Tumour calcitonin. Interaction with specific calcitonin receptors.

The human epidermoid bronchial carcinoma (BEN) cell line has been shown to have specific membrane binding sites for calcitonin and to secrete high-molecular-weight forms (ranging from 40000 to 10000) of immunoreactive calcitonin. Synthetic salmon and human calcitonins and a thyroid extract of porcine calcitonin have been shown to displace 125I-labelled salmon calcitonin from the receptors in a dose-related fashion. The binding to these receptors of calcitonins derived from the BEN cell line and a medullary thyroid carcinoma with molecular weights ranging from 28000 to 3500 (both separated by gel-filtration chromatography) has been investigated. Neither major peaks of BEN-cell-line calcitonin showed receptor binding activity. Only one form of medullary thyroid carcinoma calcitonin, that which co-eluted with synthetic calcitonin monomer on gel-filtration chromatography, caused any significant displacement of labelled hormone from the receptors.     

Calcium infusion and pentagastrin injection in diagnosis of medullary thyroid carcinoma.

Calcium infusion and pentagastrin injection were compared as tests to stimulate calcitonin secretion for the detection of medullary carcinoma of the thyroid. Plasma concentrations of immunoreactive calcitonin were measured by radioimmunoassay before and during both stimulation tests in 2 persons who had been found at operation to have medullary thyroid carcinoma, 1 relative in whom a cervical lymph node biopsy had shown medullary thyroid carcinoma and 36 asymptomatic relatives. The tests were carried out on separate days by intravenous infusion of calcium gluconate for 2 hours, to provide 3.75 mg/kg of elemental calcium per hour, and rapid intravenous injection of 0.5 microgram/kg of pentagastrin. Before stimulation immunoreactive calcitonin was undetectable in the plasma of 34 of the 36 asymptomatic persons; the 2 with elevated baseline concentrations of the hormone had a positive response to both tests. Seven others showed an increase in plasma immunoreactive calcitonin concentration only after pentagastrin injection. The two persons with initially elevated values and three of the seven with increased values after pentagastrin injection were found at subsequent operation to have focal medullary carcinoma and parafollicular cell hyperplasia; after the operation immunoreactive calcitonin was undetectable in the plasma, even after stimulation. Rapid injection of pentagastrin is more reliable than slow infusion of calcium as a stimulation test for the early detection of medullary thyroid carcinoma.     

Ectopic synthesis of high-Mr calcitonin by the BEN lung carcinoma cell line reflects aberrant proteolytic processing

Cloning and nucleotide sequence analysis of the human calcitonin mRNA from the BEN lung carcinoma cell line, a cell line known to secrete high-Mr forms of calcitonin, showed no difference in the coding region at the nucleotide level compared with calcitonin mRNA isolated from medullary thyroid carcinoma which secretes calcitonin monomer. Therefore, the secretion of high-Mr forms of calcitonin reflects the absence or limited activity of proteolytic processing enzymes within the secretory pathway of this cell line. In all other respects, as judged by RNA blotting and S1 mapping, calcitonin/alpha-CGRP expression was identical to that found in medullary thyroid carcinoma, including the differential use of an alternative splice donor site within intron 1. The BEN cell line also produces low levels of alpha-CGRP mRNA and secretes CGRP antigenic determinants. Analysis of plasma CGRP levels in 12 patients with anaplastic lung carcinoma showed elevated levels in 11 of these, suggesting that CGRP may be an important diagnostic marker for this disease.     

Immunoreactive calcitonin in leukaemia.

A radioimmunoassay was used to measure concentrations of immunoreactive human calcitonin (HCT) in plasma and leucocytes from patients with various leukaemic and myeloproliferative disorders. Plasma immunoreactive HCT concentrations were increased in 32 out of 33 patients with chronic granulocytic leukaemia (CGL) and in all eight patients with acute myeloid leukamia (AML) at presentation or in relapse. Out of 11 patients with other myeloproliferative disorders, eight had increased plasma immunoreactive HCT concentrations. Buffy-coat-cell extracts and culture media from peripheral leucocytes of patients with CGL also contained increased immunoreactive HCT concentrations. In contrast, plasma from patients with chronic lymphocytic leukaemia, acute lymphoblastic leukaemia, and AML in remission had low or undetectable immunoreactive HCT concentrations. Increased plasma and cellular concentrations of immunoreactive HCT may be a consequence of abnormal proliferation of myeloid cells and might prove to be valuable in predicting relapse in patients with myeloid leukaemias.     

