Exposure to hypomethylating agent 5-aza-2’-deoxycytidine (decitabine) causes rapid, severe DNA damage, telomere elongation and mitotic dysfunction in human WIL2-NS cells

2019 ◽  
Author(s):  
Caroline Bull ◽  
Graham Mayrhofer ◽  
Michael Fenech
Keyword(s):  
Dna Damage ◽  
Telomere Length ◽  
Chromosomal Instability ◽  
Fold Increase ◽  
Complete Medium ◽  
Global Methylation ◽  
Telomere Elongation ◽  
Chromosomal Stability ◽  
Hypomethylating Agent ◽  
Dna Hypomethylating Agent

AbstractBackground5-aza-2’-deoxycytidine (5azadC, decitabine) is a DNA hypomethylating agent used in the treatment of myelodysplastic syndromes. Due to cytotoxic side effects dose optimization is essential. This study defines and quantifies the effects of 5azadC on chromosomal stability and telomere length, at clinically relevant dosages.MethodsHuman WIL2-NS cells were maintained in complete medium containing 0, 0.2 or 1.0μM 5azadC for four days, and analysed daily for telomere length (flow cytometry), chromosomal stability (cytokinesis-block micronucleus cytome (CBMN-cyt) assay), and global methylation (%5me-C).ResultsDNA methylation decreased significantly in 1.0 μM 5azadC, relative to control (p<0.0001). Exposure to 1.0μM 5azadC resulted in 170% increase in telomere length (p<0.0001), in parallel with rapid increase in biomarkers of DNA damage; (micronuclei (MN, 6-fold increase), nucleoplasmic bridges (NPB, a 12-fold increase), and nuclear buds (NBud, a 13-fold increase) (all p<0.0001). Fused nuclei (FUS), indicative of mitotic dysfunction, showed a 5- and 13-fold increase in the 0.2μM and 1.0μM conditions, respectively (p = 0.001) after 4 days.ConclusionsThese data show that (i) clinically relevant concentrations of 5azadC are highly genotoxic; (ii) hypomethylation was associated with increased TL and DNA damage; and (iii) longer TL was associated with chromosomal instability. These findings suggest that lower doses of 5azdC may be effective as a hypomethylating agent, while potentially reducing DNA damage and risk for secondary disease.

scholarly journals Novel Germline POT1 Variant Predisposes to Childhood Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3449-3449
Author(s):  
Pia Michler ◽  
Franziska Auer ◽  
Rabea Wagener ◽  
Triantafyllia Brozou ◽  
Ulrike Anne Friedrich ◽  
...  
Keyword(s):  
Dna Damage ◽  
Acute Myeloid Leukemia ◽  
Telomere Length ◽  
Chromosomal Instability ◽  
Myeloid Leukemia ◽  
T Test ◽  
Qrt Pcr ◽  
Student’S T ◽  
Acute Myeloid ◽  
Student’S T Test

