Telomeres in cancer

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Using the Xenopus egg extract system, we investigated the involvement of DNA replication in activation of the DNA damage checkpoint. We show here that DNA damage slows replication in a checkpoint-independent manner and is accompanied by replication-dependent recruitment of ATR and Rad1 to chromatin. We also find that the replication proteins RPA and Polalpha accumulate on chromatin following DNA damage. Finally, damage-induced Chk1 phosphorylation and checkpoint arrest are abrogated when replication is inhibited. These data indicate that replication is required for activation of the DNA damage checkpoint and suggest a unifying model for ATR activation by diverse lesions during S phase.

PIG-a Gene Expression Deficiency Association with Reduced DNA Damage Checkpoint Response and Activation

DNA-damaging therapies represent the most frequently used non-surgical anticancer strategies in the treatment of human tumors. These therapies can kill tumor cells, but at the same time they can be particularly damaging and mutagenic to healthy tissues. The efficacy of DNA-damaging treatments can be improved if tumor cell death is selectively enhanced, and the recent application of poly-(ADP-ribose) polymerase inhibitors in BRCA1/2-deficient tumors is a successful example of this. DNA damage is known to trigger cell-cycle arrest through activation of DNA-damage checkpoints. This arrest can be reversed once the damage has been repaired, but irreparable damage can promote apoptosis or senescence. Alternatively, cells can reenter the cell cycle before repair has been completed, giving rise to mutations. In this review we discuss the mechanisms involved in the activation and inactivation of DNA-damage checkpoints, and how the transition from arrest and cell-cycle re-entry is controlled. In addition, we discuss recent attempts to target the checkpoint in anticancer strategies.

Photo-aged polylactic acid microplastics causes severe transgenerational decline in reproductive capacity in C. elegans: Insight into activation of DNA damage checkpoints affected by multiple germline histone methyltransferases.

Glioblastomas (GBMs) are highly lethal brain tumours with current therapies limited to palliation due to therapeutic resistance. We previously demonstrated that GBM stem cells (GSCs) display a preferential activation of DNA damage checkpoint and are relatively resistant to radiation. However, the molecular mechanisms underlying the preferential checkpoint response in GSCs remain undefined. Here, we show that L1CAM (CD171) regulates DNA damage checkpoint responses and radiosensitivity of GSCs through nuclear translocation of L1CAM intracellular domain (L1-ICD). Targeting L1CAM by RNA interference attenuated DNA damage checkpoint activation and repair, and sensitized GSCs to radiation. L1CAM regulates expression of NBS1, a critical component of the MRE11-RAD50-NBS1 (MRN) complex that activates ataxia telangiectasia mutated (ATM) kinase and early checkpoint response. Ectopic expression of NBS1 in GSCs rescued the decreased checkpoint activation and radioresistance caused by L1CAM knockdown, demonstrating that L1CAM signals through NBS1 to regulate DNA damage checkpoint responses. Mechanistically, nuclear translocation of L1-ICD mediates NBS1 upregulation via c-Myc. These data demonstrate that L1CAM augments DNA damage checkpoint activation and radioresistance of GSCs through L1-ICD-mediated NBS1 upregulation and the enhanced MRN-ATM-Chk2 signalling.

BackgroundChronic kidney disease (CKD) is highly prevalent worldwide, and its global burden is substantial and growing. CKD displays a number of features of accelerated senescence. Tubular cell senescence is a common biological process that contributes to CKD progression. Tubulointerstitial inflammation is a driver of tubular cell senescence and a common characteristic of CKD. However, the mechanism by which the interstitial inflammation drives tubular cell senescence remains unclear. This paper aims to explore the role of exosomal miRNAs derived from macrophages in the development of tubular cell senescence.MethodsAmong the identified inflammation-related miRNAs, miR-155 is considered to be one of the most important miRNAs involved in the inflammatory response. Macrophages, the primary immune cells that mediate inflammatory processes, contain a high abundance of miR-155 in their released exosomes. We assessed the potential role of miR-155 in tubular cell senescence and renal fibrosis. We subjected miR-155−/− mice and wild-type controls, as well as tubular epithelial cells (TECs), to angiotensin II (AngII)-induced kidney injury. We assessed kidney function and injury using standard techniques. TECs were evaluated for cell senescence and telomere dysfunction in vivo and in vitro. Telomeres were measured by the fluorescence in situ hybridization.ResultsCompared with normal controls, miR-155 was up-regulated in proximal renal tubule cells in CKD patients and mouse models of CKD. Moreover, the expression of miR-155 was positively correlated with the extent of renal fibrosis, eGFR decline and p16INK4A expression. The overexpression of miR-155 exacerbated tubular senescence, evidenced by increased detection of p16INK4A/p21expression and senescence-associated β-galactosidase activity. Notably, miR-155 knockout attenuates renal fibrosis and tubule cell senescence in vivo. Interestingly, once released, macrophages-derived exosomal miR-155 was internalized by TECs, leading to telomere shortening and dysfunction through targeting TRF1. A dual-luciferase reporter assay confirmed that TRF1 was the direct target of miR-155. Thus, our study clearly demonstrates that exosomal miR-155 may mediate communication between macrophages and TECs, subsequently inducing telomere dysfunction and senescence in TECs.ConclusionsOur work suggests a new mechanism by which macrophage exosomes are involved in the development of tubule senescence and renal fibrosis, in part by delivering miR-155 to target TRF1 to promote telomere dysfunction. Our study may provide novel strategies for the treatment of AngII-induced kidney injury.