Purification and partial characterization of high-molecular-weight forms of ectopic calcitonin from a human bronchial carcinoma cell line.

Studies on the high-molecular-weight immunoreactive calcitonin produced ectopically in culture by an epidermoid bronchial carcinoma cell line are reported. In cell-exposed medium, the principal component has a molecular weight of 40000 and molecules of mol.wts. 13000 and 10000 also occur. Only a trace amount of material co-eluting with 35000-mol.wt. human calcitonin is detectable. None of the calcitonins show cross-reactivity with anti-corticotropin serum. The 40000-mol.wt. immunoreactive calcitonin is readily proteolysed to the 13000- and 10000-mol.wt. components, but the 10000-mol.wt. component behaves as a comparatively stable 'core' molecule. By using immunoprecipitation and high-pressure liquid chromatography (h.p.l.c.), it is possible to prepare radiochemically homogeneous 10000-mol.wt. immunoreactive calcitonin from cells grown in the presence of individual 35S- or 3H-labelled amino acids. Peptide mapping of enzymic digests of this material by h.p.l.c. shows that it contains peptides in common with synthetic human calcitonin.     

Effect of infusion of salmon calcitonin on the secretion of somatostatin and gastrin in man

In vitro and animal studies have pointed out complex interrelations between gastrointestinal hormones and calcitonin. To analyse the acute effects of calcitonin in more detail, patients undergoing surgery were infused intravenously with synthetic salmon calcitonin, a potent analog of the human hormone. Samples were taken after 0, 30 and 60 minutes from the hepatic, portal and a peripheral vein. Somatostatin and gastrin were determined by radioimmunoassay. The mean basal levels of somatostatin in peripheral and hepatic venous plasma (14.2 and 15.6 pg/ml) were significantly lower than in portal plasma (45.6 pg/ml), indicating effective removal by the liver. After infusion of calcitonin there was a general rise in somatostatin levels and an increase in the gradient between hepatic and portal blood. Basal gastrin levels were highest in the portal vein when compared intraindividually. The differences disappeared after calcitonin infusion with a concomitant systemic reduction of gastrin levels. Thus, calcitonin is able to stimulate the secretion of somatostatin from the gastrointestinal tract and does reduce gastrin secretion, possibly via the stimulation of somatostatin secretion.     

Structure, measurement, and secretion of human glucagon-like peptide-2

By using radioimmunoassays toward the cDNA-predicted amino acid sequence of human glucagon-like peptide-2, a peptide was isolated from extracts of human ileum. By mass spectrometry and Edman sequencing, this peptide was identified as human proglucagon 126-158. High-performance liquid chromatography analyses indicated that a similar immunoreactive peptide (iGLP-2) was present in human plasma. Human plasma concentrations of iGLP-2 were elevated 3- to 4-fold at 1 to 2 h after ingestion of 800 to 1200 kcal meals.     

Isolation and determination of the amino acid sequence of chicken calcitonin I from chicken ultimobranchial glands

A radioimmunoassay for chicken calcitonin in chicken ultimobranchial glands was established utilizing a rabbit antiserum against eel calcitonin. This assay method, which is about 100 times as sensitive as the usual bioassay for hypocalcemic activity, was used for monitoring chicken calcitonin during its purification. The immunoreactivity in chicken ultimobranchial extract was separated by SP-Sephadex C-25 chromatography into two fractions. Chicken calcitonin I, which was occurred in the major immunoreactive fraction, was further purified to homogeneity as shown by reverse phase HPLC. In the end, 39 nmol of chicken calcitonin I was obtained from 3,384 chickens following a 12,000-fold purification. The complete amino acid sequence of purified chicken calcitonin I was determined to be H-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Ly s-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH2 and confirmed by synthesis. The specific biological activity of chicken calcitonin I (4,500 MRCU/mg) was identical to that of eel calcitonin, which has the highest specific biological activity among the calcitonins so far isolated. Chicken calcitonin I resembled the calcitonins from the ultimobranchial glands both of salmon and eel in sequence, biological activity, and immunological property.     

