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. 2012 Dec;4(12):952-65.
doi: 10.18632/aging.100521.

Potential anti-aging agents suppress the level of constitutive mTOR- and DNA damage- signaling

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

Potential anti-aging agents suppress the level of constitutive mTOR- and DNA damage- signaling

H Dorota Halicka et al. Aging (Albany NY). 2012 Dec.

Abstract

Two different mechanisms are considered to be the primary cause of aging. Cumulative DNA damage caused by reactive oxygen species (ROS), the by-products of oxidative phosphorylation, is one of these mechanisms (ROS concept). Constitutive stimulation of mitogen- and nutrient-sensing mTOR/S6 signaling is the second mechanism (TOR concept). The flow- and laser scanning- cytometric methods were developed to measure the level of the constitutive DNA damage/ROS- as well as of mTOR/S6- signaling in individual cells. Specifically, persistent activation of ATM and expression of γH2AX in untreated cells appears to report constitutive DNA damage induced by endogenous ROS. The level of phosphorylation of Ser235/236-ribosomal protein (RP), of Ser2448-mTOR and of Ser65-4EBP1, informs on constitutive signaling along the mTOR/S6 pathway. Potential gero-suppressive agents rapamycin, metformin, 2-deoxyglucose, berberine, resveratrol, vitamin D3 and aspirin, all decreased the level of constitutive DNA damage signaling as seen by the reduced expression of γH2AX in proliferating A549, TK6, WI-38 cells and in mitogenically stimulated human lymphocytes. They all also decreased the level of intracellular ROS and mitochondrial trans-membrane potential ΔΨm, the marker of mitochondrial energizing as well as reduced phosphorylation of mTOR, RP-S6 and 4EBP1. The most effective was rapamycin. Although the primary target of each on these agents may be different the data are consistent with the downstream mechanism in which the decline in mTOR/S6K signaling and translation rate is coupled with a decrease in oxidative phosphorylation, (revealed by ΔΨm) that leads to reduction of ROS and oxidative DNA damage. The decreased rate of translation induced by these agents may slow down cells hypertrophy and alleviate other features of cell aging/senescence. Reduction of oxidative DNA damage may lower predisposition to neoplastic transformation which otherwise may result from errors in repair of DNA sites coding for oncogenes or tumor suppressor genes. The data suggest that combined assessment of constitutive γH2AX expression, mitochondrial activity (ROS, ΔΨm) and mTOR signaling provides an adequate gamut of cell responses to evaluate effectiveness of gero-suppressive agents.

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Conflict of interest statement

The authors of this manuscript have no conflict of interests to declare.

Figures

Figure 1
Figure 1. Effect of exposure of TK6 cells to different presumed anti-aging 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), DNA was stained with DAPI; cellular fluorescence was measured by flow cytometry. Based on differences in DNA content cells were gated in the respective phases of the cell cycle, as marked by the dashed vertical lines. The percent decrease in mean 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.
Figure 2
Figure 2. Effect of treatment of TK6 cells with different presumed anti-aging drugs for 4 h on the level of constitutive phosphorylation of ribosomal protein S6 (RP-S6)
Exponentially growing TK6 cells were untreated (Ctrl) or treated with the respective agents at concentrations as shown. Phosphorylation status of ribosomal S6 protein was detected immunocytochemically with the phospho-specific Ab (AlexaFluor647), DNA was stained with DAPI; cellular fluorescence was measured by flow cytometry. Top panels: 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 mean fluorescence intensity of the treated cells in particular phases of the cell cycle, with respect to the 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. Bottom panels: Single parameter frequency histograms showing expression of phosphorylated ribosomal S6 protein (RB-S6P) in all (G1+S+G2M) cells of the respective cultures.
Figure 3
Figure 3. Effect of treatment of TK6 cells with MF, RAP or RSV for 24 h on the level of constitutive phosphorylation of S6 protein
TK6 cells were untreated (Ctrl) or treated with different concentrations of MF as well as with RAP or RSV for 24 h. Phosphorylation status of S6 was assessed as described in legend to Fig. 2. Top panels: The percent decrease in mean fluorescence intensity of the drug-treated cells in particular phases of the cell cycle is shown above the arrows. Bottom panels: Frequency histograms showing expression of RP-S6P in all cells of the respective cultures. Insets show cellular DNA content histograms of cells in these cultures.
Figure 4
Figure 4. Reduction of the level of constitutive expression of RP-S6P in A549 cells exposed to MF, BRB, Vit. D3 or RSV for 24 h
Exponentially growing in chamber slides A549 cells, were treated with the respective agents and their fluorescence was measured the laser scanning cytometry (LSC)75. Top panels show RP-S6P immunofluorescence integrated over the nuclei (reporting expression of RP-S6P in the cytoplasm located over and below the nucleus); bottom panels present RP-S6P immunofluorescence integrated over the cytoplasm aside of the nucleus. The percent decrease in expression of RP-S6P in cells in particular phases of the cell cycle (mean values) is shown above the arrows. Because stock solutions of some of these agents were made in DMSO, other in MeOH or EtOH, the equivalent quantities of these solvents were included in the respective control culture and the percent decrease shown in the panels refers to the decrease compared to these controls shown are the cells from EtOH and DMSO containing controls. The insets present DNA content frequency histograms from the respective cultures.
Figure 5
Figure 5. Effect of treatment of WI-38 cells with 2dG, MF, RAP, BRB, Vit. D3 or RSV for 24 h on the level of constitutive expression of γH2AX (left panel) and RP-S6P (right panel)
Exponentially growing cells, were treated with the respective agents at concentrations as shown in Figs. 1 and 2, RP-S6P was detected immunocytochemically and cell fluorescence was measured with the laser scanning cytometry (LSC). The bar graphs present the mean fluorescence intensity measured as an integral over the nucleus (γH2AX) or over cytoplasm (PR-S6P).
Figure 6
Figure 6. Effect of treatment of mitogenically stimulated human lymphocytes with RAP, BRB, Vit. D3, RSV or ASA for 4 h on the level of constitutive expression of RP-S6P
Peripheral blood lymphocytes were mitogenically stimulated with phytohemagglutinin (PHA) for 72 h, the cells were then treated with the respective drugs at concentrations as shown in Figs. 1 and 2 for 4 h, RP-S6 wasdetected immunocytochemically and cellular fluorescence measured by flow cytometry. The bar graphs present the mean values (+SD) of RP-S6P immunofluorescence for G1, S and G2M cell subpopulations identified by differences in DNA content (intensity of DAPI fluorescence).
Figure 7
Figure 7. Effect of the studied gero-preventive agents on constitutive level of expression of mTOR-Ser2448P, RP-S6-Ser235/236P and 4EBP1-Ser65P and their corresponding unphosphorylated forms in TK6 cells, detected by western blotting
TK6 cells were exposed to the studied agents at concentrations as shown in Figs 1 and 2 for 4 h. The protein expression level were determined by western blot analysis and the intensity of the specific immunoreactive 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).
Figure 8
Figure 8. Effects of the studied gero-preventive agents on the intercellular level of ROS
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. The cell-permeant non-fluorescent 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.
Figure 9
Figure 9. Effect of the studied gero-preventive agents on the mitochondrial transmembrane potential (ΔΨm)
TK6 cells, untreated (Ctrl) or treated for 24 h with the investigated agents were exposed for 30 min to the mitochondrial probe rhodamine 123 (Rh-123) and their fluorescence intensity was measured by flow cytometry. 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.
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