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Genotoxic Stress-Induced Senescence

  • Dorothy N. Y. Fan
  • Clemens A. Schmitt
Part of the Methods in Molecular Biology book series (MIMB, volume 1896)

Abstract

A cell’s genomic integrity is at risk when DNA-damaging stress, evoked by mitogenic oncogenes or genotoxic treatment modalities such as radiation or chemotherapy, apply. If the DNA repair machinery fails to fix the damaged site during a temporary cell-cycle arrest, or if massive genotoxic stress overwhelmed the repair capacity, cellular failsafe programs such as apoptosis or senescence will be triggered to limit aberrant propagation of these damaged and potentially harmful cells. After decades of scientific focusing on apoptosis, cellular senescence is increasingly recognized as an equally important but biologically and fundamentally different type of ultimate cell-cycle exit program, because of its lastingly persistent nature and cell-intrinsic and extrinsic roles within the tissue and tumor microenvironment. We established primary apoptosis-compromised, Bcl2-expressing Eμ-myc transgenic mouse lymphomas as a versatile and clinically relevant model system to study therapy-induced senescence (TIS). Given the lack of a single specific senescence-defining marker, we previously exploited co-staining of senescence-associated β-galactosidase (SA-β-gal) activity with immunohistochemical detection of trimethylated histone H3 lysine 9 (H3K9me3), an established S-phase gene expression-controlling, repressive chromatin mark, and the proliferation marker Ki67. This biomarker panel is instrumental to characterize cells as senescent via their high SA-β-gal activity, strong nuclear H3K9me3 expression and Ki67-negative profile. In this chapter, we demonstrate the detection of viable senescent cells by novel methods based on a fluorescent version of the SA-β-gal (fSA-β-gal) assay, combined with immuno-fluoroscence staining of H3K9me3 or Ki67, or analysis of the DNA replication status by incorporating 5-ethynyl-2′-deoxyuridine (EdU) detection into the protocol. Notably, while most senescence markers, irrespective of their specificity and sensitivity, may only be assessed in endpoint assays, we would like to emphasize here the strength of viable fSA-β-gal to track single-cell fate in senescent populations over time.

Key words

Biomarker Cell fate 5-Ethynyl-2′-deoxyuridine (EdU) Fluorescent SA-β-gal (fSA-β-gal) Genotoxic stress H3K9me3 Ki67 Senescence Senescence-associated β-galactosidase (SA-β-gal) Single cell Therapy-induced senescence (TIS) 

