Somatostatin 28(1-12) in a somatostatin-secreting human medullary thyroid carcinoma cell line

We have identified a system, the TT human medullary thyroid carcinoma cell line, which we found to contain 31.3 +/- 27.7 ng of somatostatin 28(1-12) immunoreactivity/mg protein. Radioimmunoassay of gel filtration fractions showed that the major form of immunoreactive somatostatin 28(1-12) had a molecular weight of 1,500 daltons. During reversed-phase high pressure liquid chromatography, this 1,500-dalton species coeluted with synthetic somatostatin 28(1-12). Somatostatin 28(1-12) containing forms larger than 7,000 daltons were also observed. Further studies will be required to elucidate the route of processing of prosomatostatin. The fact that the products of prosomatostatin processing in these cells are similar to those in normal tissues indicates that the TT medullary thyroid carcinoma cell line constitutes a useful model for human somatostatin gene expression.     

人甲状腺髓样癌TT细胞胃动素基因表达的调控

目的:研究人甲状腺髓样癌TT细胞系中胃动素基因表达调控.方法:通过人甲状腺髓样癌TT细胞体外培养,观察经cAMP,生长激素或甲状腺雌激素诱导后,胃动素表达的改变以及胃动素对TT细胞生长、侵袭和转移的影响.结果:甲状腺髓样癌TT细胞表达胃动素mRNA,胃动素可抑制TT细胞的生长.当胃动素基因沉默后,TT细胞转移和侵袭能力增加.当TT细胞分别经cAMP、胃动素、生长激素或甲状腺刺激素孵育48小时后,胃动素基因转录增加,降钙素基因相关肽与胃动素mRNA比值持续下降.环磷酸腺苷可降低TT细胞增殖和c-myc基因的表达.结论:人甲状腺髓样癌细胞生长活性可能与甲状腺C细胞(低的降钙素基因相关肽与胃动素比率)分化的表型有关.     

The regulation of somatostatin production in human medullary thyroid carcinoma cells by dexamethasone

There have been few studies of physiological importance on the regulation of somatostatin by hormones. We have studied the effect of the synthetic glucocorticoid dexamethasone on somatostatin production in the human medullary thyroid carcinoma TT cell line, a model for somatostatin production by the parafollicular cell. Dexamethasone inhibited somatostatin production in a dose-related manner with a maximal effect at a concentration of 10(-6) M. TT cells treated with dexamethasone (10(-6) M) showed an almost complete inhibition of somatostatin peptide production by 48 h of treatment. Molecular sizing chromatography demonstrated a decrease in both the probable somatostatin precursor (13,000 dalton) and the fully processed peptide. Analysis of mRNA content by hybridization revealed that dexamethasone also caused a decrease in detectable somatostatin mRNA. The hybridizable somatostatin mRNA decreased to approximately 50% of basal levels within 12 h of treatment. Northern blot hybridization showed a decrease in a single RNA species representing mature somatostatin mRNA. Dose-response experiments revealed inhibition of both peptide and mRNA at concentrations from 1 X 10(-8) to 1 X 10(-5) M dexamethasone. Four days after withdrawal from dexamethasone treatment, peptide and mRNA levels were higher than dexamethasone-treated controls. The sex steroid estradiol had no inhibitory effect on somatostatin production. These results suggest a potential regulator of somatostatin production and provide a system for the study of somatostatin gene regulation.     

AG490对甲状腺髓样癌TT细胞放射敏感性的影响

为了研究AG490对甲状腺髓样癌TT细胞放射敏感性,本研究选择甲状腺髓样癌TT细胞作为研究对象,并采用不同浓度AG490 (0, 25μmol/L, 50μmol/L, 100μmol/L)处理细胞,利用MTT法、流式细胞术和克隆形成实验分别对细胞增殖、凋亡及放射敏感性进行检测,并采用Western blotting法检测各组JAK2、p-STAT3、Bcl-2、Bax蛋白表达。结果表明,与A组相比,B、C、D组的抑制增殖作用增强,呈浓度和时间依赖性;随着AG490药物浓度的增加凋亡率逐渐增加;与A组相比,C组细胞存活分数显著降低;JAK2、p-STAT3和Bcl-2的蛋白表达量随着药物浓度的升高而逐渐降低,Bax蛋白表达量随着药物浓度的升高而逐渐增加。AG490对甲状腺髓样癌TT细胞增殖有抑制作用,可促进细胞凋亡,提高放射敏感性。     

