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    • In addition to apoptosis we investigated the

    2018-11-08

    In addition to apoptosis, we investigated the occurrence of DNA lesions in response to genotoxic insults. Using LORD-Q analyses, a novel sensitive technique to quantify DNA lesions (Lehle et al., 2014), we found that, despite their elevated apoptosis sensitivity, iPSCs accumulated significantly less DNA lesions than differentiated fibroblasts. Also, in response to treatment with H2O2 and UVC, oxidative nucleotide modifications such as cyclobutane pyrimidine dimers, (6-4) photoproducts, and 8-hydroxydeoxyguanosine (8-oxo-dG) were detected less frequently. Thus, our data are consistent with the hypothesis that pluripotent stem cells have superior DNA maintenance responses. Interestingly, we observed that this DNA damage protection was rapidly lost during differentiation of iPSCs. There are presumably several mechanisms that contribute to an efficient maintenance of DNA integrity in pluripotent stem cells, including the prevention of DNA damage and the removal of DNA lesions. Compared to differentiated cells, ESCs display a moderate increase (approximately 2- to 3-fold) in the expression of certain DNA repair enzymes of the homologous recombination and non-homologous end-joining pathways, which repair DNA double-strand breaks (Saretzki et al., 2008). It is interesting to note that a previous study in human ESCs (Maynard et al., 2008) found decreased levels of oxidative DNA lesions, such as 8-oxo-dG, which we assessed as an additional marker of DNA damage in iPSCs. Although the decreased 8-oxo-dG levels are suggestive of a more efficient repair of this lesion, the authors did not find elevated activities of 8-oxoguanine glycosylase, the primary cholesterol absorption inhibitor excision repair enzyme required for removing this mutagenic DNA lesion. These results indicate that base excision repair is presumably not elevated in ESCs, but rather the occurrence of oxidative DNA lesions is prevented. Our study shows that iPSCs are highly proficient in antioxidant defense, which is presumably responsible for the low frequency of oxidative DNA lesions in both the mitochondrial and nuclear genome. Notably, previous non-quantitative proteomic studies revealed an abundant expression of antioxidant enzymes, in particular several peroxiredoxins in human ESCs (Baharvand et al., 2006). Furthermore, ESC cultures generate fewer ROS than most somatic cell types, because of their lower reliance on oxidative phosphorylation and limited mitochondrial biogenesis (Prigione et al., 2010; Armstrong et al., 2010). Structural analyses of mitochondria in human ESCs demonstrated an immature network characterized by few organelles with poorly developed cristae, which however increase during differentiation (St John et al., 2006; Facucho-Oliveira et al., 2007). Thus, stem cells maintain low ROS levels not only by high antioxidant activity, but also by reduced oxygen consumption and low mitochondrial biogenesis. Our PCR-based LORD-Q method enabled us to specifically assess not only nuclear but also mtDNA lesions. Several lines of evidence suggest that, in particular, the occurrence of mtDNA lesions must be prevented for the maintenance of pluripotency. For instance, studies in a mouse model with high levels of mtDNA mutations due to a proof-reading defect of DNA polymerase γ (mtDNA mutator mice) established causal relationships among the accumulation of mtDNA mutations, stem cell exhaustion, and premature aging (reviewed in Baines et al., 2014). In our study, we found that the level of GSH, the most important cellular antioxidant, was elevated up to 4-fold in iPSCs compared to somatic fibroblasts. In addition, the mRNA levels of several peroxiredoxins, GSTs, and glutathione reductase were considerably increased. All these enzymes might be involved in antioxidant defense and detoxification, a finding reminiscent of the expression of aldehyde dehydrogenase-1, which is often used as marker of cancer stem cells (Ginestier et al., 2007). Most notable was our finding that expression of two GSH-dependent enzymes, GSTA2 and GPX2, was elevated more than 80,000- and 10,000-fold, respectively, compared to fibroblasts. We therefore investigated their possible contribution to the maintenance of genomic integrity. Our data show that overexpression of GPX2 in fibroblasts can significantly increase resistance to oxidative stress-induced DNA damage. Moreover, using RNAi, we found that knockdown of GPX2, but not GSTA2, could overcome DNA damage protection in iPSCs. However, combined depletion of GSH was required for sensitization to DNA damage, indicating that, in addition to GPX2, further antioxidant mechanisms might be involved in the protection against DNA damage in iPSCs. This assumption is supported by our finding that the expression of several antioxidant enzymes was strongly increased in iPSCs.