Thyroid hormones (THs) play critical roles in the differentiation, growth, metabolism, and physiological function of virtually all tissues. TH binds to receptors that are ligand-regulatable transcription factors belonging to the nuclear hormone receptor superfamily. Tremendous progress has been made recently in our understanding of the molecular mechanisms that underlie TH action. In this review, we present the major advances in our knowledge of the molecular mechanisms of TH action and their implications for TH action in specific tissues, resistance to thyroid hormone syndrome, and genetically engineered mouse models.

Physiological and molecular basis of thyroid hormone action.

Two sublines of a breast epithelial cell culture, MCF-10, derived from human fibrocystic mammary tissue exhibit immortality after extended cultivation in low calcium concentrations (0.03-0.06 mM) and floating transfers in low calcium (MCF-10F), or by trypsin-Versene passages in the customary (normal) calcium levels, 1.05 mM (MCF-10A). Both sublines have been maintained as separate entities after 2.3 years (849 days) in vitro and at present have been in culture for longer than 4 years. MCF-10 has the characteristics of normal breast epithelium by the following criteria: (a) lack of tumorigenicity in nude mice; (b) three-dimensional growth in collagen; (c) growth in culture that is controlled by hormones and growth factors; (d) lack of anchorage-independent growth; and (e) dome formation in confluent cultures. Cytogenetic analysis prior to immortalization showed normal diploid cells; although later passages showed minimal rearrangement and near-diploidy, the immortal cells were not karyotypically normal. The emergence of an immortal culture in normal calcium media was not an inherent characteristic of the original tissue from which MCF-10 was derived since reactivated cryo-preserved cells from cultures grown for 0.3 and 1.2 years in low calcium were incapable of sustained growth in normal calcium.

https://cancerres.aacrjournals.org/content/canres/50/18/6075.full.pdf

Isolation and Characterization of a Spontaneously Immortalized Human Breast Epithelial Cell Line, MCF-10

In a blinded experiment, we report the first allelic association of the dopamine D2receptor gene in alcoholism. From 70 brain samples of alcoholics and nonalcoholics, DNA was digested with restriction endonucleases and probed with a clone that contained the entire 3' coding exon, the polyadenylation signal, and approximately 16.4 kilobases of noncoding 3' sequence of the human dopamine D2receptor gene (λhD2G1). In the present samples, the presence of A1 allele of the dopamine D2receptor gene correctly classified 77% of alcoholics, and its absence classified 72% of nonalcoholics. The polymorphic pattern of this receptor gene suggests that a gene that confers susceptibility to at least one form of alcoholism is located on the q22-q23 region of chromosome 11. (JAMA. 1990;263:2055-2060)

https://jamanetwork.com/HttpHandlers/ArticlePdfHandler.ashx?journal=JAMA&articleId=381428&pdfFileName=jama_263_15_027.pdf

Allelic association of human dopamine D2 receptor gene in alcoholism.

The pituitary hormone thyrotropin stimulates the function, expression of differentiation and growth of thyrocytes by cyclic AMP-dependent mechanisms. Tissue hyperplasia and hyperthyroidism are therefore expected to result when activation of the adenylyl cyclase-cAMP cascade is unregulated. This is observed in several situations, including when somatic mutations impair the GTPase activity of the G protein Gsa (ref 6, 7). Such a mechanism is probably responsible for the development of a minority of monoclonal hyperfunctioning thyroid adenomas. Here we identify somatic mutations in the carboxy-terminal portion of the third cytoplasmic loop of the thyrotropin receptor in three out of eleven hyperfunctioning thyroid adenomas. These mutations are restricted to tumour tissue and involve two different residues (aspartic acid at position 619 to glycine in two cases, and alanine at position 623 to isoleucine in one case). The mutant receptors confer constitutive activation of adenylyl cyclase when tested by transfection in COS cells. This shows that G-protein-coupled receptors are susceptible to constitutive activation by spontaneous somatic mutations and may thus behave as proto-oncogenes.

Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas

The surprising finding that amplification of genomic DNA can be directed by only one oligonucleotide primer of arbitrary sequence to produce a characteristic spectrum of short DNA products of varying complexity, was applied as a strategy to detect genetic differences between organisms. This approach, DNA amplification fingerprinting (DAF), does not depend on cloning or DNA sequence information and can generate fingerprints from DNA of viral, bacterial, fungal, plant and animal origins. Primers as short as 5 nucleotides in length can produce complex banding patterns that are resolved by polyacrylamide gel electrophoresis and silver staining. Amplification fragment length polymorphisms (AFLPs) were detected between different human individuals as well as between soybean cultivars. It is anticipated that DAF will have wide application for DNA analysis.

DNA amplification fingerprinting using very short arbitrary oligonucleotide primers.

The term "DNA fingerprint" has been used to describe the extensive restriction fragment length polymorphism associated with hypervariable minisatellites present in the human genome. Until now, it was necessary to hybridize Southern blots to specific probes cloned from human genomic DNA in order to obtain individual-specific restriction patterns. The present study describes the surprising finding that the insert-free, wild-type M13 bacteriophage detects hypervariable minisatellites in human and in animal DNA, provided no competitor DNA is used during hybridization. The effective sequence in M13 was traced to two clusters of 15-base pair repeats within the protein III gene of the bacteriophage. This unexpected use of M13 renders the DNA fingerprinting technology more readily available to molecular biology laboratories.

/pdf/a-sequence-in-m13-phage-detects-hypervariable-minisatellites-4oxk6guc8f.pdf

A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA

Polymorphic dna marker on x chromosome and manic depression

In a cell-free protein-synthesizing system from a rabbit reticulocyte lysate, total RNA extracted from cultured rat pituitary tumor (GH3) cells directed, in a dose-related manner, the synthesis of proteins that were precipitated by antisera specific to rat growth hormone (somatotropin) and rat prolactin. A marked decrease in growth hormone secretion and growth hormone mRNA activity was observed when cells were grown in a medium deficient in thyroid hormone. Addition of triiodothyronine in physiologic amounts both prevented and completely reversed this effect within 48 hr. Thyroid hormone had no effect on prolactin secretion or prolactin mRNA activity. These data suggest that thyroid hormone may stimulate synthesis of growth hormone through induction of transcriptional activity. The possibility of an additional effect at the posttranscriptional level has not been excluded. Although thyroid hormone is believed to have a general effect on a variety of metabolic processes, some effects, at the molecular level, may be quite selective, as indicated by the observed changes in growth hormone but not prolactin mRNA activity. The GH3 cell model is useful in the study of triiodothyronine action because of independence from secondary hormonal effects caused by hypothyroidism and because simultaneous measurement of prolactin mRNA activity serves as a unique internal control.

http://www.pnas.org/content/74/5/2054.full.pdf

Triiodothyronine stimulates specifically growth hormone mRNA in rat pituitary tumor cells

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.

https://www.pnas.org/content/pnas/81/19/5941.full.pdf

Thyrotropin controls transcription of the thyroglobulin gene.

We have cloned overlapping segments of the human thyroglobulin gene from a genomic cosmid library. Restriction mapping and electron microscopy show that a region of 38 kb at or near the 3'-end of this gene encodes only 850 nucleotides or 10% of the messenger RNA (mRNA) sequence. The region contains five exons of 130-210 nucleotides, split by introns of 1 to 15-17 kb. This represents the lowest ratio of coding to non-coding DNA (2.2%) found thus far in any eukaryotic gene. Blot hybridization under non-stringent conditions shows the presence of only one copy of this gene in the human genome and the absence of other closely related sequences.

/pdf/the-human-thyroglobulin-gene-contains-two-15-17-kb-introns-3meqw9i8qm.pdf

Huguette Brocas

Papers

A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA

Polymorphic dna marker on x chromosome and manic depression

Triiodothyronine stimulates specifically growth hormone mRNA in rat pituitary tumor cells

Thyrotropin controls transcription of the thyroglobulin gene.

The human thyroglobulin gene contains two 15-17 kb introns near its 3'-end.