HeLa nuclear extract. We show here for the first time that homologous in vitro transcription of mammalian tRNA genes strongly depends on extragenic control regions: interestingly, as a consequence of different flanking regions, the transcription efficiencies vary by an order of magnitude among the genes for the major and the minor tRNAVal and thus reflect the concentrations of these tRNAs in vivo.

The promoter of the human sodium/iodide-symporter gene responds to retinoic acid.

It was shown previously that hNIS mRNA expression is stimulated by retinoic acid (RA) in human follicular thyroid carcinoma cell lines FTC-133 and FTC-238, and patients with thyroid carcinomas lacking iodide uptake respond to RA treatment with increased radioiodide transport. Here, in transient transfection experiments using FTC-238 cells, hNIS promoter/luciferase reporter constructs showed an up to 2.5-fold increase in transcriptional activity after incubation with 1 microM RA. Stimulation by 10 nM T3 was up to 2.4-fold. Deletion or block mutation of a putative nuclear receptor recognition site, 'DR10', abolished RA and T3 responses. Four copies of the DR10 cloned 5' to the thymidine kinase promoter gave a 2.6-fold and a 1.4-fold increase in transcriptional activity after RA and T3 stimulation, respectively. In electrophoretic mobility shifts, a wildtype DR10 oligonucleotide, but not block mutants of either DR10 halfsite, interacted with nuclear receptors. Thus, RA redifferentiation of advanced thyroid carcinomas may reinduce iodide uptake by stimulating hNIS expression and thereby make tumours accessible for radioiodide therapy again.

Innovative strategies for the treatment of thyroid cancer.

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.

Retinoic acid redifferentiation therapy for thyroid cancer.

For the treatment of differentiated thyroid cancer, surgery, radioiodide therapy, and thyrotropin-suppressive thyroxine application represent established therapeutic measures of proven efficiency, affording a good prognosis for this disease. However, in up to 30% of the cases, dedifferentiation is observed, giving rise to tumors that are refractory to conventional treatment. Eventually, this may lead to the most malignant human tumor, anaplastic thyroid carcinoma, with a life expectancy of only a few months after diagnosis. Among novel approaches for the treatment of dedifferentiated thyroid carcinomas, retinoic acid redifferentiation therapy was evaluated in several in vitro and in vivo studies. Cell culture experiments in thyroid carcinoma lines show that RA treatment affects thyroid specific functions (type I 5'-deiodinase, sodium/iodide-symporter), cell-cell or cell-matrix interaction (intercellular adhesion molecule-1, E-cadherin), differentiation markers (alkaline phosphatase, CD97), growth, and tumorigenicity. The observed changes, which involve multiple parameters that characterize a mature, functional thyrocyte, may be interpreted as partial redifferentiation. In clinical pilot studies, about 40% of the patients responded to RA application with an increased radioiodide uptake. In an evaluation of 20 RA-treated patients with well-documented data sets, 8 exhibited a decrease (4) or stabilization (4) in tumor size and/or in serum thyroglobulin levels in addition to enhanced radioiodide transport. This indicates that these patients with a long history of unresponsiveness to other treatment may have experienced an actual therapeutic benefit. These data suggest that RA redifferentiation therapy, considering especially its comparatively mild side effects, may soon represent an alternative therapeutic approach to otherwise untreatable thyroid tumors.

Reverse transcriptase-polymerase chain reaction analysis of thyrocyte-relevant genes in fine-needle aspiration biopsies of the human thyroid.

