Simultaneous localization of calcitonin mRNA and peptide in a medullary thyroid carcinoma

We report the visualization of calcitonin gene expression products at the mRNA and peptide levels on the same section of a medullary thyroid carcinoma by combined in situ hybridization and immunohistochemistry. mRNA detection was accomplished by hybridization with radioactively labeled antisense RNA probes followed by autoradiography and immunohistochemically using the avidin-biotin complex method. Best results were obtained when in situ hybridization preceded immunohistochemistry, as determined by quantitative analysis of the autoradiographs. When immunohistochemistry was performed prior to in situ hybridization, the RNase inhibitor heparin had to be added to the antibodies to retain hybridizable mRNA. The intensity of the two reactions varied in individual cells, indicating a functional heterogeneity of tumor cells with regard to calcitonin mRNA content and storage of the related immunoreactive peptide. These results, in combination with elevated serum calcitonin levels, suggest significant differences in the rate of secretion of individual tumor cells. Simultaneous localization of mRNA and its peptide within the same cell may, therefore, provide further insight into gene expression and secretory activity at the single cell level.     

Release of calcitonin by cultures of medullary carcinoma of the thyroid.

Three biopsies of medullary carcinoma of the thyroid were grown in monolayer culture. All three cultures initially released high levels of calcitonin into the medium, but the conretion from the culture cells was not stimulated when the medium calcium concentration was increased from 1.8 to 3.6 mEq/L. Four peaks of calcitonin immunoreactivity were found when the culture medium of one cell line was fractionated by gel filtration on Bio-Gel P-10. This closely corresponded to the heterogeneous molecular profile of calcitonin in the serum of this patient and other patients with medullary carcinoma of the thyroid.     

Diagnostic relevance of the amino-terminal cleavage peptide of procalcitonin (PAS-57), calcitonin and calcitonin gene-related peptide in medullary thyroid carcinoma patients

We have identified the amino-terminal cleavage peptide of procalcitonin (PAS-57) in the plasma of normal human subjects and of medullary thyroid carcinoma (MTC) patients together with calcitonin (CT) and CT gene-related peptide (CGRP). Major components on reversed-phase high-pressure liquid chromatography had the retention times of synthetic PAS-57, CT and CGRP as well as of precursor proteins. Plasma levels of PAS-57 (290 +/- 50 pgeq/ml; mean +/- S.E.M.), CT (27 +/- 8 pgeq/ml) and CGRP (8.4 +/- 0.8 pgeq/ml) were respectively 2.3-, 1.6- and 1.5-fold higher in normal men (n = 10) than in women (n = 8). In response to 1 min intravenous calcium infusions (2 mg per kilogram body weight) PAS-57 and CT were increased 3.5- and 2.7-fold (P less than 0.001), respectively, but CGRP remained unchanged. In MTC patients (n = 57) with raised levels of PAS-57 and CT, the molar ratio between PAS-57 and CT was 1.7-times higher than in normal subjects (P less than 0.01). We have found that PAS-57 is a predominant CT/CGRP gene derived product in the circulation of normal subjects and of MTC patients and a potential new MTC tumor marker.     

Biosynthesis of calcitonin by a rat medullary thyroid carcinoma cell line

The 32-amino acid form of the peptide hormone calcitonin is the product of a series of post-translational processing steps of a 13,400-dalton precursor, procalcitonin. We have now identified the steps involved in proteolytic paring of the precursor to the mature secretory form. Cultures of the CA-77 cell line were radiolabeled and the various forms of calcitonin were isolated by specific immunoprecipitation followed by fractionation on gel filtration and reversed-phase high performance liquid chromatography. Pulse-chase kinetics showed that procalcitonin was cleaved to a 6,500-dalton biosynthetic intermediate which was subsequently processed to the size of mature calcitonin (3,400 daltons). Partial microsequencing of the [35S] methionine-labeled intermediate indicated that the sequence consisted of the COOH-terminal 52 residues of procalcitonin. Partial microsequencing of the [35S]methionine- or [3H]proline-labeled 3,400-dalton species revealed that it was indistinguishable from naturally occurring, amidated calcitonin. These data define the major pathway for calcitonin biosynthesis in this neoplastic cell line and presumably in normal cells.     

