Normal Thyroid Follicular Cells Research Articles

Spontaneous de-differentiation correlates with extended lifespan in transformed thyroid epithelial cells: an epigenetic mechanism of tumour progression?

Normal thyroid follicular cells, like many highly differentiated epithelia, have limited proliferative capacity. We previously showed that this could be extended by expression of the SV40 large T oncogene, but that immortal lines always lost thyroid-specific differentiation. Detailed analysis now show that clones expressing T undergo 2 mutually exclusive fates. They either (i) remain well-differentiated, in which case they undergo irreversible growth arrest after 5 to 15 p.d., or (ii) spontaneously develop poorly differentiated sub-clones that exhibit greatly extended proliferative life spans (up to 75 p.d.). The frequency of this event (> 3 per 10(4) cell divisions) greatly exceeds that expected from somatic mutation, suggesting an epigenetic basis. This is supported by our finding of rare de-differentiated epithelial cells in normal thyroid that all generate clones with extended life spans, indistinguishable from the above, following introduction of SV40 T. Escape from early mortality in differentiated thyroid epithelium therefore requires not only loss of tumour suppressor gene function (induced here by SV40 T), but also a switch in differentiation programme, with the latter effectively converting the follicular cell into a cell type with increased intrinsic proliferative potential. The analogy between this in vitro model and the progression of thyroid cancer from the well-differentiated to the highly aggressive, anaplastic form suggests that de-differentiation may play a causal rather than a passive role in this critical switch in tumour behaviour.

Neural cell adhesion molecule expression in thyroid cancer

Neural cell adhesion molecule (NCAM) is an integral membrane glycoprotein that participates in cell-cell interactions. We examined the expression of neural cell adhesion molecule (NCAM) by immunohistochemistry in 65 cases of thyroid disease. In the majority of thyroid tumors, NCAM expression was detected in tumor cells. In chronic thyroiditis, Hurthle cells demonstrated expression of NCAM. In normal thyroid gland, neither follicular nor parafollicular cells expressed NCAM at detectable levels. All papillary carcinomas and most poorly differentiated follicular carcinomas and some follicular adenomas demonstrated NCAM expression. In thyroid carcinoma, clinical stage and recurrent laryngeal nerve paralysis did not correlate significantly with expression of NCAM. Papillary carcinomas with strong expression of NCAM were more likely to have extra capsular invasion. It is supposed that immunohistochemical detection of NCAM expression supports histologic diagnosis and offers prognostic information.

Insulin-Like Growth Factor I in Human Thyroid Tissue: Specific Localization by Immunohistochemistry and In Situ Hybridization.

Insulin-like growth factor I and II (IGF-l and IGF-ll) have been implicated in the replication of normal thyroid follicular cells in vitro. This study evaluates the distribution and abundance of immunoreactive IGF-l by histochemical analysis in human thyroid tissue with different histopathologic characteristics. We used two types of highly specific and sensitive polyclonal rabbit anti-IGF-l antibodies and one monoclonal antibody (MAb) with the immunoperoxidase technique on sections of 25 glands harboring adenomatous goiter; 11 glands with follicular adenoma (FA); 45 glands with thyroid carcinoma of papillary, follicular, and undifferentiated types; and 18 glands with Graves' disease. Immunoreactive IGF-l was present in some thyroid follicular cells of all thyroid tissues examined. The percentage of cells staining positively varies among the different processes, being lowest in normal thyroid tissues and highest in all thyroid carcinomas. The cytoplasmic pattern of IGF-l immunoreactivity also varied among the different thyroid conditions. Furthermore, using nonradioactive in situ hybridization (ISH) we detected IGF-l mRNA in the thyroid cells of adenomatous goiter. The expression was higher in the histologically hyperplastic areas. These findings provide further support for an autocrine and/or paracrine role of IGF-l in the function and/or growth of normal thyroid follicular cells and suggest that IGF-l may play a role in the dysfunctional growth of thyroid follicular cells in adenomatous goiter, thyroid carcinoma, and Graves' hyperthyroidism.

Porcine thyroid follicular cells in monolayer culture activate the iodide-responsive precursor form of transforming growth factor-beta 1.

The release of latent transforming growth factor-beta 1 (TGF-beta 1), and conversion to the biologically active peptide, has been investigated in porcine thyroid follicular cells maintained in primary monolayer culture. Analysis by radioreceptor assay of medium conditioned for 72 h by subconfluent thyroid monolayers showed that a high proportion of the expressed TGF-beta 1 peptide was in the active form. Medium conditioned by iodide (10 mumol/l)-treated follicular cells contained higher levels of both active and total TGF-beta 1 than were present in medium conditioned by untreated cells. Exposure of cells to iodide also led to a marked decrease in [methyl-3H]thymidine incorporation that was relieved by immunoadsorption with a neutralizing antiserum against the active form of TGF-beta 1. Inclusion of a low dose (80 units/l) of porcine plasmin led to a small increase in incorporation of [methyl-3H]thymidine, while higher doses of plasmin (1250-5000 units/l) or plasminogen (100 mg/l) significantly reduced [methyl-3H]thymidine incorporation. This inhibition was effectively reversed by immunoadsorption of TGF-beta 1 from the medium during the test incubations. The study therefore provides direct evidence for a stimulatory role of thyroidal iodide in enhancing the release of latent TGF-beta 1 peptide, and suggests that in normal thyroid follicular cells, as in other TGF-beta 1 producing epithelia, post-secretory processing to the biologically active molecule occurs through an endogenous cellular mechanism. It appears likely that plasmin, generated locally within the thyroid follicular microenvironment, may play a fundamental role in effecting this conversion.

