M.S. Moukhtar

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.

Albumin messenger RNA as a marker of circulating hepatocytes in hepatocellular carcinoma.

BACKGROUND/AIMS Hepatocellular carcinoma is one of the most frequent malignancies. Liver transplantation is theoretically the treatment of choice because it eliminates both the hepatocellular carcinoma and the cirrhosis. High frequency of relapse observed after liver transplantation may be explained by the existence of undetectable metastasis before transplantation. The aim of this study was to determine whether albumin messenger RNA (mRNA), a specifically hepatocyte-expressed gene, could be a marker of metastasis in hepatocellular carcinoma. METHODS Albumin mRNA from circulating malignant hepatocytes was detected in the blood by reverse transcription followed by enzymatic amplification. RESULTS Albumin mRNA was found in the blood of 3 patients with histologically proven metastatic hepatocellular carcinoma and in 9 of 21 patients with hepatocellular carcinoma and undetectable metastases, giving a percentage rate of 43, similar to the relapse rate following liver transplantation for hepatocellular carcinoma. None of the 8 patients with secondary liver cancer had detectable albumin mRNA. CONCLUSIONS These results suggest that patients with hepatocellular carcinoma and no detectable albumin mRNA in the blood may be a subgroup with a low risk of relapse following liver transplantation.

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.

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.

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 and carcinoembryonic antigen in poorly differentiated follicular carcinoma

Previous studies have shown that certain patients suffering from poorly differentiated follicular carcinoma (PDFC) of the thyroid had high levels of calcitonin (CT) and carcinoembryonic antigen (CEA) in the plasma. In this work, both CT and CEA were localized in tissue sections obtained at operation from patients suffering from PDFC. The results confirm the hypothesis that certain cases of PDFC are in fact CT‐secreting tumors and represent another type of thyroid neoplasma. Patients suffering from PDFC should be screened using both CT and CEA assays.

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.

Biosynthesis of human calcitonin: Evidence for a prohormone

Abstract Medullary carcinoma tissue was incubated in vitro in Eagles medium containing labeled cysteine or lysine. After extraction and purification by affinity chromatography using antibodies raised against synthetic human calcitonin (hCT), two major peaks of radioactivity were detected after SDS polyacrylamide gel electrophoresis. The estimated molecular weights were respectively 11 800 for the first peak and identical to that of hCT for the second. Chase experiments reduced drastically both peaks. In the incubation medium, a single labeled product was observed, comigrating with hCT. These results suggest that hCT is liberated prior to its secretion from a larger prohormone, 11 800 Mr (Apparent molecular weight).