Abstract
The detection of senescent cells has become an important area of research in the aging field. Due to the complexity of the senescence program and the lack of a unique signature for senescence, the detection of these cells remains problematic. This is especially true for in vivo detection in aged or diseased tissue samples. This chapter outlines approaches for the detection of senescent cells based upon methods established for mesenchymal cells in culture. A stepwise approach to the detection of senescent cells using multiple techniques is provided.
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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
Wright WE, Shay JW (2000) Telomere dynamics in cancer progression and prevention: fundamental differences in human and mouse telomere biology. Nat Med 6(8):849–851
Kennedy AL, McBryan T, Enders GH, Johnson FB, Zhang R, Adams PD (2010) Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci. Cell Div 5:16
Coppe JP, Rodier F, Patil CK, Freund A, Desprez PY, Campisi J (2011) Tumor suppressor and aging biomarker p16(INK4a) induces cellular senescence without the associated inflammatory secretory phenotype. J Biol Chem 286(42):36396–36403
Itahana K, Dimri G, Campisi J (2001) Regulation of cellular senescence by p53. Eur J Biochem 268(10):2784–2791
Coppe JP, Desprez PY, Krtolica A, Campisi J (2010) The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol 5:99–118
Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, Sharpless NE (2004) Ink4a/Arf expression is a biomarker of aging. J Clin Invest 114(9):1299–1307
Naylor RM, Baker DJ, van Deursen JM (2013) Senescent cells: a novel therapeutic target for aging and age-related diseases. Clin Pharmacol Ther 93(1):105–116
Hayflick L (1965) The limited in vitro lifetime of human diploid strains. Exp Cell Res 37:614–636
Hayflick L, Moorhead P (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621
Eidukevicius R, Characiejus D, Janavicius R, Kazlauskaite N, Pasukoniene V, Mauricas M, Den Otter W (2005) A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample. BMC Cancer 5:122
Gibson GE, Tofel-Grehl B, Scheffold K, Cristofalo VJ, Blass JP (1998) A reproducible procedure for primary culture and subsequent maintenance of multiple lines of human skin fibroblasts. Age (Omaha) 21(1):7–14
Jurk D, Wang C, Miwa S, Maddick M, Korolchuk V, Tsolou A, Gonos ES, Thrasivoulou C, Saffrey MJ, Cameron K, von Zglinicki T (2012) Postmitotic neurons develop a p21-dependent senescence-like phenotype driven by a DNA damage response. Aging Cell 11(6):996–1004
Besancenot R, Chaligné R, Tonetti C, Pasquier F, Marty C, Lécluse Y, Vainchenker W, Constantinescu SN, Giraudier S (2010) A senescence-like cell-cycle arrest occurs during megakaryocytic maturation: implications for physiological and pathological megakaryocytic proliferation. PLoS Biol 8(9): e1000476. doi:10.1371/journal.pbio.1000476, pii: e1000476
Lanza RP, Cibelli JB, Blackwell C, Cristofalo VJ, Francis MK, Baerlocher GM, Mak J, Schertzer M, Chavez EA, Sawyer N, Lansdorp PM, West MD (2000) Extension of cell life-span and telomere length in animals cloned from senescent somatic cells. Science 288(5466):665–669
Niedernhofer LJ, Glorioso JC, Robbins PD (2011) Dedifferentiation rescues senescence of progeria cells but only while pluripotent. Stem Cell Res Ther 2(3):28
Banito A, Rashid ST, Acosta JC, Li S, Pereira CF, Geti I, Pinho S, Silva JC, Azuara V, Walsh M, Vallier L, Gil J (2009) Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev 23(18):2134–2139
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):9363–9367
Kurz DJ, Decary S, Hong Y, Erusalimsky JD (2000) Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J Cell Sci 113(Pt 20):3613–3622
Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, Kleijer WJ, DiMaio D, Hwang ES (2006) Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell 5(2):187–195
Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O (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
Donnini S, Solito R, Cetti E, Corti F, Giachetti A, Carra S, Beltrame M, Cotelli F, Ziche M (2010) Abeta peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis. FASEB J 24(7):2385–2395
Minamino T, Miyauchi H, Yoshida T, Ishida Y, Yoshida H, Komuro I (2002) Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation 105(13):1541–1544
Cao L, Li W, Kim S, Brodie SG, Deng CX (2003) Senescence, aging, and malignant transformation mediated by p53 in mice lacking the Brca1 full-length isoform. Genes Dev 17(2):201–213
Wei W, Sedivy JM (1999) Differentiation between senescence (M1) and crisis (M2) in human fibroblast cultures. Exp Cell Res 253(2):519–522
Narita M, Narita M, Krizhanovsky V, Nunez S, Chicas A, Hearn SA, Myers MP, Lowe SW (2006) A novel role for high-mobility group a proteins in cellular senescence and heterochromatin formation. Cell 126(3):503–514
Narita M, Nunez S, Heard E, 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
Zhang R, Chen W, Adams PD (2007) Molecular dissection of formation of senescence-associated heterochromatin foci. Mol Cell Biol 27(6):2343–2358
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
Cruickshanks HA, McBryan T, Nelson DM, Vanderkraats ND, Shah PP, van Tuyn J, Singh Rai T, Brock C, Donahue G, Dunican DS, Drotar ME, Meehan RR, Edwards JR, Berger SL, Adams PD (2013) Senescent cells harbour features of the cancer epigenome. Nat Cell Biol 15(12):1495–1506
