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.     

Establishment of a new human thyroid medullary carcinoma cell line. Morphological studies

A new human medullary carcinoma cell line has been established from a thyroid tumor removed from a 76-year-old female patient. The cultured cells grew in suspension, formed round islands and did not attach to the plastic dish. The doubling time of 48 h is the shortest recorded for C-cell lines. Ultrastructural studies disclosed that the cells had a few short profiles of rough endoplasmic reticulum, numerous ribosomes and polyribosomes, a poorly developed Golgi apparatus and small secretory granules (75 nm in mean diameter). Immunohistochemical staining for somatostatin was positive. These results show that, compared with previously established C-cell lines, this cell line has a rapid growth rate, is morphologically less differentiated, but retains a hormone production potential.     

Identification of procalcitonin in a rat medullary thyroid carcinoma cell line

As a first step in studying the biosynthesis of the peptide hormone calcitonin, we have identified procalcitonin species in CA-77 cells, a newly developed rat medullary thyroid carcinoma cell line. mRNA extracted from the cells directed the synthesis of a putative procalcitonin in a reticulocyte lysate translation system containing microsomal membranes. Both this species and a radiolabeled form of immunoreactive calcitonin from intact cells had the same retention time during reverse phase high performance liquid chromatography. The putative cellular procalcitonin was also immunoprecipitated by antiserum to a synthetic peptide whose sequence constitutes the COOH-terminal 16 residues of preprocalcitonin. The polypeptide had a Mr = 13,400, as estimated by gel filtration chromatography under denaturing conditions. Microsequencing of the [35S]methionine-labeled polypeptide indicated that residues 13, 32, and 34 of procalcitonin were methionine. Similar analysis of the peptide labeled with [3H]proline indicated that residues 2 and 11 of the precursor were proline. The positions of methionine and proline could be aligned in a unique manner with the NH2-terminal half of the preprocalcitonin sequence inferred from cDNA analyses. These results indicate that procalcitonin consists of 111 amino acids and suggest that a 25-residue signal sequence is cotranslationally cleaved from preprocalcitonin. From the procalcitonin sequence we can now predict the sequence of likely biosynthetic intermediates and mature secretory products derived from the NH2-terminal as well as COOH-terminal regions of the precursor.     

Somatostatin 1-28 circulates in human plasma

The gel filtration profile of immunoreactive somatostatin in human plasma in the fasting state is not well established as a consequence of insufficient sensitivity of the combined chromatography and radioimmunoassay procedures usually employed. We here report the gel filtration profiles of plasma samples after somatostatin concentration by batchwise immunoaffinity chromatography. The results clearly and reliably document the presence of a circulating peptide in human plasma with a gel permeation chromatography profile identical to the one of synthetic somatostatin 1-28. Approximately 46% of the total somatostatin-like immunoreactivity in plasma is due to this component.     

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.     

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with K_d of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with K_d 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with K_d 0.42 was identified as rat -CGRP as was the peak with K_d 0.53. The peak with K_d 0.68 was identified as 19–37 rat -CGRP and the peak with K_d 0.85 as 28–37 rat -CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated -CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat β-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.     

Characterization of a human follicular thyroid carcinoma cell line (UCLA RO 82 W-1)

A thyroid tumor cell line has been established from the metastases of a follicular carcinoma in a female patient. Although the primary tumor released thyroglobulin (Tg) into the circulation (greater than 10,000 ng/ml), the uptake of I131 was less than 2%. After 37 replications the doubling time was 4 days and confluency was reached after 7 days from inoculation of 3 x 10(7) cells. This human thyroid tumor cell line has now been growing in culture for several years. An aneuploid chromosomal pattern was observed (62-82 chromosomes). A pair of X chromosomes was present but no Y chromosome was found which is compatible with the female origin of the cell line. EM studies revealed the presence of microvilli. Immunoperoxidase staining using specific anti-human Tg antisera indicated the presence of Tg within the cells. Nude mice developed solid-cystic tumors within 6 months after injection of the cells. The basal release of immunodetectable Tg, as measured in a perifusion system, increased in response to thyroid stimulating hormone (TSH) (P less than 0.025) or TSH combined with theophylline (P less than 0.001). Unusual isoenzyme patterns for galactose-1-phosphate-uridyltransferase (GALT) and phosphoglucomutase1 (PGM1) were detected in the tumor, compared with normal human fibroblasts and blood cells and isoenzyme patterns from the patient's lymphocytes. Because this malignant human thyroid follicular cell line has retained the ability to synthesize Tg it represents a valuable model for the study of human follicular carcinomas.     

Biosynthesis of thyrotropin-releasing hormone by a rat medullary thyroid carcinoma cell line

Prepro-thyrotropin-releasing hormone (TRH) messenger RNA was detected in the rat medullary thyroid carcinoma cell line CA77. The RNA of 1.6 kilobases comigrated with that found in rat hypothalamus. Using three radioimmunoassays specific for pro-TRH-derived peptides, we demonstrated that CA77 cells synthesize high levels of immunoreactive TRH and all of the other pro-TRH-derived peptides identified in hypothalamic tissue. The relative levels of the pro-TRH-derived peptides also indicate that CA77 cells process the TRH precursor in a manner similar to hypothalamic tissue. CA77 cells provide a promising model system for further studies of prepro-TRH gene regulation and post-translational maturation.     

Expression and processing of procholecystokinin in a rat medullary thyroid carcinoma cell line.

