Human Medullary Thyroid Carcinoma Cell Research Articles

Somatostatin, but not somatostatin receptor subtypes 2 and 5 selective agonists, inhibits calcitonin secretion and gene expression in the human medullary thyroid carcinoma cell line, TT.

Somatostatin (SRIH) analogs are commonly used to treat symptoms in medullary thyroid carcinoma (MTC), that expresses SRIH receptors (SSTR1 to SSTR5), as does the human MTC cell line TT. The aim of this work was to evaluate whether SRIH, SSTR2 and SSTR5-selective agonists influence calcitonin (CT) secretion and gene expression in the TT cell line. CT secretion was evaluated by chemiluminescence, and gene expression was analyzed by Northern blot. TT cell line proliferation was also assessed by [(3)H] thymidine ([(3)H]thy) incorporation and viable cell number count. SRIH significantly (p < 0.05) reduced [(3)H]thy incorporation (approx. 50 %), viable cell number (approx. 20 %), CT secretion (-30 %) and CT gene expression (approx. 2-fold). Exposure to the SSTR2-selective agonist, BIM-23 120, and to the SSTR5-selective agonist, BIM-23 206, did not modify CT secretion and mRNA levels in TT cells. Thus, SRIH inhibits DNA synthesis, cell proliferation, CT secretion and CT gene expression in the TT cell line, while SSTR2 and 5 selective agonists, although influencing DNA synthesis and cell proliferation, do not modify CT gene expression, suggesting that SRIH may influence gene expression acting through SSTRs other than subtypes 2 and 5. Furthermore, these findings may explain the erratic response of MTC patients in terms of CT plasma levels to treatment with SRIH analogs, like octreotide and lanreotide, which interact mainly with SSTR2 and 5.

The mdm2 proto-oncogene sensitizes human medullary thyroid carcinoma cells to ionizing radiation.

We have analysed the radiation response of a human medullary thyroid carcinoma cell line (MTT), characterized by the absence of a functional p53 protein, and the consequences of MDM2 overexpression in this process. We show that the product of the mdm2 proto-oncogene is able to sensitize MTT cells to ionizing radiation. After radiation treatment, MTT cells display histograms consistent with a G2M arrest. MTT cells expressing MDM2 (MTT-mdm2) are unable to respond to DNA damage with G2M arrest, and display a high percentage of apoptosis. MTT-mdm2 cells show high levels of E2F-1 protein, suggesting that the induction of apoptosis observed upon MDM2 overexpression could be dependent on E2F-1. This observation is further supported with assays showing that E2F-1 binding to specific DNA sequences is enhanced in MTT-mdm2 cells. Likewise, transactivation of reporter constructs exclusively dependent on E2F-1 is also elevated after transfection with MDM2. This effect can be reverted by transient transfection with p19ARF. To link the expression of E2F-1 with the induction of apoptosis, we generated clonal cell lines overexpressing E2F-1. Transfection with E2F-1 results in a low number of outgrowing colonies with reduced proliferation rates, indicating that E2F-1 is deleterious for cell growth. This negative regulation correlates with an increase in the percentage of the cell population with DNA content below 2N, suggesting that E2F-1 promotes apoptosis. Finally, overexpression of E2F-1 sensitizes MTT cells to radiation exposure. We conclude that the effects observed by MDM2 overexpression could be mediated by E2F-1.

Single-channel pharmacology of mibefradil in human native T-type and recombinant Ca(v)3.2 calcium channels.

