Thyroid Nuclei Research Articles

Rethinking the Role of Oncogenes in Papillary Thyroid Cancer Initiation

Rethinking the Role of Oncogenes in Papillary Thyroid Cancer Initiation

Thyroid carcinoma diagnosis based on a set of karyometric parameters of follicular cells.

In the works of various authors, performed with the help of morphometry, an assessment of cell changes was carried out, as a rule, by one sign of their atypia (e.g. area of cell nuclei). Moreover, the selective analysis of morphometric data for certain nosologic forms was performed. Such an approach allowed researchers to develop only additional diagnostic criteria of thyroid carcinoma. At the same time, the use of a set of quantitative signs of cell atypia as a criterion for tumor malignancy made it possible to improve oncologic diagnosis. In this study, which is a first step in the development of a quantitative approach to thyroid carcinoma diagnosis, a system of karyometric parameters was developed with the help of morphometry of cytologic material of patients with major nosologic forms of thyroid pathology (papillary and follicular carcinoma, follicular adenoma, autoimmune thyroiditis, and nodular colloid and diffuse toxic goiter). The significance of each parameter in thyroid carcinoma diagnosis was evaluated. Thyroid cells of autopsy, operative, and aspiration biopsy material were the object of the study. One hundred fifty-two preparations with histologically verified diagnosis were studied. Light morphometry was performed with the help of a semiautomatic computer analyzer of images. One hundred fifty thyroid nuclei were assessed in each preparation. Among many karyometric parameters, six were selected: mean area of thyrocyte nuclei, ratio of frequencies, and number of classes on histograms of distribution of their area, coefficient and a free term of the regression equation to a scattered diagram of perimeter and nuclear area, and the discriminant index. Boundary values of these parameters between the malignant and benign pathology were united into an expert system for thyroid carcinoma diagnosis. Clinical trials of the system were performed. On the basis of the obtained results, the informativity (accuracy, sensitivity, specificity) of its karyometric parameters was assessed. The formula for calculation of the weighting coefficient for each parameter was derived, the diagnostic index being determined by the sum of their values. An expert system was created based on a set of karyometric parameters reflecting the regularities of pathologic changes in thyrocyte nuclei with malignant and benign pathology. The informativity and weighting coefficients included into the system of parameters have been assessed, which made it possible to perform the probabilistic diagnosis of thyroid carcinoma.

Relationship between histopathologic typing and morphonuclear assessments of 238 thyroid lesions. Digital cell image analysis performed on Feulgen-stained nuclei from formalin-fixed, paraffin-embedded materials.

A digital cell image processor was used to compute 15 parameters on Feulgen-stained thyroid nuclei from 238 archival, i.e., formalin-fixed, paraffin-embedded thyroid lesions. The morphonuclear parameters were related to morphometric (nuclear area), densitometric (nuclear DNA content), and textural (chromatin pattern characteristics) features. With respect to development of a malignant condition, their variations were compared with the World Health Organization's International Histological Classification of thyroid tumors. The relationship between morphonuclear parameter assessments and tumor size and the presence or absence of metastasis at the time of diagnosis was also investigated: no relationship with respect to cytomorphonuclear assessments was found. Nuclear area and nuclear DNA content discriminated between simple multinodular goiters and multinodular goiters with adenomatous hyperplasia. In the same way, the mean parameter values describing the chromatin pattern of multinodular goiters were significantly distinct from those describing the chromatin pattern of adenomas and carcinomas. Furthermore, chromatin pattern descriptions made it possible to discriminate between cell nuclei from papillary carcinomas and follicular carcinomas, and further between follicular carcinomas and follicular adenomas. A marked variation was observed within individual cases of multinodular goiters, adenomas, and carcinomas, a feature suggesting that morphonuclear assessment is of limited value for the diagnosis of individual clinical cases. In contrast, these results show that morphometric, densitometric, and textural nuclear assessments are useful aids for thyroid tumor typing, adding interesting results with respect to thyroid tumor progression. Indeed, the primary findings demonstrate that nodules with adenomatous hyperplasia from multinodular goiter lesions and microvesicular adenomas more closely resemble follicular carcinomas than do simple multinodular goiters and normomacrovesicular adenomas. Such data might reflect a biologic progression from benign to malignant follicular thyroid lesions.

