1,721,059 research outputs found
SSTR subtypes expression pattern influences the antiproliferative effects of somatostatin and lanreotide on human medullary thyroid carinomas in vitro
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Role of complex Cdk4/Cyclin D1 in somatostatin subtype 2 receptor-mediated inhibition of cell proliferation of a medullary thyroid carcinoma cell line in vitro
No full textSomatostatin (SRIH) inhibits cell proliferation by interacting with five distinct SRIH receptor subtypes (SSTR) by several pathways in many tissues. We previously demonstrated that SRIH, by activating SHP-1, inhibits cell proliferation of the human Medullary Thyroid Carcinoma (MTC) cell line, TT, which expresses all SSTR. However, the effects of SRIH on cell cycle proteins have not been investigated, so far. We therefore investigated the effects of SRIH and of a selective SSTR2 agonist on cell cycle protein expression, mainly focusing on Cyclin D1 and its associated kinases. TT cells were serum starved for 48 h and then treated with SRIF or with a selective SSTR2 agonist, BIM-23120 for up to 60 h. Cell proliferation was verified by a colorimentric assay and by [3H]thymidine incorporation. Moreover, cell cycle progression was studied by cytofluorimetry. Cell cycle protein expression at different time points was investigated by Western blot. Cyclin D1 expression was also investigated by quantitative RT-PCR.
Our data show that SRIH and the selective SSTR2 agonist, BIM-23120, reduce cell proliferation and DNA synthesis, as well as induce a delay of the cell cycle in G2/M phase. Moreover, treatment with SRIH or with BIM-23120 decreases Cyclin D1 and cdk4 protein levels, with a parallel reduction in Rb phosphorylation levels at Ser-780. These data indicate that the subtype 2 receptor-mediated antiproliferative effect of SRIH on TT cell proliferation may be exerted through a decrease in Cyclin D1 levels, and further underline the importance of SSTR2 in mediating the antiproliferative effects of SRIH, indicating a direct and strong effect on Cyclin D1-cdk4 comple
A Novel PKC beta II Inhibitor Induces Antiproliferative Effects in Human Pancreatic Neuroendocrine Tumor Cells
No full textA Novel PKC beta II Inhibitor Induces Antiproliferative Effects in Human Pancreatic Neuroendocrine Tumor Cell
Involvement of PKCβ and PKCδ isoforms in TSH signaling pathway in thyroid cancer cell lines
No full textIt is well established that most TSH effects on the thyroid gland, including stimulation of proliferation, thyroid hormone synthesis and expression of thyroid-specific genes, are transmitted mainly by the adenylate cyclise pathway. However, in human follicular cells and in rat FRTL-5 cells, TSH can also stimulate the β-isoforms of PLC that catalyzes the hydrolysis of phosphatidyl-inositol 4,5-phosphate, yielding the second messengers DAG and inositol 1,4,5-phosphate facilitating an increase in intracellular Ca2+. In FRTL-5 cells TSH has been suggested to increase DAG via phospholipase D, which produces DAG from phosphatidylcholine hydrolysis, suggesting an alternative mechanism for TSH-dependent activation through protein kinase C (PKC).
In the present study, we characterize the PKCβ and PKCδ isoforms expression and function in human follicular carcinoma cells, FTC133, and in human transformed thyrocytes, Nthy-ori cells, in order to understand whether these PKC isoforms are involved in TSH-mediated follicular cell proliferation and apoptosis. We mainly focus on PKCβ and PKCδ isoenzymes which are the most abundantly expressed isoforms in several tissues, are the most extensively studied and have two opposing roles in regulating cell proliferation.
In the Nthy-ori cells TSH stimulated cell proliferation and protected from apoptosis with a PKC-mediated mechanism. At the contrary, TSH did not increase FTC-133 cell viability nor protected the cells from PKC-inhibitor induced apoptosis. However, in FTC-133 cells TSH induced PKC expression, as well as downstream PKC targets GSK3β and AKT phosphorylation through a PKC-mediated mechanism. Moreover, immunofluorescence showed PKCβ and PKCδ perinuclear and citosolic location. These data suggest that TSH plays different roles in normal vs neoplastic thyrocytes.
