calcitonin and Carcinoma

Stable transfectants expressing a recombinant human calcitonin receptor respond to calcitonin via increased cyclic adenosine 3',5' monophosphate (cAMP, EC50 = 0.06 nM salmon calcitonin [sCT]) and a transient mobilization of intracellular calcium ([Ca2+]i) coincident with turnover of inositol phosphate (IP; EC50 = 6 nM sCT). Millimolar increases in extracellular calcium ([Ca2+]o, EC50 = 8 mM) cause a rapid elevation in [Ca2+]i after a calcitonin dose-dependent pretreatment of cells (pretreatment EC50 = 0.2 nM sCT). Cells exhibit persistent sensitivity to increased [Ca2+]o up to 3 h after hormone exposure and even after multiple cycles of increased [Ca2+]o followed by wash. Calcitonin pretreatment of cells also allows apparent influx of elevated extracellular strontium and manganese, but little or no effect is observed on addition of barium, cadmium, or lanthanum. Human amylin (100 nM) causes a rapid and transient increase in [Ca2+]i comparable to that of calcitonin; however, no significant response to increased [Ca2+]o is observed after amylin addition. Human calcitonin gene-related product (hCGRP) (300 nM) and forskolin do not increase [Ca2+]i or activate a sensitivity to increased [Ca2+]o. Nevertheless, human amylin and human calcitonin gene-related product (hCGRP) activate adenylate cyclase with EC50s of 0.7 nM and 8 nM, respectively. The calcium-channel drugs verapamil, BAY K 8644, diltiazem, and nifedipine have little effect on [Ca2+]i increases. The calcitonin-induced transient mobilization of calcium is inhibited by treatment of cells with cholera toxin or 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8); whereas, the response to subsequent increased [Ca2+]o is inhibited by lanthanum chloride (200 microM) and lower pH (6.0).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Amyloid; Animals; Breast Neoplasms; Calcitonin; Calcitonin Gene-Related Peptide; Calcium; Calcium Channel Blockers; Carcinoma; Cations, Divalent; Cell Line; Cholera Toxin; Colforsin; Cyclic AMP; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Inositol Phosphates; Islet Amyloid Polypeptide; Receptors, Calcitonin; Recombinant Proteins; Transfection; Tumor Cells, Cultured

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 nM). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Amyloid; Animals; Base Sequence; Binding, Competitive; Breast Neoplasms; Calcitonin; Calcitonin Gene-Related Peptide; Carcinoma; Cyclic AMP; DNA, Complementary; DNA, Neoplasm; Enzyme Activation; Female; Gene Expression Regulation; Genes, Reporter; Humans; Islet Amyloid Polypeptide; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Neoplasm Proteins; Protein Conformation; Receptors, Calcitonin; Second Messenger Systems; Tumor Cells, Cultured

We report the development and validation of three microbioassays for calcitonin based on calcitonin-induced inhibition of the activity of isolated osteoclasts. Having precisely quantified osteoclast motility, spreading and bone resorptive activity, we have applied stringent analytical procedures to define assay characteristics. We have found that the appropriately transformed responses significantly regress on log dose of the peptides. Furthermore, potency estimates obtained using calcitonins from three species (human, salmon and a synthetic analogue of eel calcitonin) have been found to be consistent with those obtained using conventional calcitonin bioassays. In addition, the assays are remarkably sensitive (detection limit 10(-15) M), highly specific and precise. We have determined plasma levels of bioactive calcitonin on samples from patients with medullary thyroid carcinoma; these are several-fold lower than those obtained using our routine calcitonin radioimmunoassay. Our study thus, forms the basis of an entirely new approach for the determination of 'biologically active' calcitonin, and we envisage that such target cell-specific assays could become useful microanalytical methods.

Topics: Animals; Animals, Newborn; Biological Assay; Bone Resorption; Calcitonin; Carcinoma; Cell Movement; Eels; Humans; Osteoclasts; Radioimmunoassay; Rats; Rats, Inbred Strains; Thyroid Neoplasms

Calcitonin-like peptides have been identified in the serum of normal subjects and of medullary thyroid carcinoma (MTC) patients. Using specific homologous radioimmunoassays (RIA) in combination with reversed-phase high performance liquid chromatography and gel permeation chromatography under denaturing conditions, we have recognized major components which coeluted with human calcitonin-(1-32), PDN-21, a carboxyl-terminal flanking peptide derived from the calcitonin mRNA sequence, and salmon calcitonin-(1-32). An additional 12000 molecular weight peak possibly represents a human calcitonin-PDN-21 polyprotein. In both the human calcitonin-(1-32) (normal value less than 0.043 ngEq/ml; MTC 140 +/- 80 ngEq/ml, mean value +/- SEM) and the PDN-21 (normal value less than 0.050 ngEq/ml; MTC 33.6 +/- 16.5 ngEq/ml) RIAs, serum levels were increased in MTC patients. Circulating levels of the salmon calcitonin-like peptide were indistinguishable between normal subjects (0.038 +/- 0.006 ngEq/ml) and MTC patients (0.037 +/- 0.011 ngEq/ml).

Topics: Calcitonin; Carcinoma; Chromatography, Gel; Chromatography, High Pressure Liquid; Humans; Peptides; Polydeoxyribonucleotides; Radioimmunoassay; Thyroid Neoplasms

A salmon calcitonin-like material indistinguishable from synthetic salmon calcitonin-(1-32) on high performance liquid chromatography (HPLC) has been recognized in thyroid extracts of normal subjects and of patients with medullary carcinoma. The same peptide was detected in extracts of the periventricular mesencephalic region which included the periventricular dorsal thalamus, the subthalamus and the hypothalamus. Human calcitonin-(1-32)- and carboxyl-terminal adjacent peptide (CCAP)-like components were also found. The content of immunoreactive salmon calcitonin of the periventricular mesencephalic region (n = 6) and of normal thyroid glands (n = 6) was comparable (mean +/- SE, 0.34 +/- 0.17 ngeq/g wet tissue and 0.39 +/- 0.22 ngeq/g, respectively); and the levels were slightly, but not significantly higher in medullary thyroid carcinoma extracts (1.95 +/- 0.69 ngeq/g) (P less than 0.1). Immunoreactive human calcitonin and CCAP occurred in roughly equimolar concentrations. They were lowest in the periventricular mesencephalic region (0.26 +/- 0.09 ngeq/g and 0.46 +/- 0.10 ngeq/g, respectively), followed by normal thyroid glands (146 +/- 26 ngeq/g and 94 +/- 19 ngeq/g, respectively), and they were highest in medullary thyroid carcinoma tissue (680 +/- 372 mu geq/g and 144 +/- 125 mu geq/g, respectively).

Topics: Brain; Calcitonin; Carcinoma; Diencephalon; Humans; Mesencephalon; Peptide Fragments; Thyroid Gland; Thyroid Neoplasms