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Low oxygen delays fibroblast senescence despite shorter telomeres

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Abstract

It has been widely accepted that telomere shortening acts as a cell division counting mechanism that beyond a set critical length signals cells to enter replicative senescence. In this study, we demonstrate that by simply lowering the oxygen content of the cell culture environment 10-fold (20–2%) extends the replicative lifespan of fetal bovine fibroblasts at least five-times (30–150 days). Although, low oxygen fibroblasts display a slightly slower rate (P > 0.05) of telomere attrition than their high oxygen counterparts (171 bp versus 182 bp/PD), late passage fibroblasts (>50 PD) that have extended their replicative capacity under low oxygen conditions exhibited significantly (P < 0.05) shorter telomere lengths (11,135 ± 467 bp) compared to senescent cells (25–34 PD) cultured under high oxygen conditions (14,827 ± 1173 bp). There was a significant increase (P < 0.05) in chromosomal abnormalities with continual cell division under both high and low oxygen environments, however, fibroblasts displayed a significant reduction (P < 0.001) in chromosomal abnormalities at low oxygen tensions compared to those under 20% oxygen. These apparent protective effects on telomere shortening, delayed senescence and reduced chromosomal aberrations may be attributed to the up-regulation of telomerase activity observed for fibroblasts cultured under low oxygen. These results are consistent with the idea that a critically short telomere length may not be the sole trigger of replicative senescence, but may be regulated by the integrity of telomere structure itself and/or the amount of oxidative DNA damage in the cell.

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Abbreviations

FF:

Fetal fibroblast

PDL:

Population doubling level

ROS:

Reactive oxygen species

TERT:

Telomerase reverse transcriptase

TRF:

Terminal Restriction Fragment

References

  • Allsopp RC, Chang E, Kashefi-Aazam M, Rogaev EI, Piatyszek MA, Shay JW, Harley CB (1995) Telomere shortening is associated with cell division in vitro and in vivo. Exp Cell Res 220(1):194–200

    Article  PubMed  CAS  Google Scholar 

  • Ames BN, Shigenaga MK (1992) Oxidants are a major contributor to aging. Ann N Y Acad Sci 663:85–96

    Article  PubMed  CAS  Google Scholar 

  • Bakkenist CJ, Drissi R, Wu J, Kastan MB, Dome JS (2004) Disappearance of the telomere dysfunction-induced stress response in fully senescent cells. Cancer Res 64(11):3748–3752

    Article  PubMed  CAS  Google Scholar 

  • Balin AK, Goodman DB, Rasmussen H, Cristofalo VJ (1977) The effect of oxygen and vitamin E on the lifespan of human diploid cells in vitro. J Cell Biol 74(1):58–67

    Article  PubMed  CAS  Google Scholar 

  • Ben-Porath I, Weinberg RA (2004) When cells get stressed: an integrative view of cellular senescence. J Clin Invest 113(1):8–13

    Article  PubMed  CAS  Google Scholar 

  • Blackburn EH, Chan S, Chang J, Fulton TB, Krauskopf A, McEachern M, Prescott J, Roy J, Smith C, Wang H (2000) Molecular manifestations and molecular determinants of telomere capping. Cold Spring Harb symp quant biol 65:253–263

    Article  PubMed  CAS  Google Scholar 

  • Blanco R, Munoz P, Flores JM, Klatt P, Blasco MA (2007) Telomerase abrogation dramatically accelerates TRF2-induced epithelial carcinogenesis. Genes Dev 21(2):206–220

    Article  PubMed  CAS  Google Scholar 

  • Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279(5349):349–352

    Article  PubMed  CAS  Google Scholar 

  • Bond JA, Wyllie FS, Wynford-Thomas D (1994) Escape from senescence in human diploid fibroblasts induced directly by mutant p53. Oncogene 9(7):1885–1889

    PubMed  CAS  Google Scholar 

  • Brown JP, Wei W, Sedivy JM (1997) Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 277(5327):831–834

    Article  PubMed  CAS  Google Scholar 

  • Chen Q, Fischer A, Reagan JD, Yan LJ, Ames BN (1995) Oxidative DNA damage and senescence of human diploid fibroblast cells. Proc Natl Acad Sci USA 92(10):4337–4341

