Characterization of the new human pleomorphic undifferentiated sarcoma TP53-null cell line mfh-val2.

Pleomorphic undifferentiated sarcoma (PUS), also called malignant fibrous histiocytoma, is a soft tissue sarcoma which occurs predominantly in the extremities. Its origin is a poorly defined mesenchymal cell, which derives to histiocytic and fibroblastic cells. The patient, a 58 year-old man, presented a lesion located in the forearm composed by spindle cells and multinucleated giant cells, which expressed vimentin and adopted a histological pattern formed by irregular-swirling fascicles. Cells were cultured in vitro and a new cell line was established. We characterized this new cell line by histological analyses, cytogenetics (using G-bands and spectral karyotype technique) and cytometric analyses. Cells were grown in culture for more than 100 passages. They had elongated or polygonal morphology. The cells presented a saturation rate of 70,980 cells/cm2, a plating efficiency of 21.5% and a mitotic index of 21 mitoses per field. The cell line was tumorigenic in nude mice. The ploidy study using flow cytometry revealed an aneuploid peak with a DNA index of 1.43. A side population was detected, demonstrating the presence of stem and progenitor cells. Cytogenetics showed a hypotriploid range with many clonal unbalanced rearrangements. Loss of p53 gene was evidenced by MLPA. We describe, for the first time, the characterization of a new human PUS TP53-null cell line called mfh-val2. Mfh-val2 presents a wide number of applications as a TP53-null cell line and a great interest in order to characterize genetic alterations influencing the oncogenesis or progression of PUS and to advance in the biological investigation of this tumor.

Expression of the Chemokine Receptors CXCR3, CXCR4, CXCR7 and Their Ligands in Rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) are soft tissue malignant tumors of childhood and adolescents. The mechanisms underlying their aggressiveness are still poorly understood. Chemokines are chemotactic proteins involved in pathological processes that have been intensely studied in several types of cancers because of their influence in migration, angiogenesis, or metastases. We analyzed the expression of the chemokine receptors CXCR3, CXCR4 and CXCR7 and their ligands CXCL9, CXCL10, CXCL11 and CXCL12, in 15 RMS samples derived from nine patients. Expression was measured in tumors and primary cultures of RMS by Real-Time Polymerase Chain Reaction, immunostaining and flow cytometry. Our results show that these receptors are widely expressed in RMS. A significant difference between CXCL12/CXCR4, CXCL12/CXCR7, CXCL11/CXCR7 expression ratios was found in alveolar versus embryonal RMS and similarly between CXCL12/CXCR4 and CXCL11/CXCR3 ratios in primary versus recurrent tumors. These findings suggest a possible association between the interrelation of chemokine/chemokine-receptor and an aggressive biological behavior in RMS.

WITHDRAWN: Systems Biology and Immune Aging.

The Publisher regrets that this article is an accidental duplication of anarticle that has already been published, http://dx.doi.org/10.1016/j.imlet.2014.09.009. The duplicate article has therefore been withdrawn.

Systems Biology and immune aging.

Many alterations of innate and adaptive immunity are common in the aging population, which reflect a deterioration of the immune system, and have lead to the terms "immune aging" or "immunosenescence". Systems Biology aims to the comprehensive knowledge of the structure, dynamics, control and design that define a given biological system. Systems Biology benefits from the continuous advances in the omics sciences, based on high-throughput and high-content technologies, as well as on bioinformatic tools for data mining and integration. The Systems Biology approach is becoming gradually used to propose and to test comprehensive models of aging, both at the level of the immune system and the whole organism. In this way, immune aging may be described by a dynamic view of the states and interactions of every individual cell and molecule of the immune system and their role in the context of aging and longevity. This mini-review presents a panoramics of the current strategies, tools and challenges for applying Systems Biology to immune aging.

Correlation between EGFR amplification and the expression of microRNA-200c in primary glioblastoma multiforme.

