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Methods to Study Myc-Regulated Cellular Senescence: An Update

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The Myc Gene

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2318))

Abstract

Cellular senescence plays a role in several physiological processes including aging, embryonic development, tissue remodeling, and wound healing and is considered one of the main barriers against tumor development. Studies of normal and tumor cells both in culture and in vivo suggest that MYC plays an important role in regulating senescence, thereby contributing to tumor development. We have previously described different common methods to measure senescence in cell cultures and in tissues. Unfortunately, there is no unique marker that unambiguously defines a senescent state, and it is therefore necessary to combine measurements of several different markers in order to assure the correct identification of senescent cells. Here we describe protocols for simultaneous detection of multiple senescence markers in situ, a quantitative fluorogenic method to measure senescence-associated β-galactosidase activity (SA-β-gal), and a new method to detect senescent cells based on the Sudan Black B (SBB) analogue GL13, which is applicable to formalin-fixed paraffin-embedded tissues. The application of these methods in various systems will hopefully shed further light on the role of MYC in regulation of senescence, and how that impacts normal physiological processes as well as diseases and in particular cancer development.

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References

  1. 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. https://doi.org/10.1038/nrm2233. nrm2233 [pii]

    Article  CAS  PubMed  Google Scholar 

  2. Gorgoulis V, Adams PD, Alimonti A et al (2019) Cellular senescence: defining a path forward. Cell 179(4):813–827. https://doi.org/10.1016/j.cell.2019.10.005

    Article  CAS  PubMed  Google Scholar 

  3. Hernandez-Segura A, Nehme J, Demaria M (2018) Hallmarks of cellular senescence. Trends Cell Biol 28(6):436–453. https://doi.org/10.1016/j.tcb.2018.02.001

    Article  CAS  PubMed  Google Scholar 

  4. Kuilman T, Michaloglou C, Mooi WJ et al (2010) The essence of senescence. Genes Dev 24(22):2463–2479. https://doi.org/10.1101/gad.1971610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Munoz-Espin D, Serrano M (2014) Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol 15(7):482–496. https://doi.org/10.1038/nrm3823

    Article  CAS  PubMed  Google Scholar 

  6. Sharpless NE, Sherr CJ (2015) Forging a signature of in vivo senescence. Nat Rev Cancer 15(7):397–408. https://doi.org/10.1038/nrc3960

    Article  CAS  PubMed  Google Scholar 

  7. Lee S, Schmitt CA (2019) The dynamic nature of senescence in cancer. Nat Cell Biol 21(1):94–101. https://doi.org/10.1038/s41556-018-0249-2

    Article  CAS  PubMed  Google Scholar 

  8. Serrano M, Lin AW, McCurrach ME et al (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88(5):593–602

    Article  CAS  Google Scholar 

  9. Lowe SW, Cepero E, Evan G (2004) Intrinsic tumour suppression. Nature 432(7015):307–315. https://doi.org/10.1038/nature03098. nature03098 [pii]

    Article  CAS  PubMed  Google Scholar 

  10. Hydbring P, Bahram F, Su Y et al (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. https://doi.org/10.1073/pnas.0900121106

    Article  PubMed  Google Scholar 

  11. Juan J, Muraguchi T, Iezza G et al (2014) Diminished WNT → beta-catenin → c-MYC signaling is a barrier for malignant progression of BRAFV600E-induced lung tumors. Genes Dev 28(6):561–575. https://doi.org/10.1101/gad.233627.113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Larsson LG (2011) Oncogene- and tumor suppressor gene-mediated suppression of cellular senescence. Semin Cancer Biol 21(6):367–376. https://doi.org/10.1016/j.semcancer.2011.10.005

    Article  CAS  PubMed  Google Scholar 

  13. Mallette FA, Gaumont-Leclerc MF, Huot G et al (2007) Myc down-regulation as a mechanism to activate the Rb pathway in STAT5A-induced senescence. J Biol Chem 282(48):34938–34944. https://doi.org/10.1074/jbc.M707074200

    Article  CAS  PubMed  Google Scholar 

  14. Ruggero D, Montanaro L, Ma L et al (2004) The translation factor eIF-4E promotes tumor formation and cooperates with c-Myc in lymphomagenesis. Nat Med 10(5):484–486. https://doi.org/10.1038/nm1042

    Article  CAS  PubMed  Google Scholar 

  15. Tabor V, Bocci M, Alikhani N et al (2014) MYC synergizes with activated BRAFV600E in mouse lung tumor development by suppressing senescence. Cancer Res 74(16):4222–4229. https://doi.org/10.1158/0008-5472.CAN-13-3234

    Article  CAS  PubMed  Google Scholar 

  16. Wu CH, van Riggelen J, Yetil A et al (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. https://doi.org/10.1073/pnas.0701953104. 0701953104 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhuang D, Mannava S, Grachtchouk V et al (2008) C-MYC overexpression is required for continuous suppression of oncogene-induced senescence in melanoma cells. Oncogene 27(52):6623–6634. https://doi.org/10.1038/onc.2008.258. onc2008258 [pii]

