Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric non-coding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. Importantly, the cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, , suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.Cancer cells overcome replicative senescence by activating telomerase or the alternative lengthening of telomeres (ALT) pathway (1-3). ALT is used in ~5-15% of all human Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript cancers and is prevalent in specific cancer types, including osteosarcoma and glioblastoma (4). Currently, there are no therapies specifically targeting ALT. ALT relies on recombination to elongate telomeres (3), but how the recombinogenic state of ALT telomeres is established remains elusive. In contrast to cancer cells defective for homologous recombination (HR) and susceptible to Poly(ADP-ribose) polymerase (PARP) inhibition (5, 6), ALT-positive cells are HR-proficient (7). Thus, the reliance of ALT on recombination raises an important question as to whether recombination can be exploited in ALT-positive cancers as a means for targeted therapy.Single-stranded DNA (ssDNA) coated by replication protein A (RPA) is a key intermediate in both DNA replication and HR (8). RPA transiently associates with telomeres during DNA replication, but is released from telomeres after S phase (9, 10). The release of RPA may be an important mechanism to suppress HR at telomeres. The association of RPA with telomeres in S phase is facilitated by TERRA, the telomere repeat-containing RNA, which is also present at telomeres during this period (9,(11)(12)(13). To investigate how ALT is established, we determined whether the association of TERRA with telomeres is altered in ALT cells. TERRA colocalized with the telomere-binding protein TRF2 in telomerasepositive HeLa cervical cancer cells ( fig. S1) (9). However, in both HeLa and telomerasepositive SJSA1 osteosarcoma cells ( fig. S24B), the number of TERRA foci declined from S phase to G2 ( Considering that RPA is released from telomeres in G2/M when TERRA is repressed by ATRX (9), we examined whether ATRX is required for the release of RPA. In HeLa cells, numerous small replication-associated RPA foci (type-A RPA foci) were detected in S phase (Fig. S7). As cells progressed from S to ...

The ADP ribosyl transferase [poly(ADP-ribose) polymerase] ARTD3 (PARP3) is a newly characterized member of the ARTD(PARP) family that catalyzes the reaction of ADP ribosylation, a key posttranslational modification of proteins involved in different signaling pathways from DNA damage to energy metabolism and organismal memory. This enzyme shares high structural similarities with the DNA repair enzymes PARP1 and PARP2 and accordingly has been found to catalyse poly(ADP ribose) synthesis. However, relatively little is known about its in vivo cellular properties. By combining biochemical studies with the generation and characterization of loss-of-function human and mouse models, we describe PARP3 as a newcomer in genome integrity and mitotic progression. We report a particular role of PARP3 in cellular response to doublestrand breaks, most likely in concert with PARP1. We identify PARP3 as a critical player in the stabilization of the mitotic spindle and in telomere integrity notably by associating and regulating the mitotic components NuMA and tankyrase 1. Both functions open stimulating prospects for specifically targeting PARP3 in cancer therapy. mitotic division | poly(ADP ribosyl)ation | double-strand break repair P oly(ADP ribosyl)ation is a posttranslational modification of proteins mediated by poly(ADP ribose) polymerases (PARPs). PARPs catalyze the transfer and polymerization of ADP ribose units from NAD + to form branched polymers of ADP ribose covalently linked to heterologous acceptor proteins or PARPs themselves. PARP1, the founding and best-studied member of the PARP family, was for a long time considered to be the only enzyme that could generate poly(ADP ribose) polymers. However,

Telomerase represents a relevant target for cancer therapy. Molecules able to stabilize the G-quadruplex (G4), a structure adopted by the 3 0 -overhang of telomeres, are thought to inhibit telomerase by blocking its access to telomeres. We investigated the cellular effects of four new 2,6-pyridine-dicarboxamide derivatives displaying strong selectivity for G4 structures and strong inhibition of telomerase in in vitro assays. These compounds inhibited cell proliferation at very low concentrations and then induced a massive apoptosis within a few days in a dosedependent manner in cultures of three telomerase-positive glioma cell lines, T98G, CB193 and U118-MG. They had also antiproliferative effects in SAOS-2, a cell line in which telomere maintenance involves an alternative lengthening of telomeres (ALT) mechanism. We show that apoptosis was preceded by multiple alterations of the cell cycle: activation of S-phase checkpoints, dramatic increase of metaphase duration and cytokinesis defects. These effects were not associated with telomere shortening, but they were directly related to telomere instability involving telomere end fusion and anaphase bridge formation. Pyridine-based G-quadruplex ligands are therefore promising agents for the treatment of various tumors including malignant gliomas.

BackgroundIn mammals, new neurons are added to the olfactory bulb (OB) throughout life. Most of these new neurons, granule and periglomerular cells originate from the subventricular zone (SVZ) lining the lateral ventricles and migrate via the rostral migratory stream toward the OB. Thousands of new neurons appear each day, but the function of this ongoing neurogenesis remains unclear.Methodology/Principal FindingsIn this study, we irradiated adult mice to impair constitutive OB neurogenesis, and explored the functional impacts of this irradiation on the sense of smell. We found that focal irradiation of the SVZ greatly decreased the rate of production of new OB neurons, leaving other brain areas intact. This effect persisted for up to seven months after exposure to 15 Gray. Despite this robust impairment, the thresholds for detecting pure odorant molecules and short-term olfactory memory were not affected by irradiation. Similarly, the ability to distinguish between odorant molecules and the odorant-guided social behavior of irradiated mice were not affected by the decrease in the number of new neurons. Only long-term olfactory memory was found to be sensitive to SVZ irradiation.Conclusion/SignificanceThese findings suggest that the continuous production of adult-generated neurons is involved in consolidating or restituting long-lasting olfactory traces.

