Human Medullary Thyroid Carcinoma Research Articles

Production of CGRP in three continuous cell lines from human medullary thyroid carcinomas

Production of CGRP in three continuous cell lines from human medullary thyroid carcinomas

Discordant secretion of calcitonin and chromogranin in the human medullary thyroid carcinoma cell line.

A cell fine of human medullary carcinoma of the thyroid, abundantly producing calcitonin (Ct) and related hormones, has proved remarkably useful as an endocrine tumor model for the study of the secretion mechanism. This cell line (TT cell) was used in studies to elucidate the dynamics of the release of Ct and chromogranin (Cg) to culture medium. The studies evaluated the intracellular concentration of Ct and Cg and the concentration changes elicited by the protein kinase C activator, phorbol ester (TPA); the adenylate cyclase-associated protein kinase A activator, forskolin; and the calcium ionophore, A23187. In addition, immunogold labeling of Ct and Cg was carried out to investigate the ultrastructural changes resulting from the stimulations. All these secretagogues effected the release of Ct and Cg into the medium in a dose-dependent manner, and the rate of the increase in the Ct secretion was consistently and markedly higher than that of Cg in more than certain dosages of the secretagogues. Most cells contained secretory granules immunolabeled for both Ct and Cg, and a considerable decrease was noted in the poststimulation count of the granules containing both substances, with the cells retaining more Cg than Ct. The discordance may be explained by different secretory pathways of the two proteins or different rates of synthesis.

PCR amplification of CGRP II mRNA: Variable expression in tumoral and non-tumoral human thyroid

Two genes code for calcitonin gene-related peptides (CGRPs). One expresses by tissue-specific alternate splicing calcitonin and CGRP I mRNAs, the other CGRP II mRNA. Calcitonin is the marker of sporadic or hereditary human medullary thyroid carcinoma (MTC). CGRP II expression is not well established in normal or tumoral thyroid. After amplification by polymerase chain reaction, CGRP I and II mRNAs were detected in six cases of MTC associated with other endocrine neoplasia (MEN IIa) and in two cases of isolated MTC. CGRP I was detected in all non-C cell tumoral thyroids (6 samples), CGRP II was barely detectable in three out of six cases. CGRP II could be a specific tumoral marker of MTC.

Verapamil and cyclosporin A potentiate the effects of chemotherapeutic drugs in the human medullary thyroid carcinoma TT cell line not expressing the 170 kDa P-glycoprotein

The TT-cell line, derived from a patient with metastatic medullary thyroid carcinoma (MTC), was found to exhibit intrinsic resistance to vincristine (VCR) despite the absence of immunohistochemically detectable 170 kDa P-glycoprotein (PGP 170) associated with multidrug resistance (MDR). Verapamil and cyclosporin A, two well known resistance modifiers of MDR, were found to significantly potentiate the action of VCR (60-fold) and to a lesser degree also of VP-16 and daunorubicin (dnr). The present results suggests that resistance of MTC to chemotherapy may be at least partly circumvented by the addition resistance modifiers to chemotherapeutic regimens.

Immunoreactive Gastrin-Releasing Peptide in Medullary Thyroid Carcinoma

We have previously shown that immunoassayable concentration of somatostatin (SRIH) was elevated in 70% of 34 consecutive medullary thyroid carcinoma (MTC) tissue samples. In the present study gastrin releasing peptide (GRP)-like immunoreactivity was measured in tissue extracts from these 34 MTC (25 inherited, 7 sporadic, 2 unclassified) and in 7 normal thyroid tissue. Plasma SRIH, calcitonin (CT) and carcinoembryonic antigen were assayed in all patients. Normal thyroid tissue contained less than 61 pmol GRP per g wet weight; in contrast GRP concentration was elevated (62-7800 pmol/g) in 32/34 tumor extracts. The distribution of tissue GRP values were similar in sporadic as well as in familial MTC. We found no significant correlation between tumor GRP concentration and plasma SRIH (r = -0.05), plasma CT (r = -0.24), or plasma carcinoembryonic antigen levels (r = -0.21). Tumor concentrations of immunoreactive GRP and SRIH were positively correlated when logarithmic transformation was used (P less than 0.01). Thus GRP, as well as SRIH, is a major product of tumoral C cells in human MTC when systematically evaluated in a large number of cases.

