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Supplementary Figure 1 from Loss of the CBX7 Gene Expression Correlates with a Highly Malignant Phenotype in Thyroid Cancer

Supplementary Figure 2 from Loss of the CBX7 Gene Expression Correlates with a Highly Malignant Phenotype in Thyroid Cancer

Supplementary Methods and Figure Legends 1-2 from Loss of the CBX7 Gene Expression Correlates with a Highly Malignant Phenotype in Thyroid Cancer

Differentiated, cloned rat thyroid epithelial cells (424 cells) were infected with a wild-type and a temperature-sensitive strain of the myeloproliferative variant of the Moloney murine sarcoma virus. The thyroid cells were productively infected and transformed by both virus strains and displayed some of the typical properties of malignant cells, such as morphological changes, growth in soft agar, and in vivo tumorigenicity. The acquisition of the transformed phenotype by the virus-infected cells was accompanied by a loss of the typical differentiated features of the thyroid epithelium, such as thyroglobulin (TG) secretion, iodide uptake, and dependence for growth on six factors including thyrotropin, the physiological thyroid stimulator. TG mRNA could not be demonstrated in cells transformed by both viral strains, suggesting a block at the level of the TG gene transcription. While the transformed state of the cell clones infected with the temperature-sensitive strain could be reverted by shifting the cultures to the temperature nonpermissive for transformation (39 degrees C), no reversion of the differentiated functions took place after such a shift, showing that the v-mos oncogene irreversibly shuts off the differentiation of thyroid epithelial cells in vitro. These results demonstrate, for the first time, an oncogenic potential of the v-mos oncogene family towards differentiated epithelial cells in vitro.

The high mobility group (HMG) proteins (HMGA1a, HMGA1b, and HMGA2) bind to DNA and interact with various transcriptional factors. Therefore, they play an important role in chromatin organization. HMGA protein expression is low in normal adult tissues, but abundant during embryonic development and in several experimental and human tumors. Blockage of HMGA expression inhibits the transformation of rat thyroid PC Cl 3 cells treated with oncogene-carrying retroviruses, thus implicating HMGA in rat thyroid transformation. To better understand the role of HMGA and to establish whether its up-regulated expression is sufficient to induce the transformed phenotype, we generated PC Cl 3 cells that overexpress the protein. We demonstrate that HMGA1b protein overexpression does not transform normal rat thyroid PC Cl 3 cells, but it deregulates their cell cycle: cells enter S-phase earlier and the G(2)-M transition is delayed. HMGA1-overexpressing cells undergo apoptosis through a pathway involving caspase-3 activation, probably consequent to the conflict between mitogenic pressure and the inability to proceed through the cell cycle. Using various HMGA1b gene mutations, we found that the third AT-hook domain and the acetylation site K60 are the protein regions required for induction of apoptosis in PC Cl 3 cells. In conclusion, although HMGA1 protein overexpression is associated with the malignant phenotype of rat and human thyroid cells, it does not transform normal thyroid cells in culture but leads them to programmed cell death.

BACKGROUND: Thyroid fine-needle aspiration (FNA) samples belonging to the follicular neoplasm/suspicious for malignancy classes are controversial. The authors identified UbcH10 as a marker useful in the diagnosis of several neoplasms, including thyroid cancer. Here, analysis of UbcH10 expression by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry was applied to FNAs. METHODS: A series of 84 follicular neoplasm/suspicious for malignancy FNAs with histological follow-up (30 malignant) was prospectively collected. UbcH10 immunostaining was performed on cell blocks and compared with that of the proliferation marker Ki-67. At the mRNA level, UbcH10 was compared with CCND2 and PCSK2 expression, these latter being the best performing components of the previously reported 3-gene assay; to determine the diagnostic accuracy, the area under the curve (AUC) of the receiver operating characteristic (ROC) curve for each gene individually and in combination was evaluated. RESULTS: UbcH10 and Ki-67 shared a similar pattern; although UbcH10 expression was higher in malignant than in benign lesions (P < .001), staining was sporadic, and the cutoff value derived by the ROC analysis was too low (1.25%) for routine application. Conversely, UbcH10 expression assessment by quantitative RT-PCR was effective. UbcH10 mRNA levels associated with malignant histology were significantly higher than those associated with benign histology (P = .02). The AUC was 0.74 for UbcH10, 0.81 for CCDN2, 0.62 for PCSK2, and 0.84 for UbcH10 and CCND2 combination. CONCLUSIONS: UbcH10 quantitative RT-PCR analysis, rather than immunohistochemistry, is useful to increase the detection of malignancy in thyroid FNAs. UbcH10 may be added as a panel component in quantitative RT-PCR–based assays. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.