    Article  PubMed  CAS  Google Scholar 

  • Collins K (2000) Mammalian telomeres and telomerase. Curr Opin Cell Biol 12(3):378–383

    Article  PubMed  CAS  Google Scholar 

  • Counter CM, Hirte HW, Bacchetti S, Harley CB (1994) Telomerase activity in human ovarian carcinoma. Proc Natl Acad Sci USA 91(8):2900–2904

    Article  PubMed  CAS  Google Scholar 

  • Cristofalo VJ, Allen RG, Pignolo RJ, Martin BG, Beck JC (1998) Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation. Proc Natl Acad Sci USA 95(18):10614–10619

    Article  PubMed  CAS  Google Scholar 

  • Cristofalo VJ, Beck J, Allen RG (2003) Cell senescence: an evaluation of replicative senescence in culture as a model for cell aging in situ. J Gerontol 58(9):B776–B779; discussion 779–781

    Google Scholar 

  • d’Adda di Fagagna F, Reaper PM, Clay-Farrace L, Fiegler H, Carr P, Von Zglinicki T, Saretzki G, Carter NP, Jackson SP (2003) A DNA damage checkpoint response in telomere-initiated senescence. Nature 426(6963):194–198

    Article  PubMed  CAS  Google Scholar 

  • Davis T, Skinner JW, Faragher RG, Jones CJ, Kipling D (2005) Replicative senescence in sheep fibroblasts is a p53 dependent process. Exp Gerontol 40(1–2):17–26

    Article  PubMed  CAS  Google Scholar 

  • Duan J, Duan J, Zhang Z, Tong T (2005) Irreversible cellular senescence induced by prolonged exposure to H2O2 involves DNA-damage-and-repair genes and telomere shortening. Int J Biochem Cell Biol 37(7):1407–1420

    Article  PubMed  CAS  Google Scholar 

  • Favetta LA, Robert C, King WA, Betts DH (2004) Expression profiles of p53 and p66shc during oxidative stress-induced senescence in fetal bovine fibroblasts. Exp Cell Res 299(1):36–48

    Article  PubMed  CAS  Google Scholar 

  • Favetta LA, St John EJ, King WA, Betts DH (2007) High levels of p66shc and intracellular ROS in permanently arrested early embryos. Free Radic Biol Med 42(8):1201–1210

    Article  PubMed  CAS  Google Scholar 

  • Figueroa R, Lindenmaier H, Hergenhahn M, Nielsen KV, Boukamp P (2000) Telomere erosion varies during in vitro aging of normal human fibroblasts from young and adult donors. Cancer Res 60(11):2770–2774

    PubMed  CAS  Google Scholar 

  • Frenck RW Jr., Blackburn EH, Shannon KM (1998) The rate of telomere sequence loss in human leukocytes varies with age. Proc Natl Acad Sci USA 95(10):5607–5610

    Article  PubMed  CAS  Google Scholar 

  • Golczewski JA (1984) Effect of 2-mercaptoethylamine on proliferation and lifespan of WI-38 cells. Exp Gerontol 19(1):7–11

    Article  PubMed  CAS  Google Scholar 

  • Goldstein S (1974) Aging in vitro. Growth of cultured cells from the Galapagos tortoise. Exp Cell Res 83:297–302

    Article  Google Scholar 

  • Goldstein S, Moerman EJ, Soeldner JS, Gleason RE, Barnett DM (1978) Chronologic and physiologic age affect replicative life-span of fibroblasts from diabetic, prediabetic, and normal donors. Science 199(4330):781–782

    Article  PubMed  CAS  Google Scholar 

  • Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H, de Lange T (1999) Mammalian telomeres end in a large duplex loop. Cell 97(4):503–514

    Article  PubMed  CAS  Google Scholar 

  • Haendeler J, Hoffmann J, Diehl JF, Vasa M, Spyridopoulos I, Zeiher AM, Dimmeler S (2004) Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells. Circ Res 94(6):768–775

    Article  PubMed  CAS  Google Scholar 

  • Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345(6274):458–460

    Article  PubMed  CAS  Google Scholar 

  • Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC (1990) Telomere reduction in human colorectal carcinoma and with ageing. Nature 346(6287):866–868

