Thyroid Tumor Cell Lines Research Articles

Expression of vascular endothelial growth factor (VEGF) and its receptors in thyroid carcinomas of follicular origin: a potential autocrine loop

The aim of this study was to clarify the role of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) pathways in thyroid tumourigenesis. We examined VEGF, VEGFR-1 and VEGFR-2 expression on 34 papillary thyroid carcinomas (PTCs), 18 follicular thyroid carcinomas (FTCs), eight poorly differentiated thyroid carcinomas (PDTCs) and on a thyroid tumour-derived cell line (NPA'87) by immunohistochemistry, reverse transcriptase PCR, immunofluorescence and Western blotting. We have demonstrated that VEGF expression was significantly (P < 0.05) more prevalent in PTCs (79%) than in FTCs (50%) or PDTCs (37%). Similarly, 76% of PTCs, 83% of FTCs and 25% of PDTCs expressed VEGFR-1, whereas 68% of PTCs, 56% of FTCs and 37% of PDTCs expressed VEGFR-2. Coexpression of VEGF and its receptors was observed in 50% of PTCs, 39% of FTCs and 12% of PDTCs, raising the possibility that VEGF may signal in an autocrine loop in these neoplasias, as observed previously for other types of cancer. In agreement with the idea that autocrine VEGF signalling plays an important role in thyroid carcinogenesis, the blockade of either VEGF or its receptors with neutralizing antibodies significantly reduced cell viability and increased apoptosis levels of the VEGFR-positive thyroid tumour cell line NPA'87. Our results highlight a previously undefined VEGF autocrine action in thyroid carcinomas which could play a crucial role in tumour cell survival and could represent a useful therapeutic target for thyroid tumours.

RASSF1A and NORE1A methylation and BRAFV600E mutations in thyroid tumors

We analyzed RASSF1A and NORE1A methylation and BRAF mutation in 89 thyroid tumors, 42 non-neoplastic thyroid tissues and three thyroid tumor cell lines using polymerase chain reaction (PCR), methylation-specific PCR, Western blotting and DNA sequencing in order to study thyroid tumor pathogenesis and progression. RASSF1A promoter methylation was present in all three thyroid cell lines and in 27/78 (35%) of benign and malignant thyroid tumors. We showed for the first time that there was generally good agreement between RASSF1A methylation status and RASSF1A protein expression. We also examined for the first time NORE1A promoter region methylation in thyroid cell lines and primary tumors and showed that two of three thyroid cell lines were methylated in the NORE1A promoter region, while all primary thyroid tumors analyzed (n=51) were unmethylated. BRAF mutation was present in 38% of papillary thyroid carcinomas (PTC), including 20% of PTC with a follicular variant pattern and 67% of the tall cell variant of PTC. Hyalinizing trabecular tumors (n=23), which had nuclear features similar to PTC, did not have BRAF mutations, indicating that the presence of BRAF mutations can help to separate these two tumor types. Phospho-MEK expression was increased in the NPA cell line, which had a BRAF mutation, supporting the importance of the BRAF pathway alterations in PTC pathogenesis. These results indicate that RASSF1A epigenetic changes are an early event in thyroid tumor pathogenesis and progression and that NORE1A methylation is uncommon in primary thyroid tumors. BRAF mutation occurs later in thyroid tumor progression and is restricted mainly to PTC and anaplastic thyroid carcinoma.

