Effects of S‐adenosylhomocysteine and homocysteine on DNA damage and cell cytotoxicity in murine hepatic and microglia cell lines

Limited research has been performed on S‐adenosylhomocysteine (SAH) or homocysteine (Hcy)‐evoked cell damage in hepatic and neuronal cells. In this study, we assessed effects of SAH or Hcy on cell cytotoxicity and DNA damage in hepatic and neuronal cells and attempted to find the underlying mechanism. Cell cytotoxicity and DNA damage were evaluated in murine hepatic cells (BNL CL.2 cell line) and microglia cells (BV‐2 cell line) with SAH or Hcy treatment for 48 h. The influences of SAH or Hcy on lipid peroxidation and DNA methylation were also measured in both cell lines. SAH (5–20 μM) or Hcy (1–5 mM) dose dependently inhibited cell cytotoxicity and enhanced DNA damage in both types of cells. Furthermore, SAH treatment markedly increased intracellular SAH levels and DNA hypomethylation, whereas Hcy caused minimal effects on these two parameters at much higher concentrations. Hcy significantly induced lipid peroxidation, but not SAH. The present results show that SAH might cause cellular DNA damage in hepatic and microglia cells by DNA hypomethylation, resulting in irreversible DNA damage and increased cell cytotoxicity. In addition, higher Hcy could induce cellular DNA damage through increased lipid peroxidation and DNA hypomethylation. We suggest that SAH is a better marker of cell damage than Hcy in hepatic and microglia cells. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:349–356, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20298     

Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21- Mediated Early Senescence Signalling

Unrepaired or inaccurately repaired DNA damage can lead to a range of cell fates, such as apoptosis, cellular senescence or cancer, depending on the efficiency and accuracy of DNA damage repair and on the downstream DNA damage signalling. DNA damage repair and signalling have been studied and modelled in detail separately, but it is not yet clear how they integrate with one another to control cell fate. In this study, we have created an integrated stochastic model of DNA damage repair by non-homologous end joining and of gamma irradiation-induced cellular senescence in human cells that are not apoptosis-prone. The integrated model successfully explains the changes that occur in the dynamics of DNA damage repair after irradiation. Simulations of p53/p21 dynamics after irradiation agree well with previously published experimental studies, further validating the model. Additionally, the model predicts, and we offer some experimental support, that low-dose fractionated irradiation of cells leads to temporal patterns in p53/p21 that lead to significant cellular senescence. The integrated model is valuable for studying the processes of DNA damage induced cell fate and predicting the effectiveness of DNA damage related medical interventions at the cellular level.     

Cellular responses to ionizing radiation: effects of interrupting DNA repair with chemical agents

This review is concerned with the influence of different classes of chemical agents on cellular repair of DNA damage induced by ionizing radiation. Single-strand break rejoining is little affected by inhibitors of DNA synthesis; however, such inhibitors do lead to a persistence of double-strand breaks in the DNA, and this correlates with an enhancement of chromosome aberrations and cell killing. Experiments with antagonists of topoisomerase II suggest an intriguing role for this DNA unwinding enzyme in double-strand break repair. Interference with poly(ADP-ribose) synthesis, by means of the inhibitor 3-aminobenzamide, does not have a clear-cut effect on recovery from ionizing radiation damage. Various substances (for example, caffeine and trypsin) affect DNA repair via a modulation of the cell cycle, altering the time available to the cell for repairing potentially lethal DNA damage before such damage is 'fixed' by the process of DNA replication. Finally, disturbing cellular energy metabolism, and depressing the level of ATP, can inhibit the repair of radiation damage.     

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae.     

