A. Jullienne

The complete sequence of human preprocalcitonin.

DNA complementary to mRNA extracted from the thyroid glands of patients suffering from medullary carcinoma of the thyroid (MCT), a calcitonin‐producing tumour, was inserted in the Pst site of pBR 322 by G‐C tailing. The recombinant plasmids were used to transform Escherichia coli DP 50. Ampicillin‐resistant clones were screened using a 32P‐labelled cDNA to mRNA extracted from a case of MCT particularly rich in calcitonin (CT) mRNA. Positive clones were subsequently rescreened using a 32P poly(T) probe. Eighty clones were thus purified, and the inserts obtained by digestion with PstI were subjected to positive hybridization selection with subsequent translation in vitro. An insert stimulating synthesis of the protein and containing restriction sites compatible with the previously published complete sequence of calcitonin mRNA from rat was sequenced. This cDNA insert contained the entire coding region of 426 bp, 70 bp at the 5′‐end, and 295 bp upstream from the poly(A) tail. The complete amino acid sequence of human preprocalcitonin could thus be deduced.

Expression of glucagon‐like peptide 1 receptor in a murine C cell line Regulation of calcitonin gene by glucagon‐like peptide 1

We have characterized, by RT‐PCR amplification using specific primers, the presence of glucagon‐like peptide‐1 (GLP‐1) receptor mRNA in CA‐77 cells, a C cell line derived from a rat medullary thyroid carcinoma. Down‐regulation of the GLP‐1 receptor mRNA was observed after exposure of CA‐77 C cells with GLP‐1 (7–37). Increased secretion of both calcitonin gene‐related peptide (CGRP) and calcitonin (CT) occurred after treatment with GLP‐1 (7–37) associated with elevated steady‐state levels of CGRP and CT mRNA. GLP‐1 (7–37) increased cAMP formation in CA‐77 cells in a dose‐dependent manner; exendin (9–39), a GLP‐1 receptor antagonist, inhibited cAMP production. The GLP‐1 peptide which is produced by intestinal cells could be involved in the control of CT secretion through an entero‐thyroidal axis implying GLP‐1 receptor and increased CT gene expression.

An isoform of the human calcitonin receptor is expressed in TT cells and in medullary carcinoma of the thyroid

We amplified, using the polymerase chain reaction and calcitonin receptor (CTR) specific primers, RNA extracted from medullary thyroid carcinoma (MTC) and the derived TT cell line. Both secrete large amounts of calcitonin. Electrophoresis of amplification products revealed, in both cases, an ethidium bromide‐stained band that hybridized to a CTR probe. Sequencing the band amplified from TT cells revealed an open reading frame identical to the sequence of H‐CTR but lacking 16 amino acids in the first intracellular loop. This demonstrates the existence of an mRNA coding for a subtype of H‐CTR which is expressed in TT cells and MTC.

Human cord blood monocytes undergo terminal osteoclast differentiation in vitro in the presence of culture medium conditioned by giant cell tumor of bone.

Osteoclasts (OCs), which form by fusion of hematopoietic precursor cells, are typically present in large numbers in giant cell tumors of bone (GCTBs). These tumors may, therefore, contain cells which secrete factors that stimulate recruitment and differentiation of OC precursors. Multinucleated cells resembling OCs also form in cultures of human cord blood monocytes (CBMs) stimulated by 1.25 dihydroxyvitamin D3, but these cells lack the ability to form bone resorption pits, the defining functional characteristic of mature OCs. CBMs may thus require additional stimulation to form OCs; we therefore investigated whether GCTBs are a source of such a stimulus. CBMs were stimulated in long term (21 day) culture by medium conditioned by explants of GCTBs; media collected within 15 days of explant (early‐CM) and after 15 days (late‐CM) were employed. We also cocultured CBMs with primary GCTB‐derived stromal cells as well as immortalized bone marrow stroma‐derived cells. CBMs stimulated by early‐CM formed resorption pits on cortical bone slices; however, stimulation by late‐CM resulted in virtually no resorption. Both early‐CM and late‐CM increased CBM proliferation, but not the proportion of vitronectin receptor positive or multinucleated cells. Coculture of CBMs with stromal cells of GCTBs or bone marrow did not result in bone resorption, although these stromal cells (most expressing alkaline phosphatase but progressively losing parathyroid hormone receptor expression) expressed mRNA for cytokines involved in OC differentiation, including macrophage‐CSF, granulocyte‐macrophage‐CSF, IL‐11, IL‐6, and stem cell factor. Our results indicate that CBMs are capable of terminal OC differentiation in vitro, a process requiring 1,25 dihydroxyvitamin D3 as well as diffusible factor(s) which can be derived from GCTB. Stromal cells of GCTB may produce such factors in vivo, but do not support OC differentiation in vitro, possibly through phenotypic instability in culture.

