Induces Telomere Dysfunction Research Articles

Renal fibroblasts are involved in fibrogenic changes in kidney fibrosis associated with dysfunctional telomeres.

Tubulointerstitial fibrosis associated with chronic kidney disease (CKD) represents a global health care problem. We previously reported that short and dysfunctional telomeres lead to interstitial renal fibrosis; however, the cell-of-origin of kidney fibrosis associated with telomere dysfunction is currently unknown. We induced telomere dysfunction by deleting the Trf1 gene encoding a telomere-binding factor specifically in renal fibroblasts in both short-term and long-term life-long experiments in mice to identify the role of fibroblasts in renal fibrosis. Short-term Trf1 deletion in renal fibroblasts was not sufficient to trigger kidney fibrosis but was sufficient to induce inflammatory responses, ECM deposition, cell cycle arrest, fibrogenesis, and vascular rarefaction. However, long-term persistent deletion of Trf1 in fibroblasts resulted in kidney fibrosis accompanied by an elevated urinary albumin-to-creatinine ratio (uACR) and a decrease in mouse survival. These cellular responses lead to the macrophage-to-myofibroblast transition (MMT), endothelial-to-mesenchymal transition (EndMT), and partial epithelial-to-mesenchymal transition (EMT), ultimately causing kidney fibrosis at the humane endpoint (HEP) when the deletion of Trf1 in fibroblasts is maintained throughout the lifespan of mice. Our findings contribute to a better understanding of the role of dysfunctional telomeres in the onset of the profibrotic alterations that lead to kidney fibrosis.

Inhibition of hTERT/telomerase/telomere mediates therapeutic efficacy of osimertinib in EGFR mutant lung cancer.

The inevitable acquired resistance to osimertinib (AZD9291), an FDA-approved third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) for the treatment of patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR activating or T790M resistant mutations, limits its long-term clinical benefit. Telomere maintenance via telomerase reactivation is linked to uncontrolled cell growth and is a cancer hallmark and an attractive cancer therapeutic target. Our effort toward understanding the action mechanisms, including resistance mechanisms, of osimertinib has led to the identification of a novel and critical role in maintaining c-Myc-dependent downregulation of hTERT, a catalytic subunit of telomerase, and subsequent inhibition of telomerase/telomere and induction of telomere dysfunction in mediating therapeutic efficacy of osimertinib. Consequently, osimertinib combined with the telomere inhibitor, 6-Thio-dG, which is currently tested in a phase II trial, effectively inhibited the growth of osimertinib-resistant tumors, regressed EGFRm NSCLC patient-derived xenografts, and delayed the emergence of acquired resistance to osimertinib, warranting clinical validation of this strategy to manage osimertinib acquired resistance.

5-aza-2′-deoxycytidine induces telomere dysfunction in breast cancer cells

AimsAzacitidine, a drug that epigenetically modifies DNA, is widely used to treat haematological malignancies. However, at low doses, it demethylates DNA, and as a result, can alter gene expression. In our previous publication, we showed that low doses of azacitidine induce telomere length elongation in breast cancer cells. In this study, we aim to identify the mechanisms which lead to telomere length increases. MethodsBreast cancer cell lines representing different molecular sub-types were exposed to 5-aza-2′-deoxycytidine (5-aza) in 2 and 3D cultures, followed by DNA, RNA, and protein extractions. Samples were then analysed for telomere length, DNA damage, telomerase, and ALT activity. ResultsWe show that treatment of the cell lines with 5-aza for 72 h induced DNA damage at the telomeres and increased ALT activity 3-fold. We also identified a gene, POLD3, which may be involved in the ALT activity seen after treatment. ConclusionOur results indicate that while 5-aza is a useful drug for treating haematological cancers, surviving cancer cells that have been exposed to lower doses of the drug may activate mechanisms such as ALT. This could lead to cancer cell survival and possible resistance to 5-aza clinically.

Macrophage-derived exosomes promote telomere fragility and senescence in tubular epithelial cells by delivering miR-155

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.

Targeting Telomere Dynamics as an Effective Approach for the Development of Cancer Therapeutics.