Tankyrase (TANK1) is a human telomere-associated poly(ADP-ribose) polymerase (PARP) that binds the telomere-binding protein TRF1 and increases telomere length when overexpressed. Here we report characterization of a second human tankyrase, tankyrase 2 (TANK2), which can also interact with TRF1 but has properties distinct from those of TANK1. TANK2 is encoded by a 66-kilobase pair gene (TNKS2) containing 28 exons, which express a 6.7-kilobase pair mRNA and a 1166-amino acid protein. The protein shares 85% amino acid identity with TANK1 in the ankyrin repeat, sterile alpha-motif, and PARP catalytic domains but has a unique N-terminal domain, which is conserved in the murine TNKS2 gene. TANK2 interacted with TRF1 in yeast and in vitro and localized predominantly to a perinuclear region, similar to the properties of TANK1. In contrast to TANK1, however, TANK2 caused rapid cell death when highly overexpressed. TANK2-induced death featured loss of mitochondrial membrane potential, but not PARP1 cleavage, suggesting that TANK2 kills cells by necrosis. The cell death was prevented by the PARP inhibitor 3-aminobenzamide. In vivo, TANK2 may differ from TANK1 in its intrinsic or regulated PARP activity or its substrate specificity.

Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs) can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR) and DSB repair (DSBR) within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to γ-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline.

<div>Abstract<p>Histone deacetylase (HDAC) inhibitors potently inhibit tumor growth and are currently being evaluated for their efficacy as chemosensitizers and radiosensitizers. This efficacy is likely to be limited by the fact that HDAC inhibitors also induce cell cycle arrest. Deletion of the class I HDAC Rpd3 has been shown to specifically suppress the sensitivity of <i>Saccharomyces cerevisiae</i> DNA damage checkpoint mutants to UV and hydroxyurea. We show that in the fission yeast <i>Schizosaccharomyces pombe</i>, inhibition of the homologous class I HDAC specifically suppresses the DNA damage sensitivity of checkpoint mutants. Importantly, the prototype HDAC inhibitor Trichostatin A also suppressed the sensitivity of DNA damage checkpoint but not of DNA repair mutants to UV and HU. TSA suppressed DNA damage activity independently of the mitogen-activated protein kinase–dependent and spindle checkpoint pathways. We show that TSA delays progression into mitosis and propose that this is the main mechanism for suppression of the DNA damage sensitivity of <i>S. pombe</i> checkpoint mutants, partially compensating for the loss of the G<sub>2</sub> checkpoint pathway. Our studies also show that the ability of HDAC inhibitors to suppress DNA damage sensitivity is not species specific. Class I HDACs are the major target of HDAC inhibitors and cancer cells are often defective in checkpoint activation. Effective use of these agents as chemosensitizers and radiosensitizers may require specific treatment schedules that circumvent their inhibition of cell cycle progression. [Mol Cancer Ther 2009;8(9):2606–15]</p></div>

Rat sarcoma virus (RAS)-induced tumorigenesis has been suggested to follow a three-stage model consisting of an initial RAS activation, senescence induction, and evasion of p53-dependent senescence checkpoints. While reactive oxygen species act as second messengers in RAS-induced senescence, they are also involved in oncogenic transformation by inducing proliferation and promoting mutations. In the current work, we investigated the role of extracellular superoxide dismutase (SOD3) in RAS-induced senescence and immortalization in vitro and in vivo. We used a mouse embryonic fibroblast (MEF) primary cell model along with immortalized and transformed human cell lines derived from papillary and anaplastic thyroid cancer. Based on our data, sod3 RNA interference in H-RasV12-transduced cells markedly inhibited cell growth, while sod3 over-expression in MEFs initially caused a proliferative burst followed by the activation of DNA damage checkpoints, induction of p53-p21 signal transduction, and senescence. Subsequently, sod3-transduced MEF cells developed co-operative p21-p16 down-regulation and acquired transformed cell characteristics such as increased telomerase activity, loss of contact inhibition, growth in low-nutrient conditions, and in vivo tumorigenesis. Interestingly, as previously reported with RAS, we showed a dose-dependent response to SOD3 in vitro and in vivo involving transcriptional and non-transcriptional regulatory mechanisms. SOD3 may mediate H-RasV12-induced initiation of primary cell immortalization. Our results indicate that SOD3 influences growth signaling in primary and cancer cells downstream of the ras oncogene and could serve as a therapy target at an early tumorigenesis phase.