Electron microscopic demonstration of calcitonin in human medullary carcinoma of thyroid by the immuno gold staining method

Summary In a human medullary carcinoma of thyroid gland containing calcitonin in light microscopic demonstration by the avidin biotin complex (ABC) method characteristic secretory granules were found electron microscopically in the cytoplasm of the tumour cells. They consisted in so-called type I granules (270±25 nm) and type II granules (135±17 nm). By the immuno gold staining (IGS) method the content of many secretory granules measuring 85–270 nm (152±18 nm) in diameter could be identified as calcitonin. These granules seemed to be predominantly of type II because of their nearly corresponding size and feature. The type I grnaules were less frequent in number and they showed no or little immunoreactivity. The results indicate that the IGS-method is practicable to demonstrate the ultrastructural localization of calcitonin and to identify clearly the nature of intracytoplasmic granules in electron microscopy.     

Electron microscopic demonstration of calcitonin in human medullary carcinoma of thyroid by the immuno gold staining method

In a human medullary carcinoma of thyroid gland containing calcitonin in light microscopic demonstration by the avidin biotin complex (ABC) method characteristic secretory granules were found electron microscopically in the cytoplasm of the tumour cells. They consisted in so-called type I granules (270 +/- 25 nm) and type II granules (135 +/- 17 nm). By the immuno gold staining (IGS) method the content of many secretory granules measuring 85-270 nm (152 +/- 18 nm) in diameter could be identified as calcitonin. These granules seemed to be predominantly of type II because of their nearly corresponding size and feature. The type I granules were less frequent in number and they showed no or little immunoreactivity. The results indicate that the IGS-method is practicable to demonstrate the ultrastructural localization of calcitonin and to identify clearly the nature of intracytoplasmic granules in electron microscopy.     

Somatostatin receptor subtype 1-selective activation reduces cell growth and calcitonin secretion in a human medullary thyroid carcinoma cell line

Medullary thyroid carcinoma (MTC) is a rare and aggressive tumor and so far medical therapy has provided inconclusive results. In the human MTC cell line TT, expressing all somatostatin (SST) receptor subtypes, cell proliferation decreases with SST and SST receptor subtype 2 (sst(2)), but not sst(5), selective agonist treatment, whereas calcitonin (CT) expression and secretion are reduced by SST, but not by sst(2) and sst(5) agonists. The effectiveness of two new SST analogs, BIM-23926 and BIM-23745, selectively interacting with sst(1), was investigated in the TT cell line. DNA synthesis is significantly reduced by BIM-23926 (27-40% at 10(-10)-10(-6)M) and BIM-23745 (32-90% at 10(-8)-10(-6)M). Viable cell number is also significantly reduced by both BIM-23926 (40% at 10(-12)-10(-6)M) and BIM-23745 ( approximately 40% at 10(-10)-10(-6)M). Treatment with sst(1)-selective agonists significantly reduces CT secretion and gene expression, with a reduction of CREB phosphorylation. These findings suggest that potent sst(1)-selective agonists could have a therapeutic role in MTC.     

Bimodal regulation of secretion by cytoplasmic Ca2+ as demonstrated by the parathyroid

Bovine parathyroid cells were used to study parathyroid hormone (PTH) release and the cytoplasmic Ca2+ concentration (Cai2+). When the extracellular Ca2+ concentration was decreased from 3.0 to 0.5 mM, perifused cells reacted with rapid stimulation of PTH release. However, a further reduction of extracellular Ca2+ to less than 10 nM resulted in prompt inhibition. Both effects were readily reversible. Using the intracellular Ca2+ indicator quin-2 also as a buffer for calcium it was possible to control Cai2+ within the 20-600 nM range. PTH release was found to increase with Cai2+ up to 200 nM but was gradually suppressed above this concentration.     

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.     

Isolation of the bombesin/gastrin-releasing peptide receptor from human small cell lung carcinoma NCI-H345 cells.