To study the molecular pharmacology of low-voltage-activated calcium channels in biophysical detail, human medullary thyroid carcinoma (hMTC) cells were investigated using the single-channel technique. These cells had been reported to express T-type whole-cell currents and a Ca(v)3.2 (or alpha 1H) channel subunit. We observed two types of single-channel activity that were easily distinguished based on single-channel conductance, voltage dependence of activation, time course of inactivation, rapid gating kinetics, and the response to the calcium agonist (S)-Bay K 8644. Type II channels had biophysical properties (activation, inactivation, conductance) typical for high-voltage-activated calcium channels. They were markedly stimulated by 1 microM (S)-Bay K 8644, allowing to identify them as L-type channels. The channel termed type I is a low-voltage-activated, small-conductance (7.2 pS) channel that inactivates rapidly and is not modulated by (S)-Bay K 8644. Type I channels are therefore classified as T-type channels. They were strongly inhibited by 10 microM mibefradil. Mibefradil block was caused by changes in two gating parameters: a pronounced reduction in fraction of active sweeps and a slight shortening of the open-state duration. Single recombinant low-voltage-activated T-type calcium channels were studied in comparison, using human embryonic kidney 293 cells overexpressing the pore-forming Ca(v)3.2 subunit. Along all criteria examined (mechanisms of block, extent of block), recombinant Ca(v)3.2 interact with mibefradil in the same way as their native counterparts expressed in hMTC cells. In conclusion, the pharmacologic phenotype of these native human T-type channels--as probed by mibefradil--is similar to recombinant human Ca(v)3.2.

Substantial Production of Ghrelin by a Human Medullary Thyroid Carcinoma Cell Line

Ghrelin, an endogenous ligand for the GH secretagogue receptor, is a novel acylated peptide produced in the gastrointestinal endocrine cells as well as neuroendocrine cells in the hypothalamus. The Ser3 residue of ghrelin is modified by n-octanoic acid, a modification necessary for hormonal activity. Human medullary thyroid carcinoma is known to produce a variety of gastrointestinal and neuroendocrine peptides. In the present study we investigated ghrelin production in the thyroid gland, especially in human medullary thyroid carcinoma. PCR amplification demonstrated prepro-ghrelin gene transcripts in normal human thyroid tissue and two medullary thyroid carcinoma cell lines (human TT cells and rat 6-23 cells), but not in a rat thyroid follicular cell line. TT cells showed the expression of prepro-ghrelin mRNA of about 0.6 kb by Northern blot analysis. Furthermore, production of ghrelin in TT cells was demonstrated by RIA and immunocytochemistry. Accumulation of des-n-octanoyl ghrelin in the cultured medium of the cells was confirmed. Finally, human medullary thyroid carcinoma surgical specimens showed significantly higher des-n-octanoyl ghrelin contents than normal thyroid tissues. In conclusion, we revealed that ghrelin was produced by the human thyroid parafollicular carcinoma cell line, TT cells. These findings suggest that ghrelin is produced in the thyroid C cells as well as in medullary thyroid carcinoma and may provide opportunities to investigate its physiological role in the thyroid gland.

Substantial production of ghrelin by a human medullary thyroid carcinoma cell line.

Ghrelin, an endogenous ligand for the GH secretagogue receptor, is a novel acylated peptide produced in the gastrointestinal endocrine cells as well as neuroendocrine cells in the hypothalamus. The Ser(3) residue of ghrelin is modified by n-octanoic acid, a modification necessary for hormonal activity. Human medullary thyroid carcinoma is known to produce a variety of gastrointestinal and neuroendocrine peptides. In the present study we investigated ghrelin production in the thyroid gland, especially in human medullary thyroid carcinoma. PCR amplification demonstrated prepro-ghrelin gene transcripts in normal human thyroid tissue and two medullary thyroid carcinoma cell lines (human TT cells and rat 6-23 cells), but not in a rat thyroid follicular cell line. TT cells showed the expression of prepro-ghrelin mRNA of about 0.6 kb by Northern blot analysis. Furthermore, production of ghrelin in TT cells was demonstrated by RIA and immunocytochemistry. Accumulation of des-n-octanoyl ghrelin in the cultured medium of the cells was confirmed. Finally, human medullary thyroid carcinoma surgical specimens showed significantly higher des-n-octanoyl ghrelin contents than normal thyroid tissues. In conclusion, we revealed that ghrelin was produced by the human thyroid parafollicular carcinoma cell line, TT cells. These findings suggest that ghrelin is produced in the thyroid C cells as well as in medullary thyroid carcinoma and may provide opportunities to investigate its physiological role in the thyroid gland.