Telomere is a protective structure located at the end of chromosomes of eukaryotes, involved in maintaining the integrity and stability of the genome. Telomeres play an essential role in cancer progression; accordingly, targeting telomere dynamics emerges as an effective approach for the development of cancer therapeutics. Targeting telomere dynamics may work through multifaceted molecular mechanisms; those include the activation of anti-telomerase immune responses, shortening of telomere lengths, induction of telomere dysfunction and constitution of telomerase-responsive drug release systems. In this review, we summarize a wide variety of telomere dynamics-targeted agents in preclinical studies and clinical trials, and reveal their promising therapeutic potential in cancer therapy. As shown, telomere dynamics-active agents are effective as anti-cancer chemotherapeutics and immunotherapeutics. Notably, these agents may display efficacy against cancer stem cells, reducing cancer stem levels. Furthermore, these agents can be integrated with the capability of tumor-specific drug delivery by the constitution of related nanoparticles, antibody drug conjugates and HSA-based drugs.

TZAP overexpression induces telomere dysfunction and ALT-like activity in ATRX/DAXX-deficient cells

The appropriate regulation of telomere length homeostasis is crucial for the maintenance of genome integrity. The telomere-binding protein TZAP has been suggested to regulate telomere length by promoting t-circle and c-circle excisions through telomere trimming, yet the molecular mechanisms by which TZAP functions at telomeres are not understood. Using a system based on TZAP overexpression, we show that efficient TZAP recruitment to telomeres occurs in the context of open telomeric chromatin caused by loss of ATRX/DAXX independently of H3.3 deposition. Moreover, our data indicate that TZAP binding to telomeres induces telomere dysfunction and ALT-like activity, resulting in the generation of t-circles and c-circles in a Bloom-Topoisomerase IIIα-RMI1-RMI2 (BTR)-dependent manner.

Mammalian telomeric RNA (TERRA) can be translated to produce valine–arginine and glycine–leucine dipeptide repeat proteins

Mammalian telomeres consist of (TTAGGG)n repeats. Transcription of the C-rich strand generates a G-rich RNA, termed TERRA, containing G-quadruplex structures. Recent discoveries in several human nucleotide expansion diseases revealed that RNA transcripts containing long runs of 3 or 6 nt repeats which can form strong secondary structures can be translated in multiple frames to generate homopeptide or dipeptide repeat proteins, and multiple studies have shown them to be toxic in cells. We noted that the translation of TERRA would generate two dipeptide repeat proteins: highly charged repeating valine-arginine (VR)n and hydrophobic repeating glycine-leucine (GL)n. Here, we synthesized these two dipeptide proteins and raised polyclonal antibodies to VR. The VR dipeptide repeat protein binds nucleic acids and localizes strongly to replication forks in DNA. Both VR and GL form long 8-nm filaments with amyloid properties. Using labeled antibodies to VR and laser scanning confocal microscopy, threefold to fourfold more VR was observed in the nuclei of cell lines containing elevated TERRA as contrasted to a primary fibroblast line. Induction of telomere dysfunction via knockdown of TRF2 led to higher amounts of VR, and alteration of TERRA levels using a locked nucleic acid (LNA) GapmeR led to large nuclear VR aggregates. These observations suggest that telomeres, in particular in cells undergoing telomere dysfunction, may express two dipeptide repeat proteins with potentially strong biological properties.

Dietary selenium excess affected spermatogenesis via DNA damage and telomere-related cell senescence and apoptosis in mice.

Selenium (Se) is a vital microelement for spermatogenesis and male fertility. The aim of this study was to investigate the effects of Se on the male reproductive function and possible mechanisms. Fourty male mice were randomly divided into 0, 0.1, 0.3 and 0.9mg/kg Se supplementation groups and given with Se dietary intervention for 12 weeks. Our data showed that excessive Se intake damaged the tissue structure of testes and epididymides of the mice, resulting in decreased sperm quality and quantity. Moreover, excessive Se induced oxidative stress, causing DNA damage and activated DNA damage repair factors (Mre11/Rad50/Nbs1), and also disrupted telomere function by shortening telomere length and decreasing TERT expression. Se excess activated the senescence pathway p53/p21/p16, leading to germ cell senescence, and inhibited cell proliferation by suppressing the Sirt1/Foxo1/c-Myc pathway. All of this led to spermatogenic cell apoptosis, thereby causing a decrease of sperm quantity and quality. In conclusion, excessive Se caused reproductive toxicity via inducing telomere dysfunction due to DNA damage, leading to germ cellular senescence and apoptosis in the testes of male mice. Our research provide new proof to explain the underlying mechanism of male reproductive toxicity triggered by excessive Se intake.

