Telomeres and Telomerase – in space? Susan M. Bailey Department of Environmental & Radiological Health Sciences TELOMERES Hermann Müller (1938), studying inversions resulting
from radiation-induced DSBs, noted that the end of a
chromosome never fused to some other part of a chromosome.
Because of their special properties, he called the ends
telomeres.
end-part
McClintock (1939) noted that certain maize lines contained a
chromosome that would often break, and when breaks occurred
in both sister chromatids, broken ends could fuse to form
dicentric chromosomes. Dicentrics cause anaphase bridges and
lagging chromosomes. Normal ends not “sticky”
Genome Stability Natural DNA ends Broken DNA ends Telomeres Double-­‐Strand Breaks Homologous recombination Non-­‐homologous end-­‐joining Shortening / T-­‐SCE DNA repair/mis-­‐repair Cellular senescence Chromosome Aberrations Reduces tissue regenerative capacity AGING Mutation/instability CANCER Telomeres – protect genomic stability * DNA-­‐PK End-­‐replication problem loss of sequence
Lagging-strand requires RNA primer (8-12 nt gap)
(Watson 1972; Olovnikov 1973)
Human & mouse shorten 50-100 bp/end/division
(Harley, Nature 1990; Zhao 2008)
Position and removal of terminal RNA primer
(Chow, 2012)
Telomeres – “hallmarks of radiosensitivity” [Ayouaz et al., 2008]   Telomere shortening enhances sensitivity to IR   Wong et al., Nat Genet 2000   Telomere shortening associated with chromatin structure changes   Heterochromatin limits access of ATM and slows kinetics of repair following IR exposure   Drissi et al., Cancer Prev Res 2011   Lots of evidence that oxidative stress/accumulating ROS shortens telomeres   von Zglinicki; 1995, 2000, 2007 Telomere shortening – and lifestyle factors [Lin J et al., 2012]   Role in general cellular response program to stress; ROS 
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Increased telomere shortening with life stress (Epel, 2004) Shortening with obesity and cigarette smoking (Valdes, 2005) Physical activity helps prevent telomere shortening (Cherkas, 2008) Healthy lifestyle increases telomerase activity and telomere length (Ornish, 2008 & 2013) Pessimism shortens telomeres (O Donovan, 2008) Mediation promotes telomere maintenance (Epel, 2009) Diet and nutritional status influences telomere length (Paul, 2011) Chronic stress and disease (Blackburn & Epel, 2012); “powerfully quantifies life’s insults”   Contributes to aging phenotypes   Telomere shortening main trigger of replicative senescence (Hayflick, 1961; Harley, 1990; Blasco 1995; Bodner, 1998)   Degeneration; loss of tissue/organ function (Greider, 2009)   Reduced immune function; short telomeres associated with increased risk of common cold (Cohen, 2013)   Contributes to cancer – age is biggest risk factor   Telomere shortening proposed as informative biomarker of aging and disease   Both short and long telomeres have been associated with cancer risk, depending on tissue type   Xie H, 2013; Gramatges M, 2010; Svenson U, 2008 Telomeres – susceptible to damage, refractory to repair…   Telomeres hypersensitive to UV damage   G-­‐rich nature of telomere/oxidative base damage   Rochette et al., PLoS Genetics 2010   Telomeric DNA irreparable   I-­‐SCE1 DSB within 100kb of telomere deficient in NHEJ   Miller et al., DNA Repair 2011   X-­‐rays triggered persistent DNA-­‐damage response (TIFs)   Senescence independent of shortening   Fumagalli et al., Nature Cell Biology 2012   Independent of telomerase activity   Hewitt et al, Nature Communications 2012 Telomeres and Ionizing Radiation – much less well understood   Longer telomeres in irradiated (4 Gy X-­‐rays) vs. unirradiated cells 14 days post exposure   Neuhof et al., 2001   Shorter telomeres in peripheral blood of radiotherapy patients (variety of cancers) in