Further studies are needed to clarify the role of PKCβ and PKCδ in the TSH signaling pathway in thyroid cells
miR-15a and miR-16-1 down-regulation in pituitary adenomas
No full textMicro RNAs (miRs) are small noncoding RNAs, functioning as antisense regulators of other RNAs. miR-15a and miR-16-1 genes are located at chromosome 13q14, a region which is frequently deleted in pituitary tumors. An inverse correlation has been shown in B cell chronic lymphocytic leukemia (B-CLL) between miR-15a and miR-16-1 expression and the expression levels of arginyl-tRNA synthetase (RARS), an enzyme which associates with the cofactor p43 in the aminoacyl-tRNA synthetase complex. When secreted, p43 regulates local inflammatory response and macrophage chemotaxis, and seems to have anti-neoplastic properties in mice. We explored miR-15a and miR-16-1 expression in 10 GH-secreting and in 10 PRL-secreting pituitary macroadenomas by Northern blot, and investigated the possible correlation with in vivo and in vitro characteristics. We found that miR-15a and miR-16-1 are expressed at lower levels in pituitary adenomas as compared to normal pituitary tissue. Moreover, their expression inversely correlates with tumor diameter and with RARS expression (P < 0.05), but directly correlates with p43 secretion (P < 0.02). Therefore, miR15 and miR16 down-regulation in pituitary adenomas correlates with a greater tumor diameter and a lower p43 secretion, suggesting that these genes may, at least in part, influence tumor growth
Allelic discrimination in the diagnosis of somatic BRAF V600E mutation on fine-needle aspiration biopsies
No full textMany studies demonstrated that somatic BRAF gene mutation analysis increases diagnostic accuracy for papillary thyroid carcinoma (PTC), even from very small samples. The gold standard for point mutations research is direct sequencing, that implies DNA extraction, PCR with specific primers, sequencing reaction and run on an automatic sequencer. This is an expensive and time consuming method, and the possible contamination with wild-type DNA not coming from the nodule significantly reduces sensitivity. Allelic discrimination is a real-time PCR application that can discriminate between two alleles differing for the insertion, substitution or deletion of a single base, due to the presence of two real time Taqman probes, each labelled with a different fluorochrome (FAM for the mutated allele and VIC for the wild-type allele).
The aim of our study is to verify whether allelic discrimination can be useful in the diagnosis of BRAF somatic mutation, starting from fine needle aspiration biopsies (FNAB). In each allelic discrimination reactions three positive controls are present, one for each possible genotype; omozygous controls are oligonucleotides containining the target sequence (wild-type or mutated), whereas the eterozygous control is a 4:1 mix of wild-type omozygous control and mutated omozygous control, respectively, in order to mimic wild-type contamination. To evaluate the method sensitivity, mutated DNA has been diluted in wild-type DNA at variable concentrations (1:2, 1:4, 1:10, 1:20, 1:100) and analyzed both by direct sequencing and allelic discrimination. Allelic discrimination was more sensitive since it detected the presence of mutated DNA in all dilutions, while direct sequencing detected the mutation until 1:20 dilution.
Five hundred FNAB have been analyzed with both methods; allelic discrimination identified 55 V600E mutated samples, while direct sequencing identified only 50 V600E mutated samples. Post surgical histological examination confirmed 54 PTC and one anaplastic carcinoma. In conclusion, allelic discrimination is more sensitive (P<0.05) and more accurate (P<0.05) than direct sequencing, and its use in diagnostic procedures is very useful, even when samples (i.e. FNAB) are contaminated with wild-type DNA
Utilization of luminescent technology to develop a kinase assay: Cdk4 as a model system
No full textProtocols to assess kinase activity generally include radioactive methods, fluorescent polarization technology and the use of specific antibodies. Here, a simple, effective, non radioactive method to measure kinase activity of immunoprecipitated proteins is described. Cdk4, a cell cycle dependent enzyme, was immunoprecipitated from whole cell extracts and used in kinase reactions. This system has been developed taking advantage of the kinase-Glo reagent (Promega), based on ATP depletion technology, but with a wider range of applications. The original aim of the commercial kit is the evaluation of kinase activity of highly purified enzymes, while this system enabled the evaluation of native kinases, retrieved by immunoprecipitation. This method was highly homogeneous and did not require any kind of separation or purification as well. Moreover, it was suitable for basic research and may be useful for low-medium throughput pharmaceutical screening of chemical libraries
Magmas, a novel over-expressed gene in Pituitary rat cell lines and in human pituitary adenomas: a new pituitary bio marker?
No full textPituitary tumors are mostly benign, being locally invasive in 5-35% of cases. Deregulation of several genes has been suggested as a possible alteration underlying the development and progression of pituitary tumors. Recently we have demonstrated that Magmas is over-expressed in a mouse ACTH-secreting pituitary adenoma cell line. Here we investigate by RT-QPCR the expression of Magmas in 4 rat pituitary adenoma cell lines: three growth hormone (GH) and prolactin (PRL)-secreting cells lines (GH1, GH3, and GH4C1) and one PRL-secreting cell line (MMQ). We found that Magmas is over-expressed in GH3 (2.2 fold) and MMQ (3.6 fold) cell lines, while GH1 and GH4C1 display Magmas expression levels similar to those detected in a pool of normal rat pituitaries. These results were confirmed by Western blot analysis that showed the same expression pattern found by RT-QPCR. Magmas mRNA expression was also assessed in 29 human pituitary tissues, including 19 nonfunctioning, 8 GH-secreting, 2 PRL-secreting, and one TSH-secreting pituitary adenoma, as well as in a pool of human normal pituitary mRNAs. We found that 24 out of 29 pituitary adenomas (82.7%) displayed a Magmas mRNA expression level >2-fold than that detected in a pool of human normal pituitaries (from 2 to 30-fold), in all types of pituitary adenomas, except for the TSH-secreting pituitary adenoma. Our results suggest that Magmas could be a novel tumor selective over-expressed gene and could be a novel molecular target to be studied in order to understand pituitary adenomas pathophysiology
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