Angelica sinensis extract induces telomere dysfunction, cell cycle arrest, and mitochondria-mediated apoptosis in human glioblastoma cells.

Glioblastoma (GB) is one of the most aggressive and malignant tumors of the central nervous system. Conventional treatment for GB requires surgical resection followed by radiotherapy combined with temozolomide chemotherapy; however, the median survival time is only 12-15 months. Angelica sinensis Radix (AS) is commonly used as a traditional medicinal herb or a food/dietary supplement in Asia, Europe, and North America. This study aimed to investigate the effect of AS-acetone extract (AS-A) on the progression of GB and the potential mechanisms underlying its effects. The results indicated that AS-A used in this study showed potency in growth inhibition of GB cells and reduction of telomerase activity. In addition, AS-A blocked the cell cycle at the G0/G1 phase by regulating the expression of p53 and p16. Furthermore, apoptotic morphology, such as chromatin condensation, DNA fragmentation, and apoptotic bodies, was observed in AS-A-treated cells, induced by the activation of the mitochondria-mediated pathway. In an animal study, AS-A reduced tumor volume and prolonged lifespans of mice, with no significant changes in body weight or obvious organ toxicity. This study confirmed the anticancer effects of AS-A by inhibiting cell proliferation, reducing telomerase activity, altering cell cycle progression, and inducing apoptosis. These findings suggest that AS-A has great potential for development as a novel agent or dietary supplement against GB.

Aging results in DNA damage and telomere dysfunction that is greater in endothelial versus vascular smooth muscle cells and is exacerbated in atheroprone regions.

Aging increases the risk of atherosclerotic cardiovascular disease which is associated with arterial senescence; however, the mechanisms responsible for the development of cellular senescence in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) remain elusive. Here, we study the effect of aging on arterial DNA damage and telomere dysfunction. Aging resulted in greater DNA damage in ECs than VSMCs. Further, telomere dysfunction-associated DNA damage foci (TAF: DNA damage signaling at telomeres) were elevated with aging in ECs but not VMSCs. Telomere length was modestly reduced in ECs with aging and not sufficient to induce telomere dysfunction. DNA damage and telomere dysfunction were greatest in atheroprone regions (aortic minor arch) versus non-atheroprone regions (thoracic aorta). Collectively, these data demonstrate that aging results in DNA damage and telomere dysfunction that is greater in ECs than VSMCs and elevated in atheroprone aortic regions.

Abstract 6228: APR-246, which restores p53 function, is highly active against alternative lengthening of telomere (ALT) cell lines and PDXs