    Article  CAS  PubMed  Google Scholar 

  18. van Riggelen J, Muller J, Otto T et al (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. https://doi.org/10.1101/gad.585710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Soucek L, Whitfield J, Martins CP et al (2008) Modelling Myc inhibition as a cancer therapy. Nature 455(7213):679–683. https://doi.org/10.1038/nature07260. nature07260 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Swartling FJ, Grimmer MR, Hackett CS et al (2010) Pleiotropic role for MYCN in medulloblastoma. Genes Dev 24(10):1059–1072. https://doi.org/10.1101/gad.1907510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Campaner S, Doni M, Hydbring P et al (2010) Cdk2 suppresses cellular senescence induced by the c-myc oncogene. Nat Cell Biol 12(1):54–59. https://doi.org/10.1038/ncb2004

    Article  CAS  PubMed  Google Scholar 

  22. Grandori C, Wu KJ, Fernandez P et al (2003) Werner syndrome protein limits MYC-induced cellular senescence. Genes Dev 17(13):1569–1574. https://doi.org/10.1101/gad.1100303. 17/13/1569 [pii]

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Moser R, Toyoshima M, Robinson K et al (2012) MYC-driven tumorigenesis is inhibited by WRN syndrome gene deficiency. Mol Cancer Res 10(4):535–545. https://doi.org/10.1158/1541-7786.MCR-11-0508

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Post SM, Quintas-Cardama A, Terzian T et al (2010) p53-dependent senescence delays Emu-myc-induced B-cell lymphomagenesis. Oncogene 29(9):1260–1269. https://doi.org/10.1038/onc.2009.423. onc2009423 [pii]

    Article  CAS  PubMed  Google Scholar 

  25. Reimann M, Lee S, Loddenkemper C et al (2010) Tumor stroma-derived TGF-beta limits myc-driven lymphomagenesis via Suv39h1-dependent senescence. Cancer Cell 17(3):262–272. https://doi.org/10.1016/j.ccr.2009.12.043

    Article  CAS  PubMed  Google Scholar 

  26. Collado M, Serrano M (2006) The power and the promise of oncogene-induced senescence markers. Nat Rev Cancer 6(6):472–476. https://doi.org/10.1038/nrc1884

    Article  CAS  PubMed  Google Scholar 

  27. Tabor V, Bocci M, Larsson LG (2013) Methods to study MYC-regulated cellular senescence. Methods Mol Biol 1012:99–116. https://doi.org/10.1007/978-1-62703-429-6_8

    Article  CAS  PubMed  Google Scholar 

  28. Dimri GP, Lee X, Basile G 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):9363–9367

    Article  CAS  Google Scholar 

  29. Itahana K, Itahana Y, Dimri GP (2013) Colorimetric detection of senescence-associated beta galactosidase. Methods Mol Biol 965:143–156. https://doi.org/10.1007/978-1-62703-239-1_8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Yang NC, Hu ML (2004) A fluorimetric method using fluorescein di-beta-D-galactopyranoside for quantifying the senescence-associated beta-galactosidase activity in human foreskin fibroblast Hs68 cells. Anal Biochem 325(2):337–343. https://doi.org/10.1016/j.ab.2003.11.012

    Article  CAS  PubMed  Google Scholar 

  31. Debacq-Chainiaux F, Erusalimsky JD, Campisi J et al (2009) Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4(12):1798–1806. https://doi.org/10.1038/nprot.2009.191. nprot.2009.191 [pii]

    Article  CAS  PubMed  Google Scholar 

  32. Gary RK, Kindell SM (2005) Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts. Anal Biochem 343(2):329–334. https://doi.org/10.1016/j.ab.2005.06.003

    Article  CAS  PubMed  Google Scholar 

  33. Georgakopoulou EA, Tsimaratou K, Evangelou K et al (2013) Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues. Aging (Albany NY) 5(1):37–50. https://doi.org/10.18632/aging.100527

    Article  CAS  Google Scholar 

  34. Evangelou K, Lougiakis N, Rizou SV et al (2017) Robust, universal biomarker assay to detect senescent cells in biological specimens. Aging Cell 16(1):192–197. https://doi.org/10.1111/acel.12545

    Article  CAS  PubMed  Google Scholar 

  35. Hendrikx PJ, Martens AC, Visser JW et al (1994) Differential suppression of background mammalian lysosomal beta-galactosidase increases the detection sensitivity of LacZ-marked leukemic cells. Anal Biochem 222(2):456–460. https://doi.org/10.1006/abio.1994.1516

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Swedish Cancer Society (L.G.L.), the Swedish Childhood Cancer Society (M.A., L.G.L.), the Knut and Alice Wallenberg Foundation (L.G.L.), the O. E. och Edla Johanssons foundation (M.A.) and the Karolinska Institutet Foundations (M.A., L.G.L.).

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Author notes

  1. Fan Zhang

    Present address: Vesicode AB, Solna, Sweden

Authors and Affiliations

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

    Fan Zhang, Wesam Bazzar, Mohammad Alzrigat & Lars-Gunnar Larsson

Authors
  1. Fan Zhang
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  2. Wesam Bazzar
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  3. Mohammad Alzrigat
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  4. Lars-Gunnar Larsson
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Corresponding author

Correspondence to Lars-Gunnar Larsson .

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Editors and Affiliations

  1. Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain

    Laura Soucek

  2. Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain

    Jonathan Whitfield

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Zhang, F., Bazzar, W., Alzrigat, M., Larsson, LG. (2021). Methods to Study Myc-Regulated Cellular Senescence: An Update. In: Soucek, L., Whitfield, J. (eds) The Myc Gene. Methods in Molecular Biology, vol 2318. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1476-1_12

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  • DOI: https://doi.org/10.1007/978-1-0716-1476-1_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1475-4

  • Online ISBN: 978-1-0716-1476-1

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