Telomerase activity in human leukemic cells with or without monosomy 7 or 7q-

BACKGROUND: In bone marrow material from patients with various leukemias we noted that samples with either a deletion on the long arm of one chromosome 7 (7q-) or a monosomy 7 had a higher telomerase activity. Considering that introduction of a chromosome 7 into a cancer cell line had been reported to eliminate telomerase activity, that 7q- is a common negative prognostic finding in cancers, and that the deleted segment (band 7q31) contains an unidentified tumor suppressor gene, we wondered if this gene might be a telomerase inhibitor. RESULTS: We found no significant difference in telomerase activity between the three groups of patient samples. In contrast to reports on tumor cell lines we observed no amplification of the telomerase genes. METHODS: We analyzed telomerase activity and copy number of the telomerase genes hTERT and hTR in frozen archival bone marrow samples from leukemia patients with a referral diagnosis of AML, and either a monosomy for chromosome 7, a deletion on the long arm of chromosome 7 (7q-), or none of these aberrations. Telomerase activity was measured with a commercially available kit, and the copy number of the telomerase genes was tested by FISH. CONCLUSIONS: We found no evidence of a telomerase inhibitor in band 7q31. The lack of telomerase gene amplification found in cell lines from solid tumors could reflect that this amplification is a property of solid tumors, not of hematological cancers

Molecular Characterization of Spontaneous Mesenchymal Stem Cell Transformation

Background. We previously reported the in vitro spontaneous transformation of human mesenchymal stem cells (MSC) generating a population with tumorigenic potential, that we termed transformed mesenchymal cells (TMC). Methodology/Principal Findings. Here we have characterized the molecular changes associated with TMC generation. Using microarrays techniques we identified a set of altered pathways and a greater number of downregulated than upregulated genes during MSC transformation, in part due to the expression of many untranslated RNAs in MSC. Microarray results were validated by qRT-PCR and protein detection. Conclusions/Significance. In our model, the transformation process takes place through two sequential steps; first MSC bypass senescence by upregulating c-myc and repressing p16 levels. The cells then bypass cell crisis with acquisition of telomerase activity, Ink4a/Arf locus deletion and Rb hyperphosphorylation. Other transformation-associated changes include modulation of mitochondrial metabolism, DNA damage-repair proteins and cell cycle regulators. In this work we have characterized the molecular mechanisms implicated in TMC generation and we propose a two-stage model by which a human MSC becomes a tumor cell

The ocean sampling day consortium

Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world’s oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits

Neuronal hypoxia in vitro: Investigation of therapeutic principles of HUCB-MNC and CD133+ stem cells

Background The therapeutic capacity of human umbilical cord blood mononuclear cells (HUCB-MNC) and stem cells derived thereof is documented in animal models of focal cerebral ischemia, while mechanisms behind the reduction of lesion size and the observed improvement of behavioral skills still remain poorly understood. Methods A human in vitro model of neuronal hypoxia was used to address the impact of total HUCB-MNC (tMNC), a stem cell enriched fraction (CD133+, 97.38% CD133-positive cells) and a stem cell depleted fraction (CD133-, 0.06% CD133-positive cells) of HUCB-MNC by either direct or indirect co-cultivation with post-hypoxic neuronal cells (differentiated SH-SY5Y). Over three days, development of apoptosis and necrosis of neuronal cells, chemotaxis of MNC and production of chemokines (CCL2, CCL3, CCL5, CXCL8, CXCL9) and growth factors (G-CSF, GM-CSF, VEGF, bFGF) were analyzed using fluorescence microscopy, FACS and cytometric bead array. Results tMNC, CD133+ and surprisingly CD133- reduced neuronal apoptosis in direct co-cultivations significantly to levels in the range of normoxic controls (7% ± 3%). Untreated post-hypoxic control cultures showed apoptosis rates of 85% ± 11%. tMNC actively migrated towards injured neuronal cells. Both co-cultivation types using tMNC or CD133- reduced apoptosis comparably. CD133- produced high concentrations of CCL3 and neuroprotective G-CSF within indirect co-cultures. Soluble factors produced by CD133+ cells were not detectable in direct co-cultures. Conclusion Our data show that heterogeneous tMNC and even CD133-depleted fractions have the capability not only to reduce apoptosis in neuronal cells but also to trigger the retaining of neuronal phenotypes

Nuclear Translocation of β-Catenin during Mesenchymal Stem Cells Differentiation into Hepatocytes Is Associated with a Tumoral Phenotype