Kreiling JA, Tamamori-Adachi M, Sexton AN, Jeyapalan JC, Munoz-Najar U, Peterson AL, Manivannan J, Rogers ES, Pchelintsev NA, Adams PD, Sedivy JM (2011) Age-associated increase in heterochromatic marks in murine and primate tissues. Aging Cell 10(2):292–304
Coppe JP, Patil CK, Rodier F, Sun Y, Munoz DP, Goldstein J, Nelson PS, Desprez PY, Campisi J (2008) Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6(12):2853–2868
Goldstein S, Moerman EJ, Baxter RC (1993) Accumulation of insulin-like growth factor binding protein-3 in conditioned medium of human fibroblasts increases with chronologic age of donor and senescence in vitro. J Cell Physiol 156(2):294–302
Thweatt R, Murano S, Fleischmann RD, Goldstein S (1992) Isolation and characterization of gene sequences overexpressed in Werner syndrome fibroblasts during premature replicative senescence. Exp Gerontol 27(4):433–440
Tchkonia T, Zhu Y, van Deursen J, Campisi J, Kirkland JL (2013) Cellular senescence and the senescent secretory phenotype: therapeutic opportunities. J Clin Invest 123(3):966–972
Freund A, Orjalo AV, Desprez PY, Campisi J (2010) Inflammatory networks during cellular senescence: causes and consequences. Trends Mol Med 16(5):238–246
Rodier F, Coppe JP, Patil CK, Hoeijmakers WA, Munoz DP, Raza SR, Freund A, Campeau E, Davalos AR, Campisi J (2009) Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion. Nat Cell Biol 11(8):973–979
Freund A, Patil CK, Campisi J (2011) p38MAPK is a novel DNA damage response-independent regulator of the senescence-associated secretory phenotype. EMBO J 30(8):1536–1548
Kuilman T, Michaloglou C, Vredeveld LC, Douma S, van Doorn R, Desmet CJ, Aarden LA, Mooi WJ, Peeper DS (2008) Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell 133(6):1019–1031
Acosta JC, Banito A, Wuestefeld T, Georgilis A, Janich P, Morton JP, Athineos D, Kang TW, Lasitschka F, Andrulis M, Pascual G, Morris KJ, Khan S, Jin H, Dharmalingam G, Snijders AP, Carroll T, Capper D, Pritchard C, Inman GJ, Longerich T, Sansom OJ, Benitah SA, Zender L, Gil J (2013) A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat Cell Biol 15(8):978–990
Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C, Yee H, Zender L, Lowe SW (2008) Senescence of activated stellate cells limits liver fibrosis. Cell 134(4):657–667
Jun JI, Lau LF (2010) The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing. Nat Cell Biol 12(7):676–685
Severino V, Alessio N, Farina A, Sandomenico A, Cipollaro M, Peluso G, Galderisi U, Chambery A (2013) Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells. Cell Death Dis 4:e911
Coppe JP, Patil CK, Rodier F, Krtolica A, Beausejour CM, Parrinello S, Hodgson JG, Chin K, Desprez PY, Campisi J (2010) A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen. PLoS One 5(2):e9188
Acosta JC, Snijders AP, Gil J (2013) Unbiased characterization of the senescence-associated secretome using SILAC-based quantitative proteomics. Methods Mol Biol 965:175–184
Lerner C, Bitto A, Pulliam D, Nacarelli T, Konigsberg M, Van Remmen H, Torres C, Sell C (2013) Reduced mammalian target of rapamycin activity facilitates mitochondrial retrograde signaling and increases life span in normal human fibroblasts. Aging Cell 12(6):966–977
Bitto A, Lerner C, Torres C, Roell M, Malaguti M, Perez V, Lorenzini A, Hrelia S, Ikeno Y, Matzko ME, McCarter R, Sell C (2010) Long-term IGF-I exposure decreases autophagy and cell viability. PLoS One 5(9):e12592
Kim WY, Sharpless NE (2006) The regulation of INK4/ARF in cancer and aging. Cell 127(2):265–275
Gil J, Peters G (2006) Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 7(9):667–677
Sawicka M, Pawlikowski J, Wilson S, Ferdinando D, Wu H, Adams PD, Gunn DA, Parish W (2013) The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding. PLoS One 8(1):e53313
Bhat R, Crowe EP, Bitto A, Moh M, Katsetos CD, Garcia FU, Johnson FB, Trojanowski JQ, Sell C, Torres C (2012) Astrocyte senescence as a component of Alzheimer’s disease. PLoS One 7(9):e45069
Bitto A, Sell C, Crowe E, Lorenzini A, Malaguti M, Hrelia S, Torres C (2010) Stress-induced senescence in human and rodent astrocytes. Exp Cell Res 316(17):2961–2968
Acknowledgements
This work was supported by grants 1RO1NS078283 to C.T. and AG039799 to C.S., the Commonwealth of Pennsylvania Universal Research Enhancement Grant (CT), the Drexel University College of Medicine Research Program Planning Grant (CT), and the Drexel Aging Initiative. T.N. is the recipient of a fellowship from the Drexel Aging Initiative. Research reported in this publication is also supported by the National Institute on Aging of the National Institutes of Health under Award Number F30AG043307 (EPC). The content of this chapter is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Crowe, E.P., Nacarelli, T., Bitto, A., Lerner, C., Sell, C., Torres, C. (2014). Detecting Senescence: Methods and Approaches. In: Noguchi, E., Gadaleta, M. (eds) Cell Cycle Control. Methods in Molecular Biology, vol 1170. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0888-2_23
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DOI: https://doi.org/10.1007/978-1-4939-0888-2_23
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