Secretion of GAWK, a Presumed Product of Chromogranin B Processing, by a Human Medullary Thyroid Carcinoma Cell Line: Effect of Phorbol Ester, Forskolin and Calcium‐lonophore

Abstract GAWK was isolated from human pituitary glands and presumed to be a processing product of human Chromogranin B. In the present studies, we investigated effects of phorbol ester (TPA), forskolin and Ca(2+)-ionophore (A23187) on GAWK secretion using a human medullary thyroid carcinoma-derived cell line (TT-cell). A dose-responsive stimulation of GAWK-like immunoreactivity (LI) secretion was found in the presence of TPA as well as forskolin. A23187 caused an increase of GAWK-LI secretion and addition of TPA together with A23187 revealed an additive effect on the GAWK-LI secretion. These findings indicate that GAWK-LI is a secretory peptide in the TT-cells and suggest that A and C kinase, and possibly Ca(2+)-calmodulin transduction systems are involved in the GAWK-LI secretion by the TT-cells. TT-cells may provide a good model for the investigation of regulation of Chromogranin secretion.

Quantification of intracellular calcitonin gene transcripts in human medullary thyroid carcinoma (MTC) by in situ hybridization

The human calcitonin gene generates 2 distinct mature mRNAs by alternative RNA processing, encoding calcitonin (CT) in thyroid C-cells or a neuropeptide (CGRP) in the brain. We evaluated quantitatively by in situ hybridization the expression of the CT gene in tissue section of 5 MTCs (2 sporadic and 3 familial forms). The primary tumor of one MTC was compared to a brain metastasis. In situ hybridization was carried out with tritiated cDNA probes coding for CT and CGRP mRNA. After autoradiography the number of silver grains was counted in 400 cells by computerized analysis of digitized images and expressed as the labelling level (L.L. = grain area/cell area per day of autoradiographic exposure). This was used to calculate the relative abundance per cell of the specific messengers studied, which depends on the autoradiographic efficiency and the specific activity of the probe used. The CT mRNA content was 3.25-6.55 10(-10) micrograms equivalents in the 3 familial forms of MTC and 4.95-9.25 10(-10) micrograms equivalents for the 2 sporadic forms. The levels of CT mRNA in the brain metastasis and in the primary tumor were identical (4.10 10(-10) micrograms equivalents). CGRP mRNA expression was weaker in the sporadic and in the familial thyroid tumors (0.60-1.65 10(-10) micrograms equivalents). The content of mRNA CGRP in the brain metastasis (0.60 10(-10) micrograms equivalents) was lower than that in the primary tumor (1.05 10(-10) micrograms equivalents).(ABSTRACT TRUNCATED AT 250 WORDS)

Transcription factor levels in medullary thyroid carcinoma cells differentiated by Harvey ras oncogene: C-jun in increased

In the TT cell line of human medullary thyroid carcinoma, the viral Harvey ras (v-rasH) oncogene induces differentiation, marked by morphological changes, diminution of growth, and increased expression of the calcitonin gene. Here, we show that the transcriptional factor c-jun is increased during v-rasH induced differentiation of TT cells both at the mRNA and functional protein levels. In contrast, nuclear proteins with binding activities related to AP2, AP3, NF1/CTF, and Sp1 were unchanged in v-rasH differentiated TT cells.

Histochemical detection of the messenger RNAs coding for calcitonin and calcitonin gene‐related peptide in medullary thyroid carcinomas with radioactive and biotinylated oligonucleotide probes

The present study has been undertaken to investigate the efficiency of biotinylated synthetic oligonucleotide probes in detection by in situ hybridization of the mRNAs coding for calcitonin (CT) or calcitonin gene-related peptide (CGRP) in human medullary thyroid carcinomas (MTCs). Tissue sections fixed with formaldehyde were hybridized with 45-base long oligonucleotides, specific for CT or CGRP mRNA. Recombinant DNA probe or synthetic oligonucleotides radioactively labelled with 32P or 35S were used as controls to detect by autoradiography the corresponding mRNAs in the tumour cells. Oligonucleotide probes labelled by fixation of one biotin molecule at their 5'-end, or by incorporation of a tail of biotin-11-dUTP at their 3'-end, were used and were revealed by incubation with streptavidin-alkaline phosphatase associated with the corresponding substrate. Each biotinylated probe stained exclusively the cytoplasm of the tumour cells, the CT probe giving a much higher level of staining than the CGRP probe. The same cells were found to contain CT and CGRP mRNAs. Controls performed with either radioactive or biotinylated probes confirmed the specificity of the staining. These results demonstrate that biotinylated synthetic oligonucleotides can be used as efficient tools to investigate gene expression in tissue sections, thus avoiding the various inconveniences connected with the use of radioactive probes, especially bio-hazards, the use of autoradiography, the limited histological resolution, and the delay in obtaining results.

