A new chronological survival assay in mammalian cell culture

Department of Pathology, University of Washington, Seattle, WA, USA.
Cell cycle (Georgetown, Tex.) (Impact Factor: 4.57). 01/2012; 11(2):201-2. DOI: 10.4161/cc.11.2.18959
Source: PubMed
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Available from: Matt Kaeberlein, Sep 22, 2015
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    • "Indeed, medium acidification has been shown to stimulate chronological senescence (aging) also in cultured human cancer cells, in which the acidification is due to accumulation of lactic acid (Leontieva and Blagosklonny, 2011). Thus, it is conceivable that the important role of extracellular acidification in accelerating the chronological mode of cellular aging and, perhaps, cell-nonautonomous mechanisms underlying such role have been conserved in the course of evolution (Demidenko, 2011; Fabrizio and Wei, 2011; Kaeberlein and Kennedy, 2012; Leontieva and Blagosklonny, 2011). In support of evolutionary conservation of mechanisms by which extracellular acidification drives chronological aging of yeast and cultured human cells, the target of rapamycin signaling pathway has been shown to accelerate aging and promote medium acidification in both types of cells (Arlia-Ciommo et al., 2014a; Hu et al., 2014; Leontieva and Blagosklonny, 2011; Longo et al., 2012). "
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    ABSTRACT: Cell-autonomous mechanisms underlying cellular and organismal aging in evolutionarily distant eukaryotes have been established; these mechanisms regulate longevity-defining processes within a single eukaryotic cell. Recent findings have provided valuable insight into cell-non-autonomous mechanisms modulating cellular and organismal aging in eukaryotes across phyla; these mechanisms involve a transmission of various longevity factors between different cells, tissues and organisms. Herein, we review such cell-non-autonomous mechanisms of aging in eukaryotes. We discuss the following: (1) how low molecular weight transmissible longevity factors modulate aging and define longevity of cells in yeast populations cultured in liquid media or on solid surfaces, (2) how communications between proteostasis stress networks operating in neurons and non-neuronal somatic tissues define longevity of the nematode Caenorhabditis elegans by modulating the rates of aging in different tissues, and (3) how different bacterial species colonizing the gut lumen of C. elegans define nematode longevity by modulating the rate of organismal aging.
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    ABSTRACT: The TOR (target of rapamycin) pathway is involved in aging in diverse organisms from yeast to mammals. We have previously demonstrated in human and rodent cells that mTOR converts stress-induced cell cycle arrest to irreversible senescence (geroconversion), whereas rapamycin decelerates or suppresses geroconversion during cell cycle arrest. Here, we investigated whether rapamycin can suppress replicative senescence of rodent cells. Mouse embryonic fibroblasts (MEFs) gradually acquired senescent morphology and ceased proliferation. Rapamycin decreased cellular hypertrophy, and SA-β-Gal staining otherwise developed by 4-6 passages, but it blocked cell proliferation, masking its effects on replicative lifespan. We determined that rapamycin inhibited pS6 at 100-300 pM and inhibited proliferation with IC(50) around 30 pM. At 30 pM, rapamycin partially suppressed senescence. However, the gerosuppressive effect was balanced by the cytostatic effect, making it difficult to suppress senescence without causing quiescence. We also investigated rat embryonic fibroblasts (REFs), which exhibited markers of senescence at passage 7, yet were able to slowly proliferate until 12-14 passages. REFs grew in size, acquired a large, flat cell morphology, SA-β-Gal staining and components of DNA damage response (DDR), in particular, γH2AX/53BP1 foci. Incubation of REFs with rapamycin (from passage 7 to passage 10) allowed REFs to overcome the replicative senescence crisis. Following rapamycin treatment and removal, a fraction of proliferating REFs gradually increased and senescent phenotype disappeared completely by passage 24.
    Full-text · Article · Jun 2012 · Cell cycle (Georgetown, Tex.)
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    ABSTRACT: Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.
    Full-text · Article · Aug 2012 · Cell cycle (Georgetown, Tex.)
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