Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with a striking degree of mitochondrial hyperplasia that are particularly frequent in the thyroid gland. To understand whether specific mitochondrial DNA (mtDNA) mutations are associated with the accumulation of mitochondria, we sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid tumors of epithelial cell derivation and 5 mitochondrion-rich breast tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15 breast carcinomas, and 16 gliomas) by using recently developed technology that allows specific and reliable amplification of the whole mtDNA with quick mutation scanning. Thirteen oncocytic lesions (26%) presented disruptive mutations (nonsense or frameshift), whereas only two samples (3.8%) presented such mutations in the nononcocytic control group. In one case with multiple thyroid nodules analyzed separately, a disruptive mutation was found in the only nodule with oncocytic features. In one of the five mitochondrion-rich breast tumors, a disruptive mutation was identified. All disruptive mutations were found in complex I subunit genes, and the association between these mutations and the oncocytic phenotype was statistically significant (P ‫؍‬ 0.001). To study the pathogenicity of these mitochondrial mutations, primary cultures from oncocytic tumors and corresponding normal tissues were established. Electron microscopy and biochemical and molecular analyses showed that primary cultures derived from tumors bearing disruptive mutations failed to maintain the mutations and the oncocytic phenotype. We conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors.oncocytic tumors ͉ heteroplasmy ͉ homoplasmy ͉ damaging mutation ͉ microenvironment

The Aurora kinases are involved in the regulation of cell cycle progression, and alterations in their expression have been shown to associate with cell malignant transformation. In the present study, we demonstrated that human thyrocytes express all 3 Aurora kinases (A, B and C) at both protein and mRNA level and this expression is cell cycle-regulated. An increase in the protein level of the 3 kinases was found, with respect to normal human thyrocytes (HTU5), in the human cell lines derived from follicular (FTC-133), papillary (B-CPAP) and anaplastic (8305C) thyroid carcinomas, but not in cells derived from a follicular adenoma (HTU42). These observations were mirrored in RT-PCR experiments for Aurora-A and B. In contrast, Aurora-C mRNA levels were not significantly different among the different cell types analyzed, suggesting that posttranscriptional mechanism(s) modulate its expression. The expression at the protein level of all 3 Aurora kinases was significantly higher in 3 thyroid papillary carcinomas with respect to normal matched tissues obtained from the same patients. Similar modifications, at the mRNA level, could be observed in 7 papillary carcinoma tissues for Aurora-A and B, but not for Aurora-C. In conclusion, we demonstrated that normal human thyrocytes express all 3 members of the Aurora kinase family, and their expression is amplified in malignant thyroid cell lines and tissues. These results suggest that the Aurora kinases may play a relevant role in malignant thyroid cancers, and may represent a putative therapeutic target for thyroid neoplasms

Programmed cell death is characterized by posttranslational modifications of a limited and specific set of nuclear proteins. We demonstrate that during apoptosis of different types of tumor cells there is a monomethylation of the nuclear protein HMGA1a that is associated to its previously described hyperphosphorylation/dephosphorylation process. HMGA1a methylation is strictly related to the execution of programmed cell death and is a massive event that involves large amounts of the protein. In some tumor cells, HMGA1a protein is already methylated to an extent that depends on cell type. The degree of methylation in any case definitely increases during apoptosis. In the studied cell systems (human leukaemia, human prostate tumor, and rat thyroid transformed cells) among the low-molecular-mass HMG proteins, only HMGA1a was found to be methylated. A tryptic digestion map of HPLC-purified HMGA1a protein showed that methylation occurs at arginine 25 in the consensus G(24)R(25)G(26) that belongs to one of the DNA-binding AT-hooks of the protein. An increase of HMGA1a methylation could be related to heterochromatin and chromatin remodeling of apoptotic cells.

No abstract

Anaplastic thyroid cancers (ATC) are aggressive tumors, which exhibit cell cycle misregulations leading to uncontrolled cellular proliferation and genomic instability. They fail to respond to chemotherapeutic agents and radiation therapy, and most patients die within a few months of diagnosis. In the present study, we evaluated the in vitro effects on ATC cells of VX-680, an inhibitor of the Aurora serine/threonine kinases involved in the regulation of multiple aspects of chromosome segregation and cytokinesis. The effects of VX-680 on proliferation, apoptosis, soft agar colony formation, cell cycle, and ploidy were tested on the ATC-derived cell lines CAL-62, 8305C, 8505C, and BHT-101. Treatment of the different ATC cells with VX-680 inhibited proliferation in a time-and dose-dependent manner, with the IC 50 between 25 and 150 nM. The VX-680 significantly impaired the ability of the different cell lines to form colonies in soft agar. Analysis of caspase-3 activity showed that VX-680 induced apoptosis in the different cell lines. CAL-62 cells exposed for 12 h to VX-680 showed an accumulation of cells with R4N DNA content. Time-lapse analysis demonstrated that VX-680-treated CAL-62 cells exit metaphase without dividing. Moreover, histone H3 phosphorylation was abrogated following VX-680 treatment. In conclusion, our data demonstrated that VX-680 is effective in reducing cell growth of different ATC-derived cell lines and warrant further investigation to exploit its potential therapeutic value for ATC treatment.