Differential Pattern of Integrin Receptor Expression in Differentiated and Anaplastic Thyroid Cancer Cell Lines

Adhesion of tumor cells to the extracellular matrix (ECM) is a crucial step for the development of metastatic disease and is mediated by specific integrin receptor molecules (IRM). The pattern of metastatic spread differs substantially among the various histotypes of thyroid cancer (TC). However, IRM have only occasionally been characterized in TC until now. IRM expression was investigated in 10 differentiated (FTC133, 236, 238, HTC, HTC TSHr, XTC, PTC4.0/4.2, TPC1, Kat5) and two anaplastic TC cell lines (ATC, C643, Hth74), primary cultures of normal thyroid tissue (Thy1,3), and thyroid cancer specimens (TCS). Expression of 16 IRM (beta1-4, beta7, alpha1-6, alphaV, alphaIIb, alphaL, alphaM, alphaX) and of four IRM heterodimers (alpha2beta1, alpha5beta1, alphaVbeta3, alphaVbeta5), was analyzed by fluorescent-activated cell sorter (FACS) and immunohistochemical staining. Thyroid tumor cell adhesion to ECM proteins and their IRM expression in response to thyrotropin (TSH) was assessed. Follicular TC cell lines presented high levels of integrins alpha2, alpha3, alpha5, beta1, beta3 and low levels of alpha1, whereas papillary lines expressed a heterogenous pattern of IRM, dominated by alpha5 and beta1. ATC mainly displayed integrins alpha2, alpha3, alpha5, alpha6, beta1 and low levels of alpha1, alpha4 and alphaV. Integrin heterodimers correlated with monomer expression. Evaluation of TCS largely confirmed these results with few exceptions, namely alpha4, alpha6, and beta3. The ability of TC cell lines to adhere to purified ECM proteins correlated with IRM expression. TSH induced TC cell adhesion in a dose-dependent fashion, despite an unchanged array of IRM expression or level of a particular IRM. Thyroid carcinoma cell lines of different histogenetic background display profoundly different patterns of IRM expression that appear to correlate with tumor aggressiveness. In vitro adhesion to ECM proteins and IRM expression concur. Finally, TSH-stimulated adhesion of thyroid tumor cell lines to ECM may not be associated with altered IRM expression.

Uncommon Mutation, but Common Amplifications, of thePIK3CAGene in Thyroid Tumors

As in many other human cancers, overactivation of the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway occurs frequently in thyroid cancer, but the mechanism is not completely clear. Because activating mutations and genomic amplification of the PIK3CA gene, which encodes the p110a catalytic subunit of PI3K, are common in many cancers, we sought to investigate this phenomenon in thyroid tumors. To search for PIK3CA mutations, we isolated genomic DNA from primary thyroid tumors of various types and performed direct sequencing of the exons of PIK3CA gene that carry the most common mutations in other cancers. We used real-time quantitative PCR to investigate genomic amplification of the PIK3CA gene. We found no PIK3CA gene mutations in 37 benign thyroid adenomas, 52 papillary thyroid cancers, 25 follicular thyroid cancers, 13 anaplastic thyroid cancers, 13 medullary thyroid cancers, and seven thyroid tumor cell lines. We found a C3075T single-nucleotide polymorphism in exon 20 of this gene in two cases. With a copy number of 4 or more defined as amplification, we found PIK3CA gene amplification in four of 34 (12%) benign thyroid adenomas, three of 59 (5%) papillary thyroid cancer, five of 21 (24%) follicular thyroid cancer, none of 14 (0%) medullary thyroid cancer, and five of seven (71%) thyroid tumor cell lines. The PIK3CA gene amplification and consequent Akt activation were confirmed by fluorescence in situ hybridization and Western blotting studies using cell lines, respectively. These data suggest that mutation of the PIK3CA gene is not common, but its amplification is relatively common and may be a novel mechanism in activating the PI3K/Akt pathway in some thyroid tumors.

