Normal Thyroid Cells Research Articles

Down-regulation of an inhibitor of cell growth, transmembrane protein 34 (TMEM34), in anaplastic thyroid cancer

Ansaplastic thyroid cancer (ATC) is one of the most lethal malignancies, but the carcinogenic mechanism of ATC has not been clarified. Recently, we performed a cDNA microarray analysis and identified transmembrane protein 34 (TMEM34) that down-regulated in anaplastic thyroid cancer cell lines (ACL)s as compared to normal thyroid tissues. To investigate the role of TMEM34 in ATC carcinogenesis, we examined expression levels of TMEM34 in ACLs as well as differentiated thyroid cancers (DTC)s and normal human tissues. To explore the effect of TMEM34 in ATC development, cell-growth assays with KTA2 cells were performed. Expression of TMEM34 was down-regulated in all 11 ACLs, as compared to either normal thyroid tissues or cell lines derived from papillary or follicular thyroid cancers. TMEM34 was expressed ubiquitously in normal human tissues tested. Transfection of TMEM34 into KTA2 cells led to inhibition of cell growth. Our findings suggest that TMEM34 might be a tumor suppressor gene, associated with the development of ATC from DTC.

Carcinoma Showing Thymus-like Differentiation of the Thyroid (CASTLE): A Comparative Study

Carcinoma showing thymus-like differentiation (CASTLE) is a rare intrathyroidal neoplasm, a member of a tumor family probably arising from ectopic thymus or branchial pouch remnants. Thyroid solid cell nests (SCNs) may also be derived from branchial pouch remnants. SCNs express p63, carcinoembryonic antigen (CEA), and high molecular weight keratin (HMWK). To determine whether CASTLE and SCNs derive from similar embryologic origins/lines of differentiation, and to better differentiate CASTLE from other thyroid neoplasms, we compared p63, CD5, HMWK, and CEA staining of CASTLE and SCNs with other thyroid and thymic lesions. Seven CASTLE, 11 SCNs, 10 thymic carcinoma, 11 invasive thymoma, 12 thymoma, 28 papillary thyroid carcinoma, 4 thyroid squamous cell carcinoma, 2 childhood sclerosing carcinoma, 4 follicular adenoma, 6 follicular carcinoma, 4 poorly differentiated carcinoma, and 20 lymphocytic thyroiditis cases were analyzed. In normal thyroid, only SCNs stained for p63, HMWK, and CEA. The only CD5-positive cells in normal thyroid were T cells. Thymomas and normal thymus stained similarly to SCNs. All CASTLE and thymic carcinomas exhibited diffuse p63 and HMWK staining and all CASTLE cases and the majority of thymic carcinomas were positive for CEA and CD5. In contrast, none of the other thyroid neoplasms examined exhibited consistent staining for all 4 markers studied. These findings provide further evidence that CASTLE is distinct from other thyroid neoplasms, is probably of thymic origin, and may arise from branchial pouch remnants, the thyroid SCNs. Moreover CD5, HMWK, CEA and p63 can be used to help distinguish CASTLE from other thyroid neoplasms.

Proliferative Activity of Human Thyroid Cells in Various Age Groups and Its Correlation with the Risk of Thyroid Cancer after Radiation Exposure

The thyroid gland is vulnerable to the carcinogenic effects of ionizing radiation, and there is a well-documented inverse correlation between thyroid cancer and age at exposure, particularly for ages less than 20 yr. One of the factors responsible for this phenomenon may be more rapid cell proliferation in children. The objective of this study was to determine the proliferative rate of normal human thyroid cells in different age groups. We used immunohistochemical analysis to determine the Ki-67 proliferative index in 117 thyroid glands obtained at autopsy, including 25 fetal thyroids (11-40 wk gestation), 55 childhood thyroids (0-19 yr), and 37 adult thyroids (20-60 yr). The rate of Ki-67 labeling in the three groups was 7.4 +/- 6.10, 0.23 +/- 0.15, and 0.08 +/- 0.04% respectively, demonstrating an overall trend for diminishing proliferative activity of thyroid cells with increasing age. However, a lack of correlation was noted between the slopes of cancer risk calculated from previous studies of irradiated populations and proliferative rate in the pediatric age intervals of 0-4 and 5-9 yr, suggesting that other factors are likely to be responsible for the particularly high sensitivity to radiation-induced thyroid cancer among the youngest children. Our findings of a general decrease in proliferative activity of thyroid cells with age may explain, at least in part, the higher risks of radiation-related thyroid cancer in children compared with adults. However, the variation in the rate of cell proliferation is unlikely to be responsible entirely for this phenomenon and other factors may also be involved.