Melanin-producing medullary thyroid carcinoma with glandular differentiation

A rare case is reported of melanin-producing medullary thyroid carcinoma in a 62-year-old man. Intraoperative imprints of the thyroid tumor revealed numerous detached tumor cells containing large amounts of brown pigment. The Fontana-Masson argentaffin reaction with bleach confirmed that those granules were melanin. Histologically, the tumor was composed of two different components--a medullary area with hyalinized stroma and a follicular area. Melanin was scattered in both areas. The tumor cells in both areas were immunoreactive to carcinoembryonic antigen, calcitonin, gastrin-releasing peptide, somatostatin, met.-enkephalin, neuron-specific enolase, chromogranin and neurofilaments, and negative for thyroglobulin and S-100 protein. The histologic diagnosis was melanin-producing medullary thyroid carcinoma with glandular differentiation. Although various kinds of peptides and amines have been reported to be produced in medullary thyroid carcinoma, melanin production is quite rare; this appears to be only the third reported case.     

Immunocytochemical characterization of two thyroid medullary carcinoma cell linesin vitro

Summary The immunocytochemical characterization of cell lines originating from thyroid medullary carcinoma, i.e. human TT cells and rat rMTC 6-23 cells, was undertaken. The immunocytochemical studies were supplemented by ultrastructural studies, including ultrastructural immunocytochemistry, and by radioimmunological estimation of calcitonin secretion to the medium. In rMTC 6-23 cells (subcultures 24 to 30), no hormone presence was demonstrated immunocytochemically, which corresponded to the absence of secretory granules at the ultrastructural level. Of various proteins sought, only neuron-specific enolase could be demonstrated. Nevertheless, the cells secreted calcitonin into the medium. TT cells (passages 145 to 160) produced secretory granules. The granules contained calcitonin, calcitonin gene-related peptide, somatostatin, neurotensin, met-enkephalin, leu-enkephalin, gastrin releasing peptide, parathyroid hormone-related protein, functional proteins of the chromogranin group and synaptophysin. Other functional proteins found in the cytosol of TT cells included non-specific enolase, calbindin and tyrosine hydroxylase. Receptor for calcitriol was localized in the cell nucleus. Marker proteins were localized in the cytosol (carcinoembryonic antigen) and in the cell skeleton (-tubulin, cytokeratin). Following changes in ionized calcium levels in the medium, changes in calcitonin secretion and in immunocytochemical detectability of some hormones and functional proteins were observed. TT cells demonstrated the expression of numerous hormones and functional proteins associated with calcitonin secretion. Further, the cells in their ultrastructure, immunocytochemical and secretory characteristics, resemble more closely normal parafollicular cells of the thyroid and, in our opinion, represent a more appropriate model for functional studies.     

Differential utilization of calcitonin gene regulatory DNA sequences in cultured lines of medullary thyroid carcinoma and small-cell lung carcinoma.

Regulation of expression of the human calcitonin gene was found to differ between two tumor lines of different tissue origin, medullary thyroid carcinoma (TT line) and small-cell lung carcinoma (DMS53 line). Distal 5' DNA elements between -750 and -2000 exhibited a stronger basal activity in DMS53 than in TT cells, whereas proximal DNA sequences between -132 and -252 mediated a dramatic cyclic AMP response in TT but not DMS53 cells.     

Hybridocytochemical and immuno-ultrastructural study of calcitonin gene expression in cultured medullary carcinoma cells

The study was aimed at a morphological demonstration of calcitonin (CT) gene expression in cultured TT cells, or, more specifically, hybridocytochemical detection of CT mRNA and calcitonin gene-related peptide (CGRP) mRNA and ultrastructural localization of the two hormones. The TT cells originated from medullary carcinoma of human thyroid gland. Ultrastructural studies of TT cells demonstrated a well-developed rough endoplasmic reticulum, large Golgi apparatus and low number of secretory granules. Hybridocytochemical studies showed the presence of mRNAs for CT and CGRP in all TT cells. At the ultrastructural level, double immunolabelling demonstrated that the two hormones were always expressed together in the same secretory granules. Our results provide a significant addition to the biochemical studies performed up to now and indicate that all TT cells produce both mRNAs and both hormones in parallel.     