Cell proliferation and thyroid neoplasia

The classic experimental model of thyroid neoplasia using radiation as a mutagen followed by long-term goitrogen treatment gives rise to multiple tumours in rats. Radiation alone, with suppression of TSH produces no tumours, TSH-induced growth alone causes a low level of tumorigenesis. Cell proliferation in this model is therefore critical. Epimutation as well as mutation is important. Rodent and human thyroid tumours show a clear stepwise progression, associated with both morphological and oncogene changes. In experimental animals the finding that monoclonal adenomas and carcinomas induced in the presence of long-term high TSH retain TSH dependency suggests that the step in the tumour progression requiring the development of TSH independent growth is bypassed, explaining the frequency of tumour development in this model. Normal thyroid follicular cell growth is limited, and a genotoxic effect before the growth plateau is more effective in carcinogenesis than a genotoxic effect after a period of growth. These observations will be interpreted in relation to the importance of thyroid tumours in regulatory toxicology and the pathobiology of thyroid tumours in man.

Detection and partial purification of a potent mitogenic factor for human thyroid follicular cells

Normal adult human thyroid follicular cells have an extremely limited proliferative capacity in vitro. No previously studied mitogen, including thyrotropin (TSH) or epidermal growth factor (EGF), has in our hands resulted in a significant improvement over the 3–4% nuclear [ 3H]thymidine pulse-labelling index (LI) obtainable with 10% fetal calf serum. Here we report the detection in the conditioned medium from a sub-clone of NIH3T3 fibroblasts of a mitogenic activity capable of increasing this response up to 10-fold, to an LI of over 20%, together with an even greater relative stimulation of mitotic activity. Preliminary characterisation has excluded EGF and TGFα, and demonstrated that the activity is bound reversibly by heparin-Sepharose, thus pointing to a member of the heparin-binding fibroblast- or hepatocyte-growth factor families. This material should have wide practical application in facilitating primary culture of follicular cells, and may reveal new mechanisms of stromal-epithelial interaction regulating normal and neoplastic thyroid growth in vivo.

Stepwise transformation of primary thyroid epithelial cells by a mutant Ha‐ras oncogene: An in vitro model of tumor progression

Activating mutations of the ras oncogene family occur at high frequency in all stages of thyroid tumorigenesis, both human and experimental. To test the causal nature of this association, and to investigate the biological role of ras mutation, we introduced a mutant c-Ha-ras gene into normal rat thyroid follicular cells using an ecotropic retroviral vector. The major immediate effect was to greatly extend the proliferative lifespan of these cells in culture from less than 3 to more than 15 doublings, without any observable loss of growth-factor dependence or differentiated functions. This in vitro phenotype strongly supports an initiating role for ras mutation in the genesis of benign thyroid tumors (adenomas) in vivo. Spontaneous transformation was observed at low frequency on continuous culture of mutant ras-expressing cells, giving rise to fully immortalized, growth factor-independent, highly tumorigenic lines. Transformation was associated with (i) loss of responsiveness to the growth inhibitor TGF-beta 1, and (ii) greatly increased nuclear levels of p53 protein, which unexpectedly was not due to point mutation in the conserved regions of the p53-coding sequence. We postulate that these two phenomena are causally related to each other and to the transformed phenotype.

Calcitriol receptors in rat thyroid follicular cells (FRTL-5)

The FRTL-5 cell line is widely used as a model for normal thyroid follicular cells. These cells have retained their ability to alter cAMP production, cell proliferation, iodine uptake, and thyroglobulin synthesis in response to thyrotropin. We have previously shown that calcitriol attenuated both basal and TSH stimulated cAMP production dose-dependently in FRTL-5 cells. Cytosol fractions (105,000 g, 60 min, 4 degrees C) prepared from FRTL-5 cell homogenates possessed calcitriol-binding components with a sedimentation coefficient of approximately 3.7 S in high salt (0.3 mol/l KCl) sucrose gradients (5-20%). At 4 degrees C, specific binding increased rapidly during the first 4 h and reached a plateau after 8 h. The specific binding (18 h, 4 degrees C) was maximal at a [3H]calcitriol concentration of approximately 0.5 nmol/l. Scatchard analysis of the binding data indicated one single class of high affinity binding sites with Kd = 105 +/- 2 pmol/l and Bmax = 38.5 +/- 4.7 pmol/g cytosol protein (mean +/- SD, N = 6). In conclusion, our results suggest that the FRTL-5 cells possess functional receptors for calcitriol with the same physicochemical properties as the receptors found in normal rat tissues.