    Article  PubMed  CAS  Google Scholar 

  • Hayflick L (1965) the limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636

    Article  PubMed  CAS  Google Scholar 

  • Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621

    Article  Google Scholar 

  • Hecht A, Strahl-Bolsinger S, Grunstein M (1996) Spreading of transcriptional repressor SIR3 from telomeric heterochromatin. Nature 383(6595):92–96

    Article  PubMed  CAS  Google Scholar 

  • Holmes GE, Bernstein C, Bernstein H (1992) Oxidative and other DNA damages as the basis of aging: a review. Mutat Res 275(3–6):305–315

    PubMed  CAS  Google Scholar 

  • Holt SE, Wright WE, Shay JW (1997) Multiple pathways for the regulation of telomerase activity. Eur J Cancer 33(5):761–766

    Article  PubMed  CAS  Google Scholar 

  • Honda S, Matsuo M (1983) Shortening of the in vitro lifespan of human diploid fibroblasts exposed to hyperbaric oxygen. Exp Gerontol 18(5):339–345

    Article  PubMed  CAS  Google Scholar 

  • Hornsby PJ, Harris SE (1987) Oxidative damage to DNA and replicative lifespan in cultured adrenocortical cells. Exp Cell Res 168(1):203–217

    Article  PubMed  CAS  Google Scholar 

  • Huffman KE, Levene SD, Tesmer VM, Shay JW, Wright WE (2000) Telomere shortening is proportional to the size of the G-rich telomeric 3′-overhang. J Biol Chem 275(26):19719–19722

    Article  PubMed  CAS  Google Scholar 

  • Hyeon Joo O, Hande MP, Lansdorp PM, Natarajan AT (1998) Induction of telomerase activity and chromosome aberrations in human tumour cell lines following X-irradiation. Mutat Res 401(1–2):121–131

    PubMed  CAS  Google Scholar 

  • Jones CJ, Kipling D, Morris M, Hepburn P, Skinner J, Bounacer A, Wyllie FS, Ivan M, Bartek J, Wynford-Thomas D, Bond JA (2000) Evidence for a telomere-independent “clock” limiting RAS oncogene-driven proliferation of human thyroid epithelial cells. Mol Cell Biol 20(15):5690–5699

    Article  PubMed  CAS  Google Scholar 

  • Karlseder J, Smogorzewska A, de Lange T (2002) Senescence induced by altered telomere state, not telomere loss. Science 295(5564):2446–2449

    Article  PubMed  CAS  Google Scholar 

  • Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266(5193):2011–2015

    Article  PubMed  CAS  Google Scholar 

  • Kim NW, Wu F (1997) Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP). Nucleic Acids Res 25(13):2595–2597

    Article  PubMed  CAS  Google Scholar 

  • Kurz DJ, Decary S, Hong Y, Trivier E, Akhmedov A, Erusalimsky JD (2004) Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells. J Cell Sci 117(Pt 11):2417–2426

    Article  PubMed  CAS  Google Scholar 

  • Leteurtre F, Li X, Gluckman E, Carosella ED (1997) Telomerase activity during the cell cycle and in gamma-irradiated hematopoietic cells. Leukemia 11(10):1681–1689

    Article  PubMed  CAS  Google Scholar 

  • Li GZ, Eller MS, Firoozabadi R, Gilchrest BA (2003) Evidence that exposure of the telomere 3’ overhang sequence induces senescence. Proc Natl Acad Sci USA 100(2):527–531

    Article  PubMed  CAS  Google Scholar 

  • Lindsey J, McGill NI, Lindsey LA, Green DK, Cooke HJ (1991) In vivo loss of telomeric repeats with age in humans. Mutat Res 256(1):45–48

    PubMed  CAS  Google Scholar 

  • Marcand S, Buck SW, Moretti P, Gilson E, Shore D (1996) Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap 1 protein. Genes Dev 10(11):1297–1309

    Article  PubMed  CAS  Google Scholar 

  • Martens UM, Chavez EA, Poon SS, Schmoor C, Lansdorp PM (2000) Accumulation of short telomeres in human fibroblasts prior to replicative senescence. Exp Cell Res 256(1):291–299