Studies on in vitro effect of serotonin on calcitonin secretion by rat thyroid C cells

Summary Thyroid glands of young rats were incubated for 3 h in Eagle's solution supplemented with 5-hydroxy-l-tryptophan (5-HTP) or with serotonin. Following control incubations or incubations with serotonin, no serotonin could be demonstrated in C cells using immunocytochemical techniques. However, serotonin was demonstrated in the secretory granules of all C cells following incubation with 5-HTP. The secretory function of C cells was evaluated by ultrastructural and immunocytochemical studies, and by calcitonin radioimmunoassays of the incubation medium. Following incubation with 5-HTP, the secretory function of the majority of C cells was inhibited, and calcitonin levels in the media were decreased. Incubation with serotonin produced an increased secretory function of C cells and higher calcitonin levels in the media. The results indicate that serotonin and its direct precursor, 5-HTP, affect calcitonin secretion by rat thyroid C cells by distinct mechanisms.     

Covalent cross-linking of a photoactive derivative of calcitonin to human breast cancer cell receptors

A photoaffinity derivative of salmon calcitonin has been produced by transglutaminase-mediated incorporation of N-(beta-aminoethyl)-4-azido-2-nitroaniline into the hormone. The derivative, purified by high pressure liquid chromatography, retained the abilities to bind to cultured T47D breast cancer cells and to stimulate adenylate cyclase in these cells. In both these respects it was equipotent with synthetic salmon calcitonin. Photolysis of the 125I-labeled photoactive salmon calcitonin derivative bound to T47D cells was accompanied by specific labeling of only one component (Mr approximately 85,000) as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling was observed only upon photolysis and was inhibited by unlabeled synthetic salmon calcitonin but not by the inactive calcitonin analogue, 8-glycine human calcitonin. Reduction did not alter the apparent molecular weight of the calcitonin receptor complex. No macromolecular forms of calcitonin were produced by photolysis in the absence of T47D cells.     

Secretion of immunoreactive calcitonin by human breast carcinomas.

Twenty-three out of 28 patients with metastatic breast carcinoma and one out of 13 patients with localised disease had raised levels of plasma immunoreactive calcitonin. Monolayer cultures of breast carcinomas maintained for up to 10 weeks released immunoreactive calcitonin, and a primary breast carcinoma passaged in "nude" mice for over a year contained material immunologically and chromatographically resembling the monomeric form of human calcitonin. These studies indicate that breast carcinomas can produce calcitonin and that plasma calcitonin measurements may be useful in staging patients with breast carcinomas.     

Many monoclonal antibodies with an apparent specificity for certain lung cancers are directed against a sugar sequence found in lacto-N-fucopentaose III

Monoclonal antibodies with an apparent specificity for human small-cell carcinoma, adenocarcinoma, and squamous carcinoma of the lung are produced by some hybridomas obtained from mice and rats immunized with an established line of human small cell lung cancer. Out of 85 of these antibodies produced by independently isolated hybridomas from 15 different fusions, 21 are directed against the sugar sequence which occurs in lacto-N-fucopentaose III ceramide, in several higher glycolipids and in glycoproteins. Specificity was determined by autoradiography of thin-layer chromatograms of glycolipids, by solid-phase radioimmunoassays, and by hapten inhibition studies. All 21 antibodies are of the immunoglobulin M type.     

Heterogeneity of immunoreactive calcitonin in extracts of normal human thyroid

Immunoreactive calcitonin in serum of patients with medullary carcinoma of the thyroid and in extracts of this tumor has been found to exhibit heterogeneity when analysed by gel-chromatography and radioimmunoassay. In this study normal human thyroid tissue was homogenized in buffered saline a neutral ph and a soluble fraction was obtained after ultracentrifugation. Extracts were analysed by gel-filtration before and after treatment with detergents, with denaturing agents and after reduction and alkylation. The stability in vitro of the extracted immunoreactive calcitonin was determined at different temperatures and the changes in heterogeneity analysed by gel-filtration. The results suggest that at least part of the largest form of immunoreactive calcitonin is an artefact of the chromatographic procedure. Moieties with a molecular weight below 12000 Dalton were present and could be identical with prehormones described by others. However, no indication for a sequential degradation releasing calcitonin monomer from larger precursors was found in normal thyroid.     

Salmon calcitonin inhibits human sperm motility in vitro

We have evaluated by a stroboscopic technique the in vitro effect of salmon calcitonin and human calcitonin on the motility of human migrated spermatozoa. We report here that human calcitonin is uneffective while salmon calcitonin is a potent inhibitor of the sperm motility. This salmon calcitonin action is abolished by the preincubation of the peptide with an anti-salmon calcitonin antiserum, demonstrating the specificity of the effect. In addition, we provide evidence that the release of intracellular calcium represents a necessary step for the action of the peptide. In fact, the salmon calcitonin effect is prevented in a dose-dependent way by dantrolene sodium which inhibits the release of calcium from intracellular stores while the calcium channel blocker verapamil is unefficacious. These results suggest a potential role for calcitonin in regulating human sperm motility.     