Molecular anatomy of the DNA damage and replication checkpoints

Cell cycle checkpoints are signal transduction pathways that enforce the orderly execution of the cell division cycle and arrest the cell cycle upon the occurrence of undesirable events, such as DNA damage, replication stress, and spindle disruption. The primary function of the cell cycle checkpoint is to ensure that the integrity of chromosomal DNA is maintained. DNA lesions and disrupted replication forks are thought to be recognized by the DNA damage checkpoint and replication checkpoint, respectively. Both checkpoints initiate protein kinase-based signal transduction cascade to activate downstream effectors that elicit cell cycle arrest, DNA repair, or apoptosis that is often dependent on dose and cell type. These actions prevent the conversion of aberrant DNA structures into inheritable mutations and minimize the survival of cells with unrepairable damage. Genetic components of the damage and replication checkpoints have been identified in yeast and humans, and a working model is beginning to emerge. We summarize recent advances in the DNA damage and replication checkpoints and discuss the essential functions of the proteins involved in the checkpoint responses.     

Histone modifications in DNA damage response

DNA damage is a relatively common event in eukaryotic cell and may lead to genetic mutation and even cancer. DNA damage induces cellular responses that enable the cell either to repair the damaged DNA or cope with the damage in an appropriate way. Histone proteins are also the fundamental building blocks of eukaryotic chromatin besides DNA, and many types of post-translational modifications often occur on tails of histones. Although the function of these modifications has remained elusive, there is ever-growing studies suggest that histone modifications play vital roles in several chromatin-based processes, such as DNA damage response. In this review, we will discuss the main histone modifications, and their functions in DNA damage response.     

Repair on the Go: E. coli Maintains a High Proliferation Rate while Repairing a Chronic DNA Double-Strand Break

DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.     

Oxidative DNA damage in cultured fibroblasts from patients with hereditary glutathione synthetase deficiency

The SH compound glutathione (GSH) is involved in several fundamental functions in the cell, including protection against reactive oxygen species (ROS). Here, we studied the effect on oxidative DNA damage in cultured skin fibroblasts from patients with hereditary GSH synthetase deficiency. Our hypothesis was that GSH-deficient cells are more prone to DNA damage than control cells. Single cell gel electrophoresis (the comet assay) in combination with the formamidopyrimidine DNA glycosylase enzyme, which recognizes oxidative base modifications, was used on cultured fibroblasts from 11 patients with GSH synthetase deficiency and five control subjects. Contrary to this hypothesis, we found no significant difference in background levels of DNA damage between cells from patients and control subjects. To study the induction of oxidative DNA damage without simultaneous DNA repair, the cells were γ-irradiated on ice and DNA single-strand breaks measured. The patient and control cells were equally sensitive to induction of single strand breaks by γ-irradiation. Therefore, factors other than GSH protect DNA from oxidative damage. However, cells with a high background level of oxidative DNA damage were found to be more sensitive to ionizing radiation. This suggests that differences in background levels of oxidative DNA damage may depend on the cells' intrinsic protection against induction of oxidative damage.     

Chromatin plasticity in response to DNA damage: The shape of things to come

DNA damage poses a major threat to cell function and viability by compromising both genome and epigenome integrity. The DNA damage response indeed operates in the context of chromatin and relies on dynamic changes in chromatin organization. Here, we review the molecular bases of chromatin alterations in response to DNA damage, focusing on core histone mobilization in mammalian cells. Building on our current view of nucleosome dynamics in response to DNA damage, we highlight open challenges and avenues for future development. In particular, we discuss the different levels of regulation of chromatin plasticity during the DNA damage response and their potential impact on cell function and epigenome maintenance.     

Drosophila melanogaster: a model for the study of DNA damage checkpoint response

The cells of metazoans respond to DNA damage by either arresting their cell cycle in order to repair the DNA, or by undergoing apoptosis. This response is highly conserved across species, and many of the genes involved in this DNA damage response have been shown to be inactivated in human cancers. This suggests the importance of DNA damage response with regard to the prevention of cancer. The DNA damage checkpoint responses vary greatly depending on the developmental context, cell type, gene expression profile, and the degree and nature of the DNA lesions. More valuable information can be obtained from studies utilizing whole organisms in which the molecular basis of development has been well established, such as Drosophila. Since the discovery of the Drosophila p53 orthologue, various aspects of DNA damage responses have been studied in Drosophila. In this review, I will summarize the current knowledge on the DNA damage checkpoint response in Drosophila. With the ease of genetic, cellular, and cytological approaches, Drosophila will become an increasingly valuable model organism for the study of mechanisms inherent to cancer formation associated with defects in the DNA damage pathway.     