Tumour suppressor p53 is a transcription factor essential for DNA damage checkpoints during cellular response to stress. Mutations in the p53 gene are the most common genetic alterations found in human tumours; most pathogenetic modifications are missense mutations that abolish the p53 DNA-binding function. In the same cell type, distinct p53 missense mutations may determine different phenotypes. The PC Cl3 cell line retains several markers of thyroid differentiation in vitro. Introduction of the V143A mutant p53 allele, which abolishes the p53 DNA-binding function, leads to loss of differentiation markers as well as TSH dependency for growth. Conversely, PC Cl3 cells transfected with the S392A mutant p53 allele, presenting the mutation located outside the DNA-binding domain, show only loss of TSH dependency for growth. To identify molecular differences existing between PC Cl3 cell lines transformed by the V143A and the S392A mutant alleles, a differential proteomic approach was used. Two-dimensional gel electrophoresis analyses indicated that expression of a significant portion of protein species was modified by both p53 mutants. In fact, compared with wild-type PC Cl3 cells, modification of expression in V143A mutant cells occurred in 23.6% of the entire protein species. Conversely, modification of S392A mutant cells affected 14.0% of total proteins. Among these components, 8.3% were common to both mutants. Several of these proteins were identified by mass spectrometry procedures; some proteins, such as HSP90 and T-complex proteins, are already known to be related to p53 function.

High mobility group A 1 (HMGA1) proteins are chromatinic factors, which are absent or expressed at very low levels in normal adult tissues, while they are over-expressed in several human malignant tumors. In this study, HMGA1 protein expression was investigated by immunohistochemistry in a series of 44 epithelial ovarian specimens, which included four normal ovarian tissues, 29 primary invasive carcinomas, one metastatic ovarian tumor and 10 low malignant potential (LMP) tumors. HMGA1 staining was not detected in normal ovarian surface epithelium, which is the area from which ovarian adenocarcinoma frequently arises. HMGA1 proteins were expressed at low levels in some LMP tumors, whereas they were present in abundance in most of the primary ovarian adenocarcinomas. RT-PCR and western blot analysis correlated with immunohistochemical data. We demonstrated that the suppression of HMGA1 protein synthesis by an adenovirus carrying the HMGA1 gene in antisense orientation (Ad-Yas-GFP) inhibited the growth of two human ovarian carcinoma cell lines (OVCAR-5 and OVCAR-8). These results confirm HMGA1 over-expression as a general feature of human malignant neoplasias, including ovarian cancer and suggest that suppression of HMGA1 protein synthesis by an antisense adenoviral vector may represent a new and promising gene therapy for the treatment of ovarian cancer.

Elevated expression of HMGA1 and HMGA2 proteins is correlated with a highly malignant phenotype in several human tumors. We previously demonstrated that the block of HMGA2 protein synthesis prevented rat thyroid cell transformation by murine retroviruses. Suppression of HMGA2 synthesis was associated with lack of induction of HMGA1 proteins suggesting that both HMGA1 and HMGA2 play a role in the process of neoplastic transformation. To determine the role of the HMGA1 gene in thyroid cell transformation, we blocked HMGA1 protein synthesis by an antisense methodology. Here we report that transfection of an HMGA1 cDNA antisense construct into a normal rat thyroid cell line (FRTL-5 Cl2), followed by infection with Kirsten murine sarcoma virus (KiMSV), generated a transformed cell line that expresses high levels of the v-ras-Ki oncogene and that does not require thyroid-stimulating hormones for growth. However, this cell line does not show the malignant phenotype, i.e., it neither grows in soft agar nor induces tumors after injection in athymic mice. Moreover, the lack of the neoplastic phenotype in the virus-infected thyroid cells carrying the HMGA1 antisense construct correlates with the absence of induction of AP-1 transcriptional activity.

Overexpression of the high mobility group I (HMGI) proteins is often associated with the malignant phenotype. Moreover, many benign human tumors, mainly of mesenchymal origin, are characterized by rearrangements of the HMGI-C gene. In most cases, HMGI-C alterations involve breaks within the third intron of the gene resulting in aberrant transcripts carrying exons from 1-3, which encode the three DNA binding domains, fused to ectopic sequences. Here, we show that the expression of a truncated form of HMGI-C protein carrying only the three DNA-binding domains, or of a fusion protein carrying the three DNA-binding domains of HMGI-C and the LIM domains of the lipoma preferred partner gene (LPP) protein, causes malignant transformation of NIH3T3 cells. The unrearranged wild-type HMGI-C cDNA did not exert any transforming activity. These findings indicate that rearranged forms of HMGI-C play a role in cell transformation.

A human anaplastic thyroid cancer cell line FB-1, derived from a 68-yr-old woman who underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike cells have been proliferating stably for more than 2 yr. Karyotype analysis shows many abnormalities and many marker chromosomes have been observed. Heterotransplant of FB-1 cells into severe combined immunodeficient mice has resulted in rapidly growing tumors classified as anaplastic carcinomas, although 50% have shown areas with a trabecular pattern. FB-1 cells failed to express messenger RNA for thyroglobulin; TSH-receptor; thyroperoxidase, and placental angiogenic growth factor. Conversely, PAX8 and thyroid transcription factor 1, whose expression is thyroid specific, was kept in an FB-1 cell line at a level comparable with that observed in normal thyroid tissue. In addition, the present cell line expressed high levels of messenger RNA for high-mobility group proteins (Y) and -C. The in vitro study revealed that FB-1 cells are able to produce high levels of interleukin (IL)-8 and medium amount of IL-6, whereas no release of IL-1-α, IL-1-β, and IL-4 was observed. No modulation of cell proliferation and DNA synthesis in FB-1 cells has been observed after the addition of exogenous IL-6.

