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The Myc Gene pp 99–116Cite as

Methods to Study MYC-Regulated Cellular Senescence

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1012))

Abstract

Studies in primary and tumor cells suggest that MYC plays an important role in regulating cellular senescence, thereby impacting on tumor development. Here we describe different common methods to measure senescence in cell cultures and in tissues. These include measurement of senescence-associated β-galactosidase activity (SA-β-gal), senescence-associated heterochromatin foci (SAHFs), proliferative arrest, morphological changes, and expression and activity of proteins involved in the senescence process, such as p53 and Rb pathway proteins and secretory proteins. It is important to note that there is no unique marker that unambiguously defines a senescent state, and it is therefore necessary to combine measurements of several different markers that together determine whether cells are senescent or not. Measurement of senescence is an important aspect of studies of MYC biology and will improve our understanding of MYC function and regulation both in preclinical and clinical settings. This may form the basis for new concepts of pro-senescence therapy to combat MYC in cancer.

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References

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

    Article  PubMed  CAS  Google Scholar 

  2. Herbig U, Ferreira M, Condel L, Carey D, Sedivy JM (2006) Cellular senescence in aging primates. Science 311(5765):1257

    Article  PubMed  CAS  Google Scholar 

  3. Campisi J, d'Adda di Fagagna F (2007) Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 8(9):729–740

    Article  PubMed  CAS  Google Scholar 

  4. Collado M, Serrano M (2010) Senescence in tumours: evidence from mice and humans. Nat Rev Cancer 10(1):51–57

    Article  PubMed  CAS  Google Scholar 

  5. Kuilman T, Michaloglou C, Mooi WJ, Peeper DS (2010) The essence of senescence. Genes Dev 24(22):2463–2479

    Article  PubMed  CAS  Google Scholar 

  6. Larsson LG (2011) Oncogene- and tumor suppressor gene-mediated suppression of cellular senescence. Semin Cancer Biol 21(6):367–376

    Article  PubMed  CAS  Google Scholar 

  7. 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):593–602

    Article  PubMed  CAS  Google Scholar 

  8. Gil J, Kerai P, Lleonart M, Bernard D, Cigudosa JC, Peters G, Carnero A, Beach D (2005) Immortalization of primary human prostate epithelial cells by c-Myc. Cancer Res 65(6):2179–2185

    Article  PubMed  CAS  Google Scholar 

  9. Guney I, Wu S, Sedivy JM (2006) Reduced c-Myc signaling triggers telomere-independent senescence by regulating Bmi-1 and p16(INK4a). Proc Natl Acad Sci U S A 103(10):3645–3650

    Article  PubMed  CAS  Google Scholar 

  10. Hydbring P, Bahram F, Su Y, Tronnersjo S, Hogstrand K, von der Lehr N, Sharifi HR, Lilischkis R, Hein N, Wu S, Vervoorts J, Henriksson M, Grandien A, Luscher B, Larsson LG (2010) Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation. Proc Natl Acad Sci U S A 107(1):58–63

    Article  PubMed  CAS  Google Scholar 

  11. Mallette FA, Gaumont-Leclerc MF, Huot G, Ferbeyre G (2007) Myc down-regulation as a mechanism to activate the Rb pathway in STAT5A-induced senescence. J Biol Chem 282(48):34938–34944

    Article  PubMed  CAS  Google Scholar 

  12. Ruggero D, Montanaro L, Ma L, Xu W, Londei P, Cordon-Cardo C, Pandolfi PP (2004) The translation factor eIF-4E promotes tumor formation and cooperates with c-Myc in lymphomagenesis. Nat Med 10(5):484–486

    Article  PubMed  CAS  Google Scholar 

  13. Zhuang D, Mannava S, Grachtchouk V, Tang WH, Patil S, Wawrzyniak JA, Berman AE, Giordano TJ, Prochownik EV, Soengas MS, Nikiforov MA (2008) C-MYC overexpression is required for continuous suppression of oncogene-induced senescence in melanoma cells. Oncogene 27(52):6623–6634

