Archaeometric researches on the provenance of Mediterranean Archaic Phoenician and Punic pottery.

The aim of this study is to setup a first chemical database that could represent the starting point for a reliable classification method to discriminate between Archaic Phoenician and Punic pottery on the base of their chemical data. This database up to now can discriminate between several different areas of production and provenance and can be applied also to unknown ceramic samples of comparable age and production areas. More than 100 ceramic fragments were involved in this research, coming from various archaeological sites having a crucial importance in the context of the Phoenician and Punic settlement in central and western Mediterranean: Carthage (Tunisia), Toscanos (South Andalusia, Spain), Sulci, Monte Sirai, Othoca, Tharros (Sardinia, Italy) and Pithecusa (Campania, Italy). Since long-time archaeologists hypothesised that Mediterranean Archaic Phoenician and Punic pottery had mainly a local or just a regional diffusion, with the exception of some particular class like transport amphorae. To verify the pottery provenance, statistical analyses were carried out to define the existence of different ceramic compositional groups characterised by a local origin or imported from other sites. The existing literature data are now supplemented by new archaeometric investigations both on Archaic Phoenician ceramics and clayey raw materials from Sardinia. Therefore, diffractometric analyses, optical microscopy observations and X-ray fluorescence analyses were performed to identify the mineralogical and chemical composition of Othoca ceramics and clayey raw material. The obtained results were then compared with own literature data concerning Phoenician and Punic pottery in order to find features related to the different ceramic productions and their provenance. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were also performed on the chemical compositional data in order to discriminate ceramic groups. A very complex situation was found: imported ceramics coming from Carthage, with a large-scale distribution, were found together with a predominant local production pottery. The archaeometric results demonstrate that historical and typological approach has to be supported by scientific analyses to better understand local or Mediterranean exchanges.

Performance of EGFR mutant-specific antibodies in different cytological preparations: a validation study.

OBJECTIVES: Molecular testing for epidermal growth factor receptor (EGFR) mutations is required to select the most appropriate treatment for advanced-stage non-small cell lung cancer (NSCLC). In routine practice, cytological samples are often the only specimens available for testing. When the number of neoplastic cells is large, DNA-based assays are the gold standard. When cytological samples contain only a few neoplastic cells, immunocytochemistry (ICC) using anti-EGFR mutant-specific antibodies may be more effective. We aim to assess the specificity and sensitivity of IHC staining in cytological specimens using mutated cell lines subjected to different cytopreparations and staining methods. METHODS: HCC827 (exon 19 p.E746-A750 del) and H3255 (exon 21 p.L858R) cell lines were subjected to different fixation (air dried, alcohol or CytoLyt(®)), staining (Diff-Quik(®) or Papanicolaou) and preparation (smears or cell blocks) methods before ICC. In a second set of experiments, mutated cells were mixed with EGFR wild-type cells to obtain low-level (10%) mutated cytological samples. The intensity and percentage of cells stained were evaluated against validated molecular techniques. Moreover, the cell lines were subjected to poor growing conditions to simulate routine specimens that are less optimal than in vitro samples. RESULTS: The cytological preparations showing the most intense staining were formalin-fixed cell blocks and samples fixed with CytoLyt or alcohol, including Papanicolaou-destained samples. Conversely, air-dried slides showed the least intense staining. Mutant antibodies allowed the detection of mutated cells, even when representing only 10% of the total population. Although, in necrotic specimens, an aspecific background signal appeared, the viable cells still retained anti-mutant EGFR positivity. CONCLUSIONS: All cytological preparations are suitable for ICC using anti-EGFR mutant-specific antibodies, in particular formalin-fixed cell blocks and alcohol- or CitoLyt-fixed samples. the method is also validated to detect even a few mutant cells in less than optimal samples.

Desregulación de la expresión de microRNAs en el carcinoma papilar de tiroides / Deregulation of MicroRNAs Expression in Human Papillary Thyroid Carcinoma

El descubrimiento de los microRNAs (miRNAs) ha demostrado que estos se comportan como una poderosa clase de reguladores de la expresión génica. Al actuar a nivel postranscripcional, los miRNAs son capaces de modular la expresión de al menos un tercio de los RNA mensajeros codificados por el genoma. Aquí resumimos las principales alteraciones en la expresión de los genes para miRNAs identificados en el carcinoma papilar de tiroides. Se discuten también los mecanismos por los cuales la desregulación de estos miRNAs podrían estar involucrados en la transformación de las células foliculares tiroideas. Rev Argent Endocrinol Metab 51:205-212, 2014 Los autores declaran no poseer conflictos de interés.

