Neural stem cells exposed to BrdU lose their global DNA methylation and undergo astrocytic differentiation

Bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrdU) is a halogenated nucleotide of low toxicity commonly used to monitor DNA replication. It is considered a valuable tool for in vitro and in vivo studies, including the detection of the small population of neural stem cells (NSC) in the mammalian brain. Here, we show that NSC grown in self-renewing conditions in vitro, when exposed to BrdU, lose the expression of stem cell markers like Nestin, Sox2 and Pax6 and undergo glial differentiation, strongly up-regulating the astrocytic marker GFAP. The onset of GFAP expression in BrdU exposed NSC was paralleled by a reduced expression of key DNA methyltransferases (DNMT) and a rapid loss of global DNA CpG methylation, as we determined by our specially developed analytic assay. Remarkably, a known DNA demethylating compound, 5-aza-2′-deoxycytidine (Decitabine), had similar effect on demethylation and differentiation of NSC. Since our key findings apply also to NSC derived from murine forebrain, our observations strongly suggest more caution in BrdU uses in stem cells research. We also propose that BrdU and its related substances may also open new opportunities for differentiation therapy in oncology

Telomere transcription in Ageing

Telomeres, the ends of eukaryotic chromosomes, play a central role in the control of cellular senescence and organismal ageing and need to be protected in order to avoid being recognised as damaged DNA and activate DNA damage response pathways. Dysfunctional telomeres arise from critically short telomeres or altered telomere structures, which ultimately lead to replicative cellular senescence and chromosome instability: both hallmarks of ageing. The observation that telomeres are transcribed led to the discovery that telomeric transcripts play important roles in chromosome end protection and genome stability maintenance. Recent evidence indicates that particular long non-coding (nc)RNAs transcribed at telomeres, namely TElomeric Repeat-containing RNA (TERRA) and telomeric damage-induced long ncRNAs (tdilncRNA), play key roles in age-related pathways by actively orchestrating the mechanisms known to regulate telomere length, chromosome end protection and DNA damage signalling. Here, we provide a comprehensive overview of the telomere transcriptome, outlining how it functions as a regulatory platform with essential functions in safeguarding telomere integrity and stability. We next review emerging antisense oligonucleotides therapeutic strategies that target telomeric ncRNAs and discuss their potential for ameliorating ageing and age-related diseases. Altogether, this review provides insights on the biological relevance of telomere transcription mechanisms in human ageing physiology and pathology

NOTCH1 Inhibits Activation of ATM by Impairing the Formation of an ATM-FOXO3a-KAT5/Tip60 Complex

The DNA damage response (DDR) signal transduction pathway is responsible for sensing DNA damage and further relaying this signal into the cell. ATM is an apical DDR kinase that orchestrates the activation and the recruitment of downstream DDR factors to induce cell-cycle arrest and repair. We have previously shown that NOTCH1 inhibits ATM activation upon DNA damage, but the underlying mechanism remains unclear. Here, we show that NOTCH1 does not impair ATM recruitment to DNA double-strand breaks (DSBs). Rather, NOTCH1 prevents binding of FOXO3a and KAT5/Tip60 to ATM through a mechanism in which NOTCH1 competes with FOXO3a for ATM binding. Lack of FOXO3a binding to ATM leads to the loss of KAT5/Tip60 association with ATM. Moreover, expression of NOTCH1 or depletion of ATM impairs the formation of the FOXO3a-KAT5/Tip60 protein complex. Finally, we show that pharmacological induction of FOXO3a nuclear localization sensitizes NOTCH1-driven cancers to DNA-damage-induced cell death

Human replication protein Cdc6 is selectively cleaved by caspase 3 during apoptosis

In eukaryotes, the initiation of DNA replication involves the ordered assembly on chromatin of pre-replicative complexes (pre-RCs), including the origin recognition complex (ORC), Cdc6, Cdt1 and the minichromosome maintenance proteins (MCMs). In light of its indispensable role in the formation of pre-RCs, Cdc6 binding to chromatin represents a key step in the regulation of DNA replication and cell proliferation. Here, we study the human Cdc6 (HuCdc6) protein during programmed cell death (apoptosis). We find that HuCdc6, but not HuOrc2 (a member of the ORC) or HuMcm5 (one of the MCMs), is specifically cleaved in several human cell lines induced to undergo apoptosis by a variety of stimuli. Expression of caspase-uncleavable mutant HuCdc6 attenuates apoptosis, delaying cell death. Therefore, an important function for cleavage of HuCdc6 is to prevent a wounded cell from replicating and to facilitate death

Stable Cellular Senescence Is Associated with Persistent DDR Activation

<div><p>The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged <i>in vitro</i> culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability <i>in vitro,</i> due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.</p></div

DDR is detectable months after establishment of telomere-initiated cellular senescence.

<p><b>a.</b> In BJ cells, DDR, in the form of γH2AX foci, is detectable long time (up to three months) after establishment of telomere-initiated cellular senescence. Scale bar, 40 µm. <b>b.</b> Bar graph shows the fraction of γH2AX foci-positive cells ± s.e.m. before (young and pre-sen) and at the indicated time points after senescence establishment. Cells were considered positive if bearing more than 3 DDR foci (*** p-value <0.001). <b>c.</b> Bar graph shows the average number of γH2AX foci ± s.e.m. per cell at the indicated time points before and after senescence establishment (*** p-value <0.001; * p-value <0.05).</p

Telomere-initiated senescent cells retain active DDR foci for years after senescence establishment.

<p><b>a.</b> DDR, in the form of ATM pS1981 foci co-localizing with 53BP1 and γH2AX foci, is detectable three years after senescence establishment. Scale bar, 10 µm. Below, bar graphs show the percentage of cells positive ± s.e.m. for the indicated DDR markers, in senescent (sen), early passage proliferating (prol) or telomerized proliferating (tel) skin fibroblasts from two independent centenarian donors (cen2 and cen3). Cells were considered positive if bearing more than 3 DDR foci (*** p-value <0.001). <b>b.</b> SA-β-gal staining of the two batches, cen2 and cen3, is shown together with the percentage of BrdU-positive cells. Scale bar, 100 µm.</p

Apparent differential DNA damage response activation during IR-induced cellular senescence correlates with cell survival.

<p><b>a–b.</b> BJ and IMR-90 cells were irradiated with 20 Gy and stained for 53BP1, as a DDR marker, three, ten and thirty days later. Bar graphs show the quantification of 53BP1-positive cells and number of 53BP1 foci per cell ± s.e.m. Cells were considered positive if bearing more than 3 DDR foci (*** p-value <0.001). <b>c.</b> BJ and IMR-90 cells were irradiated or not with 20 Gy. Graphs show the average cell number ± s.d. at different time points, in irradiated cells and non-irradiated controls (* p-value <0.05; ** p-value <0.01).</p