    Article  PubMed  CAS  Google Scholar 

  14. van Riggelen J, Muller J, Otto T, Beuger V, Yetil A, Choi PS, Kosan C, Moroy T, Felsher DW, Eilers M (2010) The interaction between Myc and Miz1 is required to antagonize TGFbeta-dependent autocrine signaling during lymphoma formation and maintenance. Genes Dev 24(12):1281–1294

    Article  PubMed  Google Scholar 

  15. Soucek L, Whitfield J, Martins CP, Finch AJ, Murphy DJ, Sodir NM, Karnezis AN, Swigart LB, Nasi S, Evan GI (2008) Modelling Myc inhibition as a cancer therapy. Nature 455(7213):679–683

    Article  PubMed  CAS  Google Scholar 

  16. Wu CH, van Riggelen J, Yetil A, Fan AC, Bachireddy P, Felsher DW (2007) Cellular senescence is an important mechanism of tumor regression upon c-Myc inactivation. Proc Natl Acad Sci U S A 104(32):13028–13033

    Article  PubMed  CAS  Google Scholar 

  17. Campaner S, Doni M, Hydbring P, Verrecchia A, Bianchi L, Sardella D, Schleker T, Perna D, Tronnersjo S, Murga M, Fernandez-Capetillo O, Barbacid M, Larsson LG, Amati B (2010) Cdk2 suppresses cellular senescence induced by the c-myc oncogene. Nat Cell Biol 12(1):54–59, sup pp 51–14

    Article  PubMed  CAS  Google Scholar 

  18. Grandori C, Wu KJ, Fernandez P, Ngouenet C, Grim J, Clurman BE, Moser MJ, Oshima J, Russell DW, Swisshelm K, Frank S, Amati B, Dalla-Favera R, Monnat RJ Jr (2003) Werner syndrome protein limits MYC-induced cellular senescence. Genes Dev 17(13):1569–1574

    Article  PubMed  CAS  Google Scholar 

  19. Moser R, Toyoshima M, Robinson K, Gurley KE, Howie HL, Davison J, Morgan M, Kemp CJ, Grandori C (2012) MYC-driven tumorigenesis is inhibited by WRN syndrome gene deficiency. Mol Cancer Res 10(4):535–545

    Article  PubMed  CAS  Google Scholar 

  20. Post SM, Quintas-Cardama A, Terzian T, Smith C, Eischen CM, Lozano G (2010) p53-Dependent senescence delays Eμ-myc-induced B-cell lymphomagenesis. Oncogene 29(9):1260–1269

    Article  PubMed  CAS  Google Scholar 

  21. Reimann M, Lee S, Loddenkemper C, Dorr JR, Tabor V, Aichele P, Stein H, Dorken B, Jenuwein T, Schmitt CA (2010) Tumor stroma-derived TGF-beta limits myc-driven lymphomagenesis via Suv39h1-dependent senescence. Cancer Cell 17(3):262–272

    Article  PubMed  CAS  Google Scholar 

  22. Nardella C, Clohessy JG, Alimonti A, Pandolfi PP (2011) Pro-senescence therapy for cancer treatment. Nat Rev Cancer 11(7):503–511

    Article  PubMed  CAS  Google Scholar 

  23. Collado M, Serrano M (2006) The power and the promise of oncogene-induced senescence markers. Nat Rev Cancer 6(6):472–476

    Article  PubMed  CAS  Google Scholar 

  24. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, Peacocke M, Campisi J (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):9363–9367

    Article  PubMed  CAS  Google Scholar 

  25. Severino J, Allen RG, Balin S, Balin A, Cristofalo VJ (2000) Is beta-galactosidase staining a marker of senescence in vitro and in vivo? Exp Cell Res 257(1):162–171

    Article  PubMed  CAS  Google Scholar 

  26. Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O (2009) Protocols to detect senescence-associated beta-galactosidase (SA-βgal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4(12):1798–1806