A rat medullary thyroid carcinoma cell line, CA-77, was shown to express the cholecystokinin (CCK) gene. Measurements using a library of sequence-specific radioimmunoassays before and after enzymic treatment of extracts and chromatographic fractions showed that the cells contained 1.0 pmol of alpha-carboxyamidated cholecystokinins/10(6) cells, 0.4 pmol of glycine-extended intermediates/10(6) cells and 1.0 pmol of further C-terminal-extended pro-CCK/10(6) cells. Gel chromatography and reverse-phase h.p.l.c. revealed both sulphated and nonsulphated CCK-8 in the cells. The growth medium contained in addition alpha-amidated CCK-33, glycine-extended CCK-8 and pro-CCK. Exposure to 0.1 microM-dexamethasone for 6 days increased the cellular content and secretion of all of the described CCK peptides by 2-3-fold. The increase was first noted after 3 days of treatment. Monensin inhibited the synthesis of alpha-carboxyamidated CCK and the secretion of all of the CCK forms measured. Colchicine at a low concentration (0.2 mumol/l) apparently increased the synthesis and secretion of alpha-carboxyamidated CCK, whereas higher concentrations inhibited CCK synthesis. Finally, chloroquine inhibited the alpha-carboxyamidation of CCK. We conclude that the CA-77 cell line is a useful tool for studies of the expression and post-translational processing of pro-CCK.     

15-Hydroxyprostaglandin-dehydrogenase is involved in anti-proliferative effect of non-steroidal anti-inflammatory drugs COX-1 inhibitors on a human medullary thyroid carcinoma cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin (PG) synthesis enzymes, the cyclooxygenases (COX-1 and 2). It is suggested that these enzymes are not their only targets. We reported that in tumoral TT cell, indomethacin, in vivo and in vitro, decreases proliferation and increases activity of 15-hydroxyprostaglandin-dehydrogenase (15-PGDH), the PG catabolism key enzyme. Here, we show that the COX-1 inhibitors, selective or not, and sulindac sulfone, a non-COX inhibitor, increased 15-PGDH activity and reduced PGE2 levels. This increase was negatively correlated to the decrease in cell proliferation and suggested that 15-PGDH could be implicated in NSAIDs anti-proliferative effect. Indeed, the silencing of 15-PGDH expression by RNA interference using 15-PGDH specific siRNA enhanced TT cell proliferation and abolished the anti-proliferative effect of a representative non-selective inhibitor, ibuprofen. Moreover, a specific inhibitor of 15-PGDH activity, CAY 10397, completely reversed the effect of ibuprofen on proliferation. Consequently our results demonstrate that, at least in TT cells, 15-PGDH is implicated in proliferation and could be a target for COX-1 inhibitors specific or not. NSAIDs defined by their COX inhibition should also be defined by their effect on 15-PGDH.     

Biosynthesis of an asparagine-linked oligosaccharide-containing calcitonin by a rat medullary thyroid carcinoma cell line

Calcitonin contains an amino acid sequence that provides a potential site for glycosylation of the peptide at the asparagine at position 3. Preliminary evidence has suggested that there are glycosylated forms of calcitonin and its precursor, procalcitonin. The CA-77 rat medullary thyroid carcinoma cell line, recently developed to study calcitonin biosynthesis, was used to demonstrate the synthesis of glycosylated forms of this hormone by intact cells. Cultures were incubated in medium containing either [3H]mannose or [35S]methionine. Two species incorporating both labels were specifically immunoprecipitated when cell extracts were treated with calcitonin antibodies. Gel filtration chromatography in 6 M guanidine hydrochloride indicated that one peptide had a molecular weight of 5500, approximately 2000 daltons larger than calcitonin, while the second peptide had a molecular weight of 14 400, the approximate size of procalcitonin. Treatment of the [3H]mannose-labeled cell extract with endo-beta-N-acetylglucosaminidase H before immunoprecipitation removed the labeled sugar from the calcitonin species. Microsequence analysis of the radiolabeled immunoreactive 5500-dalton calcitonin species showed methionine at cycle 8 and mannose at cycle 3, suggesting that this peptide is calcitonin containing an N-linked oligosaccharide at Asn-3. These results suggest that in this cell line a minor but significant biosynthetic pathway exists for the production of glycosylated calcitonin from glycosylated procalcitonin.     

Establishment and characterization of a human thyroid carcinoma cell line (HOTHC) producing colony stimulating factor

A cell line designated HOTHC was established from an anaplastic carcinoma (giant cell type) of the thyroid gland of 80-year-old woman. The HOTHC line grew rapidly in multilayer without contact inhibition, and more than 120 serial passages were made within 27 months. The cells were spindle or polygonal in shape and revealed neoplastic and pleomorphic features. These cells were characterized as containing coloid droplets and poorly developed rough-endoplasmic reticulum in the cytoplasm. Doubling time was about 24 hours and plating efficiency was about 70%. The karyotype exhibits hyperploidy and marker chromosomes, and the modal chromosome number ranged between 77-90. The HOTHC cells were transplanted into the subcutis of BALB/c nude mice and produced anaplatic carcinomas (giant cell type) resembling the original tumor. The HOTHC cells produced colony stimulating factor (CSF) and caused granulocytosis in the mice.     

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6)

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.     

Establishment and characterization of a human papillary thyroid carcinoma cell line with oxyphilic differentiation (ONCO-DG 1).

In the present study the establishment and characterization of a new oxyphilic papillary thyroid carcinoma cell line--ONCO-DG1- is given. With immunohistological, histochemical and flow cytometric methods, ONCO-DG 1 cells revealed features of epithelial differentiation. Furthermore the cells formed von Kossa-positive deposits resembling psammoma bodies in monolayer and spheroid culture until late passages. The tumor cell line is now in the 40th subculture. Because of the ability to form multicellular tumor spheroids (MCTS), this cell line is a good model for examining the interaction between thyroid tumor cells and confluent human endothelial cells on extracellular matrix in vitro. It is also suitable for xenotransplantation studies, because it is tumorigenic in NMRI nude mice in vivo.