Detection of BRAF mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA).

Objective: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate sub- stitution at residue 600 (BRAF V600E ), is an ideal hallmark of papillary thyroid carcinoma (PTC). How- ever, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. Design and methods: We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation poly- morphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. Results: The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) follicular variants. No mutations of BRAF were detected in the normal thyroid tissues, nor in follicular adenomas or follicular carcinomas. No correlation was found between BRAF mutation and clinicopathologic features nor with recurrence during a post- operative follow-up period of 4–11 years. BRAF V600E significantly correlated with absence of node metastasis. Conclusions: BRAF V600E is present in PTC, both in the classic form and in follicular variant with simi- lar prevalence. No correlation was found between BRAF mutation and aggressive clinical behavior. MASA-PCR proved to be a specific, sensitive and reliable method to detect BRAF T1799A in DNA extracted from different sources, including cytologic samples obtained either fresh or from archival glass slides. We propose this method as a useful tool to improve accuracy of preoperative diagnosis identifying PTC from biopsies with indeterminate cytologic findings

Insulin stimulates fibroblast proliferation through calcium-calmodulin-dependent kinase II.

Insulin effects are mediated by multiple integrated signals generated by the insulin receptor. Fibroblasts, as most of mammalian cells, are a target of insulin action and are impor- tant actors in the vascular pathogenesis of hyperinsulinemia. A role for calcium-calmodulin-dependent kinases (CaMK) in insulin signaling has been proposed but has been under inves- tigated. We investigated the role of the CaMK isoform II in insulin signaling in human fibroblasts. A rapid and transient increase of intracellular calcium concentration was induced by insulin stimulation, followed by increase of CaMKII activity, via L type calcium channels. Concomitantly, insulin stimula- tion induced Raf-1 and ERK activation, followed by thymidine uptake. Inhibition of CaMKII abrogated the insulin-induced Raf-1 and ERK activation, resulting also in the inhibition of thymidine incorporation. These results demonstrate that in fibroblasts, insulin-activated CaMKII is necessary, together with Raf-1, for ERK activation and cell proliferation. This represents a novel mechanism in the control of insulin signals leading to fibroblast proliferation, as well as a putative site for pharmacological intervention

Insulin stimulates fibroblast proliferation through calcium-calmodulin-dependent kinase II.

Insulin effects are mediated by multiple integrated signals generated by the insulin receptor. Fibroblasts, as most of mammalian cells, are a target of insulin action and are impor- tant actors in the vascular pathogenesis of hyperinsulinemia. A role for calcium-calmodulin-dependent kinases (CaMK) in insulin signaling has been proposed but has been under inves- tigated. We investigated the role of the CaMK isoform II in insulin signaling in human fibroblasts. A rapid and transient increase of intracellular calcium concentration was induced by insulin stimulation, followed by increase of CaMKII activity, via L type calcium channels. Concomitantly, insulin stimula- tion induced Raf-1 and ERK activation, followed by thymidine uptake. Inhibition of CaMKII abrogated the insulin-induced Raf-1 and ERK activation, resulting also in the inhibition of thymidine incorporation. These results demonstrate that in fibroblasts, insulin-activated CaMKII is necessary, together with Raf-1, for ERK activation and cell proliferation. This represents a novel mechanism in the control of insulin signals leading to fibroblast proliferation, as well as a putative site for pharmacological intervention

Calcium-calmodulin-dependent kinase II (CaMKII) mediates insulin-stimulated proliferation and glucose uptake.

Cellular growth and glucose uptake are regulated by multiple signals generated by the insulin receptor. The mechanisms of individual modulation of these signals remain somewhat elusive. We investigated the role of CaMKII in insulin signalling in a rat skeletal muscle cell line, demonstrating that CaMKII modulates the insulin action on DNA synthesis and the negative feedback that down regulates glucose uptake. Insulin stimulation generated partly independent signals leading to the rapid activation of Akt, Erk-1/2 and CaMKII. Akt activation was followed by Glut-4 translocation to the plasma membrane and increase of glucose uptake. Then, IRS-1 was phosphorylated at S612, the IRS-1/p85PI3K complex was disrupted, Akt was no more phosphorylated and both Glut-4 translocation and glucose uptake were reduced. Inhibition of CaMKII abrogated the insulin-induced Erk-1/2 activation, DNA synthesis and phosphorylation of IRS-1 at S612. Inhibition of CaMKII also abrogated the down-regulation of insulin-stimulated Akt phosphorylation, Glut-4 membrane translocation and glucose uptake. These results demonstrate that: 1 — CaMKII modulates the insulin-induced Erk-1/2 activation and cell proliferation; 2 — after the initial stimulation of the IRS-1/Akt pathway, CaMKII mediates the down- regulation of stimulated glucose uptake. This represents a novel mechanism in the selective control of insulin signals, and a possible site for pharmacological intervention

