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.

Functional retinoid and thyroid hormone receptors in human thyroid-carcinoma cell lines and tissues.

Thyroid carcinomas no longer accessible to radio-iodide or TSH-suppressive T4 therapy, due to loss of thyroid-specific functions, might be sufficiently re-differentiated by retinoic acid (RA) to be treated by conventional methods again. To help evaluate the feasibility of RA re-differentiation therapy in thyroid carcinomas, we examined the functionality of RA receptors (RARs/RXRs), central RA signal mediators, in human thyroid-carcinoma cell lines as model systems. [3H]-RA binding assays with nuclear extracts from follicular thyroid-carcinoma cell lines FTC-133 and -238 revealed high-affinity binding sites for RA. Electrophoretic mobility shift and super-shift assays using a DR2 ("direct repeat" 2) RA response element demonstrated DNA-binding of RARalpha, RARgamma, RXRalpha and RXRbeta in nuclear extracts of FTC-133 and anaplastic HTh74 cells. Use of a DR5 RA response element revealed no difference in DNA binding. In supershift assays with a DR4 T3 response element, we found DNA-binding by TRalpha1, TRalpha2, and TRbeta. Northern-blot analysis showed low expression of RXRbeta mRNA in FTC-133 and of TRalpha1 mRNA in FTC-133 and FTC-238 cells. Using RT-PCR, we detected mRNA for RARalpha, RARbeta, RARgamma, RXRalpha, and RXRbeta in the 4 cell lines and in human thyroid-carcinoma samples. RARbeta mRNA was reduced in FTC-238 cells and RXRbeta mRNA was decreased in anaplastic C643 cells and 9 of 12 tumor samples. Differential RA regulation of RA-receptor-mRNA expression was observed in the various cell lines. Thus, RA and T3 nuclear receptors are present in thyroid-carcinoma cell lines or tissues, albeit with cell-line and tumor-dependent variations; in the cell lines, they were shown to be functional with respect to DNA and/or ligand binding.

Cloning and characterization of the human selenoprotein P promoter. Response of selenoprotein P expression to cytokines in liver cells.

We isolated an 18-kilobase (kb) genomic selenoprotein P clone from a human placenta library and cloned, sequenced, and characterized the 5'-flanking region of the human selenoprotein P gene. Sequence analysis revealed an intron between base pairs (bp) -13 and -14 upstream of the ATG codon and another one between bp 534 and 535 of the coding region. The major transcription start site of selenoprotein P in human HepG2 hepatocarcinoma cells was mapped to bp -70 by 5'-rapid amplification of cDNA ends and by primer extension. 1.8 kb of the 5'-flanking sequence were fused to a luciferase reporter gene. They exhibited functional promoter activity in HepG2 hepatocarcinoma and Caco2 colon carcinoma cells in transient transfection experiments. Treatment of transfected HepG2 cells with the cytokines interleukin 1beta, tumor necrosis factor alpha, and interferon gamma repressed promoter activity. Nuclear extracts of interferon gamma-treated cells bound to a signal transducer and activator of transcription response element of the promoter in gel retardation experiments. By transfection of promoter-deletion constructs, a TATA box and a putative SP1 site were identified to be necessary for selenoprotein P transcription. These data indicate that the human selenoprotein P gene contains a strong promoter that is cytokine responsive. Furthermore, selenoprotein P, secreted by the liver, might react as a negative acute phase protein.

The promoter of the human type I 5'-deiodinase gene--mapping of the transcription start site and identification of a DR+4 thyroid-hormone-responsive element.

The selenoenzyme thyroxine 5'-deiodinase type I deiodinates the prohormone thyroxine to the active thyroid hormone 3,3',5-triiodothyronine. It is thus one of the key enzymes involved in the triiodothyronine-mediated control of growth, differentiation and basal metabolism in vertebrates. We report here the identification of the transcription start site and the cloning of 1500 bases of the upstream regulatory region of the human 5'-deiodinase gene. They contain a complex triiodothyronine-responsive element at nucleotides -696 to -673, consisting of an ideal direct repeat (DR) of two AGGTCA half-sites with a spacing of four nucleotides (DR+4) and a third putative AGTTCA half-site with a spacing of another two nucleotides (DR+2). The whole DR+4+2 specifically bound to thyroid hormone receptor and retinoid X receptor in electrophoretic mobility shift assays. The DR+4+2 mediates triiodothyronine-responsiveness in cotransfection experiments of constructs containing the 5'-deiodinase upstream promoter and enhancer region fused to luciferase or chloramphenicol acetyltransferase reporter genes with expression plasmids of thyroid hormone receptor subtypes. Also, an about 2.5-fold induction of the 5'-deiodinase-promoter-luciferase-reporter construct by all-trans retinoic acid was observed in a cotransfection assay with retinoic acid receptors. Point mutation analysis of the DR+4+2 type hormone-responsive element, however, revealed that it does not alone mediate the retinoic acid effect. The transcription start point of the 5'-deiodinase gene was mapped to nucleotides -23 and -24. No CAAT or TATA box is located within the usual distance to the transcription initiation site. Two GC boxes were found at nucleotides -68 to -63 and -39 to -34. Transfection analysis revealed that the proximal 105 nucleotides in the 5'-flanking region of the 5'-deiodinase gene act as a functional core promoter. This data indicates that triiodothyronine, the end product of thyroid hormone synthesis, positively regulates one of the key enzymes in its production.

Structure of the human type I iodothyronine 5'-deiodinase gene and localization to chromosome 1p32-p33.

The human type I iodothyronine 5'-deiodinase gene encodes a member of the family of selenocysteine-containing deiodinases. These enzymes catalyze the activation of the prohormone thyroxine to 3,3',5-triiodothyronine or the degradation of thyroxine and triiodothyronine to inactive metabolites. Here we report the isolation of two genomic type I 5'-deiodinase clones from a chromosome 1-specific gridded cosmid library, the localization of the gene to chromosome 1p32-p33 by fluorescence in situ hybridization, and the determination of the complete structure of the 17.5-kb gene.