Introduction UbcH10 codes for the cancer related E2 Ubiquitin Conjugating Enzyme, an enzymatic molecule with a key role in the ubiquitin–proteasome pathway. Current studies have suggested a critical role of UbcH10 in a variety of malignancies, including human thyroid, breast, ovarian and colorectal carcinomas. The aim of this study has been to extend the analysis of UbcH10 expression to lung cancer. This neoplasia represents one of the leading cause of cancer mortality worldwide, and new tools for an accurate diagnosis/prognosis are needed. Methods The expression levels of UbcH10 were analysed in human non-small cell lung carcinoma (NSCLC) by quantitative RT-PCR and tissue microarray immunohistochemistry, and these values were correlated with the clinicopathological features of the patients affected by NSCLC. Results Our results demonstrate that UbcH10 is overexpressed in NSCLC compared to the normal lung tissue. Moreover, UbcH10 expression is significantly higher in squamous cell and large cell carcinomas than in adenocarcinomas, and directly and inversely correlated with the mutational status of p53 and EGFR , respectively. The suppression of UbcH10 expression by RNAi resulted in a drastic reduction of proliferation and migration abilities of lung carcinoma cell lines. Conclusion These results, taken together, indicate that UbcH10 overexpression has a critical role in lung carcinogenesis, and the evaluation of UbcH10 expression levels may be a new tool for the characterisation of NSCLC.

Context: Thyroid carcinoma is one of the most common malignancies of the endocrine system, and, despite the high frequency of oncogene activation in thyroid neoplastic lesions, the tumor suppressor genes involved in thyroid carcinogenesis remain unidentified. Our previous data implicated a link between the CL2/CCDC80 gene and thyroid cancer. Objective: The objective of the study was to examine the expression of the CL2/CCDC80 gene in human thyroid carcinomas in the attempt to determine whether it plays a role in thyroid carcinogenesis. Design: We evaluated the expression of CL2/CCDC80 in a large number of thyroid neoplastic tissue samples differing in degree of malignancy. We also investigated the effects of its restoration in 2 human thyroid carcinoma cell lines characterized by very low levels of CL2/CCDC80 expression. Results: CL2/CCDC80 expression was much lower in almost all the thyroid carcinomas analyzed than in normal thyroid tissues and was lowest in follicular variants of papillary carcinomas. Loss of heterozygosity partially accounted for CL2/CCDC80 down-regulation in thyroid carcinoma samples. Restoration of CL2/CCDC80 expression in the 2 human thyroid anaplastic carcinoma cell lines resulted in a higher susceptibility to apoptosis and suppression of the malignant phenotype. CL2/CCDC80 expression positively regulated the expression of E-cadherin, thereby halting cancer progression. Conclusions: These results indicate that CL2/CCDC80 is a putative tumor suppressor gene in thyroid carcinogenesis.

The RET proto-oncogene encodes a receptor with tyrosine kinase activity (RET) that is involved in several neoplastic and non-neoplastic diseases. Oncogenic activation of RET, achieved by different mechanisms, is detected in a sizeable fraction of human thyroid tumors, as well as in multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma tumoral syndromes. Germline mutations of RET have also been associated with a non-neoplastic disease, the congenital colonic aganglionosis, i.e. Hirschsprung's disease (HSCR). To analyse the impact of HSCR mutations on RET function, we have introduced into wild-type RET and activated RET(MEN2A) and RET(MEN2B) alleles three missense mutations associated with HSCR. Here we show that the three mutations caused a loss of function of RET when assayed in two model cell systems, NIH 3T3 and PC12 cells. The effect of different HSCR mutations was due to different molecular mechanisms. The HSCR972 (Arg972-->Gly) mutation, mapping in the intracytoplasmic region of RET, impaired its tyrosine kinase activity, while two extracellular mutations, HSCR32 (Ser32-->Leu) and HSCR393 (Phe393-->Leu), inhibited the biological activity of RET by impairing the correct maturation of the RET protein and its transport to the cell surface.

The effect of thyrotropin and norepinephrine on cytosolic calcium levels were evaluated in normal (PC C13) and transformed (PC E1A, PC Py and PC E1APy) rat thyroid epithelial cell lines. A different pattern of response to both norepinephrine and thyrotropin was observed among the distinct cell lines. In PC C13 the cytosolic calcium rise induced by norepinephrine, characterized by an early transient spike followed by a second phase of sustained calcium levels, was greatly enhanced by thyrotropin. The effect of norepinephrine on calcium concentrations was less affected by thyrotropin in PC C13 transformed by the adenovirus E1A oncogene. Conversely, in Polyoma middle-T transformed PC C13 the increase in cytoplasmic calcium was still sensitive to thyrotropin. The most malignant PC E1APy were totally independent of thyrotropin. © 1993.