Somatostatin Receptor Subtypes 2 and 5 Differentially Affect Proliferation in Vitro of the Human Medullary Thyroid Carcinoma Cell Line TT

Somatostatin Receptor Subtypes 2 and 5 Differentially Affect Proliferation in Vitro of the Human Medullary Thyroid Carcinoma Cell Line TT

Somatostatin receptor subtypes 2 and 5 differentially affect proliferation in vitro of the human medullary thyroid carcinoma cell line tt.

Somatostatin and its receptors (SSTR1 to SSTR5) are expressed in normal human parafollicular C cells and medullary thyroid carcinoma (MTC), but the role of SSTR subtypes in cell growth regulation is still not clear. The present study demonstrates that the human MTC cell line TT stably expresses all the SSTR subtypes and responds to SSTR2 and SSTR5 activation by subtype-selective agonists with two different patterns in terms of [(3)H]thymidine ([(3)H]thy) incorporation and cell number. The SSTR2 preferential agonists (BIM-23120, BIM-23197, BIM-23190, and BIM-23014; 10(-9)-10(-6) M), significantly suppressed [(3)H]thy incorporation (58-13%) and reduced cell proliferation (50-28%), whereas the SSTR5-selective agonist, BIM-23206 (10(-9)-10(-6) M), significantly increased [(3)H]thy incorporation in TT cells (80-175%), but failed to influence cell proliferation. SSTR2 antagonist (BIM-23627) counteracted the action of SSTR2 preferential agonists on TT cells. Furthermore, increasing concentrations of SSTR5-selective agonists, BIM-23206, dose-dependently prevented the suppression of TT cell [(3)H]thy incorporation and proliferation produced by SSTR2 preferential agonist, BIM-23120, showing an antagonism between these compounds. The following conclusions were reached: 1) the human MTC cell line TT expresses all SSTR subtypes; 2) SSTR2 activation inhibits DNA synthesis and cell proliferation, whereas SSTR5 activation increases DNA synthesis; and 3) SSTR2 preferential agonist (BIM-23120) can antagonise SSTR5-selective agonist (BIM-23206) action and vice versa. These findings suggest a tissue-specific function and a tissue-specific interaction between the two receptors.

High level, tissue-specific expression of a modified calcitonin/calcitonin gene-related peptide promoter in a human medullary thyroid carcinoma cell line

The efficient and high level expression of therapeutic genes in target cells is critical for effective gene therapy. We have developed a novel promoter by utilizing tandem repeats of a tissue-specific regulatory element from the calcitonin/calcitonin gene-related peptide (CT/CGRP) gene placed in close proximity to a basal promoter, thereby removing interstitial sequences. This promoter drives expression of reporter genes at much higher levels than the natural promoter while significantly improving specificity in thyroid C cells.

Neuronal distribution and functional characterization of the calcium channel alpha2delta-2 subunit.

The auxiliary calcium channel alpha2delta subunit comprises a family of three genes, alpha2delta-1 to 3, which are expressed in a tissue-specific manner. alpha2delta-2 mRNA is found in the heart, skeletal muscle, brain, kidney, liver and pancreas. We report here for the first time the identification and functional characterization of alpha2delta-2 splice variants and their mRNA distribution in the mouse brain. The splice variants differ in the alpha2 and delta protein by eight and three amino acid residues, respectively, and are differentially expressed in cardiac tissue and human medullary thyroid carcinoma (hMTC) cells. In situ hybridization of mouse brain sections revealed the highest expression of alpha2delta-2 mRNA in the Purkinje cell layer of the cerebellum, habenulae and septal nuclei, and a lower expression in the cerebral cortex, olfactory bulb, thalamic and hypothalamic nuclei, as well as the inferior and superior colliculus. As the in situ data did not suggest a specific colocalization with any alpha1 subunit, coexpression studies of alpha2delta-2 were carried out either with the high-voltage-gated calcium channels, alpha1C, alpha1E or alpha1A, or with the low-voltage-gated calcium channel, alpha1G. Coexpression of alpha2delta-2 increased the current density, shifted the voltage dependence of channel activation and inactivation of alpha1C, alpha1E and alpha1A subunits in a hyperpolarizing direction, and accelerated the decay and shifted the steady-state inactivation of the alpha1G current.

