Date Published: December 30, 2012
Publisher: Impact Journals LLC
Author(s): H. Dorota Halicka, Hong Zhao, Jiangwei Li, Yong-Syu Lee, Tze-Chen Hsieh, Joseph M. Wu, Zbigniew Darzynkiewicz.
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.
The cumulative DNA damage caused by reactive oxygen species (ROS), by-products of oxidative phosphorylation, for long time has been considered to be a key factor contributing both to cell aging as well as predisposing to neoplastic transformation [1-12]. Oxidative DNA damage generates significant number of DNA double-strand breaks (DSBs), the potentially deleterious lesions. DSBs can be repaired either by the homologous recombination or nonhomologous DNA-end joining (NHEJ) mechanism. Recombinatorial repair which uses newly replicated DNA as a template restores DNA rather faithfully. It can take place however when cells have already the template, namely during late-S and G2 phase. In the cells that lack a template (G1, early-S) DNA repair relies on the NHEJ which is error-prone due to a possibility of a deletion or rearrangement of some base pairs [13-17]. If the erroneously repaired DSBs are at sites of oncogenes or tumor suppressor genes this may result in somatic mutations that predispose cell to oncogenic transformation. Oxidative damage of telomeric DNA may lead to dysfunction of telomeres thereby driving cells to undergo replicative senescence [18-30].
Fig. 1 illustrates the effect of exposure of human lymphoblastoid TK6 cells for 24 h to the investigated presumed anti-aging agents on the level of constitutive expression of γH2AX. Consistent with our prior findings [52-54] the expression γH2AX in S and G2M cells is distinctly higher than in the cells of G1 phase. This is the case for both, the untreated (Ctrl) cells as well as the cells treated with these agents. It is also apparent that exposure of cells to each of the studied drugs led to the decrease in expression of γH2AX in all phases of the cell cycle. In most treated cells, however, the decline in the mean expression γH2AX was somewhat more pronounced in the S- compared to G1 – or G2M- phase cells. Analysis of DNA content frequency histograms reveals that the 24 h treatment with most of the drugs had no effect on the cell cycle distribution. The exception are the cells treated with 50 nM RP which show about 50% reduction in frequency of cells in S and G2M which would indicate partial cells arrest in G1 phase of the cell cycle. It should be noted that exposure of cells to these agents for 4 h led to rather minor (<15%) decrease in expression of γH2AX whereas the treatment for 48 h had similar effect as for 24 h (data not shown). In the prior studies we have already observed that MF at concentrations 0.1 mM – 20 mM  and Vit. D (2 nM - 10 nM) effectively reduced constitutive level of H2AX-Ser139 and ATM-Ser1981 phosphorylation. In the present study all seven agents, all reportedly having anti-aging and/or chemopreventive properties, including MF and Vit D3[59-103], have been tested with respect of their ability to affect both the level of constitutive DNA damage signaling as monitored γH2AX expression as well as constitutive level of phosphorylation of ribosomal S6 protein (RP-S6P). The data show that each of the drugs reduced both, the level of phosphorylation of both H2AX on Ser139 and RP-S6 on Ser235/236. RP-S6, a component of the 40S ribosomal subunit and the most downstream effector of mTOR signaling, is directly involved in regulation of translation  and considered to be a determinant of cell size [105,106]. As is evident from the western blotting data (Fig. 7) with an exception of RSV all the studied drugs reduced also the level of phosphorylation of mTOR, RP-S6 and 4EBP1. The latter protein is also considered to be a critical regulator of translation and cell size determinant [105-108]. Source: http://