relatively short time (~3 mo) and specifically in shortest telo fraction   Maeda et al., 2013   Short and long-­‐term IR effects also reported; dose dependent telomere shortening in Chernobyl exposed populations (clean-­‐up workers, shelter workers, controls)   Ilyenko, et al., 2011   Telomere length modulation contributes to IR-­‐induced delayed chromosome instability   X-­‐rays and low energy protons exposures induced different telomere response; Berardinelli R et al., 2013   X-­‐rays shortened telomeres (24 hr); anaphase bridges, dicentrics   High LET protons (28.5 keV/n) lengthened telomeres (24 and 96 hrs)   Implications for terrestrial – and galactic – exposures Telomeres – prevent detection as damage/DSB *DNA-­‐PK* “Bolt with two locks” [Bombarde et al., 2010] TRF2 prevents C-­‐NHEJ; DNA-­‐PK prevents A-­‐NHEJ DNA repair proteins at telomeres End-­‐capping function/structure   DNA-­‐PKcs required for effective mammalian telomere end-­‐capping function   Bailey et al., 1999; Zhang et al., 2005; Williams et al., 2009; Fabre et al., 2011   Telomere-­‐telomere & telomere-­‐DSB fusions   Dependent on Ligase IV/NHEJ   Auto-­‐phosphorylation of Thr-­‐2609 important in vivo target; likely involves Artemis activity   Interactions with hnRNPA1 & TERRA   Le et al., 2013   Telomere fusions recently demonstrated in early human breast carcinoma   Tanaka et al., 2012 Telomere proteins in repair?   TRF1, TRF2 and POT1 directly bind telomeric DNA   TRF2 not an “early responder” to IR-­‐induced DSBs   Does not co-­‐localize with damage markers   Williams et al., 2007 Nature Genetics   Telomeric proteins do influence damage response/repair endpoints and genomic stability   siRNA knockdown/depletion   Increase background and IR-­‐induced mutation frequencies (TK locus)   Induce telomere uncapping (telomere-­‐DSB fusions; T-­‐SCE) Depletion of TRF1, TRF2, or POT1 Mutation Frequencies Fe, 1 GeV/n
Gamma Rays
4000
TK- Mutant Fraction x 106
TK- Mutant Fraction x 106
4500
4000
3500
3000
2500
2000
1500
1000
500
0
0 1 2
Mock
0 1 2
0 1 2
0 1 2
0 1 2
0 1 2
POT1
TRF1
TRF2
PK
TRF2/PK
3500
3000
2500
2000
1500
1000
500
0
0 1 2
Mock
0 1 2
0 1 2
0 1 2
0 1 2
0 1 2
POT1
TRF1
TRF2
PK
TRF2/PK
Howard Liber Elevated T-­‐SCE and telomere-­‐DSB fusion following IR exposure Telomere proteins in repair -­‐ YES!   TRF1, TRF2 and POT1 deficiencies elevate mutation frequencies   Higher MF than inhibition of DNA-­‐PKcs   MF similar both quantitatively and qualitatively   No statistically significant differences between γ-­‐rays and 1 GeV/n Fe   However RBE ~3 is indicated using new immediate plating protocol   POT1 depletion significantly elevated MF   TRF2 and POT1 deficiencies result in telomere uncapping/instability   Elevated telomere fusions and T-­‐SCE   Decreased expression of TRF2 associated with increased breast cancer malignancy   Diehl et al, 2011   Telomere uncapping/fusion upon POT1 depletion consistent with POT1 mutations in subset of chronic lymphocytic leukemia (CLL) patients   Ramsay et al., 2013 Tankyrase 1 depletion – Mutation Frequencies Dregalla et al., 2010 12000
10000
Mock
DNA-PKcs siRNA
DNA-PKcs I
DNA-PKcs siRNA & I
Tankyrase siRNA
Tankyrase siRNA & DNA-PKcs I
8000
6000
4000
-
TK Mutant Fraction x 10
6
14000
2000
0
0 Gy
1 Gy
2 Gy
γ-rays
1 Gy
56
2 Gy
Fe
Inhibition of tankyrase stabilizes axin and blocks Wnt signaling [Huang et al., 2009] Implications for stem cells and links to telomerase Telomerase – reverse transcriptase   Ribonucleoprotein enzyme
  hTERC: RNA complementary template; 11 bp
  expressed in all cells; binds 3 ss overhang
  hTERT: protein component: reverse transcriptase extends 3 (G-rich) end of
DNA; regulatory subunit
  expressed at sufficient levels only in germ line (ovaries, testes) and highly
proliferative tissues (stem cells and cancer).