Chromatosomes are not produced fromTetrahymena chromatin by micrococcal nuclease digestion

The subnucleosomal organization of Tetrahymena chromatin, which has an unusual H1 histone, was investigated by NaCl extraction and micrococcal nuclease digestion of nuclei. It was found that Tetrahymena histone H1 is extracted with 0.35 M NaCl, whereas bovine thyroid H1 is not. Micrococcal nuclease digestion of Tetrahymena nuclei did not yield chromatosomes as a stable intermediate, whereas digestion of bovine thyroid nuclei did.

Existence of nuclear thyroid hormone receptors in the porcine thyroid gland and the rat thyroid cell line FRTL-5.

We have investigated whether nuclear T3 receptors exist in the thyroid cell. Nuclear proteins extracted from porcine thyroid nuclei with 0.4 mol/l KCl were incubated with [125I]T3. The mixture was then analysed by sucrose density gradient ultracentrifugation which revealed that the T3-binding proteins migrated at the same position of 3.6 S as rat liver nuclear T3 receptors. Fractionation by high performance liquid chromatography using a size exclusion column and an ion exchanger column also demonstrated elution patterns of T3-binding similar to those of the rat liver receptor. Scatchard plots of crude nuclear extracts from porcine thyroid represented a curvilinear pattern. However, when the nuclear proteins partially purified by a DEAE column chromatography were analysed, a single binding component was found; the association constant was 4.1 x 10(10) l/mol and the maximal binding capacity was 602 fmolT3/mg protein. Displacement study with several T3 analogues showed a highly selective affinity for L-T3. Cultured rat thyroid cells of the FRTL-5 line also contained a single class of saturable, high affinity T3-binding site. Subconfluent cells in 100-mm dishes were incubated with increasing amounts of [125I]T3 at 37 degrees C for 3 h and radioactive T3 in isolated nuclei was counted. Scatchard analysis of data showed that the association constant and the maximal binding capacity were 3.44 +/- 0.63 x 10(10) l/mol and 63.7 +/- 17.8 fmolT3/mg protein, respectively. These results strongly suggest that there are nuclear T3 receptors, indistinguishable from the hepatic T3 receptors, in the porcine thyroid and rat FRTL-5 cells.

Subcellular localization and binding of 125I-triiodothyronine in calf thyroid.

The subcellular distribution of 125I-T3 was studied in calf thyroid slices, under the same experimental conditions where T3 inhibits protein and RNA synthesis, labelled hormone was found mainly in the 20,000 X g supernatant. The specificity of each subcellular localization was determined by incubating the slices with 10(-5)M T3. Only in the purified nuclei a significant decrease was found, indicating a specific localization of the labelled hormone. When slices were incubated with 125I both labelled T3 and T4 were found in purified nuclei, indicating that endogenously synthesized hormones can reach thyroid nuclei. Purified thyroid nuclei were incubated with labelled T3 and increasing amounts of cold hormone. Specific binding reached a plateau after 90 min of incubation at 20 degrees C. When the displacement curves were analysed by a Scatchard plot a binding site with a Ka of 5.2 X 10(7) M-1 and a capacity of 3.0 X 10(-15) moles/microgram DNA was observed. Digestion of nuclei with trypsin and protease abolished completely the binding of 125I-T3 thus indicating the protein nature of the receptor. The hormone-receptor complex could be extracted with 0.4M KCI and eluted in the void volume after Sephadex G-25 column chromatography, similar to peripheral tissues nuclear T3 receptors. The present studies provide the first evidence for the existence of nuclear receptors for T3 in the thyroid, an event probably related to the autoregulatory mechanism.

Thyrotropin controls transcription of the thyroglobulin gene.

