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In Search of Antiaging Modalities: mTOR- & ROS/DNA

Type: Literature (pdf)

Submitter: [anonymous]

Category: Guides / Tutorials - Health / Medical

exhibition Date: 2016-06-09 15:32:35 MST

Views: 2111

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Submitter's Comment
In Search of Antiaging Modalities: Evaluation of mTOR- and ROS/DNA Damage-Signaling by Cytometry
Zbigniew Darzynkiewicz,Hong Zhao, H. Dorota Halicka, Jiangwei Li, Yong-Syu Lee, Tze-Chen Hsieh, and Joseph M. Wu
Brander Cancer Research Institute and Department of Pathology, New York Medical College,
Valhalla, New York 10595
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595

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, ΔΨPm), 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.

Keywords: DNA damage signaling; H2AX phosphorylation; berberine; cell size; cellular senescence; mitochondria

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