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    • br Materials and methods br Statistical analysis

    2018-11-08


    Materials and methods
    Statistical analysis Data are presented as mean±standard error of the mean (SEM) of at least three independent experiments. Comparisons were performed using a two-tail unpaired t-test or a one sample t-test (real time PCR).
    Results
    Discussion In this work we have in vitro expanded BMSCs and ASCs under distinct oxygen tensions and PP, to specifically evaluate and compare their DNA repair machineries as well as effects on genomic stability and mitochondrial performance. First, our gene sphingosine 1 phosphate receptor analysis suggests a gradual decline of DNA repair capacity over time, which may lead to a concerted decrease in stem cell function, emergence of genomic instability, and activation of apoptosis signaling pathways. Previous work has shown that the two HR genes BRCA1 and RAD51 are co-repressed under hypoxia through binding of E2F4/p130 complexes to their upstream promoters (Bindra et al., 2005; Bindra and Glazer, 2007b). In the case of the MMR gene MLH1, its transcriptional down-regulation under hypoxia has been shown to be associated with deacetylation and trimethylation of histone H3 on lysine 9, thus impairing binding of the SP1 transcriptional factor at the corresponding promoter region (Rodriguez-Jimenez et al., 2008). Regarding the expression of the NHEJ gene Ku80, conflicting results obtained using several cancer cell lines are reported in the literature. Meng et al. (2005) showed repression of several NHEJ gene family members following chronic hypoxia (72h at 0.2% O2). In another study, no change in Ku80 expression was detected after 48h at 0.01% O2 (Bindra et al., 2005). Concerning the tumor supressor p53 and the proto-oncogene c-MYC, they were found to be down-regulated after PP. The fact that p53 mRNA levels may decrease or remain unaltered after PP is not necessarily at odds with its role in activating senescence, since in some cases, up-regulation is only observed at the protein level (Izadpanah et al., 2008). Another study presents contradicting data, in the sense that it refers to the absence of significant changes in p53 at both mRNA and protein levels after prolonged in vitro propagation of human SCs (Noh et al., 2010). These discrepancies may in part be due to different experimental conditions (e.g. culture medium), or to the use of heterogeneous cell populations that are more or less prone to enter a senescent stage. An example found in BMSCs is the link between donor age and increasing p53 activity (Zhou et al., 2008), which supports the view that in vivo aging directly affects the proliferative and differentiative potential of cells in vitro. In the case of c-MYC, its expression levels have been reported to decrease during the stage of pre-senescence, increasing again as cells move towards a senescent, post-senescent or eventually to a transformed state (Rubio et al., 2008). Since all BMSCs and ASCs analyzed in this study showed evidence of entering a pre-senescence/senescence phase between P10 and P20 (in agreement with data from other studies (Bernardo et al., 2007)), the c-MYC down-regulation observed in these cells comes as no surprise. We also show that down-regulation of DNA repair genes under hypoxia favoured the onset of MSI, particularly within D5S346, D17S250 and D2S123. These findings are consistent with those of several groups who have reported preferential instability in these loci in a wide range of MSI-L cancer cell lines (Murphy et al., 2006; Deschoolmeester et al., 2008). This bias has led to the supposition that MSI-L and MSI-H are distinct entities, which seem to arise from distinct biological processes (Murphy et al., 2006). Our MSI analysis supports this theory and further extends it to human SCs, suggesting that the onset of the MSI-L phenomenon might follow a common pathway. Others have found that lowering the O2 percentage to 1% apparently results in an earlier onset and increase of MSI in neural stem cells and SCs, both using the Bethesda panel, as well as in other markers (Rodriguez-Jimenez et al., 2008). This fact is suggestive of an even more pronounced repression of the MMR system, and an indicator that excessively low O2 tensions should be avoided, as they might be detrimental for genetic stability.