title: In search of antiaging modalities: evaluation of mTOR- and ROS/DNA damage-signaling by cytometry.
abstract: This review presents the evidence in support of the IGF-1/mTOR/S6K1 signaling as the primary factor contributing to aging and cellular senescence. Reviewed are also specific interactions between mTOR/S6K1 and ROS-DNA damage signaling pathways. Outlined are critical sites along these pathways, including autophagy, as targets for potential antiaging (gero-suppressive) and/or chemopreventive agents. Presented are applications of flow- and laser scanning- cytometry utilizing phospho-specific Abs, to monitor activation along these pathways in response to the reported antiaging drugs rapamycin, metformin, berberine, resveratrol, vitamin D3, 2-deoxyglucose, and acetylsalicylic acid. Specifically, effectiveness of these agents to attenuate the level of constitutive mTOR signaling was tested by cytometry and confirmed by Western blotting through measuring phosphorylation of the mTOR-downstream targets including ribosomal protein S6. The ratiometric analysis of phosphorylated to total protein along the mTOR pathway offers a useful parameter reporting the effects of gero-suppressive agents. In parallel, their ability to suppress the level of constitutive DNA damage signaling induced by endogenous ROS was measured. While the primary target of each of these agents may be different the data obtained on several human cancer cell lines, WI-38 fibroblasts and normal lymphocytes suggest common downstream mechanism in which the decline in mTOR/S6K1 signaling and translation rate is coupled with a reduction of oxidative phosphorylation and ROS that leads to decreased oxidative DNA damage. The combined assessment of constitutive γH2AX expression, mitochondrial activity (ROS, ΔΨm), and mTOR signaling provides an adequate gamut of cell responses to test effectiveness of gero-suppressive agents. Described is also an in vitro model of induction of cellular senescence by persistent replication stress, its quantitative analysis by laser scanning cytometry, and application to detect the property of the studied agents to attenuate the induction of senescence. Discussed is cytometric analysis of cell size and heterogeneity of size as a potential biomarker used to asses gero-suppressive agents and longevity.
authors.affiliation: Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York, 10595.
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: H Dorota
authors.initials: HD
authors.lastname: Halicka
authors.forename: Jiangwei
authors.initials: J
authors.lastname: Li
authors.forename: Yong-Syu
authors.initials: YS
authors.lastname: Lee
authors.forename: Tze-Chen
authors.initials: TC
authors.lastname: Hsieh
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Wu
mesh_terms: D000375:Aging / Q000187:drug effects*; D001343:Autophagy / Q000235:genetics; D016922:Cellular Senescence / Q000187:drug effects / Q000235:genetics; D004249:DNA Damage / Q000235:genetics; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D047569:Laser Scanning Cytometry; D008928:Mitochondria / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D058570:TOR Serine-Threonine Kinases / Q000235:genetics*
chemical_list: D009687:Nuclear Proteins; C495327:POLDIP3 protein, human; D016601:RNA-Binding Proteins; D017382:Reactive Oxygen Species; D007334:Insulin-Like Growth Factor I; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases
keywords: DNA damage signaling; H2AX phosphorylation; berberine; cell size; cellular senescence; mitochondria; red cell distribution width; replication stress; ribosomal protein S6 phosphorylation; translation