Increase in cytoplasmic Ca2+ and stimulation of calcitonin secretion from human medullary thyroid carcinoma cells by the gastrin-releasing peptide.

Examination was made of the effects of gastrin-releasing peptide (GRP) on human medullary thyroid carcinoma cells (TT cells). GRP stimulated calcitonin(CT) release in a concentration-dependent manner at 0.1-1000 nmol/l. On adding forskolin along with GRP, CT release was greater than by GRP alone. The stimulatory effect of A23187 was not additive. Intracellular free calcium concentration ([Ca2+]i) was measured for individual TT cells loaded with fura-2. The addition of GRP caused a rapid and transient rise in [Ca2+]i in a concentration-dependent manner followed by a sustained increase in [Ca2+]i. In the medium without Ca2+, this sustained increase did not occur and the concentration of CT release from TT cells by GRP was reduced by approximately a half. GRP would thus appear to be importantly involved in the regulation of thyroid C cell function through modulation of [Ca2+]i.     

Discovery of targeting peptides for selective therapy of medullary thyroid carcinoma

BACKGROUND: Adenovirus efficiently infects a broad range of target cells, thereby preventing selective gene transfer. Moreover, several cell types and tissues including primary tumors are refractory to adenoviral infection, mainly because of low expression levels of coxsackie-adenovirus receptor (CAR). Thus, identification of cancer-selective ligands which yield gene transfer to neoplastic cells by minimizing transduction of normal cells is a key issue for successful cancer therapy. METHODS: We initially analyzed adenoviral receptor expression in human medullary thyroid carcinoma (MTC) cells. MTC cell-specific peptides were isolated by biopanning a phage display peptide library on cultured cancer cells and on tumors in vivo and further characterized. RESULTS: We found significant differences in CAR and alphav-integrin protein levels between MTC-derived TT cells in vitro and established xenograft tumors in mice, indicating a lack of alphav-integrin expression on growing tumors. MTC-specific candidates were identified by performing three rounds of subtraction. Selected phages showed up to 22-fold higher binding efficiency for TT cells when compared with wild-type M13 phage or other human cell lines and tumor tissue in vivo. Homing to TT cells of the best binding phage was clearly blocked in the presence of specific peptide, whereas no phage competition was observed with an unspecific peptide. The best binding peptide mediated efficient internalization of the phage. Importantly, specific binding and internalization was also mediated by the identified peptide within the adenoviral context. CONCLUSIONS: Our results indicate that the identified ligand should be suitable to improve selectivity of adenoviral gene transfer to medullary thyroid tumors in vivo.     

Preliminary evaluation of influence of gemcitabine (Gemzar) on proliferation and neuroendocrine activity of human TT cell line: immunocytochemical investigations

The choice treatment of medullary thyroid carcinoma (MTC) is total thyroidectomy. It is difficult to evaluate effectiveness of chemotherapy due to the rare incidence of MTC. Gemcitabine is a new drug of antimetabolite nucleoside group used in treatment of cancers since 1996. The aim of this study was to evaluate the influence of gemcitabine on proliferation and neuroendocrine activity of human TT cell line derived from MTC. The cells were exposed to gemcitabine in the concentration of 10, 25 and 50 microg/ml for 24 hours. Immunocytochemical examinations were carried out by the method of avidin-biotin peroxidase complex (ABC) according to Hsu et al. to detect calcitonin, chromogranin A, synaptophysin and neuron-specific enolase in TT cells. A concentration-dependent inhibitory influence of gemcitabine on proliferative activity of TT cells was observed. It was also shown that the immunostaining was reduced, especially in case of neuron-specific enolase. Only the reaction detecting calcitonin was enhanced in persistin     

Indomethacin inhibits cell growth of medullary thyroid carcinoma by reducing cell cycle progression into S phase