Haploinsufficiency of the Hmga1 Gene Causes Cardiac Hypertrophy and Myelo-Lymphoproliferative Disorders in Mice

The HMGA1 protein is a major factor in chromatin architecture and gene control. It plays a critical role in neoplastic transformation. In fact, blockage of HMGA1 synthesis prevents rat thyroid cell transformation by murine transforming retroviruses, and an adenovirus carrying the HMGA1 gene in the antisense orientation induces apoptotic cell death in anaplastic human thyroid carcinoma cell lines, but not in normal thyroid cells. Moreover, both in vitro and in vivo studies have established the oncogenic role of the HMGA1 gene. In this study, to define HMGA1 function in vivo, we examined the consequences of disrupting the Hmga1 gene in mice. Both heterozygous and homozygous mice for the Hmga1-null allele show cardiac hypertrophy due to the direct role of HMGA1 on cardiomyocytic cell growth regulation. These mice also developed hematologic malignancies, including B cell lymphoma and myeloid granuloerythroblastic leukemia. The B cell expansion and the increased expression of the RAG1/2 endonuclease, observed in HMGA1-knockout spleen tissues, might be responsible for the high rate of abnormal IgH rearrangements observed in these neoplasias. Therefore, the data reported here indicate the critical role of HMGA1 in heart development and growth, and reveal an unsuspected antioncogenic potential for this gene in hematologic malignancies.

Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca 2+] i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca 2+] i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca 2+] i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca 2+] i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl- sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.

Interphase chromosome folding determines spatial proximity of genes participating in carcinogenic RET/PTC rearrangements

Recurrent chromosomal rearrangements are common in cancer cells and may be influenced by nonrandom positioning of recombination-prone genetic loci in the nucleus. However, the mechanism responsible for spatial proximity of specific loci is unknown. In this study, we use an 18 Mb region on 10q11.2-21 containing the RET gene and its recombination partners, the H4 and NCOA4 (ELE1) genes, as a model chromosomal region frequently involved in RET/PTC rearrangements in thyroid cancer. RET/PTC is particularly common in tumors from children exposed to ionizing radiation. Using fluorescence in situ hybridization and three-dimensional microscopy, the locations of five different loci in this region were mapped in interphase nuclei of normal human thyroid cells. We show that RET and NCOA4 are much closer to each other than expected based on their genomic separation. Modeling of chromosome folding in this region suggests the presence of chromosome coiling with coils of approximately 8 Mb in length, which positions the RET gene close to both, the NCOA4 and H4, loci. There was no significant variation in gene proximity between adult and pediatric thyroid cells. This study provides evidence for large-scale chromosome folding of the 10q11.2-21 region that offers a structural basis for nonrandom positioning and spatial proximity of potentially recombinogenic intrachromosomal loci.

Mitogenic Effects of the Up-Regulation of Minichromosome Maintenance Proteins in Anaplastic Thyroid Carcinoma

Anaplastic thyroid carcinomas (ATC) are among the most aggressive human malignancies and are characterized by high mitotic activity. Minichromosome maintenance proteins (MCM) 2-7 are required to initiate eukaryotic DNA replication, and their overexpression has been associated with dysplasia and malignancy. In an attempt to cast light on the mechanisms governing ATC, we evaluated MCM5 and MCM7 expression in human normal, papillary (PTC), and anaplastic thyroid samples, as well as in primary culture cells and transgenic mouse models. MCM5 and MCM7 expression was high in 65% of ATC and negligible in normal thyroid tissue and papillary thyroid carcinomas. In ATC, high MCM5 and MCM7 expression was paralleled by high levels of MCM2 and MCM6. An analysis of human ATC primary cell cultures and of a transgenic mouse model of ATC confirmed these findings. An increased transcription rate accounted for MCM7 up-regulation, because the activity of the MCM7 promoter was more than 10-fold higher in ATC cells compared with normal thyroid cells. Adoptive overexpression of wild-type p53, but not of its inactive (R248W and R273H) mutants, strongly down-regulated transcription from the MCM7 promoter, suggesting that p53 knock-out contributes to MCM7 up-regulation in ATC. Treatment with small inhibitory duplex RNAs, which decrease MCM7 protein levels, reduced the rate of DNA synthesis in ATC cells. MCM proteins are overexpressed in ATC and sustain the high proliferative capacity of ATC cells.

