10. After vortexing, collect the secondary antibody from the sur- face. Denser aggregates of antibody settle at the bottom of the tube and can contribute to background artifact in the staining.
Acknowledgments
N.F.B. was funded by NIH/NCI 5R01CA74592, NIH/NCI 1R01CA190533, NIH/NIAMS 1R03AR066880, and NIH/
NCATS UL1 RR025780. T.T. was funded by NIH/NIAMS 1K01AR063203-01, NIH/NC 1R03CA191937, CCTSI Research grant from NIH/NCATS UL1 RR025780, ACS IRG 57-001-53 from the American Cancer Society and a Dermatology Foundation research grant.
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Mikhail A. Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 1534, DOI 10.1007/978-1-4939-6670-7_18, © Springer Science+Business Media New York 2017
Chapter 18
Detection of Oncogene-Induced Senescence In Vivo
Kwan-Hyuck Baek and Sandra Ryeom
Abstract
Oncogene-induced senescence or OIS is defi ned as a permanent state of proliferative arrest resulting from an activating oncogenic-lesion. OIS has been suggested to function as a cancer cell intrinsic mechanism to restrain tumor growth and has been implicated as a key mechanism preventing the progression of certain premalignant lesions in genetically engineered mouse models of cancer. The senescent phenotype can be defi ned by two criteria that include cell cycle arrest and resistance to mitogens and oncogenic transforma- tion. While the phenotype and properties of senescent cells in vitro are well described, the morphological characteristics defi ning senescence in vivo have been controversial with no specifi c marker that defi nitively proves a senescent state. Indeed, many of the published in vivo markers to identify and characterize senes- cence in an organism are unreliable and often times have been found to be nonspecifi c. However, the use of multiple markers is accepted as confi rmation of senescence in vivo. Here, we describe protocols for some of the most commonly used indicators of senescence in oncogenic Kras -induced lung adenomas including the detection of senescence-associated beta-galactosidase, expression of the tumor suppressor p19 ARF , the pres- ence of senescence-associated heterochromatin foci, and in vivo BrdU uptake to confi rm cell cycle arrest.
Key words Senescence , Oncogene-induced senescence , SA-beta galactosidase , Senescence-associated heterochromatin foci , p19 ARF , BrdU , Adenomas , Ras
1 Introduction
The term cellular senescence was fi rst used by Hayfl ick to describe the limited replicative ability of primary cells in culture [ 1 , 2 ]. The term now refers to the permanent growth arrest of viable and meta- bolically active cells and happens in vivo in response to various cel- lular stresses including telomere attrition , tumor suppressor activation, DNA damaging agents, and oncogene activation among others [ 3 – 6 ]. Telomere shortening triggers replicative senescence occurs when the Hayfl ick limit is reached in cultured cells. As cells continuously propagate, their telomeres are progressively shortened until they reach a critical minimal length upon which they undergo stable proliferative arrest [ 7 ]. Stable expression of the tumor sup- pressor p19 ARF -p53 or the p16 INK4A -retinoblastoma protein (RB) pathways has also been shown to induce senescence [ 8 , 9 ]. Early
studies found that expression of oncogenic HRAS ( Hras v12 ) alone into primary cells induced cell cycle arrest phenotypically very similar to replicative senescence [ 10 ]. This concept has since become known as oncogene-induced senescence (OIS). However, unlike replicative senescence, OIS is independent of telomere shortening [ 11 ]. Cells undergoing OIS require involvement of the p19 ARF -p53 and p16 INK4A -RB tumor suppressor pathways as functional inactivation of these tumor suppressor pathways permits cells to bypass oncogenic Ras-induced senescence [ 8 – 10 ]. OIS has now been detected in pre- malignant lesions of a number of different genetically engineered mouse models of cancer driven by oncogenic RAS or its downstream effector RAF. These diverse tumors types include melanomas , papil- lomas, lung adenomas , mammary tumors, and pancreatic tumors to cite a few examples [ 12 – 18 ].
While a number of studies support the notion that OIS is a tumor suppressive mechanism preventing tumor growth and pro- gression to malignancy [ 18 , 19 ], a major obstacle in the senescence fi eld is that the markers used to identify and characterize senes- cence in vivo are unreliable and nonspecifi c. The challenges of detecting OIS in vivo are due in part to the heterogeneity of the senescence response in different tissues, the expression of senes- cence markers in nonsenescent cells, and reagents for senescence markers that have limited effectiveness in mouse tissues among other factors [ 3 ]. Here, we demonstrate detection of senescence in an oncogenic Kras -induced genetically engineered mouse model of lung cancer [ 14 , 15 ] through the detection of SA-beta galacto- sidase (SA-β-Gal), p19 ARF expression, the presence of senescence- associated heterochromatin foci (SAHF) , and the lack of BrdU uptake.
2 Materials