Purification of the gastrin-releasing peptide (GRP) or bombesin receptor has proved elusive in part due to technical difficulties. In the present studies, the problem of oxidized radioligand was avoided by the use of 125I-GRP, which was verified to be not oxidized by high performance liquid chromatography. Specific 125I-GRP binding (at 0 degrees C) to intact human small cell lung carcinoma NCI-H345 cells which had been subjected to a dilute acid wash was 6 fmol/10(6) cells. Inhibition of GRP degradation by human H345 cell membranes through the use of phenanthroline or phosphoramidon permitted the development of binding assays for the GRP receptor in detergent-solubilized crude membrane preparations. The solubilized GRP receptor exhibited saturable, high affinity (KD = 1.3 nM), temperature-dependent specific binding averaging 402 +/- 65 fmol/mg protein (mean +/- S.E. for eight separate membrane preparations with 125I-GRP concentration = 3 nM), with a Bmax = 434 fmol/mg protein using a gel filtration binding assay. That the GRP receptor had been solubilized was demonstrated by its failure to pellet when centrifuged at 100,000 x g for 60 min, its passage through a 0.22-micron filter without loss of binding activity, and its elution in the void volume of a Sephadex G-50 gel filtration column, but within the inclusion volume of a Sephacryl S-200 column (Ve/V0 = 1.1). Isolation of the GRP receptor from human H345 cell-solubilized membranes was achieved by ligand affinity chromatography. A unique 70-kDa band on silver-stained reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis was reproducibly eluted from GRP14-27 affinity columns by an acidic high salt buffer, but binding activity was denatured by these conditions. The protein nature of the GRP receptor was demonstrated by its sensitivity to proteases after isolation. In addition, two unique bands of 65 and 70 kDa were eluted from the GRP14-27 affinity column with GRP14-27 in neutral buffer, and this eluate possessed specific 125I-GRP binding with a stoichiometry of approximately 1:1. Thus, reported here is the isolation of a functional membrane-associated, saturable, high affinity GRP receptor with temperature-dependent binding from the solubilized membranes of human H345 cells.     

Direct cytoplasmic CA(2+) responses to gastrin-releasing peptide in single beta cells

The neuropeptide gastrin releasing peptide (GRP) stimulates insulin secretion and induces oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) in clonal insulinoma cells. It is not known whether GRP affects [Ca(2+)](cyt) in normal beta cells. We investigated, in single, normal, mouse islet beta cells, the effects of GRP on [Ca(2+)](cyt), by dual wavelength spectrophotofluorometry. Beta cells were identified by their typical response to glucose or tolbutamide. At 15 mM glucose, GRP (100 nM) evoked an immediate [Ca(2+)](cyt) transient to 423 +/- 48 nM compared to 126 +/- 18 nM before GRP (P < 0.001). After the initial transient, [Ca(2+)](cyt) exhibited either a sustained elevation or oscillations. At 3.3 mM glucose, in cells with a non-oscillating [Ca(2+)](cyt), GRP stimulated a prompt increase in [Ca(2+)](cyt) (from 60 +/- 6 to 285 +/- 30 nM, P = 0.024) followed by either a sustained increase in [Ca(2+)](cyt) or [Ca(2+)](cyt) oscillations. We conclude that GRP promptly elevates [Ca(2+)](cyt) by a direct action in normal mouse pancreatic beta cells.     

In vitro stimulation of human red blood cell Ca2+-ATPase by thyroid hormone

Ca²⁺-ATPase activity in human erythrocyte ghosts previously washed to remove endogenous thyroid hormone is stimulated $\text{in}\text{vitro}$ by physiologic concentrations of thyroxine (T₄) and triiodothyronine (T₃). Two- to three-fold increases (P <0.005) in Ca²⁺-ATPase activity occurred after 60–120 minutes' exposure of membranes to iodothyronines at concentrations of T₄ and T₃ of 10^?8 M to 10^?12 M. T₄ was more active than T₃ and its activity did not depend upon prior conversion to T₃. The Ca²⁺-ATPase effect represents an extranuclear action of thyroid hormone in a human cell model.