Effects of Histone Acetylation on Sodium Iodide Symporter Promoter and Expression of Thyroid-Specific Transcription Factors

Inhibitors of histone deacetylases (HDACs) activate the sodium iodide symporter (NIS) expression in thyroid tumor cells. In this study, mechanisms accounting for these effects were investigated. Various human thyroid tumor cell lines (ARO, BCPAP, FRO, TPC-1) were treated with the HDAC inhibitors Na butyrate (NaB) and tricostatin A (TSA), and the effects on the expression of NIS and several thyroid-specific transcription factors together with the activity of NIS promoter were evaluated. TSA and NaB increased NIS mRNA levels in all cell lines. Among thyroid-specific transcription factors, only expression of PAX8 in ARO cells was increased. Down-regulation of thyroid-specific transcription factor-1 expression was observed in BCPAP and TPC-1 cell lines. Thyroid-specific transcription factor-2 mRNA was reduced in FRO, BCPAP, and TPC-1 cells. Histone acetylation had no significant effects on HEX expression. Altogether, these data indicate that the increase of NIS expression is not mediated by modification of expression of thyroid-specific transcription factors. Accordingly, in transfection experiments performed in the HeLa cell line (which does not express thyroid-specific transcription factors), treatment with TSA and NaB increased NIS promoter activity. Stimulation of NIS promoter activity was also obtained by overexpressing histone acetylating proteins pCAF and p300 in HeLa cells. Conversely, overexpression of the HDAC 1 enzyme inhibited basal activity of the NIS promoter. Effects of TSA and NaB on NIS expression were also evaluated in nonthyroid cell lines MCF-7, Hep-G2, and SAOS-2. In all cell lines TSA and NaB greatly increased NIS mRNA levels. We concluded that control of NIS expression by inhibition of HDAC appears not to be mediated by cell-specific mechanisms, suggesting it as a potential strategy to induce radioiodine sensitivity in different human tumors.

Wnt-5a has tumor suppressor activity in thyroid carcinoma

Stabilization of beta-catenin by inhibition of its phosphorylation is characteristic of an activation of the canonical Wnt/beta-catenin signaling pathway and is associated with various human carcinomas. It contrasts to an as yet incompletely characterized action of an alternative noncanonical Wnt signaling pathway on neoplastic transformation. The aim of the present study was to test the effects of a member of the noncanonical Wnt signaling pathway, Wnt-5a, in primary thyroid carcinomas and in thyroid carcinoma cell lines. Compared to normal tissue Wnt-5a mRNA expression was clearly increased in thyroid carcinomas. Immunohistochemically, a bell-shaped response was observed with low to undetectable levels in normal tissue and in anaplastic tumors whereas differentiated thyroid carcinomas showed strong positive immunostaining for Wnt-5a. Transfection of Wnt-5a in a thyroid tumor cell line FTC-133 was able to reduce proliferation, migration, invasiveness and clonogenicity in these cells. These effects of Wnt-5a are associated with membranous beta-catenin translocation and c-myc oncogene suppression and are mediated through an increase in intracellular Ca(2+) release, which via CaMKII pathways promotes beta-catenin phosphorylation. Specific inhibition of beta-catenin phosphorylation by W-7, a calmodulin inhibitor, or by KN-93, a CaMKII inhibitor, supports these findings whereas PKC inhibitors were without effect. This interaction occurs downstream of GSK-3 beta as no Wnt-5a effect was seen on the Ser(9) phosphorylation of GSK-3 beta. Our data are compatible with the hypothesis that Wnt-5a serves as an antagonist to the canonical Wnt-signaling pathway with tumor suppressor activity in differentiated thyroid carcinomas.

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.

High frequency of somatic mitochondrial DNA mutations in human thyroid carcinomas and complex I respiratory defect in thyroid cancer cell lines

Significant progress has been made to elucidate the molecular mechanisms that determine thyroid tumor development and progression. However, most investigations have mainly focused on the genetic alterations of nuclear DNA. The potential role of mitochondrial DNA (mtDNA) mutations in thyroid tumorigenesis is not well defined. In the present study, we investigated the frequency of mtDNA mutations in 24 thyroid tumor specimens (19 primary papillary thyroid carcinomas (PTC), one follicular thyroid carcinoma, and four multinodular hyperplasias) and four thyroid cancer cell lines by sequencing the entire coding regions of mitochondrial genome. Among the 19 PTC samples tested, seven (36.8%) had somatic mutations. Somatic mtDNA mutations were also detected in one of four multinodular hyperplasias examined. All the thyroid tumor cell lines carried sequence variations that change amino acid and have not been reported previously as normal sequence variants. Flow cytometry analysis of mitochondria respiratory function in the thyroid tumor cell lines revealed a severe defect in mitochondrial complex I activity. The majority of the mutations was involved in genes located in the complex I of the mitochondrial genome. The mutations were either A --> G or C --> T transitions, often resulting in a change of a moderately or highly conserved amino acid of their corresponding protein. These data suggest that mtDNA mutations may play an important role in the thyroid tumorigenesis. Given that mtDNA mutation is present in the benign multinodular hyperplasia, it might be involved in the early stage of tumor development.