  NOT sufficient in somatic cells to maintain telomere length
  telomeres shorten with cell division; aging
  Deficiency results in Dyskeratosis Congenita (DC)
  Short telomeres; progeria, bone marrow failure; pulmonary fibrosis; cancer-prone
  Common mutations in telomerase promoter/regulatory region found
associated with melanomas [Xie H et al., 2013]
  Not just genes; controlling elements also important (non-coding regions)
  Genetic variant of TERC linked to myeloma [Chubb D. et al., 2013]
Nobel Prize in Physiology or Medicine -­‐ 2009 "for the discovery of how chromosomes are protected by telomeres and telomerase" Elizabeth H. Blackburn University of California San Francisco, CA, USA Carol W. Greider Johns Hopkins University School of Medicine BalAmore, MD, USA Telomerase inhibition and cancer treatment Jack W. Szostak Harvard Medical School MassachuseDs General Hospital Boston, MA, USA Howard Hughes Medical InsAtute Scientific American: August 2009
AnE-­‐Aging Pill Targets Telomeres – TELOMERASE ACTIVATORS Could the secrets to anA-­‐aging be at the Aps of our chromosomes? WATCHING THE CLOCK: You can't stop the relentless march of Ame by casAng a spell on a clock. Telomerase: central regulator of all the hallmarks of cancer *  Non-­‐canonical roles *  independent of telomere length maintenance *  Function in *  Cell growth, proliferation *  Inhibiting anti-­‐growth signals *  Suppressing differentiation Low & Tergaonkar, 2013 Telomerase and Cancer   Replicative immortality   Hanahan & Weinberg, 2011   Cells that escape crisis almost universally express telomerase   Kim et al., 1994   Wnt/β-­‐catenin signaling implicated in regulating telomerase and TRF2 expression   Diala et al., 2013; Hoffmeyer et al., 2013   Telomerase promotes “stemness”   Proliferation, resistance to anti-­‐growth signals, invasion & metastasis   Reprogramming Telomerase & ionizing radiation?   IR elevates telomerase activity in cancer cell lines & spheres ~5 days post exposure; not DNA repair   Sawant et al., 1999; Karimi-­‐Busher et al., 2010   IR induces enrichment of cancer stem cells in epithelial cancer cell lines (breast, colon, prostate)   Phillips et al., 2006; 2010.   IR induces conditional reprogramming of non-­‐stem cancer cells into a stem-­‐like state; dedifferentiation   Lagadek et al., 2012; Yang et al., 2012   Mechanisms unknown – and what about non-­‐tumorigenic cells? Approach   Human cell lines   variety of tissue types of relevance for radiation carcinogenesis   represent a range of levels of telomerase activity   Mammary epithelial   MCF-­‐7: adenocarcinoma   MCF-­‐10a: immortalized, non-­‐tumorigenic   AG11137: primary   Hematopoietic   WTK1: B-­‐lymphoblastoid p53 deficient mutagenesis system   KG1a: Acute Myeloid Leukemia   LCL15044: normal, immortalized lymphoblastoid   US Radiologic Technologist Cohort; Alice Sigurdson (NCI)   Osteosarcoma   U20S and SAOS2: Alternative lengthening of telomeres (ALT) Assays   Real-­‐Time quantitative telomere repeat amplification protocol (qRT-­‐PCR TRAP)   Sensitive measure of telomerase activity   Flow Cytometry with tissue-­‐specific stem cell markers to distinguish/evaluate stem cell compartments   Mammary: CD44high/CD24low/-­‐ and ALDH activity   Hematopoietic: CD34+ or CD34+/CD38-­‐   Osteosarcoma: CD44+/CD133+ Telomerase Activity in various cell lines Telomerase activity post IR – Mammary epithelial cell lines 1.2 Relative Telomerase Activity 1 0.8 MCF-­‐7 0 Gy MCF-­‐7 10 Gy 0.6 MCF-­‐10a 0 Gy MCF-­‐10a 10 Gy AG11137 0 Gy 0.4 AG11137 10 Gy 0.2 0 0 24 48 72 Hours post IR exposure 96 120 Telomerase activity post IR – Hematopoietic cell lines * 1.6 1.4 KG1a 0 Gy Relative Telomerase Activity 1.2 KG1a 1 Gy 1 KG1a 4 Gy WTK1 0 Gy 0.8 WTK1 1 Gy 0.6 WTK1 4 Gy LCL15044 0 Gy 0.4 LCL15044 1 Gy 0.2 LCL15044 