References

  1. 1.
    Collado M, Blasco MA, Serrano M (2007) Cellular senescence in cancer and aging. Cell 130(2):223–233.  https://doi.org/10.1016/j.cell.2007.07.003CrossRefPubMedGoogle Scholar
  2. 2.
    Kuilman T, Michaloglou C, Mooi WJ, Peeper DS (2010) The essence of senescence. Genes Dev 24(22):2463–2479.  https://doi.org/10.1101/gad.1971610CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Salama R, Sadaie M, Hoare M, Narita M (2014) Cellular senescence and its effector programs. Genes Dev 28(2):99–114.  https://doi.org/10.1101/gad.235184.113CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    van Deursen JM (2014) The role of senescent cells in ageing. Nature 509(7501):439–446.  https://doi.org/10.1038/nature13193CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou LV, Kolettas E, Niforou K, Zoumpourlis VC, Takaoka M, Nakagawa H, Tort F, Fugger K, Johansson F, Sehested M, Andersen CL, Dyrskjot L, Orntoft T, Lukas J, Kittas C, Helleday T, Halazonetis TD, Bartek J, Gorgoulis VG (2006) Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 444(7119):633–637.  https://doi.org/10.1038/nature05268CrossRefPubMedGoogle Scholar
  6. 6.
    Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C, Schurra C, Garre M, Nuciforo PG, Bensimon A, Maestro R, Pelicci PG, d'Adda di Fagagna F (2006) Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 444(7119):638–642.  https://doi.org/10.1038/nature05327CrossRefPubMedGoogle Scholar
  7. 7.
    Chang BD, Broude EV, Dokmanovic M, Zhu H, Ruth A, Xuan Y, Kandel ES, Lausch E, Christov K, Roninson IB (1999) A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents. Cancer Res 59(15):3761–3767PubMedGoogle Scholar
  8. 8.
    Chang BD, Xuan Y, Broude EV, Zhu H, Schott B, Fang J, Roninson IB (1999) Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs. Oncogene 18(34):4808–4818.  https://doi.org/10.1038/sj.onc.1203078CrossRefPubMedGoogle Scholar
  9. 9.
    Dorr JR, Yu Y, Milanovic M, Beuster G, Zasada C, Dabritz JH, Lisec J, Lenze D, Gerhardt A, Schleicher K, Kratzat S, Purfurst B, Walenta S, Mueller-Klieser W, Graler M, Hummel M, Keller U, Buck AK, Dorken B, Willmitzer L, Reimann M, Kempa S, Lee S, Schmitt CA (2013) Synthetic lethal metabolic targeting of cellular senescence in cancer therapy. Nature 501(7467):421–425.  https://doi.org/10.1038/nature12437CrossRefPubMedGoogle Scholar
  10. 10.
    Jing H, Kase J, Dorr JR, Milanovic M, Lenze D, Grau M, Beuster G, Ji S, Reimann M, Lenz P, Hummel M, Dorken B, Lenz G, Scheidereit C, Schmitt CA, Lee S (2011) Opposing roles of NF-kappaB in anti-cancer treatment outcome unveiled by cross-species investigations. Genes Dev 25(20):2137–2146.  https://doi.org/10.1101/gad.17620611CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    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):1876–1883Google Scholar
  12. 12.
    Braig M, Lee S, Loddenkemper C, Rudolph C, Peters AH, Schlegelberger B, Stein H, Dorken B, Jenuwein T, Schmitt CA (2005) Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 436(7051):660–665.  https://doi.org/10.1038/nature03841CrossRefPubMedGoogle Scholar
  13. 13.
    Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88(5):593–602CrossRefPubMedGoogle Scholar
  14. 14.
    Schmitt CA, Fridman JS, Yang M, Lee S, Baranov E, Hoffman RM, Lowe SW (2002) A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 109(3):335–346CrossRefPubMedGoogle Scholar
  15. 15.
    Childs BG, Durik M, Baker DJ, van Deursen JM (2015) Cellular senescence in aging and age-related disease: from mechanisms to therapy. Nat Med 21(12):1424–1435.  https://doi.org/10.1038/nm.4000CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    He S, Sharpless NE (2017) Senescence in health and disease. Cell 169(6):1000–1011.  https://doi.org/10.1016/j.cell.2017.05.015CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Baker DJ, Childs BG, Durik M, Wijers ME, Sieben CJ, Zhong J, A. Saltness R, Jeganathan KB, Verzosa GC, Pezeshki A, Khazaie K, Miller JD, van Deursen JM (2016) Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature 530(7589):184–189.  https://doi.org/10.1038/nature16932CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B, Kirkland JL, van Deursen JM (2011) Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479(7372):232–236.  https://doi.org/10.1038/nature10600CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Munoz-Espin D, Canamero M, Maraver A, Gomez-Lopez G, Contreras J, Murillo-Cuesta S, Rodriguez-Baeza A, Varela-Nieto I, Ruberte J, Collado M, Serrano M (2013) Programmed cell senescence during mammalian embryonic development. Cell 155(5):1104–1118.  https://doi.org/10.1016/j.cell.2013.10.019CrossRefPubMedGoogle Scholar