Non-sulphated cholecystokinin in human medullary thyroid carcinomas

The expression of gastrin/cholecystokinin (CCK) peptides and their precursors was examined in 16 medullary carcinomas of the human thyroid. Measurements with libraries of sequence-specific radioimmunoassays before and after enzymatic cleavage of extracts and chromatographic fractions showed that the carcinomas contained 1.7 pmol carboxyamidated CCK/g tissue (median; range 0.6-21.8 pmol/g), 0.9 pmol glycine-extended precursor/g (median; range less than 0.2-2.3 pmol/g) and 2.3 pmol further COOH-terminal-extended proCCK/g (median; range 0.9-6.2 pmol/g). Neither carboxyamidated gastrins nor any progastrins could be measured. Gel and reverse-phase chromatography revealed only small molecular forms, i.e. greater than 90% of the amidated immunoreactivity eluted like non-sulphated CCK-8 or CCK-7. The results show that human medullary thyroid carcinomas synthesize CCK peptides. The predominance of non-sulphated CCK is unusual. Taken together with earlier observations from dogs and pigs, our results raise the possibility that small non-sulphated CCK peptides modulate thyroid C-cell secretion in an autocrine manner.

The intratumoral immunoassayable somatostatin concentration is frequently elevated in medullary thyroid carcinoma results in 34 cases

The current work has been performed by the Cooperative French Group of Medullary Thyroid Carcinoma (GETC). A systematic evaluation of RIA somatostatin (SRIH) was performed in 34 medullary thyroid carcinomas (MTC) (25 inherited, seven sporadic). Plasma SRIH was measured by radioimmunoassay in parallel with calcitonin (CT) and carcinoembryonic antigen (CEA). Immunoassayable SRIH was tested in fresh tumoral tissue samples from the same 34 MTC and, for comparison, in 10 nontumoral thyroid extracts (less than 6 pmol/g wet). Although plasma SRIH was only slightly elevated in two of 20 cases, tumoral SRIH was elevated in 70.6% of our MTC (10 to 3973 pmol/g). The chromatography of two tumoral extracts showed that somatostatin 14 was the major molecular form. We found no correlation (P greater than 0.1) between tumoral SRIH and the following: (1) tumor size (r = 0.227); (2) epidemiologic form of MTC (r = 0.144); (3) plasma SRIH (r = 0.045), plasma CT (R = 0.095) or (4) plasma CEA (r = 0.032). Thus, in the authors' experience, SRIH appears as a major product of tumoral C-cell in human MTC, even when plasma SRIH is normal and SRIH immunohistochemical staining is scarce. Multiple hormonal production of these tumors may explain its presence but SRIH may act also as a regulator, since negative influence of SRIH on CT is demonstrated in normal as well in tumoral conditions.

Role of voltage-dependent calcium channels in secretion of calcitonin from human medullary thyroid carcinoma cells

Extracellular calcium concentration is an important regulator of calcitonin secretion. We used primary cell cultures of human medullary thyroid carcinoma to study the role of voltage dependent calcium channels for stimulus secretion coupling. Increasing extracellular calcium concentration (1.6-5.0 mM) in the medium caused a dose dependent release of calcitonin. The calcium channel activator BAY K 8644, a dihydropyridine, stimulated calcitonin secretion in a dose dependent manner (10(-7)-10(-5) M). This effect was completely inhibited by equimolar concentrations of the calcium channel blocker nifedipine and abolished in the absence of extracellular calcium. Similarly, nifedipine suppressed the stimulatory action of extracellular calcium. The effects of calcium and BAY K 8644 with and without nifedipine suggest that calcium influx via voltage dependent calcium channels plays an important role in calcitonin secretion. The primary cell culture of human medullary thyroid carcinoma is a good model for the study of stimulus secretion coupling.