4 Gy 0 24 48 Hours Post IR Exposure 72 Ionizing Radiation Induces Stem Cell Enrichment 4 Stem Cell Enrichment 5 Days Post Exposure * 3.5 % of CD44+/CD24-­‐ Cells 3 * 2.5 2 MCF-­‐7 MCF-­‐10a 1.5 1 0.5 0 0 10 Dose MCF-­‐10A stem cell enrichment Post IR (5 days) Representative scatter plots Stem Cell Enrichment is Dose Dependent in MCF-­‐10a MCF-­‐10a CD44+/CD24-­‐ Cell Population 5 days post IR 14 * 12 % CD44+/CD24-­‐ Cells 10 8 MCF-­‐10a 6 * 4 2 0 0 2 5 gamma ray dose (Gy) 10 20 WTK1 stem cells (CD34+) Post IR (γ-­‐rays) 1.8 * 1.6 1.4 % CD34 + Cells 1.2 1 0 Gy * 0.8 1 Gy * 0.6 *
0.4 * 0.2 0 24 48 Hours Post IR 72 4 Gy Telomerase Inhibition (MST-­‐312) blocks IR-­‐induced reprogramming MST-­‐312 dose response: MCF-­‐7 Stem cells: MCF-­‐7 1.4 100 % CD44+/CD24-­‐ Cells Relative Telomerase Activity 120 80 60 40 20 1.2 1 0.8 DMSO 0.6 MST-­‐312 1 uM 0.4 MST-­‐312 3 uM 0.2 0 0 DMSO 1 uM 5 uM 10 uM 25 uM 0 50 uM gamma ray dose (Gy) Stem cells: MCF-­‐10a MST-­‐312 dose response: MCF-­‐10a 5 140.00 120.00 % CD44+/CD24-­‐ Cells Relative Telomerase Activity 10 100.00 80.00 60.00 40.00 20.00 0.00 DMSO 0.5 uM 1 uM MST-­‐312 Concentration 3 uM 4 3 DMSO 2 1 uM MST-­‐312 1 0 0 10 gamma ray dose (Gy) IR-­‐induced reprogramming   Elevation of telomerase activity (1-­‐2 days)   Enrichment of stem cell compartments (5 days)   Relevance: recent report of induction of cancer stem cells from nontumorigenic MCF-­‐10A cells [Nishi et al., 2013]   Introduction of defined factors (OCT4, SOX2, KlF4 and c-­‐Myc) transformed the bulk of cells into tumorigenic cancer stem cells   Investigating potential oncogenicity that may arise from IR-­‐
induced cellular reprogramming of normal cells   Influence of telomerase (TERT/TERC)?   Influence of DNA repair deficiencies?   Influence of telomere deficiencies? Telomeres & Telomerase in space? *  Rate at which telomeres shorten can provide an informative biomarker of aging, AND be linked to age-­‐related pathologies, including reduced immune function, loss of fertility, cardiovascular disease and cancer *  While healthy lifestyles and diets are positively correlated with telomere length and telomerase activity, chronic stress (nutritional, psychological, physical) is negatively correlated. *  Radiation exposure influences telomere length and can modulate telomerase activity; impacts a host of cancer hallmarks including DNA repair, reprogramming/stem cells *  We’re investigating what this means for astronauts Summary •  Telomeres are the natural ends of chromosomes that shorten with cellular division, and therefore with aging. It is becoming increasingly appreciated that a variety of lifestyle factors, including stress (nutritional, psychological, physical), also contribute to their demise. •  Telomerase can maintain/add telomere length, however is sufficient only in germ, stem and cancer cells to do so. Radiation exposure can modulate telomerase activity, thereby impacting telomeres and a host of cancer hallmarks. •  The rate at which telomeres shorten can provide an informative biomarker of aging – and be linked to age-­‐related pathologies, ranging from reduced immune function, loss of fertility, cardiovascular disease and cancer. •  For the astronauts, telomere maintenance represents a particularly relevant biomarker, because it reflects the combined exposures and experiences encountered during space travel. For example, an individual’s genetic susceptibility, the exposures to galactic space radiations, the stressors encountered, are all captured as changes of telomere length maintenance. Acknowledgements Colorado State University 
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Brock Sishc Christine Battaglia Chris Nelson Miles McKenna Howard Liber NASA/Wyle   Kerry George NSRL/BNL team   Adam Rusek   Peter Guida