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HTERT effects in CD8 T Lymphocytes from two healthy donors

ABSTRACT: Expression in GFP vs. GFP/hTERT transduced CD8 T Lymphocytes from Healty Donors (HD) 1 and 2 at early and late passages. Using CD8+ T lymphocyte clones over-expressing telomerase we investigated the molecular mechanisms that regulate T cell proliferation. Transduction and subcloning procedures were performed on CD8 + naive T-cell clones isolated from two different healthy individuals aged between 30 to 35 years (HD1 and HD2). T-cell cloneswere transduced to express hTERT/GFP or GFP alone. Keywords: Cell Line Comparison.

ORGANISM(S): Homo sapiens

PROVIDER: GSE5144 | GEO | 2006/06/24

SECONDARY ACCESSION(S): PRJNA96659

REPOSITORIES: GEO

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Project description:Introduction: Pleural mesothelioma (PM) is known as one of the most aggressive malignancies among all cancers, despite of usually absent tumor-driver mutations in oncogenes. It develops in a unique inflammatory tumor microenvironment (TME), which has been postulated as major contributor of PM’s highly aggressive nature. Mesothelioma-associated fibroblasts (Meso-CAFs), a main component of the TME have recently been shown to substantially stimulate several aspects of PM aggressiveness and promote the malignant transformation of pleural mesothelial cells. However, respective cell models for TME research in PM are still very limited. The most commonly used pleural mesothelial cell line Met5A has been established decades ago and patient-derived Meso-CAFs have just recently been characterized for the first time. The aim of the current study was to generate and characterize pleural mesothelial and Meso-CAF cell models with an extended life span that closely resemble the primary cells isolated from human tissue. Methods: Pleural mesothelial cells and Meso-CAFs were isolated from human tissue of pneumothorax and PM patients, respectively. Retroviral transduction was used to induce stable expression of human telomerase reverse transcriptase (hTERT) and enhanced green fluorescent protein (EGFP) in the primary cells. The established cell models were evaluated by measuring their doubling times, gene expression and protein activity levels of hTERT, as well as the absolute lengths of telomeres. The transduced cells were compared to their primary counterparts on the protein level using proteome analysis and the impact of their conditioned media (CM) on tumor cell growth was investigated by videomicroscopy. Results: All transduced derivatives exhibit elevated hTERT gene expression and protein activity, increased hTERT protein amounts in the nucleus, and moderately higher absolute telomere lengths compared to their parental primary cells. The transduction with hTERT did not elicit marked changes in the morphology of the cells, as well as in their proteomes and secretomes. The CM of primary and hTERT-transduced Meso-CAFs comparably stimulated PM cell growth, while medium conditioned by normal pleural mesothelial cells including their hTERT-transduced derivatives was not able to induce a growth stimulating effect. Conclusion: The hTERT-transduced cells closely resemble their primary counterparts, while retaining telomerase activity, thus preventing replicative senescence. The new cell models provide valuable tools for the investigation of cellular interactions cellular interactions within the TME of PM and thus may help to identify novel biomarkers for early diagnosis and to develop new therapeutic strategies.

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HTERT effects in CD8 T Lymphocytes from two healthy donors

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