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, 3H-360A (2,6-N,N′-methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-3H]. We verified the in vitro selectivity of 3H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that 3H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3′-overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with 3H-360A. We found that 3H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.

Neurogenesis decreases during aging and following cranial radiotherapy, causing a progressive cognitive decline that is currently untreatable. However, functional neural stem cells remained present in the subventricular zone of high dose-irradiated and aged mouse brains. We therefore investigated whether alterations in the neurogenic niches are perhaps responsible for the neurogenesis decline. This hypothesis was supported by the absence of proliferation of neural stem cells that were engrafted into the vascular niches of irradiated host brains. Moreover, we observed a marked increase in TGF-β1 production by endothelial cells in the stem cell niche in both middle-aged and irradiated mice. In co-cultures, irradiated brain endothelial cells induced the apoptosis of neural stem/progenitor cells via TGF-β/Smad3 signalling. Strikingly, the blockade of TGF-β signalling in vivo using a neutralizing antibody or the selective inhibitor SB-505124 significantly improved neurogenesis in aged and irradiated mice, prevented apoptosis and increased the proliferation of neural stem/progenitor cells. These findings suggest that anti-TGF-β-based therapy may be used for future interventions to prevent neurogenic collapse following radiotherapy or during aging.

The repair of DNA double-stranded breaks (DNAdsb) through non-homologous end joining (NHEJ) is a prerequisite for the proper development of the central nervous system and the adaptive immune system. Yet, mice with Xlf or PAXX loss of function are viable and present with very mild immune phenotypes, although their lymphoid cells are sensitive to ionizing radiation attesting for the role of these factors in NHEJ. In contrast, we show here that mice defective for both Xlf and PAXX are embryonically lethal owing to a massive apoptosis of post-mitotic neurons, a situation reminiscent to XRCC4 or DNA Ligase IV KO conditions. The development of the adaptive immune system in Xlf − / − PAXX −/− E18.5 embryos is severely affected with the block of B-and T-cell maturation at the stage of IgH and TCRβ gene rearrangements, respectively. This damaging phenotype highlights the functional nexus between Xlf and PAXX, which is critical for the completion of NHEJ-dependent mechanisms during mouse development. Cell Death and Differentiation (2018) 25, 444-452; doi:10.1038/cdd.2017; published online 27 October 2017All living organisms are subjected to multitude sources of DNA damage during their lifespan, either as a result of external assault or endogenous physiological processes.1 Among endogenous sources of physiological DNAdsb is the somatic rearrangement of immunoglobulin (Ig) and TCR genes in B and T lymphocytes, respectively, during the diversification of the adaptive immune system through V(D)J recombination.2 DNA double-stranded breaks (DNAdsb) are considered the most toxic lesions. DNAdsbs are repaired by two main mechanisms: the homologous recombination (HR) in cycling cells, when a sister chromatid is available as DNA repair template, and the non-homologous end joining (NHEJ) during all phases of the cell cycle.NHEJ proceeds via the simple religation of DNA ends without the need for a repair template.3 Briefly, the NHEJ is composed of seven core factors comprising the Ku70/80/ DNA-PKcs (DNA-dependent protein kinase catalytic subunit) complex, which recognizes and protects the broken DNA ends, the Artemis endo/exonuclease, which participates, when needed, in processing the DNA ends and the XRCC4/ DNA-Ligase IV/Xlf complex, which ultimately reseals the DNA break. The critical function of the NHEJ apparatus in various aspects of higher eukaryote development has been extensively perceived in several animal and human pathological conditions. As emblematic examples, loss of function of either XRCC4 or DNA ligase IV results in embryonic lethality in mice 4,5 and mutations in Artemis or DNA-PKcs result in severe combined immunodeficiency conditions in both men and mice, owing to aborted V(D)J recombination. 6 In addition, defects in NHEJ results in genetic instability and the propensity to develop various types of cancers, notably leukemia and lymphomas. Recently, a new DNA repair factor, PAXX (PAralog of XRCC4 and Xlf, also known as C9orf142 or XLS), has been identified independently by three laboratories based on bioinformatic...

Chemokines are key mediators of the selective migration of leukocytes that occurs in neurodegenerative diseases and related inflammatory processes. Astrocytes, the most abundant cell type in the CNS, have an active role in brain inflammation. To ascertain the role of astrocytes during neuropathological processes, we have investigated in two models of primary cells (human fetal and simian adult astrocytes) the repertoire of chemokines and their receptors expressed in response to inflammatory stimuli. We demonstrated that, in the absence of any stimulation, human fetal and simian adult astrocytes express mRNA for receptors APJ, BOB/GPR15, Bonzo/CXCR6, CCR2, CCR3, CCR5, CCR8, ChemR23, CXCR3/GPR9, CXCR4, GPR1, and V28/CX3CR1. Moreover, TNFalpha and IL-1beta significantly increase BOB/GPR15, CCR2, and V28/CX3CR1 mRNA levels in both models. Furthermore, TNFalpha and IFNgamma act synergistically to induce expression of the major coreceptors for HIV infection, CXCR4 and CCR5, at both the mRNA and protein levels in human and simian astrocytes, whereas CCR3 expression was not affected by cytokine treatment. Finally, TNFalpha/IFNgamma was the most significant cytokine combination in leading to a pronounced upregulation in a comparable, time-dependent manner of the production of chemokines IP-10/CXCL10, RANTES/CCL5, MIG/CXCL9, MCP-1/CCL2, and IL-8/CXCL8. In summary, these data suggest that astrocytes serve as an important source of chemokines under the dependence of a complex cytokine regulation, and TNFalpha and IFNgamma are important modulators of chemokines and chemokine receptor expression in human as well as simian astrocytes. Finally, with the conditions we used, there was no difference between species or age of tissue.