The MDM2 oncoprotein promotes apoptosis in p53-deficient human medullary thyroid carcinoma cells.

The MDM2 oncoprotein has been shown to inhibit p53-mediated growth arrest and apoptosis. It also confers growth advantage to different cell lines in the absence of p53. Recently, the ability of MDM2 to arrest the cell cycle of normal human fibroblasts has also been described. We report a novel function for this protein, showing that overexpression of MDM2 promotes apoptosis in p53-deficient, human medullary thyroid carcinoma cells. These cells, devoid of endogenous MDM2 protein, exhibited a significant growth retardation after stable transfection with mdm2. Cell cycle distribution of MDM2 transfectants [medullary thyroid tumor (MTT)-mdm2] revealed a fraction of the cell population in a hypodiploid status, suggesting that MDM2 is sufficient to promote apoptosis. This circumstance is further demonstrated by annexin V labeling. MDM2-induced apoptosis is partially reverted by transient transfection with p53 and p19ARF. Both MTT and MTT-mdm2 cells were tumorigenic when injected into nude mice. However, the percentage ofapoptotic nuclei in tumor sections derived from MDM2-expressing cells was significantly higher relative to that in the parental cell line. MDM2-mediated programmed cell death is at least mediated by a down-regulation of the antiapoptotic protein Bcl-2. Protein levels of caspase-2, which are undetectable in the parental cell line, appear clearly elevated in MTT-mdm2 cells. Caspase-3 activation does not participate in MDM2-induced apoptosis, as determined by protein levels or poly(ADP-ribose) polymerase fragmentation. The results observed in this medullary carcinoma cell line show for the first time that the product of the mdm2 oncogene mediates cell death by apoptosis in p53-deficient tumor cells.

The MDM2 Oncoprotein Promotes Apoptosis in p53-Deficient Human Medullary Thyroid Carcinoma Cells

The MDM2 oncoprotein has been shown to inhibit p53-mediated growth arrest and apoptosis. It also confers growth advantage to different cell lines in the absence of p53. Recently, the ability of MDM2 to arrest the cell cycle of normal human fibroblasts has also been described. We report a novel function for this protein, showing that overexpression of MDM2 promotes apoptosis in p53-deficient, human medullary thyroid carcinoma cells. These cells, devoid of endogenous MDM2 protein, exhibited a significant growth retardation after stable transfection with mdm2. Cell cycle distribution of MDM2 transfectants [medullary thyroid tumor (MTT)-mdm2] revealed a fraction of the cell population in a hypodiploid status, suggesting that MDM2 is sufficient to promote apoptosis. This circumstance is further demonstrated by annexin V labeling. MDM2-induced apoptosis is partially reverted by transient transfection with p53 and p19ARF. Both MTT and MTT-mdm2 cells were tumorigenic when injected into nude mice. However, the percentage of apoptotic nuclei in tumor sections derived from MDM2-expressing cells was significantly higher relative to that in the parental cell line. MDM2-mediated programmed cell death is at least mediated by a down-regulation of the antiapoptotic protein Bcl-2. Protein levels of caspase-2, which are undetectable in the parental cell line, appear clearly elevated in MTT-mdm2 cells. Caspase-3 activation does not participate in MDM2-induced apoptosis, as determined by protein levels or poly(ADP-ribose) polymerase fragmentation. The results observed in this medullary carcinoma cell line show for the first time that the product of the mdm2 oncogene mediates cell death by apoptosis in p53-deficient tumor cells.