Abstract Introduction: Current studies indicate a contribution of germline predisposition in the development of approximately 8.5% of childhood cancers (Zhang J. et al., N Engl J Med, 2015), although their apparent rate is estimated to be much higher. Understanding tumor evolution based on a predisposed cell can open unknown doors for prevention and therapy of childhood cancer e.g., leukemia. Here we present a novel rare (MAF&lt;0.1%) germline POT1 variant (Q199*) predisposing to acute myeloid leukemia (AML). POT1 as part of the telomeric shelterin complex is known to play an important role in DNA damage protection, telomere length maintenance and chromosomal stability (Calvete O. et al., Nat. Commun., 2015). POT1 variants are associated with a broad range of cancer, including myeloid and lymphoid neoplasms in adults (Lim T.L. et al., Leukemia, 2021), but not yet described for myeloid malignancies in childhood. Methods: Whole exome sequencing (WES) was implemented to identify germline variants. To assess the effect of POT1 p.Q199*, patient's fibroblast and stably transfected HEK293T cells were used as cell models. The variant's functional impact was experimentally tested performing yH2AX and 53BP1 immunofluorescence assays for DNA damage detection, qRT-PCR for telomere length measurement and telomere FISH to assess chromosomal instability. Results: Utilizing WES to detect variants within shelterin complex genes we analyzed genomic data of an unselected German parent-child cohort of children with cancer (n=60, TRIO-DD), as well as a recently published parent-child pediatric cancer cohort (n=158, TRIO-D) (Wagener R. et al., Eur. J. Hum. Genet, 2021). Here, we identified a novel germline POT1 variant in a boy affected with Myelodysplastic syndrome (MDS) and secondary AML (7q-). This novel germline variant constitutes a stop-gain mutation causing a substitution of the amino acid Glutamine by a stop codon (p.Q199*). QRT-PCR analysis within the patient's fibroblasts showed a significant (student's t-test p=0.0037) reduction of POT1 mRNA expression to ≈0.5 compared to POT1 wildtype. Western Blot analysis revealed reduced POT1 levels, confirming the loss of one POT1 allele mediated by p.Q199*. Thereupon, POT1 p.Q199* cloning and stable transfection into Hek293T cells was performed to test the variant's cooperative functionality in a controlled environment. Subsequently, POT1 p.Q199* lead to a drastically significant (student's t-test p=&lt;0.001) increase of DNA double strand breaks in transfected Hek293T cells determined by yH2AX and 53BP1 immunofluorescence assays, which is in line with a deregulated DNA damage response and inappropriate repair by non-homologous end joining. In addition, we detected dysregulation of telomere length maintenance. Here, relative telomere length measurement by means of qRT-PCR indicated significant (student's t-test p=0.019) telomere elongation in POT1 p.Q199* fibroblast cells. Furthermore telomere FISH on metaphase chromosomes was performed to analyse chromosomal stability. In POT1 p.Q199* Hek293T cells we identified a significant (student's t-test p=0.002) increase in telomere fragility compared to POT1 WT cells. Conclusion: Taken together, we present the functional effects of POT1 p.Q199* leading to a significant increase of DNA damage, telomere length and chromosomal instability. Our results on functional dysregulation strengthen a potential genetic predisposition to childhood AML mediated by germline POT1 variants. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tel1 Activation by the MRX Complex Is Sufficient for Telomere Length Regulation but Not for the DNA Damage Response in Saccharomyces cerevisiae

Genetics ◽  
2019 ◽  
Vol 213 (4) ◽  
pp. 1271-1288 ◽  
Author(s):  
Rebecca Keener ◽  
Carla J. Connelly ◽  
Carol W. Greider
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Telomere Length ◽  
Dna Damage Response ◽  
Telomere Elongation ◽  
Link Type ◽  
Epistasis Analysis ◽  
Damage Response ◽  
Length Regulation ◽  
Telomere Length Regulation

Previous models suggested that regulation of telomere length in Saccharomyces cerevisiae by Tel1(ATM) and Mec1(ATR) would parallel the established pathways regulating the DNA damage response. Here, we provide evidence that telomere length regulation differs from the DNA damage response in both the Tel1 and Mec1 pathways. We found that Rad53 mediates a Mec1 telomere length regulation pathway but is dispensable for Tel1 telomere length regulation, whereas in the DNA damage response, Rad53 is regulated by both Mec1 and Tel1. Using epistasis analysis with a Tel1 hypermorphic allele, Tel1-hy909, we found that the MRX complex is not required downstream of Tel1 for telomere elongation but is required downstream of Tel1 for the DNA damage response. Our data suggest that nucleolytic telomere end processing is not a required step for telomerase to elongate telomeres.

scholarly journals Tel1 activation by the MRX complex is sufficient for telomere length regulation but not for the DNA damage response in S. cerevisiae

2019 ◽  
Author(s):  
Rebecca Keener ◽  
Carla J. Connelly ◽  
Carol W. Greider
Keyword(s):  
Dna Damage ◽  
Telomere Length ◽  
Dna Damage Response ◽  
Mammalian Cells ◽  
Telomere Elongation ◽  
Epistasis Analysis ◽  
Damage Response ◽  
Length Regulation ◽  
Processing Event ◽  
Telomere Length Regulation

AbstractPrevious models suggested that regulation of telomere length in S. cerevisiae by Tel1(ATM) and Mec1(ATR) parallel the established pathways regulating the DNA damage response. Here we provide evidence that telomere length regulation differs from the DNA damage response in both the Tel1 and Mec1 pathways. We found that Rad53 mediates a Mec1 telomere length regulation pathway but is dispensable for Tel1 telomere length regulation, whereas in the DNA damage response Rad53 is regulated by both Mec1 and Tel1. Using epistasis analysis with a Tel1 hypermorphic allele, Tel1-hy909, we found that the MRX complex is not required downstream of Tel1 for telomere elongation but is required downstream of Tel1 for the DNA damage response. Since models that invoke a required end processing event for telomerase elongation are primarily based on the yeast pathways, our data call for a re-examination of the requirement for telomere end processing in both yeast and mammalian cells.