Isolation and partial characterization of the calcitonin gene in a lower vertebrate. Predicted structure of avian calcitonin gene-related peptide.

(Chicken) Calcitonin gene‐related peptide Amino acid sequence Nucleotide sequence Calcitonin gene

Sequence and expression of the chicken calcitonin gene

The avian calcitonin gene was isolated and sequenced; two mRNAs are expressed by tissue‐specific alternate splicing. The peptides encoded by the mRNAs are the protein precursors of either calcitonin or calcitonin gene‐related peptide (CGRP). Calcitonin is expressed predominantly in ultimobranchial bodies and CGRP in brain.

Indomethacin, a cox inhibitor, enhances 15-PGDH and decreases human tumoral C cells proliferation.

High levels of prostaglandins (PGs) are currently found in tumoral cells, due to expression of the inducible PGs synthesis enzyme, the cyclooxygenase 2 (COX 2). Non Steroidal Anti Inflammatory Drugs (NSAIDs) possess an antitumoral effect related, in a large extend, to the inhibition of this enzyme. It was recently suggested that the decreased activity of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the key enzyme catabolysing PGs, may be responsible too for experimentally induced colon tumor enhancement. We report here, for the first time, that indomethacin, an NSAID, decreased TT cell proliferation, derived from a human Medullary Thyroid Carcinoma (MTC). This effect is time and concentration-dependent. Moreover, indomethacin enhanced expression and activity of 15-PGDH. The 15-PGDH levels were negatively correlated with TT cell proliferation (r = -0.52, p < 0.001). Indomethacin, known to decrease COX levels and activity, could also act in modifying catabolism of PGs. This suggests that 15-PGDH is involved in tumoral development, and could therefore be considered as a target for NSAIDs.

Structure of chicken calcitonin predicted by partial nucleotide sequence of its precursor

DNA complementary to chicken ultimobranchial gland mRNA was cloned into the Pst I site of plasmid vector pBR322. A plasmid was selected by DNA‐mRNA hybridization. We report here the partial nucleotide sequence of chicken calcitonin mRNA and the deduced complete amino acid sequence of chicken calcitonin.

Calcitonin gene expression in normal human liver

Immunoreactive calcitonin (CT) is present in liver. This could represent hormone synthesized by liver cells, degraded or bound to specific receptors reported in this organ. We report here that the calcitonin gene is expressed in liver. We proved this by demonstrating, by PCR amplification using specific primers, the presence of calcitonin messenger in human liver and in primary cultures of human hepatocytes and detected by radioimmunoassay CT in hepatic tissues and cells. The synthesis of hormone by liver that also possesses specific receptors for CT favors the presence of an autocrine or paracrine system involving calcitonin in this organ.

Chromosomal localization of the type-I 15-PGDH gene to 4q34–q35

Abstract The gene encoding the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase, designated type-I 15-PGDH, was mapped to chromosome 4 by analyzing its segregation in a panel of human-hamster somatic cell hybrids. This gene was further localized to bands 4q34–q35 by in situ hybridization on human chromosomes.