    Article  PubMed  CAS  Google Scholar 

  27. 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):703–716

    Article  PubMed  CAS  Google Scholar 

  28. Di Micco R, Sulli G, Dobreva M, Liontos M, Botrugno OA, Gargiulo G, dal Zuffo R, Matti V, d'Ario G, Montani E, Mercurio C, Hahn WC, Gorgoulis V, Minucci S, d'Adda di Fagagna F (2011) Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer. Nat Cell Biol 13(3):292–302

    Article  PubMed  Google Scholar 

  29. Kosar M, Bartkova J, Hubackova S, Hodny Z, Lukas J, Bartek J (2011) Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16(ink4a). Cell Cycle 10(3):457–468

    Article  PubMed  CAS  Google Scholar 

  30. Bayreuther K, Rodemann HP, Francz PI, Maier K (1988) Differentiation of fibroblast stem cells. J Cell Sci Suppl 10:115–130

    Article  PubMed  CAS  Google Scholar 

  31. Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M, Benguria A, Zaballos A, Flores JM, Barbacid M, Beach D, Serrano M (2005) Tumour biology: senescence in premalignant tumours. Nature 436(7051):642

    Article  PubMed  CAS  Google Scholar 

  32. 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):335–346

    Article  PubMed  CAS  Google Scholar 

  33. Shi SR, Imam SA, Young L, Cote RJ, Taylor CR (1995) Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 43(2):193–201

    Article  PubMed  CAS  Google Scholar 

  34. Bankfalvi A, Navabi H, Bier B, Bocker W, Jasani B, Schmid KW (1994) Wet autoclave pretreatment for antigen retrieval in diagnostic immunohistochemistry. J Pathol 174(3):223–228

    Article  PubMed  CAS  Google Scholar 

  35. 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

    Article  PubMed  CAS  Google Scholar 

  36. Michaloglou C, Vredeveld LC, Soengas MS, Denoyelle C, Kuilman T, van der Horst CM, Majoor DM, Shay JW, Mooi WJ, Peeper DS (2005) BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436(7051):720–724

    Article  PubMed  CAS  Google Scholar 

  37. Reimann M, Loddenkemper C, Rudolph C, Schildhauer I, Teichmann B, Stein H, Schlegelberger B, Dorken B, Schmitt CA (2007) The Myc-evoked DNA damage response accounts for treatment resistance in primary lymphomas in vivo. Blood 110(8):2996–3004

    Article  PubMed  CAS  Google Scholar 

  38. Chen Z, Trotman LC, Shaffer D, Lin HK, Dotan ZA, Niki M, Koutcher JA, Scher HI, Ludwig T, Gerald W, Cordon-Cardo C, Pandolfi PP (2005) Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 436(7051):725–730

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Swedish Cancer Society (V.T., L.G.L.), the Swedish Childhood Cancer Society (L.G.L.), the Swedish Research Council (L.G.L.), Olle Engkvist Foundation (L.G.L.), the Karolinska Institutet Foundations (V.T., L.G.L.), and the Advanced Cancer Therapies (ACT!) consortium (L.G.L.).

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Authors and Affiliations

  1. Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden

    Vedrana Tabor, Matteo Bocci & Lars-Gunnar Larsson

Authors
  1. Vedrana Tabor
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  2. Matteo Bocci
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  3. Lars-Gunnar Larsson
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Editors and Affiliations

  1. Fundació Vall d'Hebron Institute of Onco, Barcelona, Spain

    Laura Soucek

  2. Department of Biochemistry Sanger Building, University of Cambridge, Cambridge, United Kingdom

    Nicole M. Sodir

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Tabor, V., Bocci, M., Larsson, LG. (2013). Methods to Study MYC-Regulated Cellular Senescence. In: Soucek, L., Sodir, N. (eds) The Myc Gene. Methods in Molecular Biology, vol 1012. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-429-6_8

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  • DOI: https://doi.org/10.1007/978-1-62703-429-6_8

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-428-9

  • Online ISBN: 978-1-62703-429-6

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