Thyrotropin-Stimulated DNA Synthesis and Thyroglobulin Expression in Normal and Hyperthyroid Feline Thyrocytes in Monolayer Culture

Feline hyperthyroidism is a common, spontaneous disease in older cats that is similar clinically and histopathologically to human toxic multinodular goiter (TNG). In this study, the functional response of feline normal thyroid (NT) and hyperthyroid (HT) cells grown in monolayer culture to thyrotropin (TSH) was determined. Basal levels of DNA synthesis were similar in NT and HT cells. TSH stimulated concentration-dependent DNA synthesis in NT and HT cells, with maximal stimulation seen at 1 and 10 mU/mL TSH in NT and HT cells, respectively. HT cells had higher basal levels of thyroglobulin (Tg) expression. TSH stimulated Tg expression in NT and HT cells in a concentration-dependent fashion, with maximal activity at 0.5 and 5 mU/mL TSH, respectively. These results demonstrate that NT and HT cells in monolayer culture exhibit growth and functional responses to TSH. HT cells have higher basal Tg expression than NT cells and require higher TSH concentrations to stimulate DNA synthesis and Tg expression, two measures of thyroid cell activation. These data support the idea that feline hyperthyroidism is caused by cell abnormalities, resulting in dysregulated growth and hormone synthesis, and emphasize its importance as an animal model for TNG.

Fibronectin-Induced Proliferation in Thyroid Cells Is Mediated by αvβ3 Integrin through Ras/Raf-1/MEK/ERK and Calcium/CaMKII Signals

We recently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium Ca2+/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FN-induced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2+ concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2+ concentration were mediated by FN binding to alphavbeta3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered.

Expression of Heterogeneous Nuclear Ribonucleoproteins A2 and B1 in the Thyroid Follicular Cells

Several proteins implicated in hormonogenesis of the thyroid have alternatively spliced isoforms. Alternative splicing of pre-mRNA is considered to be important to regulate the hormonal activity. Heterogeneous nuclear ribonucleoproteins (hnRNP) A2 and B1 are two of the abundant nuclear RNA-binding proteins involved in alternative splicing. We examined the expression of hnRNP A2 and B1 in the thyroid, paying particular attention to the relationship between their function and the cellular morphology. B1 was expressed more frequently in cuboidal follicular cells that are hormonally active than in flat follicular cells in normal thyroid, although A2 expression showed no significant difference in two cell types. In Graves' disease, the patients who had high serum levels of triiodothyronine and thyroxine showed significantly increased expression of B1. B1 expression did not differ significantly between normal thyroids and thyroid neoplasms, except undifferentiated (anaplastic) carcinoma. Conclusively, B1 expression varied in relation to hormonal activity in thyroid follicular cells. B1 protein is a good immunohistological marker to detect hormonal activity of follicular cells, and may provide a key to elucidate the splicing mechanisms involved in thyroid hormonogenesis.

Induction and Regulation of Fas-Mediated Apoptosis in Human Thyroid Epithelial Cells

Fas-mediated apoptosis has been proposed to play an important role in the pathogenesis of Hashimoto's thyroiditis. Normal thyroid cells are resistant to Fas-mediated apoptosis in vitro but can be sensitized by the unique combination of interferon-gamma and IL-1beta cytokines. We sought to examine the mechanism of this sensitization and apoptosis signaling in primary human thyroid cells. Without the addition of cytokines, agonist anti-Fas antibody treatment of the thyroid cells resulted in the cleavage of proximal caspases, but this did not lead to the activation of caspase 7 and caspase 3. Apoptosis associated with the cleavage of caspases 7, 3, and Bid, and the activation of mitochondria in response to anti-Fas antibody occurred only after cytokine pretreatment. Cell surface expression of Fas, the cytoplasmic concentrations of procaspases 7, 8, and 10, and the proapoptotic molecule Bid were markedly enhanced by the presence of the cytokines. In contrast, P44/p42 MAPK (Erk) appeared to provide protection from Fas-mediated apoptosis because an MAPK kinase inhibitor (U0126) sensitized thyroid cells to anti-Fas antibody. In conclusion, Fas signaling is blocked in normal thyroid cells at a point after the activation of proximal caspases. Interferon-gamma/IL-1beta pretreatment sensitizes human thyroid cells to Fas-mediated apoptosis in a complex manner that overcomes this blockade through increased expression of cell surface Fas receptor, increases in proapoptotic molecules that result in mitochondrial activation, and late caspase cleavage. This process involves Bcl-2 family proteins and appears to be compatible with type II apoptosis regulation.