Extensive infiltration of the surrounding healthy brain tissue is a critical feature in glioblastoma. Several miRNAs have been related to gliomagenesis, some of them related with the EGFR pathway. We have evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns, studied by fluorescence in situ hybridization in tissue microarrays, of 30 cases of primary glioblastoma multiforme, whose clinicopathological and immunohistochemical features have also been analyzed. MicroRNA-200c showed a very significant difference between tumors having or not EGFR amplification. This microRNA plays an important role in epithelial-mesenchymal transition, but its implication in the behavior of glioblastoma is largely unknown. With respect to EGFR status our cases were categorized into three groups: high level EGFR amplification, low level EGFR amplification, and no EGFR amplification. Our results showed that microRNA-200c and E-cadherin expression are down-regulated, while ZEB1 is up-regulated, when tumors showed a high level of EGFR amplification. Conversely, ZEB1 mRNA expression levels were significantly lower in the group of tumors without EGFR amplification. Tumors with a low level of EGFR amplification showed ZEB1 expression levels comparable to those detected in the group with a high level of amplification. In this study we provide what is to our knowledge the first report of association between microRNA-200c and EGFR amplification in glioblastomas.

Intracellular coexpression of CXC- and CC- chemokine receptors and their ligands in human melanoma cell lines and dynamic variations after xenotransplantation.

BACKGROUND: Chemokines have been implicated in tumor progression and metastasis. In melanoma, chemokine receptors have been implicated in organ selective metastasis by regulating processes such as chemoattraction, adhesion and survival. METHODS: In this study we have analyzed, using flow cytometry, the systems formed by the chemokine receptors CXCR3, CXCR4, CXCR7, CCR7 and CCR10 and their ligands in thirteen human melanoma cell lines (five established from primary tumors and eight established from metastasis from different tissues). WM-115 and WM-266.4 melanoma cell lines (obtained from a primary and a metastatic melanoma respectively) were xenografted in nude mice and the tumors and cell lines derived from them were also analyzed. RESULTS: Our results show that the melanoma cell lines do not express or express in a low degree the chemokine receptors on their cell surface. However, melanoma cell lines show intracellular expression of all the aforementioned receptors and most of their respective ligands. When analyzing the xenografts and the cell lines obtained from them we found variations in the intracellular expression of chemokines and chemokine receptors that differed between the primary and metastatic cell lines. However, as well as in the original cell lines, minute or no expression of the chemokine receptors was observed at the cell surface. CONCLUSIONS: Coexpression of chemokine receptors and their ligands was found in human melanoma cell lines. However, this expression is intracellular and receptors are not found at the cell membrane nor chemokines are secreted to the cell medium. The levels of expressed chemokine receptors and their ligands show dynamic variations after xenotransplantation that differ depending on the origin of the cell line (from primary tumor or from metastasis).

Characterization of a new human melanoma cell line with CD133 expression.

A novel human malignant melanoma cell line, designated MEL-RC08, was established from a pericranial metastasis of a malignant melanoma of the skin. The cell line has been subcultured for more than 150 passages and is tumorigenic in nude mice. Growth kinetics, cytogenetics, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control; mutations in BRAF, NRAS, C-KiT, RB, and TP53 genes; and amplification of MDM2, CDK4, and cyclin D1 have been studied. Cytogenetically, the tumor and the cell line showed a hypertriploid karyotype with many clonal numeric and structural abnormalities. DNA flow cytometry showed an aneuploid peak with a DNA index value of 1.5. Mutations in TP53 and BRAF genes were demonstrated in both tumor and cell line. Furthermore, stem cell marker CD133 expression was detected in most cells, together with other stem cell markers, suggesting the presence of cells with tumor-initiating potential in this cell line.

CCL27-CCR10 and CXCL12-CXCR4 chemokine ligand-receptor mRNA expression ratio: new predictive factors of tumor progression in cutaneous malignant melanoma.