Ectopic expression of HLA class II antigens on thyroid follicular cells: induction and transfer in vitro by autologous mononuclear leukocytes.

Ectopic expression of HLA class II antigens by thyroid follicular cells (TFC) might trigger the immune recognition of TFC-specific surface constituents by self-reactive T helper/inducer lymphocytes, with the consequent generation of organ-specific autoimmunity. To explore the alternative possibilities underlying this ectopic expression, i.e. that it either reflects a primary abnormality of the TFC or is secondarily induced by the autoimmune attack itself, we employed immunofluorescence staining of monolayers cultured from thyroid tissue of patients with autoimmune thyroid disease as well as neoplastic, nodular, and normal thyroid tissues to assess the role that autologous mononuclear leukocytes (MNC) and serum factors play in HLA class II induction and modulation in vitro. In all Graves' disease (GD) tissues that initially displayed HLA-DR (DR) antigens, this expression was transient. Primary TFC cultures from tissues with tumor-associated chronic thyroiditis (CT) showed more widespread and persistent DR expression than did cultures from GD tissues. However, in contrast with the findings in GD, there was no correlation between ectopic DR expression on TFC from the CT areas and the presence of organ-specific autoimmune markers. DR expression by TFC was detected in only one of the five nontoxic nodules studied, and involved a small proportion (approximately 20%) of the TFC. A similarly low proportion of DR-positive TFC was found in cultures from two papillary carcinomas, while much stronger DR expression was seen on TFC cultured from the contralateral lobes affected with CT. Only one of four follicular adenomas expressed DR on about 35% of the cultured TFC. In contrast with the rapid extinction of DR expression on TFC cultured from GD tissues after depletion of infiltrated MNC, DR antigen loss did not occur or was significantly delayed when parallel TFC monolayers were cocultured in the presence of autologous MNC from the intrathyroidal infiltrates. Coculture of DR-negative TFC from normal tissues with autologous peripheral blood MNC in individuals without detectable thyroid autosensitization also resulted in pronounced induction of DR expression on the TFC. Purified T lymphocytes from the same peripheral blood MNC preparations, however, did not induce DR expression on these normal TFC. Similarly, MNC obtained from two follicular adenomas, mostly macrophages, did not induce DR expression when cultured with autologous TFC from the same lesions. Addition of 15% autologous serum did not prevent progressive extinction of DR expression on TFC cultured from GD or tumor-associated CT tissues after depletion of infiltrated MNC.(ABSTRACT TRUNCATED AT 400 WORDS)

Control of human thyroid follicular cell proliferation in suspension and monolayer culture

We have used both suspension and monolayer cultures to investigate the in vitro control of normal human thyroid follicular cell proliferation as assessed by both [ 3H]TdR incorporation and autoradiography. In suspension cultures, whilst TSH alone produced no effect, inclusion of two permissive factors, namely a supraphysiological concentration of insulin (80 μg/ml) and a low concentration of FCS (1%) (which, in the absence of TSH, gave only a weak proliferative response) permitted a highly significant response to TSH at 0.1 mU/ml. A higher concentration of TSH (10 mU/ml) resulted in a reduced response. In monolayer cultures in contrast, insulin and FCS were themselves highly growth stimulatory, with no super-added effect on addition of TSH. Our data indicate that suspension culture provides a more valid in vitro model for the investigation of growth factor control of normal and neoplastic human thyroid epithelium.

Transmission and scanning electron microscopy of experimentally induced thyroid tumors in rats

Irradiation of the neck region of 10 days old Wistar rats with 300 rads X-rays and their subsequent treatment with MTU leads to the development of thyroid tumors. The ultrastructure of the thyroid tumors induced was studied by transmission (TEM) and scanning electron microscopy (SEM). In follicular carcinomas, cylindrical or high columnar cells with abundant microvilli, well-developed ergastoplasmic reticulum and a hypertrophied Golgi complex predominate. The cytoplasm of the tumor cells of papillary carcinomas was composed of large pleomorphic cisternae, irregular in shape mitochondria, dense bodies, phagolysosomes and lamellar or multivesicular structures. Scanning electron microscopy revealed in the control animals a regular pattern of thyroid follicles tied to each other by tissue bridges and covered by set of blood and lymph vessels. The relief of the follicular carcinomas was irregular indicating follicles different in size and shape. The internal surface of the neoplastic follicles was also irregular as result of deep invaginations and intrafollicular cell growth. The apical region of the normal and neoplastic thyroid follicular cells was covered with abundant microvilli protruding into the colloid.