Activation of nicotinamide N-methyltransferase gene promoter by hepatocyte nuclear factor-1beta in human papillary thyroid cancer cells.

We previously demonstrated that the human nicotinamide N-methytransferase (NNMT) gene was highly expressed in many papillary thyroid cancers and cell lines. The expression in other papillary and follicular cancers or cell lines and normal thyroid cells was low or undetectable. To gain an understanding of the molecular mechanism of this cell-specific expression, the NNMT promoter was cloned and studied by luciferase reporter gene assay. The promoter construct was expressed highly in papillary cancer cell lines, including those with higher (e.g. BHP 2-7) and lower (e.g. BHP 14-9) NNMT gene expression, and expressed weakly in follicular thyroid cancer cell lines. Further study with 5'-deletion promoter construct suggested that the NNMT promoter was regulated differently in BHP 2-7 and BHP 14-9 cells. In BHP 2-7 cells, promoter activity was dependent on an upstream sequence. In BHP 14-9 cells, sequence in the basal promoter region contributed notably to the overall promoter activity. RT-PCR or Western blot analysis indicated that hepatocyte nuclear factor-1beta (HNF-1beta) was expressed in only papillary cancer cell lines with high NNMT gene expression. HNF-1beta was not expressed or expressed very weakly in other papillary, follicular, and Hurthle cancer cell lines and primary cultures of normal thyroid cells and benign thyroid conditions. A HNF-1 binding site was identified in the NNMT basal promoter region. Mutations in this site decreased NNMT promoter activity in the HNF-1beta-positive BHP 2-7 cells, but not in the HNF-1beta-negative BHP 14-9 cells. HNF-1beta bound to the HNF-1 site specifically as a homodimer as determined by gel retardation assays with HNF-1beta-specific antibody. Cotransfection of a HNF-1beta expression plasmid increased NNMT promoter activity significantly in both HNF-1beta-positive and -negative thyroid cancer cell lines and Hep G2 liver cancer cells. Furthermore, transient expression of HNF-1beta in BHP 14-9 cells increased endogenous NNMT protein levels. In summary, HNF-1beta functions as a transcription activator for NNMT gene expression in some papillary thyroid cancer cells.

Normal human thyroid cells from the ARAMIS line follow the general concept of growth or differentiation: a study with thyroglobulin as a marker.

A cell line of functional normal human adult thyroid cells was isolated 3 years ago. This cell line was used as a model to study human thyroglobulin (hTgb) production quantified in 5% fetal calf serum Click-RPMI medium and in the presence of insulin and thyrotropin (two hormones [2H]) to get maximal hTgb production. In this paper we demonstrate that these cells (line ARAMIS) follow the general dualistic opposition between growth and differentiation. Thyroglobulin cell production in 2H-stimulated cells is dependent on cell density and is not constant with time of culture in growing and in quiescent cells. High serum levels required for cell proliferation antagonize thyroglobulin cell production. Furthermore, in the absence of cell proliferation, the longer the cells stay in the stationary phase in 2H-medium, the better they produce thyroglobulin with time. The longer the cells stay in the stationary phase in 1H (insulin) medium, the higher will be the total thyroglobulin production and the initial rate in thyroglobulin production after TSH addition. The longer the cells stay in the stationary phase in 1H (insulin) medium, the higher are cyclic adenosine monophosphate (cAMP) levels after thyrotropin (TSH) stimulation. This is observed both for initial rates and total production. Neither insulin nor cell-cell interactions occurring during the stationary phase modify basal cAMP levels. Altogether the data demonstrate that in cell culture conditions that exclude proliferation, a gain in TSH sensitivity appears versus time in insulin-stimulated quiescent normal human thyroid cells during their stationary phase. This improved differentiating status appears to be TSH- and cAMP-independent. It could be an insulin or insulin-like growth factor-1 (IGF-1)-dependent trophic effect promoting an increase in TSH-receptor number or sensitivity. But once again and as mentioned previously, we cannot exclude from the data that cell to cell interactions between silent and "informed" cells (in other words, autocrine phenomena) could result, with time, in the recruitment of silent resting cells, explaining the gain in TSH sensitivity.