Aims: The UbcH10 ubiquitin-conjugating enzyme plays a key role in regulating mitosis completion. We have previously reported that UbcH10 overexpression is associated with aggressive thyroid, ovarian and breast carcinomas. The aim of this study was to investigate UbcH10 expression in human lymphomas. Methods and results: Cell lines and tissue samples of Hodgkin’s lymphoma (HL) and of non-Hodgkin’s lymphoma (NHL) were screened for UbcH10 expression at transcriptional and translational levels. UbcH10 expression was related to the grade of malignancy. In fact, it was low in indolent tumours and high in a variety of HL and NHL cell lines and in aggressive lymphomas. It was highest in Burkitt’s lymphoma, as shown by quantitative real-time polymerase chain reaction and by tissue microarray immunohistochemistry. Flow cytometry of cell lines confirmed that UbcH10 expression is cell-cycle dependent, steadily increasing in S phase, peaking in G2/M phase and dramatically decreasing in G0/G1 phases. We also showed that UbcH10 plays a relevant role in lymphoid cell proliferation, since blocking of its synthesis by RNA interference inhibited cell growth. Conclusions: Taken together, these results indicate that UbcH10 is a novel lymphoid proliferation marker encompassing the cell cycle window associated with exit from mitosis. Its overexpression in aggressive lymphomas suggests that UbcH10 could be a therapeutic target in this setting.

Anaplastic thyroid carcinoma (ATC) is considered one of the most aggressive malignancies, having a poor prognosis and being refractory to conventional chemotherapy and radiotherapy. Alteration in histone deacetylase (HDAC) activity has been reported in cancer, thus encouraging the development of HDAC inhibitors, whose antitumor action has been shown in both solid and hematological malignancies. However, the molecular basis for their tumor selectivity is unknown. To find an innovative therapy for the treatment of ATCs, we studied the effects of deacetylase inhibitors on thyroid tumorigenesis models. We show that HDACs 1 and 2 are overexpressed in ATCs compared with normal cells or benign tumors and that HDAC inhibitors induce apoptosis selectively in the fully transformed thyroid cells. Our results indicate that these phenomena are mediated by a novel action of HDAC inhibitors that reduces tumor necrosis factor-related apoptosis-inducing ligand protein degradation by affecting the ubiquitin-dependent pathway. Indeed, the combined treatment with HDAC and proteasome inhibitors results in synergistic apoptosis. These results strongly encourage the preclinical application of the combination deacetylase-proteasome inhibitors for the treatment of ATC.Oncogene advance online publication, 5 October 2009; doi:10.1038/onc.2009.306.

Using gene expression profiling, we found that the CBX7 gene was drastically down-regulated in six thyroid carcinoma cell lines versus control cells. The aims of this study were to determine whether CBX7 is related to the thyroid cancer phenotype and to try to identify new tools for the diagnosis and prognosis of thyroid cancer. We thus evaluated CBX7 expression in various snap-frozen and paraffin-embedded thyroid carcinoma tissues of different degrees of malignancy by quantitative reverse transcription-PCR and immunohistochemistry, respectively. CBX7 expression progressively decreased with malignancy grade and neoplasia stage. Indeed, it decreased in an increasing percentage of cases going from benign adenomas to papillary (PTC), follicular, and anaplastic (ATC) thyroid carcinomas. This finding coincides with results obtained in rat and mouse models of thyroid carcinogenesis. CBX7 loss of heterozygosity occurred in 36.8% of PTC and in 68.7% of ATC. Restoration of CBX7 expression in thyroid cancer cells reduced growth rate, with a retention in the G1 phase of the cell cycle, suggesting that CBX7 can contribute to the proliferation of the transformed thyroid cells. In conclusion, loss of CBX7 expression correlates with a highly malignant phenotype in thyroid cancer patients. [Cancer Res 2008;68(16):6770–8]

Previous studies suggest the expression of UbcH10 gene, that codes for a protein belonging to the ubiquitin-conjugating enzyme family, as a valid indicator of the proliferative and aggressive status of tumors of different origin. Therefore, to look for possible tools to be used as diagnostic markers in astrocytic neoplasias, we investigated UbcH10 expression in normal brain, gliosis and low-grade and high-grade astrocytic tumors by immunohistochemistry. UbcH10 expression was observed in low-grade astrocytoma and in glioblastoma. Our data indicate a clear correlation between UbcH10 expression and the histological grade of the astrocytic tumors. Moreover, the analysis of UbcH10 expression allows the differentiation between gliotic and malignant tissues. Finally, since proteasome inhibitors have recently been considered as possible drugs in the chemotherapy of various tumors, our results would suggest new perspectives for the treatment of brain malignancies based on the suppression of the UbcH10 function.