Structure and functional characterization of a novel human low-voltage activated calcium channel.

We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca2+ channel alpha1 subunit, alpha1H, from a human medullary thyroid carcinoma cell line. The alpha1H subunit is structurally similar to previously described alpha1 subunits. Northern blot analysis indicates that alpha1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba2+ currents recorded from human embryonic kidney 293 cells transiently expressing alpha1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (tau = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing alpha1H. Single-channel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of approximately 9 pS. These channels are blocked by Ni2+ (IC50 = 6.6 microM) and the T-type channel antagonists mibefradil (approximately 50% block at 1 microM) and amiloride (IC50 = 167 microM). Thus, alpha1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca2+ channels.

Post-transcriptional silencing of RET occurs, but is not required, during raf-1 mediated differentiation of medullary thyroid carcinoma cells.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor of the calcitonin secreting thyroid C-cells. Somatic and germline mutations in the RET proto-oncogene are associated with sporadic and inherited cases of MTC, respectively. The human MTC cell line, TT, can be differentiated by activated raf-1. This differentiation is characterized, in part, by down-regulation of the RET proto-oncogene. We now show that raf-1 induction is followed by activation of the downstream kinases MEK1/2 and ERK1/2 and that differentiation is dependent on activation of MEK1/2. The concurrent down-regulation of RET appears to involve altered nuclear compartmentalization and transport of RET mRNA. Although RET is down-regulated during raf-1 mediated differentiation, overexpression of activated RET alleles which resist down-regulation does not alter the raf-1 mediated differentiation response. These data suggest that RET down-regulation is associated with, but not required, for raf-1 mediated MTC cell differentiation and that the raf-1 signal transduction pathway plays a dominant role in promoting MTC cell differentiation.

Analysis of the structure and expression of the chicken gene encoding a homolog of the human RREB-1 transcription factor

Ras proteins are involved in a number of signal transduction pathways including the mitogen-activated kinase cascade. Activated MAPKs translocate to the nucleus and phosphorylate transcription factors such as c-myc, TCF and AP-1. Recently, a Ras-responsive element binding transcription factor, RREB-1, was cloned from a human medullary thyroid carcinoma cell line. RREB-1 is a zinc finger protein that binds to a Ras-responsive element in the promoter of the human calcitonin gene. We report the cloning of the chicken homologue to human RREB-1. Amino-acid alignment demonstrates that chicken and human RREB-1 are 53% identical and 69% similar. Genomic southern analysis indicates that chicken rreb-1 is a single-copy gene in the chicken genome. We demonstrate that chicken and human rreb-1 display the same tissue distribution, being expressed in all tissues examined except the brain. Interestingly, chicken RREB-1 has an extended N-terminus and contains 16 zinc fingers of the TFIIIA subclass, in comparison to human RREB-1 which was reported to contain only four zinc fingers. The size discrepancy between the two predicted gene products is further discussed. An unusual structural feature of RREB-1 is the widely spaced arrangement of the zinc fingers.

43 S9788 reverses in vitro and in in vivo the doxorubicin-induced toxicity in a human medullary thyroid carcinoma cell line

43 S9788 reverses in vitro and in in vivo the doxorubicin-induced toxicity in a human medullary thyroid carcinoma cell line

Expression of a calcium-sensing receptor in a human medullary thyroid carcinoma cell line and its contribution to calcitonin secretion.

An extracellular Ca(2+)-sensing mechanism consisting of a G protein-coupled receptor linked to phosphoinositide turnover and inhibition of PTH secretion, has recently been identified in bovine parathyroid cells. In C cells, voltage-dependent L-type calcium channels are thought to be involved in calcium-sensing mechanisms, but evidence exists for additional calcium-sensing mechanisms, such as via a calcium-sensing receptor (CaSR). Using the human medullary C cell carcinoma cell line TT, which lacks L-type calcium channels, we found that Ca2+ or cations specific for the CaSR lead to the release of calcium ions from intracellular stores and to an increase in calcitonin secretion. By molecular cloning we isolated the complete protein-coding complementary DNA of a CaSR from human TT cells, which are derived from a human medullary thyroid carcinoma. The CaSR is derived from the same CaSR gene expressed in the parathyroid gland. In addition, TT cells contain an alternative receptor form of CaSR, CaSRb. These findings provide strong evidence for the presence of a functional CaSR in the human C cell line TT. This receptor contributes not only to the inhibition of PTH secretion in the parathyroid, but also to the stimulation of calcitonin secretion in C cells.