Human medullary thyroid carcinoma: characterization of the serotonergic and neuronal properties of a neurectodermally derived cell line

Parafollicular (PF) cells have been found to be a good model system for the study of serotonergic cellular mechanisms relevant to neurons. PF cells are derived from the same region of the neural crest that gives rise to the neurons of the gut and are capable of extending neurofilament-bearing neuritic processes. PF cells also synthesize 5-hydroxytryptamine (5-HT) and costore 5-HT in the same vesicles as the specific 5-HT-binding protein, 45 kDa SBP. A hypothesis has been advanced that PF cells and enteric neurons share a common developmental precursor. The present investigation was undertaken in order to determine whether a human medullary thyroid carcinoma (MTC) cell line, which is derived from PF cells, sufficiently mimics PF cells that it can be substituted for them in investigations of serotonergic cellular biology. In contrast to PF cells, MTC cells can be propagated in vitro to provide adequate amounts of material for biochemical studies. MTC cells were found to contain neuropeptides, including calcitonin, calcitonin gene-related peptide, and somatostatin, which have also been reported to be present in PF cells and enteric neurons. MTC cells also were observed to store endogenous 5-HT, to be able to synthesize 3H-5-HT from 3H-L-tryptophan, and to take up 3H-5-HT from the ambient medium by a carrier-mediated mechanism very similar to that of serotonergic neurons. In addition, the longterm accumulation of 3H-5-HT in MTC cells was antagonized by reserpine, suggesting that the cells contain 5-HT storage vesicles that, like the synaptic vesicles of serotonergic neurons, are characterized by a reserpine-sensitive transporter of biogenic amines. MTC cells also contain type A, but not type B, monoamine oxidase. Finally, MTC cells were found to contain both 45 and 56 kDa SBP. MTC cells thus retain a great many of the properties of PF cells, and, like PF cells, they are serotonergic cells with characteristics similar to serotonergic neurons. Substantial differences were found in the content of immunoreactive 5-HT and neuropeptides in individual MTC cells. Moreover, the release of newly synthesized 5-HT to the medium exceeded the ability of the cells to store the amine. Studies of the ultrastructure of the MTC cells revealed a limited and highly variable number of secretory granules, probably accounting for their limited 5-HT storage capacity and for the heterogeneity of immunostaining with antisera to 5-HT or neuropeptides.(ABSTRACT TRUNCATED AT 400 WORDS)

101 Chromosomes in cell-lines of a human medullary thyroid carcinoma

101 Chromosomes in cell-lines of a human medullary thyroid carcinoma

Human Medullary Thyroid Carcinoma: Initial Characterization and In Vitro Differentiation of Two New Cell Lines

Medullary carcinoma of the thyroid (MCT), a tumor of calcitonin (CT)-secreting C cells, can display a variable malignant potential with poor prognosis linked to decreased cell differentiation. In vitro study of MCT has been hampered by the fact that few cell lines derived from this neoplasm have been available for study. Herein are reported the characteristics of two new lines derived from human MCT that are tumorigenic in athymic mice and do not secrete CT. After treatment with various concentrations of retinoic acid (a vitamin A derivative) and cyclic adenosine monophosphate, both lines exhibit the traits of more differentiated cells with a decrease in cellular proliferation and an increase in cytoplasmic CT content as shown by in situ immunoperoxidase staining. These cell lines should prove of great value in the study of the biology of MCT and the mechanisms underlying induced differentiation in this type of tumor.

Abnormal expression of the MOS proto-oncogene in human thyroid medullary carcinoma

We have been studying the expression of a range of proto-oncogenes in human thyroid tumour tissue by using Northern blot analysis. We have demonstrated the expression of a MOS mRNA of 1 kb in all thyroid samples. Furthermore, in a medullary carcinoma sample we also observed additional mRNA species of 1.7 and 2.2 kb. Southern blot analysis of DNA prepared from the same tumour sample did not reveal a rearrangement of the gene. These findings are the first report of MOS expression in any human tissue, and indicate that MOS oncogene activation might be important in the development of some thyroid tumours.