High Growth Rate of Benign Thyroid Nodules Bearing RET/PTC Rearrangements

Context: Benign thyroid nodules display a broad range of behaviors from a stationary size to a progressive growth. The RET/PTC oncogene has been documented in a fraction of benign thyroid nodules, besides papillary thyroid carcinomas, and it might therefore influence their growth. Objective: The aim of the present work was to evaluate whether RET/PTC in benign thyroid nodules associates with a different nodular growth rate. Study Design: In this prospective multicentric study, 125 subjects with benign nodules were included. RET rearrangements were analyzed in cytology samples; clinical and ultrasonographic nodule characteristics were assessed at the start and at the end of the study. Results: RET/PTC was present in 19 nodules. The difference between the mean baseline nodular volume of the RET/PTC− and RET/PTC+ nodules was not significant. After 36 months of follow-up, the RET/PTC+ group (n = 16) reached a volume higher than the RET/PTC− group (n = 90) (5.04 ± 2.67 vs. 3.04 ± 2.26 ml; P = 0.0028). We calculated the monthly change of nodule volumes as a percentage of baseline. After a mean follow-up of 36.6 months, the monthly volume increase of nodules bearing a RET rearrangement was 4.3-fold that of nodules with wild-type RET (1.83 ± 1.2 vs. 0.43 ± 1.0% of baseline volume; P < 0.0001). Conclusions: Benign thyroid nodules bearing RET rearrangements grow more rapidly than those with wild-type RET. Searching for RET rearrangements in benign thyroid nodules might be useful to the clinician in choosing the more appropriate and timely therapeutic option

Identification and Functional Characterization of a Novel Mutation in theNKX2-1Gene: Comparison with the Data in the Literature

Background: NKX2-1 mutations have been described in several patients with primary congenital hypothyroidism, respiratory distress, and benign hereditary chorea, which are classical manifestations of the brain-thyroid-lung syndrome (BTLS). Methods: The NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS, and a novel monoallelic mutation was identified in the affected patients. We introduced the mutation in an expression vector for the functional characterization by transfection experiments using both thyroidal and lung-specific promoters. Results: The mutation is a deletion of a cytosine at position 834 (ref. sequence NM-003317) (c.493delC) that causes a frameshift with formation of an abnormal protein from amino acid 165 and a premature stop at position 196. The last amino acid of the nuclear localization signal, the whole homeodomain, and the carboxy-terminus of NKX2-1 are all missing in the mutant protein, which has a premature stop codon at position 196 (p.Arg165Glyfs*32). The p.Arg165Glyfs*32 mutant does not bind DNA, and it is unable to transactivate the thyroglobulin (Tg) and the surfactant protein-C (SP-C) promoters. Interestingly, a dose-dependent dominant negative effect of the p.Arg165Glyfs*32 was demonstrated only on the Tg promoter, but not on the SP-C promoter. This effect was also noticed when the mutation was tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared with the data present in the literature and demonstrated that, so far, it is very difficult to establish a specific correlation among NKX2-1 mutations, their functional consequence, and the clinical phenotype of affected patients, thus suggesting that the detailed mechanisms of transcriptional regulation still remain unclear. Conclusions: We describe a novel NKX2-1 mutation and demonstrate that haploinsufficiency may not be the only explanation for BTLS. Our results indicate that NKX2-1 activity is also finely regulated in a tissue-specific manner, and additional studies are required to better understand the complexities of genotype-phenotype correlations in the NKX2-1 deficiency syndrome