Abstract
The characteristic features of senescent cells such as their “flattened” appearance, enlarged nuclei and low saturation density at the plateau phase of cell growth, can be conveniently measured by image-assisted cytometry such as provided by a laser scanning cytometer (LSC). The “flattening” of senescent cells is reflected by a decline in local density of DNA-associated staining with 4,6-diamidino-2-phenylindole (DAPI) and is paralleled by an increase in nuclear area. Thus, the ratio of the maximal intensity of DAPI fluorescence per nucleus to the nuclear area provides a very sensitive morphometric biomarker of “depth” of senescence, which progressively declines during senescence induction. Also recorded is cellular DNA content revealing cell cycle phase, as well as the saturation cell density at plateau phase of growth, which is dramatically decreased in cultures of senescent cells. Concurrent immunocytochemical analysis of expression of p21WAF1, p16INK4a, or p27KIP1 cyclin kinase inhibitors provides additional markers of senescence. These biomarker indices can be expressed in quantitative terms (“senescence indices”) as a fraction of the same markers of the exponentially growing cells in control cultures.
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References
Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636
Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345:458–460
Kuilman T, Michaloglou C, Mooi WJ, Peeper DS (2010) The essence of senescence. Genes Dev 24:2463–2479
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 5:741–747
Sherr CJ, DePinho RA (2000) Cellular senescence: mitotic clock or culture shock? Cell 102:407–410
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 88:593–602
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 436:725–730
Gewirtz DA, Holt SE, Elmore LW (2008) Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation. Biochem Pharmacol 76:947–957
Litwiniec A, Grzanka A, Helmin-Basa A, Gackowska L, Grzanka D (2010) Features of senescence and cell death induced by doxorubicin in A549 cells: organization and level of selected cytoskeletal proteins. J Cancer Res Clin Oncol 136:717–736
Banito A, Gil J (2010) Induced pluripotent stem cells and senescence: learning the biology to improve the technology. EMBO Rep 11:353–359
Cristofalo VJ, Pignolo RJ (1993) Replicative senescence of human fibroblast-like cells in culture. Physiol Rev 73:617–638
Cho S, Hwang ES (2011) Fluorescence-based detection and quantification of features of cellular senescence. Methods Cell Biol 103:149–188
Funayama R, Ishikawa F (2007) Cellular senescence and chromatin structure. Chromosoma 116:431–440
Narita M, Lowe SW (2004) Executing cell senescence. Cell Cycle 3:244–246
Zhang R, Adams PD (2007) Heterochromatin and its relationship to cell senescence and cancer therapy. Cell Cycle 6:784–789
Li Q, Ma L, Zhang Z, Tong T (2007) SAHF: a new biomarker of cellular senescence. Prog Biochem Biophys 11:1123–1128
Qian Y, Chen X (2010) Tumor suppression by p53: making cells senescent. Histol Histopathol 25:515–526
Shen H, Maki CG (2010) Persistent p21 expression after Nutlin-3a removal is associated with senescence-like arrest in 4 N cells. J Biol Chem 285:23105–23114
Korotchkina LG, Demidenko ZN, Gudkov AV, Blagosklonny MV (2009) Cellular quiescence caused by the Mdm2 inhibitor nutlin-3A. Cell Cycle 8:3777–3781
Demidenko ZN, Korotchkina LG, Gudkov AV, Blagosklonny MV (2010) Paradoxical suppression of cellular senescence by p53. Proc Natl Acad Sci U S A 107:9660–9664
Mah LJ, El-Osta A, Karagiannis TC (2010) GammaH2AX as a molecular marker of aging and disease. Epigenetics 5:129–136
Mallette FA, Ferbeyre G (2007) The DNA damage signaling pathway connects oncogenic stress to cellular senescence. Cell Cycle 6:1831–1836
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 92:9363–9367
Itahana K, Campisi J, Dimri GP (2007) Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase assay. Methods Mol Biol 371:21–31
Nowicki TS, Zhao H, Darzynkiewicz Z, Moscatello A, Shin E, Schantz S, Tiwari RK, Geliebter J (2011) Downregulation of uPAR inhibits migration, invasion, proliferation, FAK/PI3K/Akt signaling and induces senescence in papillary thyroid carcinoma cells. Cell Cycle 10:100–107
Zhao H, Halicka HD, Traganos F, Jorgensen E, Darzynkiewicz Z (2010) New biomarkers probing depth of cell senescence assessed by laser scanning cytometry. Cytometry A 77:999–1007
Kamentsky LA, Kamentsky LD (1991) Microscope-based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data. Cytometry 12:381–387
Darzynkiewicz Z, Bedner E, Li X, Gorczyca W, Melamed MR (1999) Laser-scanning cytometry: a new instrumentation with many applications. Exp Cell Res 249:1–12
Henriksen M, Miller B, Newmark J, Al-Kofahi Y, Holden E (2011) Laser scanning cytometry and its applications: a pioneering technology in the field of quantitative imaging cytometry. Methods Cell Biol 102:161–205
Gong J, Traganos F, Darzynkiewicz Z (1995) Growth imbalance and altered expression of cyclins B1, A, E, and D3 in MOLT-4 cells synchronized in the cell cycle by inhibitors of DNA replication. Cell Growth Differ 6:1485–1493
Tanaka T, Halicka HD, Huang X, Traganos F, Darzynkiewicz Z (2006) Constitutive histone H2AX phosphorylation and ATM activation, the reporters of DNA damage by endogenous oxidants. Cell Cycle 5:1940–1945
Zhao H, Tanaka T, Halicka HD, Traganos F, Zarebski M, Dobrucki J, Darzynkiewicz Z (2007) Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports aging-related processes. Cytometry A 71:905–914
Zhao H, Traganos F, Dobrucki J, Wlodkowic D, Darzynkiewicz Z (2009) Induction of DNA damage response by the supravital probes of nucleic acids. Cytometry A 75:510–519
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Supported by NCI grant RO1 28 704 and Robert A. Welke Cancer Research Foundation.
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Zhao, H., Darzynkiewicz, Z. (2013). Biomarkers of Cell Senescence Assessed by Imaging Cytometry. In: Galluzzi, L., Vitale, I., Kepp, O., Kroemer, G. (eds) Cell Senescence. Methods in Molecular Biology, vol 965. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-239-1_5
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DOI: https://doi.org/10.1007/978-1-62703-239-1_5
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