The availability of rat thyroglobulin cDNA clones was exploited to study the regulation of thyroglobulin gene transcription by thyrotropin (TSH). Groups of rats were subjected to treatments leading to reduction or increase in the rat serum TSH (rTSH) levels. Thyroid gland nuclei were isolated, incubated in vitro in the presence of 32P-labeled uridine triphosphate, and thyroglobulin transcripts were quantitated by hybridization to immobilized rat thyroglobulin cDNA clones. Transcription of the thyroglobulin gene was found to be very active in thyroid nuclei from control animals. It represented about 10% of total RNA polymerase II activity. Chronic hyperstimulation of the thyroid glands with endogenous rTSH was achieved in rats treated with the goitrogen propylthiouracil. No significant increase of thyroglobulin gene transcription could be measured in thyroid nuclei from these animals. On the contrary, a dramatic decrease in thyroglobulin gene transcription was observed in those animals in which endogenous rTSH levels had been suppressed by hypophysectomy or by the administration of triiodothyronine. Injection of exogenous bovine TSH in such animals readily restored transcriptional activity of the gene. Our results identify transcription as an important regulatory step involved in TSH action. They suggest that normal TSH levels induce close to maximal expression of the thyroglobulin gene but that continuous presence of TSH is required in order to maintain the gene in an activated state.

Increased Micrococcal Nuclease Sensitivity and/or Solubility in Nuclei from Graves' Disease Thyroid Tissue

The sensitivity of nuclei to micrococcal nuclease was compared in thyroid tissue obtained from euthyroid patients with solitary cold nodules and from patients with Graves' disease. A significant increase in the solubility and/or the sensitivity of nuclei to the nuclease was found in thyroid tissue from patients with Graves' disease. Electrophoretic analysis of DNA in chromatin solubilized by the nuclease revealed that the amount of oligonucleosomal DNA was increased, and that of polynucleosomal DNA was even more increased, in nuclei from Graves' thyroids than in those from normal thyroids. Polyacrylamide gel electrophoretic analysis in Triton acid-urea showed that the extent of histone acetylation in nuclei from Graves' thyroids was almost the same as in those from normal thyroids. These findings suggest that the state of chromatin organization in Graves' thyroid nuclei is different from that in normal thyroid nuclei and is independent of the extent of histone acetylation.

A study on the rna polymerase activities in bovine thyroid nuclei isolated in isotonic sucrose, hypertonic sucrose or in anhydrous glycerol media: Influence of the isolation procedure on the electrophoretic pattern of acid soluble nuclear proteins

1. 1. After differential pelleting of bovine thyroid bound RNA polymerase II was the more enriched enzyme activity in the nuclear fraction, and coincided best with the DNA profile. 2. 2. The RNA polymerase I + III activity was compared in nuclear fractions isolated either in 0.25 M sucrose (wet tissue) or in anhydrous glycerol (lyophilized tissue) or in 2.4 M sucrose (lyophilized tissue). 3. 3. Although the nuclei were more resistant to the isolation porcedure in glycerol, more proteins were extracted by that procedure than during the isolation in 2.4 M sucrose. 4. 4. With the 2.4 M sucrose method a twofold enrichment of RNA polymerase I + III activity in respect to DNA occurred in the nuclei pointing to an exclusive localization of these activities within the nucleus. 5. 5. Using the same isolation procedure the different classes of histones were better resolved upon polyacrylamide gelelectrophoresis.

Characterization and transcription of bovine thyroid chromatin

1. 1. Thyroidal chromatin, isolated from bovine thyroid nuclei, has been characterized and the endogenous RNA polymerase activity was studied. 2. 2. The template activity of chromatin was investigated for exogenous homologous RNA polymerase I and II. 3. 3. The influence of chromosomal proteins on the transcription process was described.

Effect of thyrotropin on the sensitivity of thyroid nuclear deoxyribonucleic acid to digestion by micrococcal nuclease.

