Individual capacity for repair of DNA damage and potential uses of stem cell lines for clinical applications:a matter of (genomic) integrity

Public and private human stem cell banking institutions are currently hosting hundreds of thousands partially characterized cell populations, including a significant number of human pluripotentstem cell lines. To be considered for use in clinical applications, stem cell preparations must undergo rigorous testing in order to ensure safety for the recipient. With development of the methodologies for in vitro derivation, ex vivo maintenance and expansion of stem cells and targeted differentiation of multipotent and pluripotent stem cells, many novel issues were added to the list of safety concerns of cell and tissue preparations. These issues are related to the potential changes that may occur in the course of in vitro propagation of stem cells and cell-derived products, how these changes may affect the quality of the preparation; and the potential effects on the recipient. Only a limited number of studies about the role of subtle variations of individual capacity for repair of genotoxic damage in maintenance in vitro of human stem cells are currently available. Nevertheless, the assessment of individual repair capacity may play a crucial role in the safety of use of human stem cells, as it constitutes a major factor in the risk of occurrence of genomic alterations that may seriously compromise the quality of the product. This article reviews the available data about the role of individual capacity for DNA damage repair in different human stem cell types and the potential adverse effects that may occur with the use of cell preparations with inferior repair capacity

A Rac switch regulates random versus directionally persistent cell migration

Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3′-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis

Hyperglycemia attenuates fibroblast contractility via suppression of TβRII receptor modulated α-smooth muscle actin expression

AbstractNonhealing wounds are a common complication in patients suffering from diabetes with hyperglycemia being the most deteriorating factor for this serious pathological condition. Despite the great body of data, the molecular mechanisms by which high glucose affects cellular physiology are still poorly defined. Here we used primary human foreskin fibroblasts cultured in normo- and hyperglycemic conditions to study the mechanisms leading to altered cell contractility. Our results demonstrated that 25 mmol/L glucose effectively reduced fibroblasts ability to contract fibrin gels, and this physiological change was accompanied by a decrease in alpha-smooth muscle actin expression and the percentage of spontaneously differentiated myofibroblasts in the population of high glucose-treated fibroblasts. These changes were a result of hyperglycemia-induced attenuation of TGF-β1 signaling, involving specific suppression of TGF-β receptor type II but not type I expression. Decreased production of the receptor abolished the ability of exogenously added TGF-β1 to induce Smad2/3 phosphorylation in the presence of high glucose concentrations. Our results identify TGF-β receptor type II as hyperglycemia expression-sensitive receptor and add further aspect to the complex way in which high glucose can affect the wound healing process

Characterization of the individual capacity for repair of genotoxic damage of a Bulgarian hESC line and two commonly used stabilized cell lines

AbstractThe number of stem cell lines worldwide grows steadily, including disease-specific lines and lines derived from specific tissues. The proportion of stem cell lines that were established in strictly xeno-free conditions is rapidly increasing, but it is still unclear whether these lines may be maintained in vitro for prolonged periods of time with satisfactory survival rates and minimal loss of their ‘stemness’ properties. The efficiency of repair of DNA damage has recently emerged as an important factor for maintenance of stem cells in culture with minimal genomic changes and preservation of the undifferentiated state. In this study we investigated the individual capacity for repair of DNA damage/maintenance of genomic integrity and additional markers in one human embryonic stem cell (hESC) line derived in Bulgaria from a discarded embryo and two of the human T-leukaemia cell lines commonly used for research purposes (T-1301 and Jurkat E6-1). Knowledge about the status of the studied cancer cell lines may be valuable for research purposes. Data about the individual repair capacity and the genetic risk for common late-onset diseases of newly established hESC lines as well as hESC lines currently in use may become a valuable tool in the assessment of the applicability of pluripotent human cell lines for research purposes, clinical trials and, potentially, clinical applications

Anti-cancer effect of Petasites hybridus L. (Butterbur) root extract on breast cancer cell lines

In this study, we present initial data for the potential anti-cancer effects of standardized Bulgarian Petasites hybridus root extract against breast cancer cells. The results showed that Butterbur extract (BE) caused a dose-dependent selective reduction in viability with concomitant increase of apoptosis in human breast cancer cells after 72 h treatment. We found the highest cytotoxicity of BE to MDA-MB-231 cells (IC50 – 520 μg/mL), followed by the MCF-7 cancer cell line (IC50 – 865 μg/mL). At the same time, the extract exhibited very low cytotoxicity to the non-tumorigenic L929 cell line (two-fold higher inhibitory concentration – 1252 μg/mL). As a result of our experiments we made the conclusion that Butterbur root extract shows selective high cytotoxicity and nucleus alterations in cancer cells comparing to non-cancerous cells

Resveratrol Affects Sphingolipid Metabolism in A549 Lung Adenocarcinoma Cells

Resveratrol is a naturally occurring polyphenol which has various beneficial effects, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, and neuroprotective effects, among others. The anti-cancer activity of resveratrol has been related to alterations in sphingolipid metabolism. We analyzed the effect of resveratrol on the enzymes responsible for accumulation of the two sphingolipids with highest functional activity—apoptosis promoting ceramide (CER) and proliferation-stimulating sphingosine-1-phosphate (S1P)—in human lung adenocarcinoma A549 cells. Resveratrol treatment induced an increase in CER and sphingosine (SPH) and a decrease in sphingomyelin (SM) and S1P. Our results showed that the most common mode of CER accumulation, through sphingomyelinase-induced hydrolysis of SM, was not responsible for a CER increase despite the reduction in SM in A549 plasma membranes. However, both the activity and the expression of CER synthase 6 were upregulated in resveratrol-treated cells, implying that CER was accumulated as a result of stimulated de novo synthesis. Furthermore, the enzyme responsible for CER hydrolysis, alkaline ceramidase, was not altered, suggesting that it was not related to changes in the CER level. The enzyme maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was downregulated, and its expression was reduced, resulting in a decrease in S1P levels in resveratrol-treated lung adenocarcinoma cells. In addition, incubation of resveratrol-treated A549 cells with the SK1 inhibitors DMS and fingolimod additionally downregulated SK1 without affecting its expression. The present studies provide information concerning the biochemical processes underlying the influence of resveratrol on sphingolipid metabolism in A549 lung cancer cells and reveal possibilities for combined use of polyphenols with specific anti-proliferative agents that could serve as the basis for the development of complex therapeutic strategies