Currently, fine-needle aspiration cytology is a valuable tool in the routine diagnosis of suspicious thyroid nodules. We present a very sensitive method for the molecular analysis of the expression of several genes important for normal thyroid function in parallel to the cytological diagnosis. We adapted reverse transcriptase polymerase chain reaction (RT-PCR) to amplify thyroid-typical mRNAs in samples of thyroid carcinoma cells as small as those obtained by fine-needle aspiration biopsy (FNAB), ie, 100-1000 cells, and applied this procedure to four routinely taken FNABs. Gene products such as thyroglobulin (Tg), thyroid-stimulating hormone-receptor (TSHr), sodium/iodide-symporter (NIS), type I iodothyronine-5'-deiodinase (DI), and type II iodothyronine-5'-deiodinase (DII) were analyzed. To establish RT-PCR protocols, serial dilutions of follicular thyroid carcinoma cells, FTC-133, which express these genes at low levels, were initially used for RNA isolation. Successful RNA isolation and reverse transcription were checked by the amplification of beta-actin mRNA. We detected the mRNAs coding for Tg in as little as 10 cells, for NIS in 100 cells, and for TSHr, DI, and DII in 10,000 cells. After preparing cytological smears of four routinely taken FNABs, all above-mentioned thyroid-typical mRNAs were observed by using the material remaining in the needle for RNA isolation followed by RT-PCR. This method offers the possibility of obtaining two different types of information from the same routinely taken thyroid FNAB: the cytological diagnosis and the expression pattern of several diagnostically relevant genes. Therefore, a more specific diagnosis could be rendered in the preoperative state, and may lead to more specific therapy.

Regulation of the sodium/iodide symporter by retinoids--a review.

Decrease or loss of iodide uptake, due to impaired expression and/or function of the sodium/iodide-symporter (NIS), is a major obstacle to the treatment of advanced thyroid carcinomas by radioiodide therapy. Several approaches are being evaluated to optimise or restore sufficient iodide transport in those cases, among them retinoid therapy. Retinoids with their growth-inhibiting and differentiation-inducing properties have been repeatedly used for treatment and chemoprevention of various cancers. In thyroid carcinoma cell lines they trigger changes in gene expression that may be interpreted as partial redifferentiation. Especially, they stimulate NIS mRNA expression and iodide uptake in human follicular thyroid carcinoma cells. Moreover, they also increase NIS expression and function in human mammary tumour cells. In a clinical pilot study to evaluate the feasibility of retinoid redifferentiation in the case of otherwise untreatable thyroid cancers, 21 of 50 patients showed an increase of radioiodide uptake after 5 weeks. This indicates that increasing NIS activity and radioiodide uptake by retinoic acid redifferentiation may be a therapeutic alternative for thyroid cancers refractory to other therapeutic modalities and probably also for mammary cancer.

Implications of the molecular characterization of the sodium-iodide symporter (NIS).

The recently cloned sodium-iodide symporter (NIS) represents a key molecule for thyroid function by efficiently accumulating iodide from the circulation into the thyrocyte against an electrochemical gradient. This uptake requires energy, is coupled to the action of Na+/K+-ATPase, and stimulated by TSH, the main hormone regulating thyroid-specific functions. NIS mutations are found in congenital hypothyroidism, and potential defects in the NIS gene, its expression, or function of the NIS protein are currently under investigation in various thyroid diseases. Increased NIS expression has been found in autonomous adenoma and Graves' disease, decreased levels of NIS protein and/or mRNA were observed in Hashimoto's disease, cold nodules, most thyroid cancers and cell lines derived therefrom. Autoantibodies directed against NIS have been identified in autoimmune thyroid disease and blocking antibodies isolated from sera of patients with Hashimoto's disease inhibit NIS function in NIS-transfected CHO cells. NIS mRNA expression can be up-regulated by retinoic acid in human thyroid carcinoma cell lines whereas retinoic acid treatment decreases NIS expression and function in differentiated rat thyroid FRTL-5 cells. Apart from thyrocytes, NIS is also expressed in other tissues known to transiently accumulate radioiodide, albeit at much lower levels, requiring RT-PCR for detection of the transcript. Diagnostic and therapeutic implications of the recent cloning of the human NIS gene such as development of NIS-directed drugs, ligands, antibodies, vaccines, gene therapeutic approaches combining NIS targeting and expression together with the long-established, efficient and safe method of radioiodide therapy are discussed both for application to thyroid related diseases and carcinoma, and non-thyroid benign and malignant diseases. Apart from these therapeutic and diagnostic perspectives the availability of the NIS gene will also open new opportunities to develop sensitive and homologous diagnostic test systems to identify factors involved in autoimmune thyroid disease, evolution of goitre, adenoma and thyroid cancer as well as NIS-directed new drugs. Advanced and sophisticated molecular diagnostic approaches (RT-PCR from fine needle aspirations, screening for mutations, analysis of gene defects) are already developed for NIS and will complement or overcome some established procedures in thyroid diagnostics.