Control of phosphatase and tensin homolog (PTEN) gene expression in normal and neoplastic thyroid cells.

The lipid phosphatase, phosphatase and tensin homolog (PTEN), is a key element in controlling cell growth and survival and has a well established role as tumor suppressor protein in many neoplasia. Several data indicate that silencing of PTEN gene expression may be relevant in follicular thyroid cell transformation. Thus, in the present study regulation of PTEN gene expression in thyroid cells was investigated. Cotransfection experiments indicated that in normal FRTL-5 rat thyroid cells, PTEN promoter activity was increased by overexpression of the transcription factor early growth response protein-1 (Egr-1). Moreover, Western blot experiments indicated that when Egr-1 expression was up-regulated by treating FRTL-5 cells with H2O2, an increase in PTEN expression was also observed. TSH induced opposite modifications on PTEN and Egr-1 protein levels. Moreover, acute or chronic TSH stimulation determined distinct effects. In fact, acute TSH stimulation (30 and 60 min) induced a decrease in PTEN, but an increase in Egr-1 protein levels. These effects were cAMP dependent; in fact, they were mimicked by forskolin. A chronic TSH treatment (5 d) stimulated PTEN protein expression, whereas Egr-1 protein was down-regulated. In contrast to normal thyroid cells, when the thyroid tumor cell lines ARO and BCPAP were exposed to H2O2, neither Egr-1 nor PTEN protein levels were increased. Acute stimulation of ARO and BCPAP cells with forskolin increased Egr-1, but not PTEN, protein levels. Therefore, thyroid tumor cell lines show alteration of PTEN gene expression regulation. RT-PCR experiments performed on human thyroid tumors showed that the absence of Egr-1 mRNA is always paralleled by the absence of PTEN mRNA. Thus, modification of the Egr-1-dependent mechanisms may play a role in the silencing of PTEN gene expression occurring during thyroid cell transformation.

Activation of the hepatocyte growth factor (HGF)-Met system in papillary thyroid cancer: biological effects of HGF in thyroid cancer cells depend on Met expression levels.

Met, the receptor for hepatocyte growth factor (HGF), is overexpressed in approximately 90% papillary thyroid carcinomas. To investigate the role of the HGF-Met system in these tumors, we examined HGF and Met expression in a variety of primary cultures, normal or malignant thyroid cells, and tissue specimens and analyzed the different HGF effects (promotion of mitogenesis, branching morphogenesis, and cell motility and invasion). In cancer specimens, HGF was produced at high levels by tumor stromal cells, and Met was constitutively phosphorylated in malignant cells. No HGF production was found in a panel of malignant thyroid cancer cells. Biological effects of HGF were examined in papillary cancer cell cultures with either high or low Met expression. High-Met cells were more sensitive to the growth effect of HGF (ED50 = 3-5 ng/ml and 10-15 ng/ml in high- or low-Met cells, respectively). Moreover, only high-Met cells underwent branching morphogenesis in response to HGF. In contrast, high-Met cells showed a reduced migration. Met down-regulation by small interfering RNAs resulted in enhanced cell migration and abrogation of branching morphogenesis in response to HGF. Conversely, Met overexpression impaired cell migration while favoring branching morphogenesis and cell adherence to substrate. These data suggest that both Met and HGF are overexpressed in papillary thyroid carcinomas, that Met is frequently activated in these carcinomas and may favor tumor growth, and that the abundance of Met expression may differently regulate cell growth, morphogenesis, and migration in response to HGF.

Functional expression of the CXCR4 chemokine receptor is induced by RET/PTC oncogenes and is a common event in human papillary thyroid carcinomas.

To identify genes involved in the transformation of thyroid follicular cells, we explored, using DNA oligonucleotide microarrays, the transcriptional response of PC Cl3 rat thyroid epithelial cells to the ectopic expression of the RET/PTC oncogenes. We found that RET/PTC was able to induce the expression of CXCR4, the receptor for the chemokine CXCL12/SDF-1alpha/beta. We observed that CXCR4 expression correlated with the transforming ability of the oncoprotein and depended on the integrity of the RET/PTC-RAS/ERK signaling pathway. We found that CXCR4 was expressed in RET/PTC-positive human thyroid cancer cell lines, but not in normal thyroid cells. Furthermore, we found CXCR4 expression in human thyroid carcinomas, but not in normal thyroid samples by immunohistochemistry. Since CXCR4 has been recently implicated in tumor proliferation, motility and invasiveness, we asked whether treatment with SDF-1alpha was able to induce a biological response in thyroid cells. We observed that SDF-1alpha induced S-phase entry and survival of thyroid cells. Invasion through a reconstituted extracellular matrix was also supported by SDF-1alpha and inhibited by a blocking antibody to CXCR4. Taken together, these results suggest that human thyroid cancers bearing RET/PTC rearrangements may use the CXCR4/SDF-1alpha receptor-ligand pathway to proliferate, survive and migrate.

