18. DAB is a hazardous chemical and harmful if swallowed, inhaled, or placed in contact with the skin. When fi nished using the DAB solution , place in 10 % bleach to neutralize and dispose of in an appropriate manner.
Acknowledgments
This work was funded by Grants SAF2010-18829 and SAF2013- 49082- P to W.M.K. from the Spanish Ministry for Economy and Competitiveness, the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) from the Generalitat de Catalunya, and CRG core funding.
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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_20, © Springer Science+Business Media New York 2017
Chapter 20
Induction and Detection of Oncogene-Induced Cellular Senescence in Drosophila
Mai Nakamura and Tatsushi Igaki
Abstract
Cellular senescence is induced by various cellular stresses, including activation of the Ras oncogene. In Drosophila imaginal epithelia, clones of cells expressing oncogenic Ras (Ras V12 ) show several markers of cellular senescence, such as elevation of SA-β-gal activity, upregulation of the Cdk inhibitor Dacapo (Dap), and heterochromatinization. However, these cells do not undergo cell cycle arrest or exhibit a DNA dam- age response (DDR), cellular hypertrophy, or a senescence-associated secretory phenotype (SASP), other essential markers of cellular senescence. However, we found that inducing mitochondrial dysfunction within Ras V12 -expressing cells caused all above-mentioned aspects of cellular senescence. This provided the fi rst evidence that cellular senescence occurs in invertebrates and is intriguing because mitochondrial dysfunction is frequently observed in human cancers. Here, we describe the procedures for the induction and detection of cellular senescence in Drosophila epithelia.
Key words Cellular senescence , Drosophila , SASP , Ras , Mitochondrial dysfunction
1 Introduction
Cellular senescence is an irreversible cell cycle arrest that can be induced by various oncogenic stresses such as activation of the oncogene Ras . Therefore, cellular senescence has been considered to act as a tumor suppression mechanism. In contrast to this view, recent studies have indicated that cellular senescence can act as a tumor promoter, as senescent cells highly express various oncogenic secreted proteins such as growth factors and infl ammatory cyto- kines , a phenomenon called the senescence-associated secretory phenotype (SASP) or the senescence-messaging secretome (SMS) [ 1 – 7 ]. We have found in Drosophila imaginal epithelia that muta- tions in mitochondrial respiratory complexes, alterations common to human cancers , combined with Ras activation (Ras V12 / mito −/− ) triggers secretion of the infl ammatory cytokine Unpaired (Upd, an IL-6 homologue) which induces tumor growth and progression in neighboring tissue [ 8 – 12 ]. This nonautonomous tumor
progression driven by Upd is reminiscent of the SASP . Indeed, we found that Ras V12 / mito −/− cells exhibit all hallmarks of cellular senescence: elevation of senescence-associated β-galactosidase (SA-β-gal) activity, upregulation of the Cdk inhibitor Dacapo (Dap, a p21/p27 homologue), cell cycle arrest , heterochromatinization , DNA damage response (DDR), cellular hypertrophy, and the SASP [ 13 ]. Intriguingly, although Ras activation alone is suffi cient to cause cellular senescence in mammalian cultured cells, it only causes elevation of SA-β-gal activity, Cdk inhibitor expression, and het- erochromatinization in Drosophila epithelia. This suggests that mitochondrial dysfunction acts as an essential factor or enhancer of the oncogene-induced cellular senescence in vivo. Here, we describe detailed procedures for the induction and detection of cellular senescence in Drosophila imaginal epithelia .
2 Materials
Drosophila melanogaster : Culture fl ies at 25 °C in vials containing a standard fl y food.
Drosophila larvae: Collect all larvae at third instar and dissect them in 1× PBS.
To induce cellular senescence in the eye imaginal disc, prepare fl ies with the following genotypes. In these fl ies, GFP-labeled Ras V12 / mito −/− clones are generated in the eye-antennal disc.
Pdsw k10101 and CoVa tend are loss-of-function mutations for the genes encoding components of the mitochondrial respiratory com- plexes I and IV, respectively. Fluorescently labeled mitotic clones are induced in the imaginal disc by the MARCM system [ 14 – 16 ].
Genotypes:
eyFLP5, Act>y+>Gal4, UAS-GFP/UAS-Ras V12 ; FRT82B, Tub-Gal80/FRT82B, CoVa tend ( see Fig. 1A, E )
Tub-Gal80, FRT40A, UAS-Ras V12 /P{w +mc =lacW}
Pdsw k10101 , FRT40A; eyFLP6, Act>y+>Gal4, UAS-GFP/+ ( see Fig. 1B–D, F )