Effects of retinoids and role of retinoic acid receptors in human thyroid carcinomas and cell lines derived therefrom.

Retinoic acids (RAs), well characterized regulators of proliferation and differentiation, partly re-differentiate follicular thyroid carcinoma cell lines (FTC-133, FTC-238, and HTC-TSHr) as well as SV40-transfected immortalized thyroid cell lines (ori3 and 7751). This is indicated by the stimulation of type I 5'-deiodinase and other differentiation markers. As demonstrated by RT-PCR, electrophoretic mobility shift, and [3H]-retinoic acid binding assays, thyroid carcinoma cell lines express RA receptor mRNAs and functional ligand- and DNA-binding receptor proteins able to mediate RA-dependent signal transduction. Together, these properties make these thyroid-derived cell lines useful in vitro models for studying the effects of an RA re-differentiation therapy of thyroid cancer.

Thyroid hormone receptors and type I iodothyronine 5'-deiodinase activity of human thyroid toxic adenomas and benign cold nodules.

The majority of thyroid adenomas are of clonal origin. In a subset of toxic adenomas (TAs) and cold nodules (CNs) activating mutations in the thyrotropin (TSH) receptor or G s -alpha gene may explain the altered functions in these benign tumours. The present study was undertaken to investigate the status of functional thyroid hormone receptors, major thyroid hormone signal mediators, in both the human TAs and CNs in comparison with a normal thyroid tissue from the same patient. Electrophoretic mobility shift assays using a DR4 ("direct repeats" 4), a thyroid hormone responsive element (TRE) of human type I iodothyronine 5'-deiodinase demonstrated the DNA-binding of thyroid hormone receptors (TRs) in thyroid tissue nuclear extracts. A significant increase (p < 0.05) in the functional binding properties of TRs to the DR4 thyroid hormone responsive element was found in TAs when compared to normal thyroid tissue. Contrary, a marked diminution in the TR-TRE complex formation was found in CNs in comparison with normal thyroid tissue. In addition, functional activity of the iodothyronine 5'-deiodinase (5'DI) was analyzed in benign tumours, thyroid TAs and CNs in comparison with that of normal thyroid tissue. A significantly increased (p < 0.01) activity of 5'DI was demonstrated in TAs, and in contrast, decreased values of the enzyme activity were found in CNs when compared to a normal tissue. From the data it is suggested that both the status of TR-TRE complex formation and the activity of the 5'DI may be altered in benign tumours of human thyroid gland.

Redifferentiation therapy of differentiated thyroid carcinoma with retinoic acid: basics and first clinical results.

Retinoic acids (RA) regulate growth and differentiation of normal epithelial tissue. They have been employed in anticancer treatment and showed positive effects in hematopoetic and various epithelial tumors. Experimental data with follicular thyroid tumor cells showed strong evidence of induction of differentiated cell function and antiproliferative effects. Based on these data a consecutive series of 10 patients with advanced thyroid carcinoma were treated with 13-cis-retinoic acid (Roaccutan) 1.5 mg/kg body weight for six weeks. Follow-up demonstrated renewed uptake of radioiodine in 4 of 10 patients allowing performance of further radioiodine therapy. Reduction in tumor size due to antiproliferative effects of RA could not yet be verified.

Expression of selenoproteins in various rat and human tissues and cell lines.

