Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports aging‐related processes

Abstract

The ongoing DNA damage caused by reactive oxygen species generated during oxidative metabolism is considered a key factor contributing to cell aging as well as preconditioning cells to neoplastic transformation. We postulated before that a significant fraction of constitutive histone H2AX phosphorylation (CHP) and constitutive activation of ATM (CAA) seen in untreated normal and tumor cells occurs in response to such DNA damage. In the present study, we provide further evidence in support of this postulate. The level of ATM activation and H2AX phosphorylation, detected immunocytochemically, has been monitored in WI-38, A549, and TK6 cells treated with H 2 O 2 as well as growing under conditions known or suspected to affect the level of endogenous oxidants. Thirty-to 60-min exposure of cells to 100 or 200 lM H 2 O 2 led to an increase in the level of H2AX phosphorylation and ATM activation, particularly pronounced (nearly fivefold) in S-phase cells. Cell growth for 24-48 h under hypoxic conditions (3% O 2 ) distinctly lowered the level of CHP and CAA while it had minor effect on cell cycle progression. Treatment (4 h) with 0.1 or 0.3 mM 3-bromopyruvate, an inhibitor of glycolysis and mitochondrial oxidative phosphorylation, reduced the level of CHP (up to fourfold) and also decreased the level of CAA. Growth of WI-38 cells in 2% serum concentration for 48 h led to a 25 and 30% reduction in CHP and CHA, respectively, compared with cells growing in 10% serum. The antioxidant vitamin C (2 mM) reduced CHP and CAA by 20-30% after 24 h of treatment, while the COX-2 inhibitor celecoxib (5 lM) had a minor effect on CHP and CAA, though it decreased the level of H 2 O 2 -induced H2AX phosphorylation and ATM activation. In contrast, dichloroacetate known to shift metabolism from anaerobic to oxidative glycolysis through its effect on pyruvate dehydrogenase kinase enhanced the level of CHP and CAA. Our present data and earlier observations strongly support the postulate that a large fraction of CHP and CAA occurs in response to DNA damage caused by metabolically generated oxidants. Cytometric analysis of CHP and CAA provides the means to measure the effectiveness of exogenous factors, which either through lowering aerobic metabolism or neutralizing radicals may protect DNA from such damage. ' International Society for Analytical CytologyKey terms H2AX phosphorylation; ATM activation; reactive oxygen species; hypoxia; hydrogen peroxide; 3-bromopyruvate; dichloroacetate; celecoxib; aging; caloric restriction; mitochondria IN living cells, nuclear DNA is constantly being damaged by endogenous reactive oxidants generated during aerobic respiration in mitochondria (1-6). Both, the firstand the second-generation reactive intermediates, the byproducts of oxidative phosphorylation reactions, can diffuse from mitochondria, reach the nuclear DNA, and induce its damage (7,8). It has been estimated that DNA exposure to endogenous oxidants during a single cell cycle of about 24-h duration results in~5,000 DNA single-strand lesi...

Remarkable enhancement of cytotoxicity of onconase and cepharanthine when used in combination on various tumor cell lines

“…28 In the case of Cep, activation of NFκB, which is a redox-sensitive transcription factor, 70 seems to be prevented by the antioxidant action of this alkaloid. [50][51][52][53][54] Antioxidant properties of Onc 56 could additionally lower the redox potential within the cell providing further hindrance in activation of NFκB. Simultaneous inactivation of NFκB by diverse mechanisms as offered by Onc and Cep, respectively, may be so effective that it completely eliminates expression of the "survival genes."…”

“…[50][51][52] We have recently reported that Cep effectively scavenges endogenous oxidants generated during aerobic metabolism, protecting DNA from oxidative damage. 53,54 The radicals scavenging properties of Cep may mediate several of the effects listed above, such as inhibition of the lipid peroxidation, 43 protection from carcinogens, 49 anti-inflammatory properties 41 or protection from radiotherapyinduced leucopenia. 39 It was recently reported that Cep sensitizes human oral carcinoma cells to radiation by inhibiting activation of NFκB.…”

Cytomics and nanobioengineering

“…These include cell shrinkage, nuclear condensation, internuclosomal DNA cleavage, and apoptotic signaling cascades that may be detected by multicolor immune fluorescence cytometry. Moreover, cytometry offers the possibility to measure intravitally mitochondrial membrane potential, generation of reactive oxygen species, Ca 21 content, caspase activity, and phosphatidylserine externalization (82,83).…”

“…DNA damage, if not repaired, accumulates with age which contributes to cell senescence and preconditions to neoplasia development. The introduction of the sensitive DNA damage reporters for cytometric assessment may facilitate and accelerate drug development (82).…”

“…He focused on three molecules which could be used as DNA damage indicators (82,83,86,87): ATM (Ataxia telangiectasia mutated) protein kinase becomes activated (A-ATM) in response to DNA damage particularly when the damage involves formation of DNA double-strand breaks (DSBs). Upon exposure of cells to ionizing radiation activation of ATM was shown to correlate strongly with number of DSBs.…”