Non-canonical Functions of Telomerase Reverse Transcriptase: Emerging Roles and Biological Relevance

2020 ◽  
Vol 20 (6) ◽  
pp. 498-507 ◽  
Author(s):  
Connor A.H. Thompson ◽  
Judy M.Y. Wong
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Reverse Transcriptase ◽  
Cancer Cells ◽  
Telomere Length ◽  
Telomere Maintenance ◽  
Telomerase Reverse Transcriptase ◽  
Alternative Mechanism ◽  
Dependent Manner ◽  
Mitochondrial Integrity

Increasing evidence from research on telomerase suggests that in addition to its catalytic telomere repeat synthesis activity, telomerase may have other biologically important functions. The canonical roles of telomerase are at the telomere ends where they elongate telomeres and maintain genomic stability and cellular lifespan. The catalytic protein component Telomerase Reverse Transcriptase (TERT) is preferentially expressed at high levels in cancer cells despite the existence of an alternative mechanism for telomere maintenance (alternative lengthening of telomeres or ALT). TERT is also expressed at higher levels than necessary for maintaining functional telomere length, suggesting other possible adaptive functions. Emerging non-canonical roles of TERT include regulation of non-telomeric DNA damage responses, promotion of cell growth and proliferation, acceleration of cell cycle kinetics, and control of mitochondrial integrity following oxidative stress. Non-canonical activities of TERT primarily show cellular protective effects, and nuclear TERT has been shown to protect against cell death following double-stranded DNA damage, independent of its role in telomere length maintenance. TERT has been suggested to act as a chromatin modulator and participate in the transcriptional regulation of gene expression. TERT has also been reported to regulate transcript levels through an RNA-dependent RNA Polymerase (RdRP) activity and produce siRNAs in a Dicer-dependent manner. At the mitochondria, TERT is suggested to protect against oxidative stress-induced mtDNA damage and promote mitochondrial integrity. These extra-telomeric functions of TERT may be advantageous in the context of increased proliferation and metabolic stress often found in rapidly-dividing cancer cells. Understanding the spectrum of non-canonical functions of telomerase may have important implications for the rational design of anti-cancer chemotherapeutic drugs.

scholarly journals Telomeres and replicative cellular aging of the human placenta and chorioamniotic membranes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsung-Po Lai ◽  
Mark Simpson ◽  
Krunal Patel ◽  
Simon Verhulst ◽  
Jungsik Noh ◽  
...  
Keyword(s):  
Dna Damage ◽  
Preterm Birth ◽  
Telomere Length ◽  
Human Placenta ◽  
Replicative Senescence ◽  
Preterm Births ◽  
Full Term ◽  
Cellular Aging ◽  
Telomere Dysfunction ◽  
Replicative Aging

AbstractRecent hypotheses propose that the human placenta and chorioamniotic membranes (CAMs) experience telomere length (TL)-mediated senescence. These hypotheses are based on mean TL (mTL) measurements, but replicative senescence is triggered by short and dysfunctional telomeres, not mTL. We measured short telomeres by a vanguard method, the Telomere shortest length assay, and telomere-dysfunction-induced DNA damage foci (TIF) in placentas and CAMs between 18-week gestation and at full-term. Both the placenta and CAMs showed a buildup of short telomeres and TIFs, but not shortening of mTL from 18-weeks to full-term. In the placenta, TIFs correlated with short telomeres but not mTL. CAMs of preterm birth pregnancies with intra-amniotic infection showed shorter mTL and increased proportions of short telomeres. We conclude that the placenta and probably the CAMs undergo TL-mediated replicative aging. Further research is warranted whether TL-mediated replicative aging plays a role in all preterm births.

scholarly journals Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines

2019 ◽  
Vol 476 (4) ◽  
pp. 629-643 ◽  
Author(s):  
Isabelle Gerin ◽  
Marina Bury ◽  
Francesca Baldin ◽  
Julie Graff ◽  
Emile Van Schaftingen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Oxidative Dna Damage ◽  
Fold Increase ◽  
Metabolic Effects ◽  
Phosphoglycerate Mutase ◽  
Wild Type ◽  
Metabolic Disturbances ◽  
Phosphoglycolate Phosphatase ◽  
Damage Repair

Abstract Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.