Abstract Introduction: Most cancers proliferate by activating telomerase (TA+) while 10% of cancers utilize alternate lengthening of telomeres (ALT). ALT has been associated with resistance to DNA damaging agents, p53 loss-of-function (p53LOF), ATRX mutations, and very poor survival. ATM kinase, which activates functional p53, is constitutively activated in ALT cancers (Science Translational Medicine 18:eabd5750, 2021). We hypothesized that the constitutive activation of ATM kinase in ALT cancers would confer high sensitivity to pharmacological reactivation of p53 function. Methods: We used patient-derived ALT (telomeric DNA C-circle positive) and TA+ neuroblastoma, sarcoma, colorectal, and breast cancer cell lines and xenografts treated with the clinical-stage p53 reactivator eprenetapopt (APR-246) and irinotecan. In vitro cytotoxicity was assayed by DIMSCAN digital imaging microscopy, ATM activation by immunofluorescence microscopy, and protein expression by western blotting. Results: ALT p53LOF cell lines were significantly (p<0.05) less sensitive to DNA-damaging agents relative to TA+ p53LOF comparators. Constitutive phosphorylation (activation) of ATM kinase and the DNA damage marker 53BP1 were observed at telomeres in ALT but not TA+ cell lines. APR-246 induced p53 targets p21 and NOXA and was significantly more cytotoxic (p<0.001) for ALT relative to TA+ cell lines, regardless of TP53 status. Overexpression of p53 in a TP53-null ALT cell line increased sensitivity (p<0.0001) to APR-246. Knockdown of p53 or ATM kinase in ALT TP53 mutated and wild-type cell lines antagonized (p<0.0001) APR-246 activity. Induction of telomere dysfunction in a TA+ p53LOF neuroblastoma cell line using dominant-negative TRF2 (a shelterin protein that blocks ATM activation at telomeres) activated ATM and sensitized cells to APR-246 (p<0.01). APR-246 enhanced the cytotoxicity of irinotecan (as SN38) in ALT cell lines in vitro to a higher degree than in TA+ p53LOF cell lines (p<0.05). Single-agent APR-246 significantly (p<0.0001) increased event-free survival (EFS) of mice with ALT xenografts relative to controls. APR-246 enhanced (p<0.0001) the activity of irinotecan in 3 neuroblastoma, 2 rhabdomyosarcoma, 1 colorectal, and 1 triple-negative breast ALT xenograft models with most mice (47/56) achieving complete responses and an EFS of >100 days (45/56) compared to no complete responses (median EFS ~ 34 days) in mice treated with only irinotecan (p<0.0001). APR-246 + irinotecan had no significant effect relative to irinotecan alone (p=0.08) on EFS of mice with TA+ p53LOF xenografts (median EFS ~ 40 days). Conclusion: ALT cancers of a variety of histologies are highly resistant to DNA damaging agents, have p53LOF, and constitutive activation of ATM kinase, which confers high sensitivity to p53 reactivation by APR-246. APR-246 warrants clinical testing in patients with ALT cancers. Citation Format: Shawn J. Macha, Balakrishna Koneru, Trevor Burrow, Charles Zhu, Dzmitry Savitski, Jonas Nance, Kristyn McCoy, Cody Eslinger, C Patrick Reynolds. APR-246, which restores p53 function, is highly active against alternative lengthening of telomere (ALT) cell lines and PDXs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6228.

Enhanced radiosensitivity by 6-thio-dG via increasing telomere dysfunction and ataxia telangiectasia mutated inhibition in non-small cell lung cancer

ObjectiveTo investigate the radiosensitivity of 6-thio-dG and its underlying molecular mechanisms in non-small cell lung cancer (NSCLC). MethodsH1299 and A549 NSCLC cells were pretreated with 6-thio-dG for one week and then exposed to γ-irradiation. Cell proliferation and survival were quantified using clonogenic assays. DNA damage was assessed using immunofluorescence for γH2AX. Telomere dysfunction-induced foci analysis was performed by the co-localization of telomere signals (FISH) and γH2AX. Telomere fusion was defined as two telomere signals merged into one at the chromosome by immuno-FISH in metaphase spreads. Proteins related to the DNA damage response were detected using Western blot analysis. Apoptosis wasanalyzed using flow cytometry and Western blot. ResultsThe presence of 6-thio-dG increased the radio sensitivity of H1299 and A549 ​cells (P<0.05), but had no effect on the normal human lung fibroblast line, MRC5. 6-thio-dG pretreatment significantly reduced the clonogenic potential induced by γ-ray irradiation and aggravated genomic DNA and telomeric DNA damage (P<0.05). In addition, 6-thio-dG pretreatment effectively increased γ-ray irradiation induced telomere dysfunction (P<0.05), resulting in disruption of chromosome stability and inhibition of the ATM pathway, thereby impairing genomic DNA and telomeric DNA repair, which was closely associated with enhanced drug-mediated radiation-induced apoptosis. Conclusions6-thio-dG increases the radiosensitivity of NSCLC by inhibiting ATM and inducing telomere dysfunction, which can potentially be used as a strategy for radiotherapy for NSCLC.

