DNA‐PK suppresses a p53‐independent apoptotic response to DNA damage

p53 is required for DNA damage‐induced apoptosis, which is central to its function as a tumour suppressor. Here, we show that the apoptotic defect of p53‐deficient cells is nearly completely rescued by inactivation of any of the three subunits of the DNA repair holoenzyme DNA‐dependent protein kinase (DNA‐PK). Intestinal crypt cells from p53 nullizygous mice were resistant to radiation‐induced apoptosis, whereas apoptosis in DNA‐PK_cs/p53, Ku80/p53 and Ku70/p53 double‐null mice was quantitatively equivalent to that seen in wild‐type mice. This p53‐independent apoptotic response was specific to the loss of DNA‐PK, as it was not seen in ligase IV (Lig4)/p53 or ataxia telangiectasia mutated (Atm)/p53 double‐null mice. Furthermore, it was associated with an increase in phospho‐checkpoint kinase 2 (CHK2), and cleaved caspases 3 and 9, the latter indicating engagement of the intrinsic apoptotic pathway. This shows that there are two separate, but equally effective, apoptotic responses to DNA damage: one is p53 dependent and the other, engaged in the absence of DNA‐PK, does not require p53.     

Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation.     

DNA fragmentation during thymic apoptosis is catalyzed by DNase γ

The physiological and pathological importance of cell death by apoptosis has recently been recognized. One of the hallmarks of apoptosis is the enzymatic cleavage of genomic DNA into nucleosomal oligomers. The identification of an endonuclease responsible for apoptosis might help to explain how this cell suicide is regulated and why DNA is cleaved. Here, we found that γ type of DNase was retained in apoptotic rat thymocyte nuclei. Homogeneously purified DNase γ (Mr = 33 kDa) from the apoptotic nuclei was revealed to be a Ca²⁺/Mg²⁺-dependent endonuclease and inhibited by Zn²⁺. This enzyme cleaved chromosomal DNA with 3′-hydroxyl (OH) and 5′-phosphoryl (P) ends. The cleavage ends and its divalent cation dependencies match those observed in apoptotic thymocytes induced by X-irradiation or glucocorticoid treatment, indicating that this endonuclease is a central component of the thymic apoptosis machinery.     

Tunicamycin and brefeldin a induce in plant cells a programmed cell death showing apoptotic features

Summary The recent identification ofDAD (defender against apoptotic death) gene in plants suggests that the N-linked glycosylation of proteins could be an important control point of plant programmed cell death. In this paper we describe the effects of Tunicamycin, an inhibitor of N-linked protein glycosylation, and Brefeldin A, an inhibitor of protein trafficking from the Golgi apparatus, on sycamore (Acer pseudoplatanus L.) cell cultures. These two chemicals proved able to induce a strong acceleration of the cell death; changes in cell and nucleus morphology; an increase in DNA fragmentation, detectable by a specific immunological reaction; and the presence of oligonucleosomal-size fragments (laddering) in DNA gel electrophoresis. Moreover, Brefeldin A, but not Tunicamycin, strongly stimulated the production of hydrogen peroxide. These results indicate that also in plants chemicals interfering with the activities of endoplasmic reticulum and of Golgi apparatus strongly induce a form of programmed cell death showing apoptotic features.     

Helicobacter hepaticus Cytolethal Distending Toxin Causes Cell Death in Intestinal Epithelial Cells via Mitochondrial Apoptotic Pathway

Background: Helicobacter hepaticus, the prototype for enterohepatic Helicobacter species, colonizes the lower intestinal and hepatobiliary tracts of mice and causes typhlocolitis, hepatitis, and hepatocellular carcinoma in susceptible mouse strains. Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus. CDT of several Gram‐negative bacteria is associated with double‐stranded DNA breaks resulting in cell cycle arrest and death of a wide range of eukaryotic cells in vitro. We previously observed H. hepaticus CDT (HhCDT) mediated apoptosis in INT407 cells. However, the exact mechanism for the induction of the apoptotic pathway by HhCDT is unknown. The objective of this study was to identify the apoptotic signaling pathway induced by HhCDT in INT407 cells. Materials and Methods: INT407 cells were incubated with or without recombinant HhCDT for 0–72 hours. H2AX phosphorylation and apoptotic parameters were analyzed. Results: H2AX was phosphorylated 24 hours postexposure to HhCDT. Expression of pro‐apoptotic Bax protein was upregulated after 24 hours, while Bcl₂ expression decreased. Cytochrome c was released from mitochondria after 12–24 hours of exposure. Concurrently, caspase 3/7 and 9 were activated. However, pretreatment of INT407 cells with caspase inhibitor (Z‐VAD‐FMK) inhibited the activation of caspase 3/7 and 9. Significant activity of caspase 8 was not observed in toxin treated cells. Activation of caspase 3/7 and caspase 9 confirms the involvement of the mitochondrial apoptotic pathway in HhCDT‐treated cells. Conclusion: These findings show, for the first time, the ability of HhCDT to induce apoptosis via the mitochondrial pathway.     