Expression of Matrix Metalloproteinases and Their Specific Inhibitors in Normal and Different Human Thyroid Tumor Cell Lines

In the present study we investigated, by means of zymography and reverse transcription-polymerase chain reaction (RT-PCR), the expression of different matrix metalloproteinases (MMPs) and of the specific tissue inhibitor of metalloproteinases [TIMPs] in human cell lines derived from normal thyrocytes (HTU5), follicular adenoma (HTU42), and follicular (FTC-133), papillary (B-CPAP), and anaplastic (CAL-62, 8305C) thyroid carcinomas. We demonstrated that normal thyrocytes constitutively express MMP-1, MMP-2, MMP-10, MMP-14, and TIMP-1, TIMP-2, TIMP-3, and TIMP-4, and this pattern of expression is profoundly modified in all thyroid tumor-derived cell lines. Analysis of the gelatinolytic activity in the different cell supernatants showed that the expressions of MMP-2 and MMP-9 are, respectively, increased or induced in all the neoplastic cell lines, except in CAL-62. Caseinolytic activity was found only in the supernatants of the 8305C and B-CPAP cells. Using RTPCR analysis we detected an increased expression of MMP-1 in cell lines derived from papillary and from one (8305C) of the two anaplastic carcinomas. MMP-13 mRNA was expressed only in the 8305C, FTC-133, and BCPAP cells. Among stromelysins, MMP-3 mRNA could not be detected in any cell line, while MMP-10 mRNA was expressed in all of them, although at variable levels. MMP-11 mRNA was absent in normal and follicular adenoma derived thyrocytes and induced in all carcinoma cell types. The expression of MMP-14 (MT1-MMP) mRNA was found significantly increased in all thyroid tumor cell lines with respect to HTU5 and HTU42 cells. The expression of TIMP-1 and TIMP-2 mRNAs was maintained in all cell lines tested, while that of TIMP-3 was lost in both anaplastic carcinoma cell lines and that of TIMP-4 was absent in the CAL-62. In conclusion, our data demonstrated a differential expression of MMPs and TIMPs in different thyroid tumor cell types with respect to normal thyrocytes. In particular, the induction of MMP-11 in all thyroid-derived carcinoma cell lines studied and of MMP-13 in all but one may represent, if confirmed in other thyroid tumor-derived cell lines and in thyroid tumor tissues, a new marker of thyrocyte transformation.

Cytotoxic effects of gemcitabine-loaded liposomes in human anaplastic thyroid carcinoma cells.