MicroRNAs (miRNAs) small (~22 nt) single-stranded RNA molecules that are not further translated into proteins. They can act as negative regulators of the protein-coding gene expression and may impact cell differentiation, proliferation and survival. They have been implicated in carcinogenesis. The purpose of this review is to summarize the main alterations of miRNA expression identified in thyroid papillary carcinomas, and discuss the mechanisms by which miRNA deregulation might be involved in thyroid cell transformation. Rev Argent Endocrinol Metab 51:205-212, 2014 No financial conflicts of interest exist.

HAND1 gene expression is negatively regulated by the High Mobility Group A1 proteins and is drastically reduced in human thyroid carcinomas.

HMGA1 proteins exert their major physiological function during embryonic development and play a critical role in neoplastic transformation. Here, we show that Hand1 gene, which codes for a transcription factor crucial for differentiation of trophoblast giant cells and heart development, is upregulated in hmga1 minus embryonic stem cells. We demonstrate that HMGA1 proteins bind directly to Hand1 promoter both in vitro and in vivo and inhibit Hand1 promoter activity. We have also investigated HAND1 expression in human thyroid carcinoma cell lines and tissues, in which HMGA proteins are overexpressed, with respect to normal thyroid; an inverse correlation between HMGA1 and HAND1 expression was found in all thyroid tumor histotypes. A correlation between HAND1 gene repression and promoter hypermethylation was found in anaplastic carcinomas but not in other thyroid tumor histotypes. Therefore, we can hypothesize that HMGA1 overexpression plays a key role on HAND1 silencing in differentiated thyroid carcinomas and that promoter hypermethylation occurs in later stages of thyroid tumor progression. Finally, the restoration of the HAND1 gene expression reduces the clonogenic ability of two human thyroid carcinoma-derived cell lines, suggesting that HAND1 downregulation may have a role in the process of thyroid carcinogenesis.

Analysis of UbcH10 expression represents a useful tool for the diagnosis and therapy of astrocytic tumors.

Previous studies suggest the expression of UbcH10 gene, that codes for a protein belonging to the ubiquitin-conjugating enzyme family, as a valid indicator of the proliferative and aggressive status of tumors of different origin. Therefore, to look for possible tools to be used as diagnostic markers in astrocytic neoplasias, we investigated UbcH10 expression in normal brain, gliosis and low-grade and high-grade astrocytic tumors by immunohistochemistry. UbcH10 expression was observed in low-grade astrocytoma and in glioblastoma. Our data indicate a clear correlation between UbcH10 expression and the histological grade of the astrocytic tumors. Moreover, the analysis of UbcH10 expression allows the differentiation between gliotic and malignant tissues. Finally, since proteasome inhibitors have recently been considered as possible drugs in the chemotherapy of various tumors, our results would suggest new perspectives for the treatment of brain malignancies based on the suppression of the UbcH10 function.

Specific microRNAs are downregulated in human thyroid anaplastic carcinomas.

Thyroid carcinomas comprise a broad spectrum of tumors with different clinical behaviors. On the one side, there are occult papillary carcinomas (PTC), slow growing and clinically silent, and on the other side, rapidly growing anaplastic carcinomas (ATC), which are among the most lethal human neoplasms. We have analysed the microRNA (miR) profile of ATC in comparison to the normal thyroid using a microarray (miRNACHIP microarray). By this approach, we found an aberrant miR expression profile that clearly differentiates ATC from normal thyroid tissues and from PTC analysed in previous studies. In particular, a significant decrease in miR-30d, miR-125b, miR-26a and miR-30a-5p was detected in ATC in comparison to normal thyroid tissue. These results were further confirmed by northern blots, quantitative reverse transcription-PCR analyses and in situ hybridization. The overexpression of these four miRs in two human ATC-derived cell lines suggests a critical role of miR-125b and miR-26a downregulation in thyroid carcinogenesis, since a cell growth inhibition was achieved. Conversely, no effect on cell growth was observed after the overexpression of miR-30d and miR-30a-5p in the same cells. In conclusion, these data indicate a miR signature associated with ATC and suggest the miR deregulation as an important event in thyroid cell transformation.

UbcH10 expression may be a useful tool in the prognosis of ovarian carcinomas.