    Article  PubMed  CAS  Google Scholar 

  • Martin SG, Laroche T, Suka N, Grunstein M, Gasser SM (1999) Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast. Cell 97(5):621–633

    Article  PubMed  CAS  Google Scholar 

  • Mastromonaco GF, Perrault SD, Betts DH, King WA (2006) Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer. BMC Dev Biol 6(1):41

    Article  PubMed  CAS  Google Scholar 

  • Masutomi K, Yu EY, Khurts S, Ben-Porath I, Currier JL, Metz GB, Brooks MW, Kaneko S, Murakami S, DeCaprio JA, Weinberg RA, Stewart SA, Hahn WC (2003) Telomerase maintains telomere structure in normal human cells. Cell 114(2):241–253

    Article  PubMed  CAS  Google Scholar 

  • Meier A, Fiegler H, Munoz P, Ellis P, Rigler D, Langford C, Blasco MA, Carter N, Jackson SP (2007) Spreading of mammalian DNA-damage response factors studied by ChIP-chip at damaged telomeres. EMBO J 26(11):2707–2718

    Article  PubMed  CAS  Google Scholar 

  • Morin GB (1989) The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59(3):521–529

    Article  PubMed  CAS  Google Scholar 

  • Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, Meyne J, Ratliff RL, Wu JR (1988) A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci USA 85(18):6622–6626

    Article  PubMed  CAS  Google Scholar 

  • Nishi H, Nakada T, Kyo S, Inoue M, Shay JW, Isaka K (2004) Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT). Mol Cell Biol 24(13):6076–6083

    Article  PubMed  CAS  Google Scholar 

  • Noda A, Ning Y, Venable SF, Pereira-Smith OM, Smith JR (1994) Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res 211(1):90–98

    Article  PubMed  CAS  Google Scholar 

  • Olovnikov AM (1973) A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol 41(1):181–190

    Article  PubMed  CAS  Google Scholar 

  • Ouellette MM, Liao M, Herbert BS, Johnson M, Holt SE, Liss HS, Shay JW, Wright WE (2000) Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase. J Biol Chem 275(14):10072–10076

    Article  PubMed  CAS  Google Scholar 

  • Packer L, Fuehr K (1977) Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 267(5610):423–425

    Article  PubMed  CAS  Google Scholar 

  • Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J (2003) Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 5(8):741–747

    Article  PubMed  CAS  Google Scholar 

  • Passos JF, Saretzki G, Ahmed S, Nelson G, Richter T, Peters H, Wappler I, Birket MJ, Harold G, Schaeuble K, Birch-Machin MA, Kirkwood TB, von Zglinicki T (2007) Mitochondrial dysfunction accounts for the stochastic heterogeneity in telomere-dependent senescence. PLoS Biol 5(5):e110

    Article  PubMed  CAS  Google Scholar 

  • Petersen S, Saretzki G, von Zglinicki T (1998) Preferential accumulation of single-stranded regions in telomeres of human fibroblasts. Exp Cell Res 239(1):152–160

    Article  PubMed  CAS  Google Scholar 

  • Rohme D (1981) Ageing and the fusion sensitivity potential of human cells in culture: relation to tissue origin, donor age, and in vitro culture level and condition. Mech Ageing Dev 16(3):241–253

    Article  PubMed  CAS  Google Scholar 

  • Saito H, Hammond AT, Moses RE (1995) The effect of low oxygen tension on the in vitro-replicative life span of human diploid fibroblast cells and their transformed derivatives. Exp Cell Res 217(2):272–279

    Article  PubMed  CAS  Google Scholar 

  • Saretzki G, Sitte N, Merkel U, Wurm RE, von Zglinicki T (1999) Telomere shortening triggers a p53-dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments. Oncogene 18(37):5148–5158

    Article  PubMed  CAS  Google Scholar 

  • Seimiya H, Tanji M, Oh-hara T, Tomida A, Naasani I, Tsuruo T (1999) Hypoxia up-regulates telomerase activity via mitogen-activated protein kinase signaling in human solid tumor cells. Biochem Biophys Res Commun 260(2):365–370