BackgroundIdentification of effective systemic antineoplastic drugs against anaplastic thyroid carcinomas has particularly important implications. In fact, the efficacy of the chemotherapeutic agents presently used in these tumours, is strongly limited by their low therapeutic index.MethodsIn this study gemcitabine was entrapped within a pegylated liposomal delivery system to improve the drug antitumoral activity, thus exploiting the possibility to reduce doses to be administered in cancer therapy. The cytotoxic effects of free or liposome-entrapped gemcitabine was evaluated against a human thyroid tumour cell line. ARO cells, derived from a thyroid anaplastic carcinoma, were exposed to different concentrations of the drug. Liposomes formulations were made up of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-MPEG (8:3:1 molar ratio). Cell viability was assessed by both trypan bleu dye exclusion assay and fluorimetric analysis of cell DNA content.ResultsA cytotoxic effect of free gemcitabine was present only after 72 h incubation (ARO cell mortality increased of approximately 4 fold over control at 1 μM, 7 fold at 100 μM). When gemcitabine was encapsulated in liposomes, a significant effect was observed by using lower concentrations of the drug (increased cell mortality of 2.4 fold vs. control at 0.3 μM) and earlier exposure time (24 h).ConclusionThese findings show that, in vitro against human thyroid cancer cells, the gemcitabine incorporation within liposomes enhances the drug cytotoxic effect with respect to free gemcitabine, thus suggesting a more effective drug uptake inside the cells. This may allow the use of new formulations with lower dosages (side effect free) for the treatment of anaplastic human thyroid tumours.

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.

Bone sialoprotein, matrix metalloproteinase 2, and alpha(v)beta3 integrin in osteotropic cancer cell invasion.

Bone sialoprotein (BSP) interacts separately with both matrix metalloproteinase 2 (MMP-2) and integrin alpha(v)beta3 and is overexpressed in many metastatic tumors. Its role in tumor biology, however, remains unclear. We investigated whether BSP enhances cancer cell invasiveness by forming a trimolecular complex with MMP-2 and cell-surface integrin alpha(v)beta3. Invasiveness of breast, prostate, lung, and thyroid tumor cell lines was measured with a modified Boyden chamber assay. Binding and co-localization of BSP, MMP-2, and integrin alpha(v)beta3 were investigated with immunoprecipitation and in situ hybridization. All statistical tests were two-sided. Treatment with BSP increased invasiveness of many breast, prostate, lung, and thyroid cancer cells through Matrigel in a dose-dependent manner. BSP at 50 nM increased the invasiveness of SW-579 thyroid cancer cells (95.2 units, 95% confidence interval [CI] = 90.4 to 100 units) by approximately 10-fold compared with that of untreated control SW-579 cells (9.1 units, 95% CI = 5.7 to 12.5 units) (P<.001). Addition of an inactive mutated BSP, in which BSP's integrin-binding RGD tripeptide was altered, or addition of integrin alpha(v)beta3-blocking antibodies resulted in invasiveness equivalent to that of untreated cells. Inhibiting cellular MMP-2 activity with chemical inhibitors or a specific antibody also blocked BSP-enhanced invasiveness. Osteopontin and dentin matrix protein 1, proteins related to BSP that also bind integrin alpha(v)beta3 and form complexes with other MMPs (but not MMP-2), did not enhance invasiveness. Immunoprecipitation showed that a complex containing BSP, integrin alpha(v)beta3, and MMP-2 formed in vitro. Addition of BSP increased the amount of MMP-2 bound by cells in an integrin-dependent fashion. Co-expression of BSP, integrin alpha(v)beta3, and MMP-2 in papillary thyroid carcinoma cells was shown by in situ hybridization. Cancer cells appear to become more invasive when BSP forms a cell-surface trimolecular complex by linking MMP-2 to integrin alpha(v)beta3.

Early occurrence of RASSF1A hypermethylation and its mutual exclusion with BRAF mutation in thyroid tumorigenesis.

Follicular epithelial cell-derived thyroid tumors are common neoplasms comprised mainly of benign thyroid adenomas, follicular thyroid cancers, and papillary thyroid cancers (PTCs). Hypermethylation of the tumor suppressor gene RASSF1A and activating mutation of BRAF gene have been reported recently in thyroid cancers. To additionally investigate the roles of these two epigenetic/genetic alterations in thyroid tumor progression, we examined their occurrences and relationship in both benign and malignant thyroid neoplasms. With real-time quantitative methylation-specific PCR, we found that 4 of 9 (44%) benign adenomas, 9 of 12 (75%) follicular thyroid cancers tumors, and 6 of 30 (20%) of PTC tumors harbored promoter methylation in > or = 25% of RASSF1A alleles. Additional quantitative analysis revealed RASSF1A methylation only in BRAF mutation-negative PTCs. A similar inverse correlation of RASSF1A methylation with BRAF mutation was seen in thyroid tumor cell lines. Our results, therefore, suggest that epigenetic inactivation of RASSF1A through aberrant methylation is an early step in thyroid tumorigenesis. Like the previously reported mutually exclusive relationship between BRAF mutation and other Ras pathway components such as RET/PTC rearrangement, a mutually exclusive relationship also exists between BRAF mutation and RASSF1A methylation in thyroid tumorigenesis.