Udgivelsesdato: June Abstract In the last years, an increasing number of experiments has provided compelling evidence for a casual role of Ras protein mutations, resulting in their constitutive activation, in thyroid carcinogenesis. However, despite the clear involvement of Ras proteins in thyroid carcinogenesis, the nature of most of the target genes, whose expression is modulated by the Ras-induced signaling pathways and that are ultimately responsible for Ras-induced cellular transformation, remains largely unknown. To analyze Ras-dependent modulation of gene expression in thyroid cells we took advantage of a differentiated rat thyroid cell line, FRTL-5. As a model for Ras-dependent thyroid transformation, we used FRTL-5 cells infected with the Kirsten murine sarcoma virus, carrying the v-Ki-Ras oncogene. The infected cells (FRTL-5 v-Ki-Ras) have lost expression of the thyroid differentiation markers and also are completely transformed. We hybridized two different Affimetrix chips containing probe sets interrogating both known rat genes and ESTs for a total of more than 17,000 sequences using mRNA extracted from FRTL-5 and FRTL-5 v-Ki-Ras cell lines. We identified about 50 genes whose expression was induced and about 40 genes whose expression was downregulated more than 10-fold by Ras. We confirmed the differential expression of many of these genes in FRTL-5 v-Ki-Ras as compared to parental cells by using alternative techniques. Remarkably, we investigated the expression of some of the Ras-regulated genes in human thyroid carcinoma cell lines and tumor samples, our results, therefore, providing a new molecular profile of the genes involved in thyroid neoplastic transformation.

We have recently reported the isolation of a rat cDNA encoding a receptor-type tyrosine phosphatase, which appears to be a marker of thyroid differentiation. To elucidate the molecular mechanisms underlying r-PTPeta expression in normal thyroid cells both in vitro and in vivo, we investigated the regulation of r-PTPeta expression in cultured thyrocytes (the rat cell line PC Cl 3) and in an animal model of TSH-dependent thyroid goitrogenesis. In vitro studies showed that mRNA expression of r-PTPeta in thyroid cells is induced in a time- and dose-dependent manner by the activation of growth- and differentiation-linked PKA pathways (TSH and forskolin), whereas it is down-regulated by the activation of the proliferative dedifferentiating PKC-dependent transduction pathway (TPA). However, the regulation of r-PTPeta expression by TSH and TPA, respectively, is observed only in normal thyroid cells, but is lost in transformed thyroid cells. In vivo studies with thiouracil-fed rats demonstrated that increased serum levels of TSH up-regulated r-PTPeta mRNA expression in parallel with the stimulation of thyroid growth and function. The reduction of blood TSH levels due to iodide refeeding to goitrous rats determined a marked down-regulation of r-PTPeta expression, in parallel with involution of thyroid hyperplasia. Taken together these results demonstrate that the phosphatase r-PTPeta is regulated by the two main thyroid regulatory pathways and suggest that it may play an important role in the growth and differentiation of thyroid cells.

Overexpression of the high mobility group I (HMGI) proteins is often associated with the malignant pheno- type. Moreover, many benign human tumors, mainly of mesenchymal origin, are characterized by rearrange- ments of the HMGI-C gene. In most cases, HMGI-C alterations involve breaks within the third intron of the gene resulting in aberrant transcripts carrying exons from 1 ± 3, which encode the three DNA binding domains, fused to ectopic sequences. Here, we show that the expression of a truncated form of HMGI-C protein carrying only the three DNA-binding domains, or of a fusion protein carrying the three DNA-binding domains of HMGI-C and the LIM domains of the lipoma preferred partner gene (LPP) protein, causes malignant transformation of NIH3T3 cells. The unrearranged wild- type HMGI-C cDNA did not exert any transforming activity. These ®ndings indicate that rearranged forms of HMGI-C play a role in cell transformation.

The effects of the stable expression of E1A and/or middle T oncogenes on the proliferative activity of PC Cl3 normal thyroid cells are reported. The proliferation of PC Cl3 cells is mainly regulated by insulin and TSH in a stimulatory way and by somatostatin in an inhibitory fashion. The transformed cell lines, named PC Py and PC E1A Py, show an autonomous pattern of proliferation. The blockade of phosphotyrosine phosphatase activity with vanadate increased the proliferation rate of PC Cl3 under basal and stimulated conditions and completely prevented the inhibitory activity of somatostatin, suggesting that in PC Cl3 cells, a tonic tyrosine phosphatase activity regulates basal and stimulated proliferation, and that a somatostatin-dependent increase in this activity may represent a cytostatic signal. Conversely, in both PC Py and PC E1A Py, vanadate did not modify basal and stimulated proliferation. We analyzed tyrosine phosphatase activity in the different cell lines basally and under conditions leading to the arrest of cell proliferation: confluence (contact inhibition), growth factor deprivation (starvation), and somatostatin treatment. Under basal conditions, tyrosine phosphatase activity was significantly lower in PC Py and PC E1APy cell lines than that in the normal cells. The inhibition of the proliferation induced by contact inhibition or somatostatin treatment was accompanied by an increase in tyrosine phosphatase activity only in PC Cl3 cells. The reduction in tyrosine phosphatase activity in PC E1APy cells correlated with a significant reduction in the expression of R-PTPη, a tyrosine phosphatase cloned from PC Cl3 cells. Conversely, the expression of another receptor-like PTP, PTPμ, was unchanged. Thus, PTPη may be a candidate to mediate inhibitory signals (i.e. activation of somatostatin receptors or cell to cell contact) on the proliferative activity of PC Cl3 cells, and the reduction of its expression in the transformed cell lines may lead to an alteration in the control of cell proliferation.