Elevated plasma and tissue concentrations of beta-endorphin and beta-lipotropin associated with an ectopic ACTH-producing lung tumor

beta-Endorphin- and ACTH-like immunoreactive peptides were measured in plasma and certain tissues of a patient suffering from an oat-cell-carcinoma of the lungs and Cushing's syndrome. Using sensitive radioimmunoassays in combination with gel-filtration, highly elevated levels of beta-endorphin, beta-lipotropin (beta-LPH), alpha-melanotropin (alpha-MSH) and ACTH were found.     

Identification of putative serum glycoprotein biomarkers for human lung adenocarcinoma by multilectin affinity chromatography and LC-MS/MS

Glycoproteins in human serum play fundamental roles in many biological processes, and also have clinical value as biomarkers for disease progression and treatment. In this study, we isolated glycoproteins from the sera of three healthy individuals and three lung adenocarcinoma patients using multilectin affinity chromatography. The recovered glycoproteins were subjected to treatment with peptide-N-glycosidase F (PNGase F) and in-gel digestion by trypsin. Tryptic peptides were analyzed by nano-LC coupled to ESI-MS/MS and the MS/MS spectra were processed by Bioworks 3.2 and an in-house bioinformatics tool, ProtAn. Approximately 90% of the proteins identified contained more than one potential glycosylation site. Comparison of the serum glycoproteome of healthy and adenocarcinoma individuals revealed 38 cancer-selective proteins. Among them, 60% have previously been reported as low abundance proteins in human sera. We identified several cancer-selective proteins that have been previously characterized as potential indicators of lung cancer in serum or plasma, including haptoglobin (HP), inter-alpha-trypsin inhibitor heavy chain 4 (ITI-H4), complement C3 precursor, and leucine-rich alpha-2-glycoprotein. In addition, plasma kallikrein (KLKB1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITI-H3) were identified as being potentially elevated in the lung cancer group, and were validated by Western blot analysis. Furthermore, approximately 18 kDa plasma kallirein protein fragment was detected at high levels in 25 out of 28 adenocarcinoma patients, while one of the eight normal individuals showed moderate positive. The results suggest that KLKB1 represents a potential candidate serum biomarker of lung cancer.     

Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.     

Monoclonal antibodies that distinguish non-small cell from small cell lung cancer

Two murine IgG2Ak monoclonal antibodies (703D4, 704A 1) were produced and characterized after immunization with a human large cell lung cancer line (NCI-H 157). These antibodies detect different epitopes on 31 kilodalton [35S]methionine incorporating protein(s). Radiobinding and immunohistochemical studies show these antibodies bind to most (11/13) human non-small cell lung cancer (adenocarcinoma, epidermoid, and large cell), but not to small cell lung cancer (0/11) tumors tested. The epitopes these antibodies recognized are also expressed on human melanomas (7/8), two other tumors (osteogenic sarcoma, renal cell carcinoma), but not on many other human tumors (breast, colon, neuroblastoma, lymphoid), and not on a panel of normal adult human tissues. Because the antigen(s) are preserved after fixation and because of their ability to distinguish lung cancer types from each other and normal tissues, they should be of clinical, as well as of biologic interest.     

Effect of salmon calcitonin on renal excretion of adenosine 3', 5' monophosphate in man.

Intravenous infusion of salmon calcitonin in six healthy subjects produced an increase in the plasma levels and urinary excretion of cyclic AMP. Cyclic AMP clearance diminished but remained higher than inulin clearance. Salmon calcitonin was also infused in six hypertensive patients with normal glomerular filtration rate. Arterial and renal venous plasma concentration of cyclic AMP were clearly raised. The difference between both these concentrations was not significant in the control periods but became marked during the treatment and post treatment periods demonstrating a net extraction of cyclic AMP from plasma by the kidneys. Renal extraction of cyclic AMP was lower than its urinary excretion in the control periods whereas it was clearly higher after salmon calcitonin was given. This shows that salmon calcitonin stimulates the production of cyclic AMP in extra-renal tissues and that the excess of cyclic AMP formed is catabolized by the kidneys.