IFNgamma sensitization to TRAIL-induced apoptosis in human thyroid carcinoma cells by upregulating Bak expression.

TRAIL preferentially induces apoptosis in tumor cells and virus-infected cells. Unlike other tumor necrosis factor family members, TRAIL does not kill cells from most normal tissues and has thus been proposed as a promising new cancer treatment. Our study demonstrated that IFNgamma combined with TRAIL can trigger apoptosis in vitro in several resistant thyroid tumor cell lines, such as thyroid anaplastic carcinoma cells (ARO cells), while either agent alone exerts only a minimal effect. We further tested this effect on a mouse thyroid tumor model, when in vivo tumor growth was also significantly inhibited by this combination. The mechanism of how IFNgamma sensitized thyroid carcinoma cells to TRAIL-induced apoptosis was investigated by screening global gene alterations in ARO cells treated with IFNgamma. Microarray data revealed that a proapoptotic gene, Bak, is markedly upregulated by IFNgamma, and this was confirmed by RNase protection assay. Western blot analysis also showed a significant increase in Bak at the protein level. Upregulation of Bak and sensitization for apoptosis by IFNgamma was blocked by overexpression of antisense Bak in ARO cells. Furthermore, overexpression of Bak sensitized ARO cell to TRAIL-induced apoptosis without the need for IFNgamma pretreatment. This suggests that Bak is a regulatory molecule involved in IFNgamma-facilitated TRAIL-mediated apoptosis in thyroid cancer cells.

Transcriptional regulation of human sodium/iodide symporter gene: a role for redox factor-1.

The transcriptional regulation of the human sodium/iodide symporter (NIS) gene in normal and transformed thyroid cells is a crucial issue in attempting to restore iodide uptake and use radioiodine as a therapeutic treatment of thyroid cancer. Previous investigations have shown that the multifunctional protein apurinic apyrimidinic endonuclease/redox factor 1 (APE/Ref-1) plays an important role in regulation of thyroid-specific gene transcription. In this study, we investigated the effects of APE/Ref-1 on human NIS promoter activity. Cotransfection experiments performed in nonthyroid HeLa cells demonstrated that APE/Ref-1 exerts both PAX8-dependent and PAX8-independent effects. In fact, in the absence of PAX8, overexpression of APE/Ref-1 enhanced NIS promoter activity 2-fold. When the expression plasmid of APE/Ref-1 was transfected together with an expression plasmid for PAX8, a strong cooperative effect was detected with an increase of NIS promoter activity 9-fold over control. The PAX8-independent effect of APE/Ref-1 was specific for the NIS promoter, resulting not present for the promoter of the thyroperoxidase (TPO) gene. It was, at least in part, due to the up-regulation of the transcriptional activity of the ubiquitous factor early growth response-1 (Egr-1). In the thyroid tumor cell lines TPC-1 and B-CPAP, APE/Ref-1 was not effective by itself, and it also failed to increase PAX8 stimulation on NIS promoter activity. These data demonstrate a role for APE/Ref-1 protein in the transcriptional regulation of NIS gene expression by itself and in cooperation with PAX8. However, restoring the PAX8-APE/Ref-1 expression in tumor cells may not be sufficient to obtain adequate levels of NIS gene expression.