A 1,10-phenanthroline derivative selectively targeting telomeric G-quadruplex induces cytoprotective autophagy, causing apoptosis of gastric cancer cells

AimsThis study aimed to evaluate the ability of compound 13d to induce autophagy and to promote apoptosis of tumor cells and its interaction mechanism. Materials and methodsUsing CCK-8 assay, transwell assay, fluorescence resonance energy transfer melting analysis (FRET), transmission electron microscopy, flow cytometry assay, immunofluorescence assay, Western blot analysis, and wound healing assay. Key findingsThe results indicated that compound 13d could induce autophagy and apoptosis of gastric cancer cells. Moreover, the findings of CCK-8 assay, colony formation, migration and invasion assay, and wound healing assay revealed that compound 13d would effectively inhibit cell proliferation, migration, and invasion. Its IC50 value is about 2.4 μM against gastric cancer cells, which is similar to positive drug‑platinum. 13d specific induction of telomere G-quadruplex formation was proved in extracellular FRET melting assay, and indirectly affected telomerase activity. G-quadruplex formation promoted cell apoptosis and autophagy. Upon incorporating the autophagy inhibitors 3-MA and HCQ, the expression of the autophagy marker protein LC3 was then checked, suggesting that the compound 13d influences the autophagy flux. Furthermore, knocking down the autophagy-related gene Atg5 to reduce the level of autophagy enhances the anti-tumor activity and increases apoptotic cells' proportion. Mechanistic experiments have shown that blocking the Akt/m-TOR signal pathway plays a crucial role in autophagy and G-quadruplex induced telomere dysfunction. DNA damage is the leading cause of autophagy. Compound 13d combined with autophagy inhibitor can inhibit tumor cells more effectively. SignificanceOur findings demonstrate that compound 13d as a telomeric G-quadruplex ligand induces Telomere dysfunction, DNA damage response, autophagy, and apoptosis in gastric cancer cells by blocking the Akt/m-TOR signaling pathway.

Oxidative Stress Induces Telomere Dysfunction and Shortening in Human Oocytes of Advanced Age Donors.

Research from the past decades provided strong evidence that in humans the pool of oocytes starts to decline already before the birth of a female individual, and from menarche to menopause the oocyte is exposed to different environmental stimuli. Since more and more women of the 21st century in developed countries wish to postpone the first pregnancy to their thirties, higher rates of miscarriage and chromosomal non-disjunction might occur. In oocytes of advanced maternal age, meaning above 35 years of age, characteristics such as chromosomal instabilities/abnormalities, spindle defects, decreased mitochondrial function and telomere shortening become more prevalent than in younger counterparts. Telomere attrition belongs to the so-called “hallmarks of aging” which are also relevant for the female germ-line cells. In oocytes, telomeres shorten with advancing maternal age due to the effects of reactive oxygen species and not upon replicative senescence, similar to how it is common in dividing cells.

Terpyridine platinum compounds induce telomere dysfunction and chromosome instability in cancer cells.

Telomerase/telomere-targeting therapy is a potentially promising approach for cancer treatment because even transient telomere dysfunction can induce chromosomal instability (CIN) and may be a barrier to tumor growth. We recently developed a dual-HAC (Human Artificial Chromosome) assay that enables identification and ranking of compounds that induce CIN as a result of telomere dysfunction. This assay is based on the use of two isogenic HT1080 cell lines, one carrying a linear HAC (containing telomeres) and the other carrying a circular HAC (lacking telomeres). Disruption of telomeres in response to drug treatment results in specific destabilization of the linear HAC. Results: In this study, we used the dual-HAC assay for the analysis of the platinum-derived G4 ligand Pt-tpy and five of its derivatives: Pt-cpym, Pt-vpym, Pt-ttpy, Pt(PA)-tpy, and Pt-BisQ. Our analysis revealed four compounds, Pt-tpy, Pt-ttpy, Pt-vpym and Pt-cpym, that induce a specific loss of a linear but not a circular HAC. Increased CIN after treatment by these compounds correlates with the induction of double-stranded breaks (DSBs) predominantly localized at telomeres and reflecting telomere-associated DNA damage. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges (CBs) in late mitosis and cytokinesis. These terpyridine platinum-derived G4 ligands are promising compounds for cancer treatment.

Pt-ttpy, a G-quadruplex binding platinum complex, induces telomere dysfunction and G-rich regions DNA damage.