  20. 20.
    Storer M, Mas A, Robert-Moreno A, Pecoraro M, Ortells MC, Di Giacomo V, Yosef R, Pilpel N, Krizhanovsky V, Sharpe J, Keyes WM (2013) Senescence is a developmental mechanism that contributes to embryonic growth and patterning. Cell 155(5):1119–1130.  https://doi.org/10.1016/j.cell.2013.10.041CrossRefPubMedGoogle Scholar
  21. 21.
    Demaria M, Ohtani N, Youssef SA, Rodier F, Toussaint W, Mitchell JR, Laberge RM, Vijg J, Van Steeg H, Dolle ME, Hoeijmakers JH, de Bruin A, Hara E, Campisi J (2014) An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev Cell 31(6):722–733.  https://doi.org/10.1016/j.devcel.2014.11.012CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Ritschka B, Storer M, Mas A, Heinzmann F, Ortells MC, Morton JP, Sansom OJ, Zender L, Keyes WM (2017) The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration. Genes Dev 31(2):172–183.  https://doi.org/10.1101/gad.290635.116CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Mosteiro L, Pantoja C, Alcazar N, Marion RM, Chondronasiou D, Rovira M, Fernandez-Marcos PJ, Munoz-Martin M, Blanco-Aparicio C, Pastor J, Gomez-Lopez G, De Martino A, Blasco MA, Abad M, Serrano M (2016) Tissue damage and senescence provide critical signals for cellular reprogramming in vivo. Science 354(6315).  https://doi.org/10.1126/science.aaf4445CrossRefPubMedGoogle Scholar
  24. 24.
    Milanovic M, Fan DNY, Belenki D, Dabritz JHM, Zhao Z, Yu Y, Dorr JR, Dimitrova L, Lenze D, Monteiro Barbosa IA, Mendoza-Parra MA, Kanashova T, Metzner M, Pardon K, Reimann M, Trumpp A, Dorken B, Zuber J, Gronemeyer H, Hummel M, Dittmar G, Lee S, Schmitt CA (2017) Senescence-associated reprogramming promotes cancer stemness. Nature 553:96–100.  https://doi.org/10.1038/nature25167CrossRefPubMedGoogle Scholar
  25. 25.
    Narita M, Nunez S, Heard E, Narita M, Lin AW, Hearn SA, Spector DL, Hannon GJ, Lowe SW (2003) Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 113(6):703–716CrossRefPubMedGoogle Scholar
  26. 26.
    Shah PP, Donahue G, Otte GL, Capell BC, Nelson DM, Cao K, Aggarwala V, Cruickshanks HA, Rai TS, McBryan T, Gregory BD, Adams PD, Berger SL (2013) Lamin B1 depletion in senescent cells triggers large-scale changes in gene expression and the chromatin landscape. Genes Dev 27(16):1787–1799.  https://doi.org/10.1101/gad.223834.113CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O et al (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92(20):9363–9367CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Sharpless NE, Sherr CJ (2015) Forging a signature of in vivo senescence. Nat Rev Cancer 15(7):397–408.  https://doi.org/10.1038/nrc3960CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Fan DN, Schmitt CA (2017) Detecting markers of therapy-induced senescence in cancer cells. Methods Mol Biol 1534:41–52.  https://doi.org/10.1007/978-1-4939-6670-7_4CrossRefPubMedGoogle Scholar
  30. 30.
    Schmitt CA, Rosenthal CT, Lowe SW (2000) Genetic analysis of chemoresistance in primary murine lymphomas. Nat Med 6(9):1029–1035.  https://doi.org/10.1038/79542CrossRefPubMedGoogle Scholar
  31. 31.
    van Oijen MG, Medema RH, Slootweg PJ, Rijksen G (1998) Positivity of the proliferation marker Ki-67 in noncycling cells. Am J Clin Pathol 110(1):24–31CrossRefPubMedGoogle Scholar
  32. 32.
    Zolzer F, Streffer C (1995) Cell cycle-dependent expression of Ki-67 antigen in human melanoma cells subjected to irradiation and/or hyperthermia. Radiat Res 143(1):98–101CrossRefPubMedGoogle Scholar
  33. 33.
    Schmitt CA, Fridman JS, Yang M, Baranov E, Hoffman RM, Lowe SW (2002) Dissecting p53 tumor suppressor functions in vivo. Cancer Cell 1(3):289–298CrossRefPubMedGoogle Scholar
  34. 34.
    Schmitt CA, Lowe SW (2002) Apoptosis and chemoresistance in transgenic cancer models. J Mol Med (Berl) 80(3):137–146.  https://doi.org/10.1007/s00109-001-0293-3CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Hematology, Oncology and Tumor Immunology, Molekulares Krebsforschungszentrum – MKFZCharité – University Medical CenterBerlinGermany
  2. 2.German Cancer Research Center (Deutsches Krebsforschungszentrum [DKFZ])HeidelbergGermany
  3. 3.Deutsches Konsortium für Translationale Krebsforschung (German Cancer Consortium), Partner Site BerlinBerlinGermany
  4. 4.Max-Delbrück-Center for Molecular MedicineBerlinGermany

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