Application of the Cre/loxP system to enhance thyroid-targeted expression of sodium/iodide symporter.

Radioiodide uptake activity mediated by the human Na(+)/I(-) symporter (hNIS) in thyroid follicular cells is the basis for effective (131)I therapy in thyroid cancer. However, radioiodide therapy is not effective in patients with thyroid cancer displaying low or absent hNIS expression. This study assessed the Cre/loxP system for enhancing thyroid-targeted hNIS expression driven by the thyroglobulin (Tg) promoter. The following three recombinant adenoviruses (rAd) were constructed: rAd-Tg-hNIS drives hNIS expression by the Tg promoter; rAd-Tg-Cre drives Cre expression by the Tg promoter; and rAd-CMV-loxP-hNIS drives hNIS expression by the cytomegalovirus (CMV) promoter after Cre-mediated excision of an intervening loxP-GFP-Zeo-loxP. Immortalized normal and malignant rat thyroid cell lines and primary cultures of normal human thyroid and human follicular adenoma cells were investigated. We found that the relative promoter activity of Tg vs. CMV is critical for the efficacy of the Cre/loxP system. In cells with weak Tg promoter activity, coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS induced higher hNIS expression than single infection of rAd-Tg-hNIS. Finally, Tg promoter activity was partially restored in malignant thyroid cells by forced expression of the paired domain-containing transcription factor (Pax-8), allowing the Cre/loxP system to mildly enhance radioiodide uptake.

TC-1 is a novel tumorigenic and natively disordered protein associated with thyroid cancer.

A novel gene, thyroid cancer 1 (TC-1), was found recently to be overexpressed in thyroid cancer. TC-1 shows no homology to any of the known thyroid cancer-associated genes. We have produced stable transformants of normal thyroid cells that express the TC-1 gene, and these cells show increased proliferation rates and anchorage-independent growth in soft agar. Apoptosis rates also are decreased in the transformed cells. We also have expressed recombinant TC-1 protein and have undertaken a structural and functional characterization of the protein. The protein is monomeric and predominantly unstructured under conditions of physiologic salt and pH. This places it in the category of natively disordered proteins, a rapidly expanding group of proteins, many members of which play critical roles in cell regulation processes. We show that the protein can be phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, and the activity of both of these kinases is up-regulated when cells are stably transfected with TC-1. These results suggest that overexpression of TC-1 may be important in thyroid carcinogenesis.

Hypothyroidism.

Hypothyroidism.

Inhibition of Nuclear Factor-κB Cascade Potentiates the Effect of a Combination Treatment of Anaplastic Thyroid Cancer Cells

Nuclear transcription factor-kappaB (NF-kappaB) is a transcriptional complex that is rapidly activated in the course of an immediate early response of cells after exposure to different stresses including ionizing radiation (IR). To overcome the limitation of radiation therapy for thyroid cancers, we studied the response of the NF-kappaB cascade to IR in cultured normal human thyroid cells and various thyroid cancer cell lines. Exposure to IR resulted in a dose-dependent increase of DNA-binding activity of p65 and p50 subunits in all types of thyroid cells. Specific inhibitors of NF-kappaB or phosphorylation deficient mutant inhibitory protein IkappaBalpha reduced thyroid cancer cell survival after exposure to IR and enhanced IR-induced cell death in a model undifferentiated thyroid cancer cell line. Tumors harboring mutant IkappaBalpha implanted into nude mice exhibited delayed growth rate and increased radiosensitivity. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and annexinV-propidium iodide staining revealed the increase of radiation-induced apoptosis in the cells with inhibited NF-kappaB signaling. Our results indicate that radiosensitivity of transformed thyroid cells is due in part to elevated basal activity and rapid induction of the active form of NF-kappaB. We therefore suggest that inhibition of NF-kappaB could be an effective modality for radiation therapy of advanced human thyroid cancers.