Abstract 424: Vascular Smooth Muscle Cell Expression of S100a12 in Vivo Induces Enhanced Oxidative Stress & Vascular Remodeling

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Marion Hofmann Bowman ◽  
Jeannine Wilk ◽  
Gene Kim ◽  
Yanmin Zhang ◽  
Jalees Rehman ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Smooth Muscle ◽  
Vascular Remodeling ◽  
Oxidative Dna Damage ◽  
Fold Increase ◽  
Brdu Incorporation ◽  
Elastic Fibers ◽  
Nuclear Staining

S100A12 is a small calcium binding protein that is a signal transduction ligand of the receptor for advance glycation endproducts (RAGE). S100A12, like RAGE, is expressed in the vessel wall of atherosclerotic vasculature, particularly in smooth muscle cells (SMC). While RAGE has been extensively implicated in inflammatory states such as atherosclerosis, the role of S100A12 is less clear. We tested the hypothesis that expression of human S100A12 directly exacerbates vascular inflammation. Several lines of Bl6/J transgenic mice (tg) expressing human S100A12 in SMC under control of the SM22a promoter were generated. Primary aortic SMC from tg and wild type (wt) littermates were isolated and analyzed for (i) proliferation using MTS/Formazan Assay and BrdU incorporation, (ii) oxidative stress using using flow cytometry with MitoSOX antibody, oxidative DNA damage using immunofluorescence microscopy with anti-8-oxo-dG antibody, and NF-kB activation measured by EMSA and (iii) cytokine expression measured by IL-6 ELISA. Furthermore, the aortas from tg and wt mice were examined. Results: Tg but not wt SMC expressed S100A12 protein. Tg SMC had a significant 1.9 to 2.7 fold increase in conversion of MTS into Formazan at 24–96 hours likely reflective of increased metabolic activity since BrdU incorporation into DNA was less in tg compared to wt SMC (4% vs 21% positive BrdU nuclei, p <0.05). Tg SMC showed significantly higher levels of mitochondrial generated ROS, nuclear staining for oxidative DNA damage which was not detected in the nuclei of wt SMC’s, and a 2.5 fold increase in NFkB activity. IL-6 production at baseline was higher in tg SMC’s (615 vs 213 pg/ml, p< 0.05) and increased dramatically after LPS treatment (10 ng/ml) in tg SMC’s (2130 vs 415 pg/ml). Histologic examination of the thoracic aorta at 10 weeks of age revealed increased collagen deposition in the aortic media with fragmentation and disarray of elastic fibers. In vivo ultrasound revealed a progressive dilation of the aortic arch from age 10 weeks to 16 weeks of age (1.27 to 1.60 mm, p<0.05) in tg but not in wt littermate mice (1.30 to 1.33 mm, p=0.1). These data reveal the novel finding that targeted expression of human S100A12 in SMC modulates oxidative stress, inflammation and vascular remodeling.

scholarly journals Effects of male telomeres on probability of paternity in sand lizards

2018 ◽  
Vol 14 (8) ◽  
pp. 20180033 ◽  
Author(s):  
Angela Pauliny ◽  
Emily Miller ◽  
Nicky Rollings ◽  
Erik Wapstra ◽  
Donald Blomqvist ◽  
...  
Keyword(s):  
Experimental Data ◽  
Dna Damage ◽  
Sperm Competition ◽  
Telomere Length ◽  
Sperm Motility ◽  
Female Identity ◽  
Laboratory Population ◽  
Lacerta Agilis

Standardized swim-up trials are used in in vitro fertilization clinics to select particularly motile spermatozoa in order to increase the probability of a successful fertilization. Such trials demonstrate that sperm with longer telomeres have higher motility and lower levels of DNA damage. Regardless of whether sperm motility, and successful swim-up to fertilization sites, is a direct or correlational effect of telomere length or DNA damage, covariation between telomere length and sperm performance predicts a relationship between telomere length and probability of paternity in sperm competition, a prediction that for ethical reasons cannot be tested on humans. Here, we test this prediction in sand lizards ( Lacerta agilis ) using experimental data from twice-mated females in a laboratory population, and telomere length in blood from the participating lizards. Female identity influenced paternity (while the mechanism was not identified), while relatively longer male telomeres predicted higher probability of paternity. We discuss potential mechanisms underpinning this result.

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