Comparative analysis of two thyroid tumor cell lines by fluorescence in situ hybridization and comparative genomic hybridization

Tumors and tumor-derived cell lines are typically chromosomally complex and heterogeneous. These features complicate the description of their karyotype. As a first approach to the chromosomal characterization of the two near-triploid thyroid tumor cell lines, BCPAP and FTC133, the techniques of fluorescence in situ hybridization and comparative genomic hybridization were used and compared. Most of the results obtained by the two methods were in good agreement. The follicular-derived cell line FTC133 showed more extensive chromosome variation between cells than the papillary-derived cell line BCPAP. Both cell lines had significant gains in part or whole of chromosomes 1, 11, and 20 and losses in chromosomes 16, 21, and 22. BCPAP cells also had gains in chromosomes 4 and 5 and losses in chromosomes 7, 9, and 10; FTC133 cells had gains in chromosomes 6, 7, 8, 14, 15, and 19. Chromosomes 4 and 5 were the most stable in BCPAP cells; in the FTC133 cells, chromosomes 7 and 19 showed the greatest segregational stability. The results have been discussed in terms of possible karyotype evolution. Moreover, it has been possible to compare the sensitivity limits of the two techniques in the analysis of polyploid tumors.

Follicular growth of a thyroid carcinoma cell line (KAT-4) with abnormal E-cadherin and impaired epithelial barrier.

Loss of the epithelial phenotype is a well-established phenomenon during progression of carcinomas to a more malignant state. In the present study, we describe a human thyroid tumor cell line (KAT-4), established from a poorly differentiated carcinoma, which displays exceptional features. In culture, the KAT-4 cells had a fast proliferation rate that was not restricted by high cell density, resulting in multilayered growth. Unexpectedly, the cells expressed normal levels of epithelial markers, e.g., cytokeratin, occludin, and E-cadherin, showed apical-basolateral polarization of the plasma membrane including microvilli and junction complexes, and formed intercellular lumens resembling thyroid follicles. Yet, when grown on filter, the cells were unable to establish a tight paracellular barrier. Moreover, E-cadherin expressed at the cell surface consisted of two peptides with abnormal size (135 and 95 kd, respectively) as compared to mature E-cadherin (120 kd) in nonneoplastic thyrocytes. Northern blot analysis and examination of immunoreactivity, glycosylation, and catenin binding suggested that E-cadherin was aberrant because of altered posttranscriptional processing. Thus, the KAT-4 thyroid carcinoma cell line has a unique phenotype, with maintained epithelial morphology despite dysfunctioning tight junctions, abnormal E-cadherin, and loss of contact-inhibited growth, that is not previously identified in other wild-type tumor cell lines.

Cloning and Characterization of the Human Heparanase-1 (HPR1) Gene Promoter: ROLE OF GA-BINDING PROTEIN AND Sp1 IN REGULATINGHPR1 BASAL PROMOTER ACTIVITY

Heparanase-1 (HPR1) is an endoglycosidase that specifically degrades the heparan sulfate chains of proteoglycan, a component of blood vessel walls and the extracellular matrix. Recent studies demonstrated that HPR1 expression is increased in a variety of malignancies and may play a critical role in tumor metastases. The HPR1 gene and its genomic structure have been recently cloned and characterized. To understand the mechanisms of HPR1 gene expression and regulation, we first mapped the transcription start site of the HPR1 gene and found that HPR1 mRNA was transcribed from the nucleotide position 101 bp upstream of the ATG codon. A 3.5-kb promoter region of the HPR1 gene was cloned. Sequence analysis revealed that the TATA-less, GC-rich promoter of the HPR1 gene belongs to the family of housekeeping genes. This 3.5-kb promoter region exhibited strong promoter activity in two thyroid tumor cell lines. Truncation analysis of the HPR1 promoter identified a minimal 0.3-kb region that had strong basal promoter activity. Truncation and mutational analysis of the HPR1 promoter revealed three Sp1 sites and four Ets-relevant elements (ERE) significantly contributing to basal HPR1 promoter activity. Binding to the Sp1 sites by Sp1 and to the ERE sites by GA-binding protein (GABP) was confirmed by electrophoretic mobility shift assay and competition and supershift electrophoretic mobility shift assays. Cotransfection of Sp- and GABP-deficient Drosophila SL-2 cells with the HPR1 promoter-driven luciferase construct plus the expression vector encoding the Sp1, Sp3, or GABP gene induced luciferase gene expression. Mutation or truncation of the Sp1 or ERE sites reduced luciferase expression in both SL-2 cells and thyroid tumor cell lines. Coexpression of GABPalpha/beta and Sp1 or Sp3 further increased luciferase reporter gene expression. Our results collectively suggest that Sp1 cooperates with GABP to regulate HPR1 promoter activity.