Expression of a calcium-sensing receptor in a human medullary thyroid carcinoma cell line and its contribution to calcitonin secretion

Expression of a calcium-sensing receptor in a human medullary thyroid carcinoma cell line and its contribution to calcitonin secretion

Primary structure of a novel ABC transporter with a chromosomal localization on the band encoding the multidrug resistance-associated protein

Complementary DNA clones encoding a novel protein, ABC-C, with the typical structural features of the ABC transporter family were identified in a human medullary thyroid carcinoma cell line. The transporter consists of 1704 amino acid residues with two homologous repeats, each harboring six putative transmembrane helices and an ATP-binding cassette motif. The mRNA is expressed highest in normal lung, but also in varying amounts in other tissues and in C-cell carcinoma. The ABC-C gene is mapped on chromosome 16p13.3, in close physical proximity to another ABC transporter, the multidrug resistance-associated protein. This related protein is assumed to confer resistance to chemotherapeutic drugs in small cell lung carcinoma. The genomic clustering of both transporters, typical also for other members of the ABC family, supports the notion that ABC-C may be involved in development of resistance to xenobiotics.

Cytogenetic characterization of three human and three rat medullary thyroid carcinoma cell lines

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor of the thyroid C-cells. MTC may arise as a sporadic tumor or as a component of one of three autosomal dominant familial cancer syndromes, MEN 2A, MEN 2B, or familial MTC. Recent studies have identified mutations of the RET protooncogene in the proximal long arm of chromosome 10, which are thought to be causative for these syndromes. To facilitate the search for other genes involved in the development of MTC, we characterized cytogenetically three human MTC cell lines and three rat MTC cell lines. The human cell lines studied were TT and RO-H85-1, previously reported, and an uncharacterized cell line, MZ-CRC-1, derived from a malignant pleural effusion from a patient with metastatic MTC. The rat MTC cell lines characterized were CA-77, 6–23C 6, and 44-2. Cytogenetic abnormalities present in the human and rat cell lines were compared with 13 reported cytogenetic studies of human MTC tumors and three other cytogenetically analyzed MTC cell lines. The human 9q/rat 3 and human 3p/rat 15 chromosomes were affected in six of the comparable cell lines and tumors. These findings suggest human chromosome regions 9q and 3p may contain genes involved in the pathogenesis of MTC.

Structure and functional expression of a new member of the tetrodotoxin-sensitive voltage-activated sodium channel family from human neuroendocrine cells.

A member of a new subclass of the voltage-activated sodium channel genes has been cloned from the human medullary thyroid carcinoma (hMTC) cell line. The cDNA of hNE-Na (human neuroendocrine sodium channel) encodes a 1977 amino acid protein which phylogenetically represents a link between sodium channels isolated from skeletal muscle and brain. The hNE-Na alpha subunit was transiently expressed in human embryonic kidney cells either alone or in combination with the human sodium channel beta 1 subunit. The channel exhibited rapid activation and inactivation kinetics, and was blocked by tetrodotoxin and cadmium with IC50 values of 24.5 nM and 1.1 mM, respectively. Action potentials were generated in cells expressing high levels of hNE-Na. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated its expression in hMTC cells, in a C-cell carcinoma, and in thyroid and adrenal gland. Transcripts were not identified in pituitary gland, brain, heart, liver or kidney, indicating that the hNE-Na is a sodium channel solely expressed in neuroendocrine cells.