In this study, we report the effects of somatostatin on the proliferation of PC Cl3 thyroid cell line and the intracellular mechanisms involved. We also evaluated the possible alterations, induced by E1A oncogene transformation on the intracellular pathways mediating somatostatin inhibition of cell proliferation. We showed that somatostatin was able to powerfully inhibit insulin- and insulin + TSH-dependent cell proliferation by inducing a block in the G1/S progression in the cell cycle. These cytostatic effects were completely reverted by vanadate, suggesting that somatostatin may induce antiproliferative effects through the modulation of phosphotyrosine phosphatases. In the E1A-transformed cell line, somatostatin was completely ineffective. The lack of somatostatin inhibitory effects on cell proliferation were not due to alterations in the expression of somatostatin receptors, which were regularly expressed and coupled to adenylyl cyclase activity, but were dependent on an alteration in their coupling with the phosphotyrosine phosphatase. In fact, although in PC Cl3 cells somatostatin increased by 100% phosphotyrosine phosphatase activity, it was completely ineffective in E1A-expressing cells. In conclusion we demonstrated that somatostatin activates phosphotyrosine phosphatases in PC Cl3 thyroid cells to inhibit cell proliferation and that the stable expression of E1A oncogene in these cells completely abolishes this antiproliferative effect.

The aim of the present study was to characterize the adrenergic receptors mediating the effects of norepinephrine on PC C13 rat thyroid cells and identify the molecular mechanisms by which TSH regulates the noradrenergic response. We studied TSH regulation of norepinephrine-induced cytosolic calcium increase by means of the fluorescent probe fura-2. In PC C13 cells grown and maintained in a medium containing TSH (PC C13 6H), norepinephrine caused a higher increase in cytosolic calcium than in PC C13 starved from TSH 5 days before the experiments (PC C13 5H). In both group of cells the calcium response to norepinephrine was concentration dependent and reduced by the removal of extracellular calcium ions. Reintroduction of TSH in the culture medium of the PC C13 5H cells induced the recovery of the norepinephrine-stimulated intracellular calcium rise similarly to that in the native PC C13 6H. This effect was complete after a 48-h incubation period and was abolished by the simultaneous treatment of the cells with the protein synthesis inhibitor cycloheximide, suggesting that TSH may stimulate the synthesis of alpha 1-adrenergic receptors in PC C13 cells. Because in these cells we found that TSH increased cAMP levels as well as inositol phosphate production, we tested whether the activation of a protein kinase-A and/or protein kinase-C was involved in TSH regulation of the adrenergic response. We found that the treatment of PC C13 5H cells with forskolin restored the effect of norepinephrine on the calcium level, and that KT5720, an inhibitor of the protein kinase-A, was able to prevent the recovery of the noradrenergic response induced by the readdition of TSH to the culture medium of PC C13 5H. Conversely, treatment of PC C13 5H cells with the protein kinase-C activator phorbol 12-myristate 13-acetate was ineffective. Norepinephrine also stimulated inositol phosphate production in PC C13 6H and, to a lesser extent, in PC C13 5H, but it did not affect the cAMP levels in the two groups of cells. To characterize alpha 1-adrenergic receptor subtypes mediating the effects of norepinephrine in PC C13 cells, we used antagonists of alpha 1A and alpha 1B receptors (WB4101 and chlorethylclonidine respectively).(ABSTRACT TRUNCATED AT 400 WORDS)

We have molecularly cloned a rearranged gene from a cDNA library of tertiary NIH 3T3 transfectants, obtained with the DNA from human thyroid papillary carcinomas. This cloned cDNA resulted from two sequences, one, at the 5’ end, deriving from an unknown gene, by us denominated H-4, and the other one, at the 3’ end, deriving from the tyrosine kinase domain of the human proto-retgene. The rearrangement was present in the original tumor DNA and therefore was not an artefact of the transfection technique, as it was observed frequently in other cases of proto-retactivation. The rearrangement was also a frequent occurrence with other cases of human thyroid papillary carcinomas, as it was detected in 33 out of 177 papillary carcinomas. By contrast, none of 109 other thyroid tumors, nor about 300 non thyroid tumors, showed retactivation. Activation was shown to occur via a paracentric intrachromosomal inversion of the long arm of chromosome 10 with breakpoints coincident with the regions were retand H-4 are located. While proto-retcodes for a tyrosine kinase-containing membrane receptor whose ligand is not yet known, the chimeric oncogene codes for a cytoplasmatic product which does not need the ligand anymore to exert its tyrosine kinase activity. In situhybridization, as well as Northern blot hybridization studies, have demonstrated thatproto-m expression is confined normally to tissues of neuroectodermal derivation and, during the embryological mouse development, also to the cartilage and to secretory organs, such as the kidney and the liver. Therefore, it is likely that the proto-retproduct has a key role in the differentiation of neuroectodermal and also of other essential organs.