Pt-ttpy (tolyl terpyridin-Pt complex) covalently binds to G-quadruplex (G4) structures in vitro and to telomeres in cellulo via its Pt moiety. Here, we identified its targets in the human genome, in comparison to Pt-tpy, its derivative without G4 affinity, and cisplatin. Pt-ttpy, but not Pt-tpy, induces the release of the shelterin protein TRF2 from telomeres concomitantly to the formation of DNA damage foci at telomeres but also at other chromosomal locations. γ-H2AX chromatin immunoprecipitation (ChIP-seq) after treatment with Pt-ttpy or cisplatin revealed accumulation in G- and A-rich tandemly repeated sequences, but not particularly in potential G4 forming sequences. Collectively, Pt-ttpy presents dual targeting efficiency on DNA, by inducing telomere dysfunction and genomic DNA damage at specific loci.

Telomere uncapping as a possible mechanism for chemotherapy‐induced vascular toxicity

Cardiovascular toxicity is one of the detrimental side effects of chemotherapy that limits treatment. Specifically, chemotherapeutics such as doxorubicin (DOXO) can induce endothelial dysfunction, including reduced endothelial nitric oxide production, cellular senescence, and changes in genes that regulate telomeres, all of which may contribute to toxicity. Telomeres are DNA–protein structures that cap chromosome ends. When telomeres are damaged, they become uncapped and recognized as damaged DNA leading to cellular senescence. Importantly, telomere dysfunction is a biomarker of aging and is associated with numerous diseases including cardiovascular disease. To examine the effects of chemotherapy on vascular endothelial cells (ECs), we used two common chemotherapeutics, DOXO and etoposide (ETOP), both of which act as topoisomerase II inhibitors. Moreover, DOXO intercalates with DNA base pairs resulting in DNA strand breaks. However, whether chemotherapeutics induce telomere dysfunction associated with senescence and, therefore, promote endothelial dysfunction remains elusive. Here, we test the hypothesis that DOXO and ETOP induce endothelial cell telomere uncapping using doses of DOXO (0.1uM) and ETOP (10uM) shown by us and others to induce senescence. We examined telomere uncapping using colocalization of 53BP1, a double-stranded DNA (dsDNA) break marker, and telomeres, by performing immunofluorescence fluorescent in situ hybridization (IF-FISH) on human umbilical vein ECs. At confluence, ECs were treated with either DOXO or ETOP for 24 hours, and 24 and 48 hours later, ECs were collected for RNA isolation or were fixed for IF-FISH. To evaluate senescence, we examined mRNA expression of the senescence marker p21. p21 expression tended to be elevated after 24 hours of treatment (both p > 0.09) and was significantly elevated 48 hours after both DOXO and ETOP treatment (Fig. 1, p<0.001). These results corroborate prior findings that chemotherapeutics such as DOXO and ETOP induce senescence. Twenty-four hours post-treatment, the percentage of cells with ≥ 1 dsDNA break was elevated to 72.5 ± 6.3% in DOXO (n=175 cells) and 70.4 ± 13.6% in ETOP (n=97 cells) treated ECs compared to 13.2 ± 4.8% in the vehicle control group (n=310 cells, Fig. 2a, both p<0.0001). Moreover, the percentage of cells with ≥ 1 Telomere dysfunction-induced Foci (TIF) also increased to 46.6 ± 5.3% in DOXO treated ECs, and 52.2 ± 9.5% in ETOP treated ECs, compared to 7.6 ± 2.8% in the vehicle control group (Fig. 2b, p<0.0001). To determine if dsDNA breaks and TIFs were associated with DOXO and ETOP induced EC senescence, we performed a bivariate correlation analysis. For both treatments, the percentage of ECs with ≥ 1 dsDNA break and ≥ 1 TIF were correlated to p21 mRNA expression at 48 hours post treatment (DOXO: r=0.77 and r=0.78, p<0.01; ETOP: r=0.88 and r=0.91, all p<0.01). These data suggest that DOXO and ETOP may lead to senescence induced endothelial dysfunction via telomere uncapping, providing mechanistic insight into the vascular consequences of chemotherapy.

MiR-376a Provokes Rectum Adenocarcinoma Via CTC1 Depletion-Induced Telomere Dysfunction.

