Isolation of human type 2 deiodinase gene promoter and characterization of a functional cyclic adenosine monophosphate response element.

We analyzed the structure and function of the 5' flanking region of the human type 2 deiodinase (hD2) gene. Two major transcription start sites were identified at -470/-474 from the ATG. The 5' flanking region of hD2 gene efficiently directed transcription in transient transfection studies, using luciferase as reporter gene, in HEK 293 cells. Basal transcriptional activity was significantly reduced by deleting the region containing a canonical cAMP-responsive element (CRE) located -766/-759 from ATG. Forskolin treatment significantly increased luciferase activity in cells transfected with CRE-containing constructs. This effect was abolished in constructs that did not contain CRE or contained the mutagenized CRE. Northern blot analysis in JEG-3 cells revealed that the hD2 messenger RNA was markedly increased after stimulation with cAMP agonist. The electrophoretic mobility shift assay with hD2-CRE probe and HEK 293 nuclear extract showed the occurrence of a DNA-protein complex, which was competed by specific unlabeled oligonucleotides and supershifted by the anti-CREB and anti-CRE modulator-1 antibodies. A-CREB, a dominant negative inhibitor of CREB, completely inhibited forskolin induction of the hD2 promoter. CREB protein, once cotransfected with hD2 promoter construct and pKA in F9 teratocarcinoma cells, which are unresponsive to cAMP, was able to stimulate the hD2 gene transcription. These results indicate the existence of a functional promoter within the 5' flanking region of hD2 gene which is characterized by the presence of a CRE. The specific involvement of CREB in the cAMP-mediated hD2 gene promoter induction also has been demonstrated.

Increased TGFbeta type II receptor expression suppresses the malignant phenotype and induces differentiation of human neuroblastoma cells.

TGFbeta can modulate neuroblastoma (NB) cell proliferation and differentiation in vitro. In this study we used a NB cell line (LAN-5) which has been shown to partially respond to TGFbeta and to present high levels of TGFbeta receptor type I and low levels of receptor type II (TbetaRII) on the cell surface. To evaluate the role of TbetaRII in mediating TGFbeta effects, LAN-5 cells were transfected with an expression vector containing the human full-length TbetaRII cDNA or with the empty vector pcDNA3. Compared to control CLV3 cells (transfected with empty plasmid) and parental LAN-5 cells, isolated neomycin-resistant clones (CL1 and CL3) expressed higher levels of TbetaRII, had reduced cell growth rate in vitro, and were unable to form tumors in vivo. Furthermore, isolated clones modified their morphology, assuming a terminally differentiated neuronal phenotype. Immunocytochemical staining demonstrated a basal increased expression of neural-specific markers, such as axonal growth-associated protein (GAP43) and neurofilaments (NF200). TGFbeta treatment further increased the synthesis of NF200 and GAP43 in the transfected clones as revealed by Western blot analysis. These data indicate that TbetaRII overexpression potentiates the TGFbeta signal transduction pathway, reverting NB cell neoplastic phenotype with the reduction of proliferation rate and the induction of terminal maturation.

Overexpression of transforming growth factor beta-type II receptor reduces tumorigenicity and metastastic potential of K-ras-transformed thyroid cells.

Expression of type II receptor of transforming growth factor beta (TbetaRII) is necessary for this factor to inhibit the growth of thyroid epithelial cells. In rat thyroid transformed cells, the resistance to transforming growth factor beta (TGFbeta) is associated with a decreased expression of TbetaRII mRNA and protein. Reduced TbetaRII expression has also been found in human thyroid differentiated and undifferentiated carcinomas. To investigate the role of TbetaRII in modulating the tumorigenic potential of k-ras-transformed thyroid cells, we transfected these cells with an expression vector carrying the human TbetaRII gene, regulated by an inducible promoter. Isolated clones, overexpressing TbetaRII, showed a reduction in the anchorage-dependent and -independent cell growth, compared with control k-ras-transformed cells. When transplanted in athymic nude mice, the transfected clones presented a decrease in tumorigenicity with respect to the highly malignant parental cells. Moreover, the diminished tumorigenic ability of the clones studied was accompanied by a statistically significant reduction in spontaneous and lung artificial metastases. Taken together, our data demonstrate that TbetaRII acts as a potent tumor suppressor gene when overexpressed in malignant thyroid cells.

Oncogenes and antioncogenes involved in human thyroid carcinogenesis.

The development of cancer is due to the accumulation of multiple somatic mutations, in some cases following germline mutations, which occur in hereditary malignancies such as retinoblastomas or multiple endocrine neoplasia (MEN 2A and B). Genetic alterations or changes in the expression of growth regulatory genes can lead to the initiation of malignant transformation and to eventual tumor progression. Cells that have undergone these cumulative alterations in either the structure or expression of these regulatory genes generally possess a selective growth and/or metastatic advantage over other normal non-transformed cells. Thus, activation of dominantly transforming oncogenes by point mutations, gene amplification, chromosomal translocation or insertional mutagenesis can lead to uncontrolled cellular growth or to a disruption in normal differentiation or apoptosis. Equally contributory to the process of malignant progression is the inactivation of recessive tumor suppressor genes due to point mutations and/or loss of heterozygosity in one allele, which can ultimately lead to a reduction of homozygosity in both alleles. Thyroid tumors in humans represent a particularly suitable multistage model of epithelial tumorigenesis. In fact, even though most thyroid neoplasms originate from a single cell type, i.e. the thyroid follicular cell, they include a broad spectrum of tumors with different phenotypic characteristics and variable biological and clinical behaviour. Multiple degrees of malignancies have been defined: from the benign colloid adenomas through the slowly progressive differentiated papillary and follicular carcinomas to the invariably fatal anaplastic carcinomas, although these histological changes are not necessarily sequential. In this review an effort has been made to summarize and integrate new data published on genetic lesions and altered expression of genes involved in the tumorigenesis of the follicular type of thyroid cancer. We have focused our interest only on gene alterations inducing gain or loss of function, that have been studied in vivo in human thyroid tumor specimens by the use of different techniques, such as PCR mediated DNA analyses, sequencing, mRNA level evaluation and protein expression by immunohistochemical staining.