CXCR4, CCR7 and CCR10 chemokine receptors are known to be involved in melanoma metastasis. Our goal was to compare the relative intratumoral mRNA expression of these receptors with that of their corresponding chemokine ligands, CXCL12, CCL19, CCL21, and CCL27 across the full spectrum of human melanoma progression: thin and thick primary melanomas, as well as "in transit", lymph node, and distant metastases. Expression was quantified by real-time RT-PCR in 103 melanoma samples: 51 primary tumors and 52 metastases. Particular emphasis was focused on chemokine ligand-receptor expression ratios. Immunohistochemistry was performed to identify the cell types expressing these molecules. CXCL12-CXCR4 and CCL27-CCR10 ratios were higher in thin than in thick primary melanomas, and all four chemokine-receptor ratios were higher in primary tumors than in melanoma metastases. CCL27-CCR10 and CXCL12-CXCR4 expression ratios in primary tumors were inversely associated with the development of distant metastases, and improved the predictive value of tumor thickness for distant metastasis, which is important since chemokine ligand-receptor ratios are not affected by the endogenous gene employed for normalizing mRNA expression. Both receptor and ligand immunolabeling were detected in neoplastic cells suggesting autocrine mechanisms. Our results support the concept that low CCL27/CCR10 and CXCL12/CXCR4 intratumoral mRNA ratios are associated with melanoma progression, and in combination with Breslow thickness, are the best predictive factors for the development of distant metastases in primary cutaneous melanoma.

Cytomics: A multiparametric, dynamic approach to cell research.

Cytomics aims to determine the molecular phenotype of single cells. Within the context of the -omics, cytomics allows the investigation of multiple biochemical features of the heterogeneous cellular systems known as the cytomes. Cytomics can be considered as the science of single cell-based analyses that links genomics and proteomics with the dynamics of cell and tissue function, as modulated by external influences. Inherent to cytomics are the use of sensitive, scarcely invasive, fluorescence-based multiparametric methods and the event-integrating concept of individual cells to understand the complexity and behaviour of tissues and organisms. Among cytomic technologies, flow cytometry, confocal laser scanning microscopy and laser capture microdissection are of great relevance. Other recent technologies based on single cell bioimaging and bioinformatic tools become important in drug discovery and toxicity testing, because of both high-content and high-troughput. The multiparametric capacity of cytomics is very useful for the identification, characterization and isolation of stem cell populations. In our experience, flow cytometry is a powerful and versatile tool that allows quantitative analysis of single molecules, prokaryotic and eukaryotic cells for basic, biotechnological, environmental and clinical studies. The dynamic nature of cytomic assays leads to a real-time kinetic approach based on sequential examination of different single cells from a population undergoing a dynamic process, the in fluxo level. Finally, cytomic technologies may provide in vitro methods alternative to laboratory animals for toxicity assessment.

Establishment and characterization of a continuous human chondrosarcoma cell line, ch-2879: comparative histologic and genetic studies with its tumor of origin.

Chondrosarcomas are malignant cartilage-forming tumors that represent the second most common malignant solid tumor of bone. These biologically poorly understood neoplasms vary considerably in clinical presentation and biologic behavior. Chemotherapy and radiation therapy are generally ineffective. Here we describe the establishment and characterization of a new human chondrosarcoma cell line named ch-2879, and we compare the cell line with its tumor of origin. The cell line was established from a recurrent grade 3 chondrosarcoma of the chest wall and characterized by growth kinetics and morphologic studies. Immunocytochemistry and RT-PCR were performed to examine the expression of cartilage-specific phenotypes. Genetic characterization was performed using cytogenetics, fluorescence in situ hybridization, flow cytometry, and molecular techniques for analysis of the genes implicated in cell cycle control, amplification of MDM2, CDK4, and Cyclin D1, and mutations in the p53 gene. ch-2879 cells were subcultured for more than 80 passages. They expressed vimentin, HNK-1, HBA-71, Ki-67, cyclin D1, Fli-1, S-100, p21, p27, and p53 and were negative for cytokeratin, EMA, p14, p16, MDM2, Rb, and c-erb-b2 antigens. Cytogenetically the recurrent tumor showed a hyperhaploid karyotype with clonal numerical and structural abnormalities. The sole structural abnormality was a chromosome derivative of a t(1;21) translocation. The cell line at passage 3 showed two populations: the hyperhaploid and an exactly duplicated, hypotriploid population. After the 18th passage, only the hypotriploid population was present. The cells expressed collagen 2. Molecular comparison of the primary and recurrent tumor evidenced an in vivo molecular change consisting of a deletion of 9p21 genes in the recurrence, probably caused by a selection process. Because of its gene expression profile, including expression of genes implicated in chondrogenesis in uncoated plastic dishes, this cell line may prove useful for cellular and molecular studies as well as studies of chondrosarcoma characterization and treatment.