Wnt/β-catenin pathway controls biochemical processes related to cell differentiation. In committed cells the alteration of this pathway has been associated with tumors as hepatocellular carcinoma or hepatoblastoma. The present study evaluated the role of Wnt/β-catenin activation during human mesenchymal stem cells differentiation into hepatocytes. The differentiation to hepatocytes was achieved by the addition of two different conditioned media. In one of them, β-catenin nuclear translocation, up-regulation of genes related to the Wnt/β-catenin pathway, such as Lrp5 and Fzd3, as well as the oncogenes c-myc and p53 were observed. While in the other protocol there was a Wnt/β-catenin inactivation. Hepatocytes with nuclear translocation of β-catenin also had abnormal cellular proliferation, and expressed membrane proteins involved in hepatocellular carcinoma, metastatic behavior and cancer stem cells. Further, these cells had also increased auto-renewal capability as shown in spheroids formation assay. Comparison of both differentiation protocols by 2D-DIGE proteomic analysis revealed differential expression of 11 proteins with altered expression in hepatocellular carcinoma. Cathepsin B and D, adenine phosphoribosyltransferase, triosephosphate isomerase, inorganic pyrophosphatase, peptidyl-prolyl cis-trans isomerase A or lactate dehydrogenase β-chain were up-regulated only with the protocol associated with Wnt signaling activation while other proteins involved in tumor suppression, such as transgelin or tropomyosin β-chain were down-regulated in this protocol. In conclusion, our results suggest that activation of the Wnt/β-catenin pathway during human mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype

Identification of target genes for wild type and truncated HMGA2 in mesenchymal stem-like cells

Background The HMGA2 gene, coding for an architectural transcription factor involved in mesenchymal embryogenesis, is frequently deranged by translocation and/or amplification in mesenchymal tumours, generally leading to over-expression of shortened transcripts and a truncated protein. Methods To identify pathways that are affected by sarcoma-associated variants of HMGA2, we have over-expressed wild type and truncated HMGA2 protein in an immortalized mesenchymal stem-like cell (MSC) line, and investigated the localisation of these proteins and their effects on differentiation and gene expression patterns. Results Over-expression of both transgenes blocked adipogenic differentiation of these cells, and microarray analysis revealed clear changes in gene expression patterns, more pronounced for the truncated protein. Most of the genes that showed altered expression in the HMGA2-overexpressing cells fell into the group of NF-κB-target genes, suggesting a central role for HMGA2 in this pathway. Of particular interest was the pronounced up-regulation of SSX1, already implicated in mesenchymal oncogenesis and stem cell functions, only in cells expressing the truncated protein. Furthermore, over-expression of both HMGA2 forms was associated with a strong repression of the epithelial marker CD24, consistent with the reported low level of CD24 in cancer stem cells. Conclusions We conclude that the c-terminal part of HMGA2 has important functions at least in mesenchymal cells, and the changes in gene expression resulting from overexpressing a protein lacking this domain may add to the malignant potential of sarcomas

Epithelial to Mesenchymal Transition of a Primary Prostate Cell Line with Switches of Cell Adhesion Modules but without Malignant Transformation

Background: Epithelial to mesenchymal transition (EMT) has been connected with cancer progression in vivo and the generation of more aggressive cancer cell lines in vitro. EMT has been induced in prostate cancer cell lines, but has previously not been shown in primary prostate cells. The role of EMT in malignant transformation has not been clarified. Methodology/Principal Findings: In a transformation experiment when selecting for cells with loss of contact inhibition, the immortalized prostate primary epithelial cell line, EP156T, was observed to undergo EMT accompanied by loss of contact inhibition after about 12 weeks in continuous culture. The changed new cells were named EPT1. EMT of EPT1 was characterized by striking morphological changes and increased invasion and migration compared with the original EP156T cells. Gene expression profiling showed extensively decreased epithelial markers and increased mesenchymal markers in EPT1 cells, as well as pronounced switches of gene expression modules involved in cell adhesion and attachment. Transformation assays showed that EPT1 cells were sensitive to serum or growth factor withdrawal. Most importantly, EPT1 cells were not able to grow in an anchorage-independent way in soft agar, which is considered a critical feature of malignant transformation. Conclusions/Significance: This work for the first time established an EMT model from primary prostate cells. The results show that EMT can be activated as a coordinated gene expression program in association with early steps of transformation. The model allows a clearer identification of the molecular mechanisms of EMT and its potential role in malignant transformation

Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was required to bring about matrix-endothelial interactions and for xenografted hMSC -BD11 cells to optimally recruit host vasculature

Human telomerase activity regulation

Telomerase has been recognized as a relevant factor distinguishing cancer cells from normal cells. Thus, it has become a very promising target for anticancer therapy. The cell proliferative potential can be limited by replication end problem, due to telomeres shortening, which is overcome in cancer cells by telomerase activity or by alternative telomeres lengthening (ALT) mechanism. However, this multisubunit enzymatic complex can be regulated at various levels, including expression control but also other factors contributing to the enzyme phosphorylation status, assembling or complex subunits transport. Thus, we show that the telomerase expression targeting cannot be the only possibility to shorten telomeres and induce cell apoptosis. It is important especially since the transcription expression is not always correlated with the enzyme activity which might result in transcription modulation failure or a possibility for the gene therapy to be overcome. This review summarizes the current state of knowledge of numerous telomerase regulation mechanisms that take place after telomerase subunits coding genes transcription. Thus we show the possible mechanisms of telomerase activity regulation which might become attractive anticancer therapy targets