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Review
. 2014 May;85(5):386-99.
doi: 10.1002/cyto.a.22452. Epub 2014 Feb 22.

In search of antiaging modalities: evaluation of mTOR- and ROS/DNA damage-signaling by cytometry

Zbigniew Darzynkiewicz  1 , Hong Zhao, H Dorota Halicka, Jiangwei Li, Yong-Syu Lee, Tze-Chen Hsieh, Joseph M Wu
Affiliations
Review

In search of antiaging modalities: evaluation of mTOR- and ROS/DNA damage-signaling by cytometry

Zbigniew Darzynkiewicz et al. Cytometry A. 2014 May.

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.

Keywords: DNA damage signaling; H2AX phosphorylation; berberine; cell size; cellular senescence; mitochondria; red cell distribution width; replication stress; ribosomal protein S6 phosphorylation; translation.

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Figures

Figure 1
Figure 1
Schematic presentation of the key pathways associated with cellular senescence and aging linking mTOR- and DNA damage-signaling as well as marking of sites for potential antiaging intervention. Signaling from several upstream pathways activated by insulin, IGFs, growth hormone (GH), or amino acids converges on, and activates, mTOR (raptor). mTOR stimulation triggers activation of S6K1 that results in phoshorylation of RP-S6 and 4EBP1, the factors indicative of activation of initiation and continuation of translation, This leads to cell growth, and particularly when cell division is postponed (e.g., because of replication stress), to growth imbalance (hypertrophy) characterized by the increased ratio of protein and RNA to DNA content, the hallmark of cellular senescence. As translation requires constant generation of energy (ATP), the oxidative phosphorylation in mitochondria persistently produces ROS. mTOR stimulation, thus, is inherently associated with generation of ROS. The oxidative DNA damage caused by endogenous ROS, when it occurs in sites coding for oncogenes or tumor suppressor genes, may lead to neoplasic transformation. Oxidative DNA damage of the telomeric DNA may cause telomere dysfunction and lead to replicative senescence, while lipid peroxidation is also a gero-promoting event, one of the typical features of the cellular senescence phenotype. The potential targets for antiaging modalities are marked with the Chinese symbol of longevity (see the text). One of the most attractive targets is AMPK whose activation inhibits mTOR signaling. Among AMPK activators that show gero-suppressive properties are metformin and berberine. The antioxidants, by scavenging ROS, have primarily chemopreventive properties but by preventing oxidative telomeric DNA damage and lipid per-oxidation they also attenuate the aging process. The gero-suppressive role of autophagy, which often is seen to be activated by inhibitors of mTOR, is also well recognized (86). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 2
Figure 2
Effect of exposure of TK6 cells to different reported antiaging drugs on the level of constitutive expression of γH2AX. Exponentially growing TK6 cells were untreated (Ctrl) or treated with the respective agents for 24 h at concentrations as shown. Expression of γH2AX in individual cells was detected immunocytochemically with the phospho-specific Ab (AlexaFluor647; AF647), DNA was stained with DAPI; cellular fluorescence was measured by flow cytometry as described (72). Cells were gated in the respective phases of the cell cycle based on differences in DNA content, as marked by the dashed vertical lines. The percent decrease in mean γH2AX fluorescence intensity of the treated cells in particular phases of the cell cycle, with respect to the respective untreated controls, is shown above the arrows. Inserts present DNA content frequency histograms from the individual cultures. The dashed skewed lines show the background level, the mean fluorescence intensity of the cells stained with secondary Ab only (72).
Figure 3
Figure 3