Little is known about the mechanisms of action of polypeptide hormones on chromatin structure and nuclear function. We have employed micrococcal nuclease to examine the effect of TSH on the accessibility of DNA in thyroid nuclei. Brief digestion of nuclear suspensions with 0.05-0.2 U/ml micrococcal nuclease at 26-28 C decreased their opacity at 600 nm. The decrease in opacity was linear with increasing nuclear concentration up to 0.2 mg/ml DNA. This response to nuclease was enhanced in nuclear suspensions prepared from thyroid slices that had been incubated with TSH (50 mU/ml) for 5 h (P less than 0.001). To determine whether TSH also increased the digestion of DNA, we measured the amount of DNA released into 1200 X g supernatants by nuclease treatment of nuclei prepared from control and TSH-treated slices. When TSH-treated nuclei (110 micrograms/100 microliters) were digested with 0.2 U micrococcal nuclease/ml at 37 C for 30 sec, a mean of 12.6 micrograms +/- 3.6 (SD) DNA appeared in the supernatant, as compared to 8.4 micrograms +/- 1.98 DNA from control nuclei (P less than 0.05). This increase in the insensitivity of nuclear DNA to micrococcal nuclease may reflect some conformational change in chromatin in response to TSH. Since micrococcal nuclease sensitivity may reflect transcriptional competence of DNA, we speculate that polypeptide hormones may enhance the accessibility of DNA to RNA polymerase or to endogenous stimulators of transcription.

DNA-Dependent rna polymerases from bovine thyroid: Catalytic properties and template specificities

1. 1. DNA-dependent RNA polymerases I and II have been purified starting from bovine thyroid nuclei yielding a purification factor of 230 for the RNA polymerase I and a purification factor 3212 for RNA polymerase II. RNA polymerase II was further characterized by gel electrophoresis and amino-acid analysis. 2. 2. Kinetics and optimal assay conditions for both RNA polymerases were studied. 3. 3. The template efficiency of a number of DNA preparations was investigated. 4. 4. Rifamycin AF 013 and heparin act as initiation inhibitors. 5. 5. Polyamines were shown to enhance the rate of chain elongation.

RNA synthesis in isolated bovine thyroid nuclei and nucleoli. alpha-Amanitin effect, a hint to the existence of a specific regulatory system.

DNA dependent RNA polymerase activities in isolated bovine thyroid nuclei and nucleoli have been studied. They retain their RNA synthetic activity for an extended period of time. This RNA synthetic activity is sensitive to actinomycin D and requires the presence of all four ribonucleoside triphosphates. The optimal conditions have been determined. Polyacrylamide gel electrophoresis reveals that the RNA synthesized has a size distribution ranging from 34S to 4S. The production of 18S-8S RNA is very sensitive to low concentrations of alpha-amanitin. However, in isolated bovine thyroid nuclei (not in nucleoli) this drug displays an effect on all RNA classes produced. The alpha-amanitin induced drastic decrease of [3H]-UMP incorporation in RNA of all sizes synthesized by isolated bovine thyroid nuclei is discussed.

311 Thyroid Antibodies and HL-A Antigens in Children with Thyroid Disease

Antibodies to thyroid cytoplasm (T.C.A.), to thyroid nuclei (ANA) and to thyroglobulin (T.G.A.) were determined in 30 children with thyrotoxicosis (Tx), in 13 with euthyroid goiter (EG), in 9 with hypothyroidism and goiter (H.G.) and in 6 with hypothyroidism with no goiter (H). Histocompatibility (HL-A) antigens were also studied. The results (as percentages) were: Patients with Tx had increased HLA-B8 (45% of controls 25% p <0.05) whereas the other thyroid patients did not. Siblings, one with Tx and one with juvenile diabetes were HLA identical (B8+) whereas an unaffected sibling shared none of the types. Families of Tx had increased Tx, rheumatoid arthritis and S.L.E. In summary, Tx is strongly inherited and related to HLA-B8. Patients with B8 have more T.G.A. and higher titers of T.C.A., but show less tendency to remit. We speculate that Tx is an antibody mediated disease and thyroiditis a predominantly cell mediated disorder. Supported by NIH grants AM19286 and AM05745.

Effect of thyrotropin on ornithine decarboxylase and of polyamines on RNA polymerase in the thyroid.