The UbcH10 gene codes for a protein that belongs to the ubiquitin-conjugating enzyme family. Previous studies of our group suggest UbcH10 expression as a valid indicator of the proliferative and aggressive status of thyroid carcinomas. Therefore, to better understand the process of ovarian carcinogenesis, and to look for possible tools to be used as prognostic markers in these neoplasias, we decided to extend the analysis of the UbcH10 expression to the ovarian neoplastic disease. We found that the UbcH10 gene was upregulated in some ovarian carcinoma cell lines analysed. Then, immunohistochemical studies demonstrate that UbcH10 expression significantly correlates with the tumor grade and the undifferentiated histotype of the ovarian carcinomas. Furthermore, a significant relationship between UbcH10 expression and overall survival was observed. Finally, the block of UbcH10 protein synthesis by RNA interference inhibited the growth of ovarian carcinoma cell lines, suggesting a role of UbcH10 overexpression in ovarian carcinogenesis. Therefore, all these data taken together suggest the possibility to use UbcH10 detection as a marker for the diagnosis and prognosis of these neoplastic diseases and open the perspective of a therapy of some ovarian carcinomas based on the suppression of the UbcH10 synthesis and/or function.

MicroRNA deregulation in human thyroid papillary carcinomas.

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in a wide range of basic processes such as cell proliferation, development, apoptosis and stress response. It has recently been found that they are also abnormally expressed in many types of human cancer. We analyzed the genome-wide miRNA expression profile in human thyroid papillary carcinomas (PTCs) using a microarray (miRNACHIP microarray) containing hundreds of human precursor and mature miRNA oligonucleotide probes. Using this approach, we found an aberrant miRNA expression profile that clearly differentiates PTCs from normal thyroid tissues. In particular, a significant increase in miRNA (miR)-221, -222 and -181b was detected in PTCs in comparison with normal thyroid tissue. These results were further confirmed by northern blot and quantitative RT-PCR analyses. Moreover, RT-PCR revealed miR-221, -222 and -181b overexpression in fine needle aspiration biopsies corresponding to thyroid nodules, which were eventually diagnosed as papillary carcinomas after surgery. Finally, miR-221, -222 and -181b overexpression was also demonstrated in transformed rat thyroid cell lines and in mouse models of thyroid carcinogenesis. Functional studies, performed by blocking miR-221 function and by overexpressing miR-221 in human PTC-derived cell lines, suggest a critical role of miR-221 overexpression in thyroid carcinogenesis. In conclusion, these data, taken together, indicate an miRNA signature associated with PTCs, and suggest miRNA deregulation as an important event in thyroid cell transformation.

UbcH10 overexpression may represent a marker of anaplastic thyroid carcinomas.

The hybridisation of an Affymetrix HG_U95Av2 oligonucleotide array with RNAs extracted from six human thyroid carcinoma cell lines and a normal human thyroid primary cell culture led us to the identification of the UbcH10 gene that was upregulated by 150-fold in all of the carcinoma cell lines in comparison to the primary culture cells of human normal thyroid origin. Immunohistochemical studies performed on paraffin-embedded tissue sections showed abundant UbcH10 levels in thyroid anaplastic carcinoma samples, whereas no detectable UbcH10 expression was observed in normal thyroid tissues, in adenomas and goiters. Papillary and follicular carcinomas were only weakly positive. These results were further confirmed by RT-PCR and Western blot analyses. The block of UbcH10 protein synthesis induced by RNA interference significantly reduced the growth rate of thyroid carcinoma cell lines. Taken together, these results would indicate that UbcH10 overexpression is involved in thyroid cell proliferation, and may represent a marker of thyroid anaplastic carcinomas.

A differential proteomic approach to identify proteins associated with thyroid cell transformation.

Tumour suppressor p53 is a transcription factor essential for DNA damage checkpoints during cellular response to stress. Mutations in the p53 gene are the most common genetic alterations found in human tumours; most pathogenetic modifications are missense mutations that abolish the p53 DNA-binding function. In the same cell type, distinct p53 missense mutations may determine different phenotypes. The PC Cl3 cell line retains several markers of thyroid differentiation in vitro. Introduction of the V143A mutant p53 allele, which abolishes the p53 DNA-binding function, leads to loss of differentiation markers as well as TSH dependency for growth. Conversely, PC Cl3 cells transfected with the S392A mutant p53 allele, presenting the mutation located outside the DNA-binding domain, show only loss of TSH dependency for growth. To identify molecular differences existing between PC Cl3 cell lines transformed by the V143A and the S392A mutant alleles, a differential proteomic approach was used. Two-dimensional gel electrophoresis analyses indicated that expression of a significant portion of protein species was modified by both p53 mutants. In fact, compared with wild-type PC Cl3 cells, modification of expression in V143A mutant cells occurred in 23.6% of the entire protein species. Conversely, modification of S392A mutant cells affected 14.0% of total proteins. Among these components, 8.3% were common to both mutants. Several of these proteins were identified by mass spectrometry procedures; some proteins, such as HSP90 and T-complex proteins, are already known to be related to p53 function.