Indomethacin, a non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit the growth of medullary thyroid carcinoma (MTC) cells in vitro. However, the mechanism of inhibition of MTC cell growth by indomethacin and its potency have yet to be revealed. We examined the effect of indomethacin on three different MTC cell lines (TT cells, DRO 81-1 cells and HRO 85-1 cells) and two non-MTC cells. The mechanism of indomethacin action in MTC cells was investigated by analyzing intracellular prostaglandin level, apoptosis, and cell cycle in TT cells. Indomethacin inhibited cell growth of all three MTC cell lines but not normal thyroid cells or anaplastic thyroid carcinoma cells. Indomethacin at 10 microM or greater showed a dose response inhibition of cell growth. Indomethacin at 25 muM, a putative therapeutic serum indomethacin level, showed potency similar to 100 to 200 nM sunitinib, a receptor tyrosine kinase inhibitor. To examine whether prostaglandin depletion might determine the inhibition of MTC cell growth, we created different prostaglandin E2 (PGE2) levels in TT cells using three different NSAIDs. A profound PGE2 depletion by indomethacin-ester, a potent cyclooxygenase (COX) II inhibitor, showed the least inhibition of cell growth. Indomethacin did not increase apoptosis of TT cells. Indomethacin, but not naproxen or indomethacin-ester, reduced cell cycle progression into S phase; this was unrelated to the degree of PGE2 depletion. The expression of phosphorylated retinoblastoma (pRb) protein that shifts cells from G(1) to S phase was reduced after exposure to indomethacin. In conclusion, indomethacin has specific anti-tumor effect on MTC cells, probably by reducing cell cycle progression into S phase rather than by prostaglandin depletion. Since no drug therapy is currently available for MTC, indomethacin may be one of the therapeutic candidates.     

The presence of somatostatin receptors in malignant neuroendocrine tumor tissue predicts responsiveness to octreotide.

In 77 percent of patients suffering from a malignant carcinoid syndrome, administration of the somatostatin analog, octreotide (SMS 201-995, Sandostatin) induced clinical improvement coupled with a decrease in 24-hour urinary 5-hydroxyindole acetic acid (5-HIAA). This finding prompted an evaluation to determine the correlation between the presence of somatostatin receptors in tumor tissue and the response to octreotide in patients with advanced, metastatic, neuroendocrine tumors. In tissues of 31 tumors (20 carcinoid, eight islet-cell carcinoma, three medullary thyroid carcinomas), the presence of somatostatin receptors was analyzed by binding of the somatostatin analog 125I-Tyr3-SMS 201-995 and autoradiography. Receptors were detected in 16 of 20 samples of carcinoid tissues; all but one patient with receptor-positive tumors improved clinically after treatment with octreotide, and the urine 5-HIAA level was reduced a median of 63 percent (range, 39-94 percent) compared to values before treatment. Of the receptor-negative carcinoid patients, only one showed clinical improvement, which was minimal, and there was a negligible reduction in 5-HIAA after octreotide therapy. All eight patients with metastatic islet-cell carcinomas were positive for somatostatin receptors. Symptomatic improvement and a > 50 percent decrease in the level of at least one of the pathologically elevated marker hormones was seen in all eight. None of the three patients with medullary carcinoma of the thyroid had a decrease in calcitonin, and all three were initially somatostatin receptor-negative. We conclude that the presence of somatostatin receptors in malignant neuroendocrine tumor tissue appears to correlate with the response to octreotide therapy. Analysis of somatostatin receptors in malignant neuroendocrine carcinoma tissue should be included in future prospective clinical trials of this synthetic peptide.     

Identification of the endocrine cells detected by the monoclonal antibody HISL-19 in human tissues.

The human endocrine cells reacting with the monoclonal antibody HISL-19 were identified with hormone antisera of proven specificity using a double immunostaining procedure. The epitope for HISL-19 was found in all types of pituitary cells except ACTH cells, in thyroid C cells, in all types of adrenal medullary and pancreatic islet cells and in somatostatin and pancreatic polypeptide cells of the gastrointestinal mucosa. No staining was found in parathyroid cells and in most gastrointestinal endocrine cells. Either paranuclear focal accumulation or diffuse cytoplasmic distribution of immunoreactive material were found. The spectrum of HISL-19 immunoreactive cells was found to be only in part complementary to that of cells immunoreactive for chromogranin A. Thus, it is concluded that the monoclonal antibody HISL-19 is a useful addition to other immunohistochemical markers for the detection of cells showing neuroendocrine features.