P44/p42-MAP kinase expression in papillary thyroid carcinomas

Background. Recent studies suggest that mitogen-activated protein (MAP) kinase contributes to the growth and tumorigenesis of human epithelial cancers. Furthermore, blockade of this pathway may inhibit the growth of epithelial cancers. To determine whether MAP kinase is activated in human papillary thyroid carcinomas (PTCs), we analyzed the presence of native MAP kinase (MAPK) and activated phosphorylated MAP kinase (pMAPK) in papillary thyroid cancers and thyroid carcinoma cell lines. Methods. Protein from paired specimens of 10 patients with PTC was analyzed by immunoblot for MAPK and pMAPK. In addition, MAPK protein expression and cell growth were analyzed in 3 thyroid tumor cell lines treated with a mitogen extracellular kinase inhibitor, U0126. Results. All 10 PTCs had equal expression of MAPK in the tumors and adjacent normal tissue. Six of the 10 tumors demonstrated increased expression of the pMAPK in the tumor specimen compared to the adjacent normal tissue. Interestingly, 3 of 4 patients without differential expression had multifocal PTC. The pMAPK was expressed constitutively in 3 thyroid cancer cell lines. The MAPK inhibitor treatment decreased pMAPK expression and decreased serum-induced growth in all 3 cell lines. Conclusions. MAP kinase activation is common in PTCs and may offer a potential target for growth inhibition of PTCs. (Surgery 2001;130:936-40.)

PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells mediated by p27kip1.

The dual-specificity phosphatase PTEN/MMAC1/TEP1 has recently been identified as the tumor suppressor gene most frequently mutated and/or deleted in human tumors. Germline mutations of PTEN give rise to Cowden Disease (CD), an autosomal dominantly-inherited cancer syndrome which predisposes to increased risk of developing breast and thyroid tumors. However, PTEN mutations have rarely been detected in sporadic thyroid carcinomas. In this study, we confirm that PTEN mutations in sporadic thyroid cancer are infrequent as we found one point mutation and one heterozygous deletion of PTEN gene in 26 tumors and eight cell lines screened. However, we report that PTEN expression is reduced both at the mRNA and at the protein level - in five out of eight tumor-derived cell lines and in 24 out of 61 primary tumors. In most cases, decreased PTEN expression is correlated with increased phosphorylation of the PTEN-regulated protein kinase Akt/PKB. Moreover, we demonstrate that PTEN may act as a suppressor of thyroid cancerogenesis as the constitutive re-expression of PTEN into two different thyroid tumor cell lines markedly inhibits cell growth. PTEN-dependent inhibition of BrdU incorporation is accompanied by enhanced expression of the cyclin-dependent kinase inhibitor p27kip1 and can be overcome by simultaneous co-transfection of an excess p27kip1 antisense plasmid. Accordingly, in a subset of thyroid primary carcinomas and tumor-derived cell lines, a striking correlation between PTEN expression and the level of p27kip1 protein was observed. In conclusion, our findings demonstrate that inactivation of PTEN may play a role in the development of sporadic thyroid carcinomas and that one key target of PTEN suppressor activity is represented by the cyclin-dependent kinase inhibitor p27kip1.