<div>Abstract<p><b>Purpose:</b> Checkpoint kinase 1 (Chk1) plays a critical role in the activation of mitotic spindle checkpoint and DNA damage checkpoint. We examined the preclinical use of the Chk1 inhibitor PF-00477736 as a docetaxel-sensitizing agent. Specifically, we investigated the correlation between PF-00477736–mediated modulation of biomarkers and the sensitization of docetaxel efficacy.</p><p><b>Experimental Design:</b><i>In vitro</i> and <i>in vivo</i> studies using COLO205 and other cell lines were done to assess PF-00477736–induced enhancement of docetaxel efficacy and effects on associated biomarkers.</p><p><b>Results:</b> PF-00477736 significantly enhanced the docetaxel-induced efficacy in tumor cells and xenografts. Docetaxel induced dose- and time-dependent increase in the levels of phosphorylated Chk1 (Ser<sup>345</sup>), phosphorylated histone H3 (Ser<sup>10</sup>), and γH2AX foci and promoted the cytoplasmic localization of phosphorylated Cdc25C (Ser<sup>216</sup>). PF-00477736 cotreatment suppressed docetaxel-induced changes in phosphorylated histone H3 and cytoplasmic phosphorylated Cdc25C (Ser<sup>216</sup>) levels and concurrently sensitized the docetaxel-induced apoptosis. Docetaxel alone or in combination with PF-00477736 induced significant antiproliferative activity in xenografts, shown via [<sup>18</sup>F]FLT-PET imaging. However, changes in [<sup>18</sup>F]FLT uptake did not reflect the potentiation of docetaxel efficacy. In contrast, bioluminescence imaging showed that PF-00477736 sensitized docetaxel-induced suppression of tumor survival.</p><p><b>Conclusions:</b> Docetaxel triggers mitotic spindle checkpoint activation at low concentrations and activates both the DNA damage checkpoint and the spindle checkpoint at high concentrations. In combination with docetaxel, PF-00477736 abrogates the mitotic checkpoint, as well as the DNA damage checkpoint, and results in sensitization to docetaxel. Chk1 inhibitor PF-00477736 offers a therapeutic potential for the enhancement of taxane therapy.</p></div>

Centrosome amplification and telomere shortening, which are commonly detected in human cancers, have been implicated in the induction of chromosome instability in tumorigenesis. The functions of these two structures are closely related to DNA damage repair machinery, and some factors that operate in the maintenance of telomeres also take part in the regulation of centrosome status, suggesting they are functionally linked. We report that TEIF (telomerase transcriptional elements-interacting factor), a transactivator of the hTERT (human telomerase reverse transcriptase subunit) gene, is distributed in the centrosome throughout the cell cycle, but its transport into the centrosome is increased under some conditions, and its distribution is dependent on its C-terminal domain. Experimental modulation of TEIF expression through overexpression, polypeptide expression or depletion affected centrosome status and increased abnormalities of cell mitosis. Localization of TEIF to the centrosome was also stimulated by treatment with genotoxic agents and experimental telomere dysfunction, accompanying centrosome amplification. Moreover, we demonstrated that the expression level of TEIF is not only closely correlated with centrosome amplification in soft tissue sarcomas but it is also significantly related to tumor histologic grade. Our data confirmed TEIF functions as a centrosome regulator. Its participation in DNA damage response, including telomere dysfunction and tumorigenesis, indicates TEIF is likely to be a factor involved in linking centrosome amplification and telomere dysfunction in cancer development.

Telomere shortening limits the proliferation of primary human cells in culture by the induction of senescence. There is evidence for telomere shortening in most human tissues during aging and chronic disease. In addition, telomerase mutations are associated with shortened telomeres, impaired organ maintenance, and a shortened lifespan in humans. Studies on telomerase knockout mice have shown that telomere shortening predominantly affects maintenance of organ systems with high rates of cell turnover, indicating that telomere function is important for organs that are constantly replenished from stem cells. These studies also showed that telomere dysfunction induces different cellular outcomes in vivo including cell cycle arrest, apoptosis, and impairment of stem cell function. However, mice with dysfunctional telomeres did not exhibit an accumulation of senescent cells, indicating that, in contrast to in vitro cultures, senescent cells do not persist in vivo in mice. In this chapter, we summarize our current knowledge on consequences of telomere dysfunction on stem cells and tissue aging.

The relationship between cancer and DNA damage is intimate and multifaceted. Damage to DNA can cause cancer, major treatment modalities including radiation therapy and chemotherapy can cure or slow down cancer through their genotoxic effects, and germline and somatic defects in the DNA damage response (DDR) machinery may predispose to or promote tumorigenesis, respectively (1, 2). Furthermore, replication stress with the ensuing endogenous DNA damage elicited by oncogenes in nascent tumor cells leads to activation of DNA damage checkpoints and cellular senescence or cell death, thereby providing an intrinsic biological barrier against tumor progression (3–5). The PNAS article by Redon et al. (6) adds yet another dimension to the interplay between DNA damage and cancer: a surprising discovery that cancer can elicit long-range signals that evoke serious types of DNA damage in otherwise healthy tissues in various parts of the body distant to sites of the growing tumor.