Two major forms of RNA polymerase were partially purified from calf thyroid nuclei. Type I showed greatest activity in 30 mM(NH4)2SO4, transcribed native DNA best and was resistant to the inhibitor alpha-amanitin. Type II showed greatest activity in 100 mM(NH4)2SO4, transcribed denatured DNA template best and was sensitive to low concentrations of alpha-amanitin...

Characterization of protein phosphokinase activities in horse thyroid nuclei

The distribution of protein phosphokinase (EC 2.7.1.37) activities has been established in horse thyroid nuclei. The presence of several enzyme activities has been demonstrated, two of which are clearly distinct. The first one acts on histone as substrate and is activated by cyclic AMP. Physico-chemical properties of this nuclear cyclic AMP-dependent histone kinase and of the cytosol histone kinase are different, demonstrating the absence of a contamination from the cytosol. The second enzyme acts on casein as substrate and is not stimulated by cyclic AMP or cyclic GMP. The findings are consistent with the observation of thyrotropin stimulation of histone phosphorylation in thyroid nuclei.

Non-random appearance of Y-chromatin-like fluorescence in the nuclei of thyroid and brain and its chromosomal background.

Non-random distribution of fluorescing chromatin bodies resembling Y chromatin was observed among the cell populations of various organs obtained by autopsy of 52 adults. This type of Y-chromatin-like fluorescence is more frequent in thyroid and brain than in other organs. The Y-chromatin-like bodies of thyroid nuclei are not closely correlated with the brilliantly fluorescing autosomal blocks of lymphocyte mitoses of the same persons, as it was demonstrated in surgical cases.

The nuclear lipids of bovine hypertrophic thyroid

A highly purified preparation of bovine hypertrophie thyroid nuclei was isolated by means of an adapted method of Widnell, C. C. and Tata, J. R. (1964) Biochem. J. 92, 313–317, followed by a centrifugation step in a zonal rotor. The total lipid content amounted to 2.1 pg/nucleus, approximately 90% of it being phospholipids. 60% of the total neutral lipid fraction was cholesterol. The phospholipid pattern showed 57% phosphatidylcholine, 22% phosphatidylethanolamine, 12% phosphatidylserine + phosphatidylinositol and 2% sphingomyelin. No glycolipids could be detected.

Subcellular structure of bovine thyroid. VI. A study of bovine thyroid nuclei by density gradient centrifugation in a zonal A-XII rotor.

METHODS Isolation of nuclei. - For this purpose an adapted method of WIDNELL & TATA (1964) was used. Bovine thyroids were obtained fresh from the slaughterhouse and transported chilled in crushed ice (DIERICK & HILDERSON, 1967). After removal of adhering adipose and connective tissue the glands were sliced, washed repeatedly in 0.32 M sucrose solution (3 mM with respect to MgCl,, adjusted to pH 7.4 with NaHCO,) and homogenized in a Ten Broeck homogenizer. The homogenate was filtered through two layers of cheese cloth. Per 25 ml filtered homogenate 15 ml of

Absence of respiratory activity in bovine thyroid nuclei.

AbstractMukherjee & Narayanaswami (1972) report the presence of respiratory activity in pig brain nuclei. They conclude that, in those nuclei, oxidative energy-yielding metabolic pathways exist that serve as sources of nuclear ATP. Two kinds of nuclear fractions were isolated by these authors: a crude nuclear pellet and a more purified nuclear fraction. The purified fraction was obtained by centrifuging the crude nuclear pellet first in 0.44 M sucrose (pH 7.7, 3 mM MgCl2) and subsequently through a two step discontinuous sucrose gradient (0.88-1.0 m). In the final preparation 8 % of the original proteins were recovered. No contamination by other particulate subfractions could be demonstrated by phase contrast microscopy. The final RNA: DNA ratio was 0.56. During the purification procedure removal of extranuclear enzymes occurred. However, the final preparation still contained 14 % of total glutamic dehydrogenase and succinic dehydrogenase and 8 % of monoamine oxidase. In this purified nuclear preparation ...