    Article  PubMed  CAS  Google Scholar 

  • Serrano M, Hannon GJ, Beach D (1993) A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366(6456):704–707

    Article  PubMed  CAS  Google Scholar 

  • Shay JW, Pereira-Smith OM, Wright WE (1991) A role for both RB and p53 in the regulation of human cellular senescence. Exp Cell Res 196(1):33–39

    Article  PubMed  CAS  Google Scholar 

  • Shay JW, Wright WE, Brasiskyte D, Van der Haegen BA (1993) E6 of human papillomavirus type 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts. Oncogene 8(6):1407–1413

    PubMed  CAS  Google Scholar 

  • Smith JR, Venable S, Roberts TW, Metter EJ, Monticone R, Schneider EL (2002) Relationship between in vivo age and in vitro aging: assessment of 669 cell cultures derived from members of the Baltimore longitudinal study of aging. J Gerontol 57(6):B239–B246

    Google Scholar 

  • Smogorzewska A, van Steensel B, Bianchi A, Oelmann S, Schaefer MR, Schnapp G, de Lange T (2000) Control of human telomere length by TRF1 and TRF2. Mol Cell Biol 20(5):1659–1668

    Article  PubMed  CAS  Google Scholar 

  • Sohal RS, Allen RG (1990) Oxidative stress as a causal factor in differentiation and aging: a unifying hypothesis. Exp Gerontol 25(6):499–522

    Article  PubMed  CAS  Google Scholar 

  • Sokal RR, Rohlf FJ (1981) Biometry: the principles and practice of statistics in biological research. W. H. Freeman, San Francisco, pp xviii, 859 p

  • Steinert S, Shay JW, Wright WE (2000) Transient expression of human telomerase extends the life span of normal human fibroblasts. Biochem Biophys Res Commun 273(3):1095–1098

    Article  PubMed  CAS  Google Scholar 

  • Stewart SA, Ben-Porath I, Carey VJ, O’Connor BF, Hahn WC, Weinberg RA (2003) Erosion of the telomeric single-strand overhang at replicative senescence. Nat Genet 33(4):492–496

    Article  PubMed  CAS  Google Scholar 

  • te Poele RH, Okorokov AL, Jardine L, Cummings J, Joel SP (2002) DNA damage is able to induce senescence in tumor cells in vitro and in vivo. Cancer Res 62(6):1876–1883

    PubMed  CAS  Google Scholar 

  • Therkelsen AJ, Nielsen A, Koch J, Hindkjaer J, Kolvraa S (1995) Staining of human telomeres with primed in situ labeling (PRINS). Cytogenet Cell Genet 68(1–2):115–118

    Article  PubMed  CAS  Google Scholar 

  • van Steensel B, Smogorzewska A, de Lange T (1998) TRF2 protects human telomeres from end-to-end fusions. Cell 92(3):401–413

    Article  PubMed  Google Scholar 

  • Vaziri H, Benchimol S (1998) Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol 8(5):279–282

    Article  PubMed  CAS  Google Scholar 

  • von Zglinicki T, Pilger R, Sitte N (2000) Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts. Free Radic Biol Med 28(1):64–74

    Article  Google Scholar 

  • von Zglinicki T, Saretzki G, Docke W, Lotze C (1995) Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res 220(1):186–193

    Article  Google Scholar 

  • Yu GL, Bradley JD, Attardi LD, Blackburn EH (1990) In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature 344(6262):126–132

    Article  PubMed  CAS  Google Scholar 

  • Zhu J, Wang H, Bishop JM, Blackburn EH (1999) Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening. Proc Natl Acad Sci USA 96(7):3723–3728

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The Barbara Graham Memorial Trust, Natural Sciences and Engineering Research Council (NSERC) of Canada, Canadian Institutes of Health Research (CIHR), and the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) supported this research.

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  1. Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada, N1G 2W1

    Dean H. Betts, Steven D. Perrault & W. Allan King

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  1. Dean H. Betts
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Correspondence to Dean H. Betts.

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Betts, D.H., Perrault, S.D. & King, W.A. Low oxygen delays fibroblast senescence despite shorter telomeres. Biogerontology 9, 19–31 (2008). https://doi.org/10.1007/s10522-007-9113-7

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