We recently detected a novel activated oncogene by transfection analysis on NIH 3T3 cells in five out of 20 primary human thyroid papillary carcinomas and in the available lymph node metastases. We designated this transforming gene FTC (for papillary thyroid carcinoma). Here we describe the molecular cloning and sequencing of the gene. The new oncogene resulted from the rearrangement of an unknown amino,terminal sequence to the tyrosine kinase domain of the ret proto-oncogene. This gene rearrangement was detected in all of the transfectants and In all of the original tumor DNAs, but not in normal DNA of the same patients, thus indicating that this genetic lesion occurred in vivo and is specific to somatic tumors. Moreover, the transcript coded for by the fused gene was detected in an additional FTC-positive human papillary carcinoma for which mRNA was available. © 1990.

Rat FRTL-5 and PC-Cl-3 thyroid cells are continuously cultured, clonal lines which require thyrotropin to grow and function. Both can be efficiently transformed when infected with RNA or DNA viruses carrying oncogenes or when directly transfected with activated oncogenes. Transformation, assayed by the appearance of cell growth in agar and by tumorigenicity in syngeneic rats or nude mice, is associated with the loss of thyrotropin-dependent cell division and thyrotropin-regulated functions such as thyroglobulin synthesis. In 16 clones of FRTL-5 or PC-CI-3 cells transformed with different oncogenes, we show that loss of thyrotropin-dependent growth and function correlates with the loss of thyrotropin receptor gene expression, measured with a rat thyrotropin receptor cDNA probe. © 1990 Academic Press, Inc.

Both cytoplasmic and membrane-bound protein kinase C activities are increased in: Harvey-Sarcoma Virus, infected thyroid epithelial cells. The cytoplasmic kinase C increase is found in the chromatographic fraction eluted at lower salt concentration (100 mM NaCl-S100), while the more acidic protein fraction eluted at higher salt concentration (350 mM NaCl-S350) is virtually absent. Although the cytoplasmic S100 fraction from the control and ras-virus infected cells display a comparable PBt2 binding activity, they are different in the Ca+2-dependence and the TPA down regulation. In addition, the membranes from the control and ras-virus infected cells are different phosphate acceptors in place of the Hl histones. © 1988 Academic Press, Inc.

The activity of the adenylate cyclase catalytic subunit is higher in Harvey and Kirsten Murine Sarcoma Viruses-infected thyroid epithelial cells than in uninfected control cells either in the presence of Mg2+ alone or following stimulation by Mn2+ or forskolin. The higher activity is associated with an increased cAMP cellular content. The Gpp(NH)p and F- anion are more effective positive modulators in the control than in the virus infected cells: these results exclude therefore that the ras p21 proteins can act as the G-protein alpha-subunit and suggest that they negatively interfere with the G-protein modulation of the adenylate cyclase system. The activity of the adenylate cyclase catalytic subunit is higher in Harvey and Kirsten Murine Sarcoma Viruses-infected thyroid epithelial cells than in uninfected control cells either in the presence of Mg2+ alone or following stimulation by Mn2+ or forskolin. The higher activity is associated with an increased cAMP cellular content. The Gpp(NH)p and F- anion are more effective positive modulators in the control than in the virus infected cells: these results exclude therefore that the ras p21 proteins can act as the G-protein alpha-subunit and suggest that they negatively interfere with the G-protein modulation of the adenylate cyclase system. © 1987.

Oncogenes of the ras family have been isolated from different human and experimental carcinomas by transfecting high molecular weight DNA onto NIH 3T3 fibroblasts. The activated ras genes differ from normal ones by point mutations in specific regions of the genes (1, 2) . This finding has allowed to establish new more sophisticated and more sensitive techniques to evidenciate point mutations of the ras genes in human tumors. In fact, using a combinations of techniques, including specific in vitro gene amplification by the polymerase chain reaction (PCR) and mutation detection by cleavage at single base mismatches by RNAase A in DNA:RNA and RNA:RNA heteroduplexes, it has been possible to evidentiate point mutations of the ras-Ki oncogene in 21 out of 22 human carcinomas of the exocrine pancreas and in the 40% of the human colorectal cancers (3, 4) . However, a problem that could be raised is whether or not these mutations in the ras genes are the unique events responsible for the carcinogenesis process. In fact, studies of chemical carcinogenesis, as well as epidemiological analysis of malignancies in humans (5, 6, 7), strongly suggest that the neoplastic transformation is a multistage process and the observation that two different oncogenes are required in concert for malignant conversion of nonestablished rat cells confirms this point of view (8, 9).