Cell wall reconstruction and DNA damage repair play a key role in the improved salt tolerance effects of He-Ne laser irradiation in tall fescue seedlings

The improved salt tolerance effects of He–Ne laser were further studied through the estimation of ROS levels, cell viability, DNA damage phenomena, physicochemical properties, and monosaccharide compositions of cell wall polysaccharides in tall fescue seedlings. Salt stress produced deleterious effects on seedlings growth and development. ROS levels and genomic DNA damage were markedly increased compared with controls. Physicochemical activities and monosaccharide proportions of cell wall polysaccharide were also pronouncedly altered. He–Ne laser irradiation improved plant growth retardation via increasing cell viability and reverting physicochemical parameters. According to the results of Fourier transform infrared (FTIR) scanning spectra and DNA apopladder analysis, He–Ne laser was showed to efficiently ameliorate cell wall polysaccharide damage and DNA fragmentation phenomena. The treatment with DNA synthesis inhibitor further demonstrated that DNA damage repair was correlated with the improvement effects of the laser. Therefore, our data illustrated that He–Ne laser irradiation resulted in cell wall reconstruction and genomic DNA injury repair in vivo in salt-stressed seedlings, then enhanced salt tolerance probably via interactions between plant cell wall and related resistance gene expression pattern.     

Heterogeneity in premature senescence by oxidative stress correlates with differential DNA damage during the cell cycle

The development of cellular senescence both by replication and by oxidative stress is not homogenous in cultured primary human fibroblasts. To investigate whether this is due to the heterogeneity in the susceptibility of DNA in different phases of the cell cycle, we subjected synchronised cells to oxidative stress and examined the extent of DNA damage and its long-term effects on the induction of cellular senescence. Here, we first show marked heterogeneity in DNA damage as detected by markers of double strand breaks caused by oxidative stress in an asynchronous human fibroblast culture. Cell cycle synchronization followed by oxidative stress demonstrated that DNA in S-phase is most susceptible to oxidative stress whereas DNA in the quiescent phase is most resistant. DNA repair is an ongoing process after sensing DNA damage; reparable DNA damage is repaired even in cells that contain persistent DNA damage. The extent of persistent DNA damage is tightly correlated with permanent cessation of DNA replication and SA-beta-gal activity. Oxidative stress encountered by cells in S-phase resulted in more persistent DNA damage, more permanent cell cycle arrest and the induction of premature senescence.     

Interactions of radioprotectors and oxygen in cultured mammalian cells. II. Effects of dithiothreitol on radiation-induced DNA damage and comparison with cell survival

The effects of the sulfhydryl-containing compound dithiothreitol (DTT) on radiation-induced DNA damage have been studied using two different assays: DNA unwinding hydroxyapatite chromatography and alkaline filter elution. DNA damage as measured by both assays for cells irradiated in air shows drug concentration-dependent radioprotection reaching high levels (dose reduction factor, DRF = 3) at high DTT concentrations. The pattern and degree of protection against DNA damage are the same as shown previously for cell survival. However, when cells are irradiated in hypoxia, DNA damage as measured by the unwinding technique is decreased less by low DTT concentrations than is survival, but DNA damage is decreased to a much greater extent (DRF = 3) at high concentrations of DTT (compared to DRF = 1.5 for cell survival). DNA damage as measured by the alkaline elution assay after hypoxic irradiation is decreased to a much greater extent at all concentrations of DTT with DRF = 1.6 at 1 mM and increasing to DRF = 4.5 at high levels of DTT. These results are discussed in terms of the different types of DNA damage produced in cells irradiated in air versus hypoxia and the differences in types of damage measured by the two different DNA assays and cell survival.     

Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos

In most cells, the DNA damage checkpoint delays cell division when replication is stalled by DNA damage. In early Caenorhabditis elegans embryos, however, the checkpoint responds to developmental signals that control the timing of cell division, and checkpoint activation by nondevelopmental inputs disrupts cell cycle timing and causes embryonic lethality. Given this sensitivity to inappropriate checkpoint activation, we were interested in how embryos respond to DNA damage. We demonstrate that the checkpoint response to DNA damage is actively silenced in embryos but not in the germ line. Silencing requires rad-2, gei-17, and the polh-1 translesion DNA polymerase, which suppress replication fork stalling and thereby eliminate the checkpoint-activating signal. These results explain how checkpoint activation is restricted to developmental signals during embryogenesis and insulated from DNA damage. They also show that checkpoint activation is not an obligatory response to DNA damage and that pathways exist to bypass the checkpoint when survival depends on uninterrupted progression through the cell cycle.     

Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage

Aging and carcinogenesis coincide with the accumulation of DNA damage and mutations in stem and progenitor cells. Molecular mechanisms that influence responses of stem and progenitor cells to DNA damage remain to be delineated. Here, we show that niche positioning and Wnt signaling activity modulate the sensitivity of intestinal stem and progenitor cells (ISPCs) to DNA damage. ISPCs at the crypt bottom with high Wnt/β‐catenin activity are more sensitive to DNA damage compared to ISPCs in position 4 with low Wnt activity. These differences are not induced by differences in cell cycle activity but relate to DNA damage‐dependent activation of Wnt signaling, which in turn amplifies DNA damage checkpoint activation. The study shows that instructed enhancement of Wnt signaling increases radio‐sensitivity of ISPCs, while inhibition of Wnt signaling decreases it. These results provide a proof of concept that cell intrinsic levels of Wnt signaling modulate the sensitivity of ISPCs to DNA damage and heterogeneity in Wnt activation in the stem cell niche contributes to the selection of ISPCs in the context of DNA damage.     

Viral manipulation of DNA repair and cell cycle checkpoints

Recognition and repair of DNA damage is critical for maintaining genomic integrity and suppressing tumorigenesis. In eukaryotic cells, the sensing and repair of DNA damage are coordinated with cell cycle progression and checkpoints, in order to prevent the propagation of damaged DNA. The carefully maintained cellular response to DNA damage is challenged by viruses, which produce a large amount of exogenous DNA during infection. Viruses also express proteins that perturb cellular DNA repair and cell cycle pathways, promoting tumorigenesis in their quest for cellular domination. This review presents an overview of strategies employed by viruses to manipulate DNA damage responses and cell cycle checkpoints as they commandeer the cell to maximize their own viral replication. Studies of viruses have identified key cellular regulators and revealed insights into molecular mechanisms governing DNA repair, cell cycle checkpoints, and transformation.     

Why do Neurons Enter the Cell Cycle?

Increasing evidence indicates that postmitotic, terminally differentiated neurons activate the cell cycle before death. The purpose of this cell cycle activation, however, remains elusive. In proliferating cells, cell cycle machinery is a major contributor to the DNA damage response, which is comprised of growth arrest. In quiescent cells such as terminally differentiated neurons, cell cycle-associated events may also be part of the DNA damage response. A link between DNA damage and repair, cell cycle regulation and cell death is becoming increasingly recognized for cycling cells but remains elusive for quiescent cells. Neurons are particularly susceptible to oxidative stress due to the high rate of oxidative metabolism in the brain and the low level of antioxidant enzymes compared to other somatic tissues. This is supported by fact that the intracellular end point of many neurotoxic stimuli is oxidative stress, which also represents a major cause of the neuropathology underlying a variety of neurodegenerative diseases. DNA is perhaps the major target of oxyradicals. Thus, oxidative stress may cause DNA damage, which is countered by a complex defense mechanism, the DNA damage response, which involves not only the elimination of DNA damage, but its coordination with other cellular processes such as cell-cycle progression, together directing to preserve genomic integrity. The function of such response is the removal of DNA damage by DNA repair pathways, or the elimination of damaged cells via apoptosis. The present review discusses the idea that the cell cycle machinery is a critical element of the DNA damage response not only in cycling, but also quiescent cells, and may bear the same function: to repair the damage or initiate apoptosis if the damage is too extensive to be repaired.