Human mesenchymal stem cells with enhanced telomerase activity acquire resistance against oxidative stress-induced genomic damage.

BACKGROUND: Human mesenchymal stem cells (MSC) are important tools for several cell-based therapies. However, their use in such therapies requires in vitro expansion during which MSCs quickly reach replicative senescence. Replicative senescence has been linked to macromolecular damage, and especially oxidative stress-induced DNA damage. Recent studies on the other hand, have implicated telomerase in the cellular response to oxidative damage, suggesting that telomerase has a telomere-length independent function that promotes survival. METHODS: Here, we studied the DNA damage accumulation and repair during in vitro expansion as well as after acute external oxidative exposure of control MSCs and MSCs that overexpress the catalytic subunit of telomerase (hTERT MSCs). RESULTS: We showed that hTERT MSCs at high passages have a significant lower percentage of DNA lesions as compared to control cells of the same passages. Additionally, less damage was accumulated due to external oxidative insult in the nuclei of hTERT overexpressing cells as compared to the control cells. Moreover, we demonstrated that oxidative stress leads to diverse nucleus malformations, such as multillobular nuclei or donut-shaped nuclei, in the control cells whereas hTERT MSCs showed significant resistance to the formation of such defects. Finally, hTERT MSCs were found to possess higher activities of the basic antioxidant enzymes, superoxide dismutase and catalase, than control MSCs. DISCUSSION: On the basis of these results, we propose that hTERT enhancement confers resistance to genomic damage due to the amelioration of the cell's basic antioxidant machinery.

Expression analysis of B-Raf oncogene in V600E-negative benign and malignant tumors of the thyroid gland: correlation with late disease onset.

B-Raf, a member of the Raf serine/threonine kinase family, is an intermediate molecule in the mitogen-activated protein kinase pathway, which relays extracellular signals from the cell membrane to the nucleus via a cascade of phosphorylation events, ultimately promoting cancer development. This pathway is usually activated in human neoplasias. The purpose of this study was to investigate the role of B-Raf in thyroid pathology. We scanned for the presence of mutations at codon 600 (V → E) of the B-Raf gene, using a PCR-RFLP assay. In tumors with no mutation (32 benign and malignant thyroid tumors) and in their adjacent normal tissue, we measured the expression levels of B-Raf gene, using a quantitative real-time PCR (qPCR) assay. B-Raf expression in V600E-negative tumors deviated from the normal pattern, since it was overexpressed in 42 % of benign samples and downregulated in 54 % of malignant specimens. Hashimoto's thyroiditis also seemed to play an important role, since benign specimens with Hashimoto's thyroiditis had a 2.2-fold higher B-Raf expression than samples without thyroiditis (1.71 ± 0.63 vs. 0.78 ± 0.13). Statistical analysis revealed that B-Raf deregulation postponed disease onset by more than 10 years in both benign and malignant thyroid (benign: 55.6 ± 3.9 vs. 45.3 ± 3.3, p = 0.049; malignant: 52.2 ± 3.5 vs. 33.0 ± 7.9, p = 0.020). From the above results, we deduce that in the absence of mutation activation, B-Raf overexpression or downregulation is a protective event, since it delays the development of both malignant and benign thyroid tumors.