The reduction of intracellular level of ROS measured by DCF fluorescence and of mitochondrial potential (ΔΨm) measured by rhodamine 123 (Rh-123) binding, by the reported gero-suppressive agents. Top panels: TK6 cells untreated (Ctrl) or treated for 24 h with the investigated agents, were exposed for 30 min to H2DCF-DA and their fluorescence intensity was measured by flow cytometry (125). The cell-permeant nonfluorescent H2DCF-DA upon cleavage of the acetate moiety by intercellular esterases and oxidation by ROS is converted to strongly fluorescent DCF and thus reports the ROS abundance. Left panel shows the frequency histograms of the untreated (Ctrl) as well MF- and RAP-treated cells (note exponential scale of the DCF fluorescence). Right panel presents the mean values (+SD) of DCF fluorescence of the untreated (Ctrl) and treated cells. Bottom panels: The cells were untreated (Ctrl) or treated as above then exposed for 30 min to the mitochondrial probe rhodamine 123 (Rh-123), and their fluorescence intensity was measured by flow cytometry (72). Left panel shows the frequency histograms of the untreated (Ctrl) as well MF- and RAP-treated cells (note exponential scale of the DCF fluorescence). Right panel presents the mean values (+SD) of Rh-123 fluorescence of the investigated cells (72,127).
Figure 4
Figure 4
The reduction of constitutive phosphorylation of ribosomal protein S6 (RP-S6) in TK6 cells treated with antiaging drugs. Exponentially growing TK6 cells were untreated (Ctrl) or treated with the respective agents at concentrations as shown, for 24 h. Based on differences in DNA content cells were gated in the respective phases of the cell cycle, as marked by the dashed vertical lines (Ctrl). The percent decrease in the RP-S6P mean fluorescence intensity of the treated cells in particular phases of the cell cycle, with respect to the same phases of the untreated cells, is shown above the arrows. The dashed skewed lines show the background level, the mean fluorescence intensity of the cells stained with secondary Ab only.
Figure 5
Figure 5
The expression of mTOR-Ser2448P, RP-S6-Ser235/236P, and 4EBP1-Ser65P and their respective total protein content, as detected by Western blotting. TK6 cells were exposed to the gero-suppressive agents at concentrations as shown in Figure 2 for 24 h. The protein expression was determined by Western blot analysis using either the phospho-specific (red) or total protein Abs (black) Abs and the intensity of the specific immuno-reactive bands were quantified by densitometry and normalized to actin (loading control). The numbers indicate the n-fold change in expression of the respective phospho-proteins in the drug-treated cultures with respect to the untreated cells (Ctrl). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 6
Figure 6
Decreased ratio of phosphorylated RP-S6 (RP-S6P) to total RP-S6 (RP-S6T) in A549 cells treated with different concentration of BRB. A549 cells were untreated (Ctrl) or treated for 24 h with BRB at concentration as shown, RP-S6P was then detected using phospho-specific primary Ab and secondary Ab tagged with AlexaFluor 647 (AF647) while total RP-S6 (RP-S6T) was detected using primary RP-S6 (not phospho-specific) Ab and secondary Ab AlexaFluor 488 (AF488), DNA was stained with DAPI. Intensity of cellular fluorescence was measured by laser scanning cytometry (LSC) and the ratiometric analysis of AF647/AF488 fluorescence intensities was carried out using the LSC software (128). The numbers on the left of the panels show the ratio of the mean values of integrated intensity of AF647 to AF488 fluorescence for all cells. The figures on right present the percent decrease of the ratio of BRB-treated cells vis-à-vis the Ctrl.
Figure 7
Figure 7