Structural characterization of a high-molecular-mass form of calcitonin [procalcitonin-(60-116)-peptide] and its corresponding N-terminal flanking peptide [procalcitonin-(1-57)-peptide] in a human medullary thyroid carcinoma.

Four peptides derived from procalcitonin were isolated in high yield from an extract of a human medullary thyroid carcinoma. The peptides were identified as procalcitonin-(1-57)-peptide, procalcitonin-(60-91)-peptide (calcitonin), procalcitonin-(60-116)-peptide and procalcitonin-(96-116)-peptide (katacalcin). Determination of the amino acid sequence of procalcitonin-(1-57)-peptide has demonstrated that the Ala25-Ala26 bond in preprocalcitonin is the site of cleavage of the signal peptide. Procalcitonin-(60-116)-peptide represents calcitonin extended from its C-terminus by the sequence Gly-Lys-Lys-Arg-katacalcin, and its formation is indicative of an aberrant pathway of procalcitonin processing in the tumour cells.

Calcitonin gene-related peptide and calcitonin secretion from a human medullary thyroid carcinoma cell line: effects of ionomycin, phorbol ester and forskolin.

Calcitonin gene-related peptide (CGRP) and calcitonin are secreted together from medullary thyroid carcinoma (MTC) cells. Interactions of cytosolic free calcium concentration (Cai2+) and the protein kinase C and A pathways on the secretion of immunoreactive CGRP and calcitonin have been investigated in a human MTC cell line. Ionomycin (10 mumol/l) raised the concentration of Cai2+, concomitant with a transient stimulation of the secretion of CGRP and calcitonin. 12-O-tetradecanoylphorbol-13-acetate (TPA; 16 nmol/l) did not affect the concentration of Cai2+, but caused a gradual rise of the secretion of CGRP and calcitonin. Combined addition of 10 mumol ionomycin/l and 16 nmol TPA/l resulted in additive stimulation of CGRP and calcitonin secretory responses. Forskolin (10 mumol/l) alone did not change the concentration of Cai2+, marginally enhanced (P greater than 0.1) the release of CGRP and calcitonin and increased by 23-fold the cellular levels of cyclic AMP (cAMP). Ionomycin and TPA did not change cellular cAMP. Forskolin synergistically enhanced (P less than 0.01) the ionomycin-induced early phase as well as the TPA-induced late phase of the CGRP and calcitonin secretory responses. In conclusion, increased concentrations of Cai2+ together with protein kinase C and A activation mediate the secretion of CGRP and calcitonin in MTC cells.

Calcitonin and chromogranin A localization in medullary carcinoma of the thyroid by immunoelectron microscopy.

We used a post-embedding immunoelectron microscopy method, using protein A-gold, to detect calcitonin and chromogranin A immunoreactivity in three cases of human medullary thyroid carcinoma. Because the epoxy-embedded tissue had been fixed (glutaraldehyde or formaldehyde) and osmicated before embedment, the proteins were identified in optimally preserved tissue. Uranyl and lead staining was used after immunolabeling, so that the tissue was also optimally contrasted. The morphological advantage provided by osmication was tested by labeling rat thyroid gland C-cells for calcitonin. The protein A-gold technique allowed localization of both antigens to the contents of membrane-bound secretory granules in the tumor cells. In one case, labeling density for each antigen was measured over several intercellular compartments and the interstitium. Calcitonin, but not chromogranin A, reactivity was also identified in intracellular amyloid fibrils in two cases, showing that the constant region of calcitonin is preserved in amyloid deposits related to the tumor cells.

Isolation and structural characterization of thymosin-beta 4 from a human medullary thyroid carcinoma.

An extract of a tumour metastases from a human medullary thyroid carcinoma contained a high concentration (at least 2.9 nmol/g wet weight) of the immunoregulatory peptide, thymosin-beta 4. The peptide was isolated as a mixture of two components with free and blocked NH2-terminal amino acid residues, the latter form predominating (approximately 98% of the total). The primary structure of the peptide was established by automated Edman degradation after cleavage with cyanogen bromide. The amino acid sequence of human thymosin-beta 4 was identical to thymosin-beta 4 previously isolated from calf thymus. Further studies are warranted to determine whether thymosin-beta 4 production is a useful marker for thyroid and other tumours.