Using DNA transfection analysis1 on NIH3T3 cells2, activated human oncogenes have been isolated from a variety of fresh solid tumours3. Thyroid neoplasias show a wide range of lesions varying from slowly progressive well-differentiated tumours to anaplastic highly malignant neoplasms4. Therefore they represent an attractive model to investigate the role of oncogene activation in different stages of the neoplastic state. Here we report the detection of transforming activity in DNAs extracted from five thyroid papillary carcinomas and two of their respective lymph-nodal metastases. © 1987 Nature Publishing Group.

Transformation of rat thyroid epithelial cells by Kirsten murine sarcoma virus results in the block of certain thyroid differentiated functions, such as synthesis and secretion of thyroglobulin. Our studies, performed by a run-on assay, demonstrate that this block occurs at the transcriptional level. We also demonstrate the de novo methylation of two methylation-sensitive sites, located within the 5' end regulatory sequences of the thyroglobulin gene, in transformed cells, in the absence of any rearrangement of the gene. These two methylation-sensitive sites were methylated also in a rat thyroid cell line transformed by another retrovirus and in two normal cell lines which do not express the thyroglobulin gene.

Differentiated, cloned rat thyroid epithelial cells (424 cells) were infected with a wild-type and a temperature-sensitive strain of the myeloproliferative variant of the Moloney murine sarcoma virus. The thyroid cells were productively infected and transformed by both virus strains and displayed some of the typical properties of malignant cells, such as morphological changes, growth in soft agar, and in vivo tumorigenicity. The acquisition of the transformed phenotype by the virus-infected cells was accompanied by a loss of the typical differentiated features of the thyroid epithelium, such as thyroglobulin (TG) secretion, iodide uptake, and dependence for growth on six factors including thyrotropin, the physiological thyroid stimulator. TG mRNA could not be demonstrated in cells transformed by both viral strains, suggesting a block at the level of the TG gene transcription. While the transformed state of the cell clones infected with the temperature-sensitive strain could be reverted by shifting the cultures to the temperature nonpermissive for transformation (39 degrees C), no reversion of the differentiated functions took place after such a shift, showing that the v-mos oncogene irreversibly shuts off the differentiation of thyroid epithelial cells in vitro. These results demonstrate, for the first time, an oncogenic potential of the v-mos oncogene family towards differentiated epithelial cells in vitro.

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A system of epithelial cells is described in which it is possible to study the number and the nature of genes capable of conferring the malignant phenotype. Two fully differentiated, hormone-responsive cell lines from rat thyroid glands are presented which are susceptible to one-step or two-step transformation upon infection with several murine acute retroviruses. After infection, both cell lines became independent from their thyrotropic hormone requirement for growth. However, complete transformation was achieved with one of the cell lines (FRTL-5 Cl 2), whereas the other cell line (PC Cl 3) failed to grow in agar and to give rise to tumors in vivo. The latter cell line was susceptible to complete transformation upon cooperation of the v-ras-Ha and the human c-myc oncogenes.

Two rat thyroid epithelial differentiated cell lines, PC Cl 3 and PC myc, were infected with the polyoma murine leukemia virus (PyMLV) carrying the Middle-T-antigen gene of polyomavirus. After infection, both cell lines acquired the typical markers of neoplastic transformation; however, the PC myc cells showed a greater malignant phenotype. Furthermore, the thyroid differentiated functions were completely suppressed in PC myc cells transformed by PyMLV, whereas they were, at least partially, retained in PC Cl 3 cells transformed by PyMLV, and in particular, thyroglobulin synthesis and secretion were not affected at all. Since no differences in the expression of the middle-T-antigen gene were observed in the two PyMLV-transformed cell lines, the different properties shown by these two infected cell lines must be ascribed to the expression of the c-myc oncogene.

Transformation of a rat thyroid epithelial cell line (FRTL5-C12) with Kirsten and Harvey murine sarcoma viruses (carrying the ras oncogenes) results in elevated levels of three perchloric acid-soluble nuclear phosphoproteins. These three proteins are also induced to high levels in the PC-C13 thyroid epithelial cell line when transformed by the myeloproliferative sarcoma virus (carrying the v-mos oncogene) and when transformed by transfection with the c-myc proto-oncogene followed by infection with the polyoma leukaemia virus (PyMuLV) carry the polyoma middle T antigen gene. Neither c-myc or PyMuLV alone induced high levels of the three nuclear proteins. Untransformed thyroid fibroblasts have high levels of two of the three proteins and can be transformed by PyMuLV alone resulting in the appearance of the third protein. Transformation with Harvey sarcoma virus also results in the induction of the third protein. The three phosphoproteins have been purified by h.p.l.c. and shown to be related to the HeLa protein HMGI already described. The results of these studies indicate that elevated levels of these HMGI-like proteins are associated with neoplastic transformation and/or with an undifferentiated phenotype.