Various rat and human tissues and cell lines naturally exposed to endogenous or exogenous oxidative stress were examined for their pattern of selenoprotein transcripts. Selenoprotein P mRNA was mainly expressed in rat kidney, testis, liver and lung. In testis, a high phospholipid hydroperoxide glutathione peroxidase (PHGPx) but only a weak cytosolic glutathione peroxidase (cGPx) signal was obtained. In kidney, spleen, heart, liver and lung cGPx mRNA levels were higher than those of PHGPx and for both only weak signals were obtained with brain mRNA. The Northern blot results concerning the tissue distribution of cGPx in the rat were fully supported by activity measurements. None of the human tissues revealed a PHGPx mRNA signal, whereas selenoprotein P transcripts were present in all human tissues with the highest abundance in heart, liver, and lung, tissues which also exhibited strong cGPx signals. The gastrointestinal glutathione peroxidase (GPx-GI) was only expressed in human liver and colon liver. Liver, the organ that showed the broadest repertoire of selenoproteins, has to cope with reactive oxygen intermediates produced during detoxification reactions. Human cell lines of the myeloic system that may be exposed to oxidative stress during inflammatory processes showed distinct cGPx signals: epithelial cells showed low cGPx signals. Similar cGPx mRNA levels were found in normal human thyroid tissue and thyroid carcinoma cells. Among the human cell lines selenoprotein P expression was detected in HepG2 and HTh74 thyroid cells. Our data confirm the necessity of getting specific information on distinct tissue- and cell-specific patterns of selenoprotein expression as endpoints of selenium supply and biological function of the selenoprotein family. Analysis of total selenium contents of tissues or body fluids only provides integrative information on the global selenium status of individuals.

Adjuvant arthritis in the rat is associated with decreased binding of nuclear receptors to thyroid hormone responsive element in spleen extracts.

In vertebrates, thyroid hormone and its cognate nuclear receptors are involved in a complex arrangement of physiological and developmental function. Since thyroid hormone has also been shown to affect immune responses, we investigated the DNA binding status of T3 receptors of spleen nuclear extracts in a) rats with adjuvant arthritis (AA); b) adrenalectomized rats (ADX), and c) animals with adjuvant arthritis followed by adrenalectomy (AA + ADX). A marked diminution in the functional binding of nuclear thyroid hormone receptors to DR4 thyroid hormone responsive DNA element was found in the spleens of AA and AA + ADX rats when compared to a control group or ADX rats. The data based on in vivo experiments suggest that the nuclear receptor--thyroid hormone responsive element complex status within the cell nucleus may be altered in adjuvant arthritis.

Increased I-131 uptake in local recurrence and distant metastases after second treatment with retinoic acid.

We present a case of increased I-131 uptake in a patient with papillary thyroid carcinoma with local recurrence and distant metastases after a second treatment with retinoic acid as a sign of redifferentiation of the tumor cells. When fine-needle aspiration cytology before and after a second course of retinoic acid treatment were compared, signs of tumor cell redifferentiation were found. This was accompanied by biochemical reexpression of thyroid marker proteins.

[Selenoproteins in bone, gastrointestinal tract and thyroid gland of the human]. / Selenoproteine im Knochen, Gastrointestinaltrakt und in der Schilddrüse des Menschen.

BASIS: Selenium is an essential trace element, which is incorporated as selenocysteine (secys) into specific proteins in a regulated fashion. In the presence of a hairpin loop structure within the 3' untranslated region of the mRNA the opal stop codon UGA is coding for selenocysteine. Selenoprotein functions are dependent on secys incorporation. Members of the family of deiodinases as well as the family of glutathione peroxidases, selenoprotein P and thioredoxin reductase are selenoproteins. DISCUSSION: Bone, the intestine and the thyroid rely on antioxidant systems against potential cell and DNA damage through endogenous and environmental peroxides and reactive oxygen species (ROS) potentially promoting inflammation and tumorigenesis. Optimized cell defense through antioxidant selenoproteins requires optimal selenium supplementation of the organism. We have analyzed the expression of selenoproteins in these tissues, thus providing molecular tools to further elucidate optimal selenium supply on a cellular level. CONCLUSION: Clinical intervention studies that focus on the development of disease must confirm the relevance of optimized selenium supply for the pathogenesis, prevention and therapy of metabolic bone disease as well as chronic (autoimmune) inflammation and tumorigenesis in the thyroid and intestine.