Induction of premature cellular senescence of A549 cells measured by laser scanning cytometry. Human pulmonary lung adenocarcinoma A549 cells were untreated (Ctrl) or to induce cellular senescence were treated with 2-nM DNA topoisomerase II inhibitor mitoxantrone (Mxt) for 48 or 72 h. Panel A shows bivariate distributions of nuclear area versus intensity of maximal pixel of fluorescence revealed by measurement of nuclear DNA (DAPI) fluorescence. Intensity of maximal pixel is a marker of chromatin condensation and in the untreated cells has the highest value and marks mitotic (M) and immediately postmitotic (pM) G1 cells, which also have low value of DAPI area. In the senescing cells, while nuclear area increases, the intensity of maximal pixel decreases (,–134). These morphometric changes reflect enlargement of the projected nuclear area and decreased DAPI local staining per unit area, due to flattened cellular appearance, the hallmark of cellular senescence (–133). The insets show DNA content frequency histograms of cells from the respective cultures. Panel B: Bar plots reporting mean values (+SD) of nuclear (DNA, DAPI) area, DNA (DAPI) maximal pixel, and ratio of maximal pixel to nuclear area, respectively, of cells from control and Mxt treated cultures. Panel C: Bivariate distributions (DNA content vs p21) reporting expression of p21WAF1 with respect to the cell cycle phase; the figures show the n-fold increase in mean expression of p21 of G1 and G2M cells from the Mxt-treated cultures with respect to respective cells in Ctrl. Panel D: Bivariate distributions of DNA content versus senescence-associated galactosidase (SA-β-gal) activity. Figures indicate percent of SA-β-gal positive (above the threshold marked by the horizontal lines) cells. Insets show the frequency distribution of SA-β-gal positive cells; the figures in insets show the n-fold increase in the mean activity of SA-β-gal in Mxt-treated cultures over Ctrl (1.0). Panel E: Images of cells growing in the absence (left) and presence of 2 nM Mxt for 72 h (right) stained to detect activation of SA-β-gal activity recorded by laser scanning cytometer (Research Imaging Cytometer iCys); 50 µm bars mark the length scale.
Figure 8
Figure 8
Attenuation of Mxt-induced senescence of A549 cells by berberine (BRB) as measured by cell morphometric features, expres-sion of p21WAF1, γH2AX, and RP-S6P. Exponentially growing A549 cells were untreated (Ctrl) or treated with 2 nM Mxt in the absence and presence of BRB at concentration as shown, for 5 days. Top panels: Morphometric analysis, reporting changes in nuclear area (DNA– DAPI) versus maximal pixel of DAPI fluorescence. The ratio of maximal pixel to nuclear area (Mp:A) is expressed as a fraction of that of the untreated cells; shown also is the percent increase in Mp:A in the BRB-treated cultures with respect to cells growing with Mxt alone (with the arrows). Second horizontal panels: bivariate distributions of p21 versus cellular DNA content; the figures (x =) present the increase (n-fold) in the mean expression of p21 for all cells with respect to the untreated cells. The percent reduction in p21 in cultures with BRB with respect to Mxt alone is shown with the arrows. Third horizontal panels: expression of γH2AX versus DNA content; the figures (x =) represent the increase (n-fold) in the mean expression of γH2AX with respect to untreated cells (1.0). The percent reduction in expression of γH2AX in cultures grown with BRB with respect to cells growing in the presence of Mxt alone is presented with the arrows. Bottom panels: expression of RP-S6P versus DNA content. The figures illustrate the change (n-fold) with respect to the untreated (Ctrl) cells. The percent reduction in expression of rpS6P in cultures with BRB with respect to cells treated with Mxt alone is shown with the arrows.
Figure 9
Figure 9
Suppression of RP-S6 phosphorylation and reduction of size of human lymphoblastoid of TK6 cells maintained at 5–60 µM BRB concentration. Exponentially growing TK6 cells were untreated (Ctrl) or treated with BRB for 24 h. Top panels: the bivariate distributions of RP-S6P versus DNA content. Figures show percent decrease in expression of the mean RP-S6P for cells at G1, S, and G2M phases of the cell cycle, respectively, in the presence of BRB compared to untreated cells. Insets show the frequency histograms of RP-S6P expression for all cells in culture. Bottom panels: Bivariate distributions of cellular forward light scatter (FLS) versus DNA content. Percent reduction of mean value of forward light scatter FLS of G1, S, or G2M of cells growing in the presence of BRB with respect to the untreated cells is shown with the arrows.
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