CTC1 is a component of the mammalian CST (CTC1–STN1–TEN1) complex which plays essential roles in resolving replication problems to facilitate telomeric DNA and genomic DNA replication. We previously reported that the depletion of CTC1 leads to stalled replication fork restart defects. Moreover, the mutation in CTC1 caused cancer-prone diseases including Coats plus (CP) or dyskeratosis congenita (DC). To better understand the CTC1 regulatory axis, the microRNAs (miRNAs) targeting to CTC1 were predicted by a bioinformatics tool, and the selected candidates were further confirmed by a dual-luciferase reporter assay. Here, our current results revealed that miR-376a significantly reduced CTC1 expression at the transcription level by recognizing CTC1 3′-UTR. In addition, the overexpression of miR-376a induced telomere replication defection and resulted in direct replicative telomere damage, which could be rescued by adding back CTC1. Telomere shortening was also observed upon miR-376a treatment. Furthermore, for the clinical patient samples, the high expression of miR-376a was associated with the deregulation of CTC1 and a poor outcome for the rectum adenocarcinoma patients. Together, our results uncovered a novel role of miR-376a in stimulating rectum adenocarcinoma progression via CTC1 downregulating induced telomere dysfunction.

Transcriptional and Proteomic Characterization of Telomere-Induced Senescence in a Human Alveolar Epithelial Cell Line.

Cellular senescence due to telomere dysfunction has been hypothesized to play a role in age-associated diseases including idiopathic pulmonary fibrosis (IPF). It has been postulated that paracrine mediators originating from senescent alveolar epithelia signal to surrounding mesenchymal cells and contribute to disease pathogenesis. However, murine models of telomere-induced alveolar epithelial senescence fail to display the canonical senescence-associated secretory phenotype (SASP) that is observed in senescent human cells. In an effort to understand human-specific responses to telomere dysfunction, we modeled telomere dysfunction-induced senescence in a human alveolar epithelial cell line. We hypothesized that this system would enable us to probe for differences in transcriptional and proteomic senescence pathways in vitro and to identify novel secreted protein (secretome) changes that potentially contribute to the pathogenesis of IPF. Following induction of telomere dysfunction, a robust senescence phenotype was observed. RNA-seq analysis of the senescent cells revealed the SASP and comparisons to previous murine data highlighted differences in response to telomere dysfunction. We conducted a proteomic analysis of the senescent cells using a novel biotin ligase capable of labeling secreted proteins. Candidate biomarkers selected from our transcriptional and secretome data were then evaluated in IPF and control patient plasma. Four novel proteins were found to be differentially expressed between the patient groups: stanniocalcin-1, contactin-1, tenascin C, and total inhibin. Our data show that human telomere-induced, alveolar epithelial senescence results in a transcriptional SASP that is distinct from that seen in analogous murine cells. Our findings suggest that studies in animal models should be carefully validated given the possibility of species-specific responses to telomere dysfunction. We also describe a pragmatic approach for the study of the consequences of telomere-induced alveolar epithelial cell senescence in humans.

BDE-209 and DBDPE induce male reproductive toxicity through telomere-related cell senescence and apoptosis in SD rat

Decabrominated diphenyl ether (BDE-209) and decabromodiphenyl ethane (DBDPE) are common flame retardants utilized in many kinds of electronic and textile products. Due to their persistence and bioaccumulation, BDE-209 and DBDPE extensively exist in the surrounding environment and wild animals. Previous studies have indicated that BDE-209 could induce male reproductive toxicity, whereas those of DBDPE remains relatively rare. In this study, we investigated the effects of both BDE-209 and DBDPE on reproductive system in male SD rats, and explored the potential mechanisms under the reproductive toxicity of BDE-209 and DBDPE. Male rats were orally administered with BDE-209 and DBDPE (0, 5, 50 and 500 mg/kg/day) for a 28-day exposure experiment. The current results showed that BDE-209 and DBDPE led to testicular damage in physiological structure, decreased the sperm number and motility, and increased the sperm malformation rates in rat. Moreover, BDE-209 and DBDPE could damage the telomeric function by shortening telomere length and reducing telomerase activity, which consequently caused cell senescence and apoptosis in testis of rat. This could contribute to the decline of sperm quality and quantity. In conclusion, BDE-209 and DBDPE led to reproductive toxicity by inducing telomere dysfunction and the related cell senescence and apoptosis in testis of SD rat. Comparatively, BDE-209 had more severe effects on male reproduction. Our findings may provide new insight into the potential deleterious effects of BFRs on male reproductive health.