The dark side of circulating nucleic acids

Free circulating or cell‐free DNA (cfDNA), possibly from dying cells that release their contents into the blood as they break down, have become of major interest as a source for noninvasive diagnostics. Recent work demonstrated the uptake of human cfDNA in mouse cells in vitro and in vivo, accompanied by the activation of a cellular DNA damage response (DDR) and the appearance of apoptotic proteins in the host cells. By acting as a source of mobile genetic elements, cfDNA could be a continuous source of DNA mutagenesis of healthy cells in the body throughout life, promoting progressive cellular aging in vivo. As such, cfDNA may causally contribute to multiple aging‐related diseases, such as cancer, diabetes, and Alzheimer's disease.     

In vitro DNA binding activity and molecular docking reveals pierisin-5 as an anti-proliferative agent against gastric cancer

Abstract

Pierisin-5 is a DNA dependent ADP ribosyltransferase (ADRT) protein from the larvae of Indian cabbage white butterfly, Pieris canidia. Interestingly, Pierisin-5 ADP-ribosylates the DNA as a substrate, but not the protein and subsequently persuades apoptotic cell death in human cancer cells. This has led to the investigation on the DNA binding activity of Pierisin-5 using in vitro and in silico approaches in the present study. However, both the structure and the mechanism of ADP-ribosylation of pierisin-5 are unknown. In silico modeled structure of the N-terminal ADRT catalytic domain interacted with the minor groove of B-DNA for ribosylation with the help of β-NAD⁺ which lead to a structural modification in DNA (DNA adduct). The possible interaction between calf thymus DNA (CT-DNA) and purified pierisin-5 protein was studied through spectral–spatial studies and the blue shift and hyperchromism in the UV–Visible spectra was observed. The DNA adduct property of pierisin-5 protein was validated by in vitro cytotoxic assay on human gastric (AGS) cancer cell lines. Our study is the first report of the mechanism of DNA binding property of pierisin-5 protein which leads to the induction of cytotoxicity and apoptotic cell death against cancer cell lines.

Communicated by Ramaswamy H. Sarma     

Taxol-induced apoptotic cell death in suspension cultures of Taxus cuspidata

Taxol caused apoptotic cell death of Taxus cuspidata in suspension cultures. Typical morphological and biochemical changes of apoptosis were observed by microscopy and total DNA agarose gel electrophoresis. Taxus cuspidata responded to the added Taxol by increasing the biosynthesis of Taxol. The percentage of apoptotic cells in total cells increased with the concentration of added Taxol. With Taxol added at 10 mg l^–1, the maximum concentration of Taxol produced was 23 mg l^–1, 3 times higher than that of the control culture.     

Localization of Apoptosis (Programmed Cell Death) in Mice by Administration of Lipopolysaccharide

Localization of apoptotic cells by administration of lipopolysaccharide into mice was studied by using the in situ specific labeling of fragmented DNA. This method clearly stained the nuclei of thymocytes at the cortex of the thymus. The nuclei of cells in the bone marrow and in the spleen were also positively stained. It was suggested that the cortex in the thymus is where the LPS-induced programmed cell death occurs.     

In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice

In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria‐targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro‐apoptotic and pro‐inflammatory redox signaling pathways.     

Apoptosis in the Initial Leaf of Etiolated Wheat Seedlings: Influence of the Antioxidant Ionol (BHT) and Peroxides

Apoptosis was observed in the initial leaf of 5-8-day-old etiolated wheat seedlings. A condensation of cytoplasm in apoptotic cells, formation of myelin-like structures, specific fragmentation of cytoplasm, appearance in vacuoles of specific vesicles containing subcellular organelles, condensation and margination of chromatin in the nucleus, and internucleosomal fragmentation of nuclear DNA are ultrastructural features of apoptosis in the initial wheat leaf. Single-membrane vesicles detected in vacuoles of the leaf cells resemble in appearance the vacuolar vesicles in the coleoptile apoptotic cells described earlier (Bakeeva, L. E., et al. (1999) FEBS Lett., 457, 122-125); they contain preferentially plastids but not mitochondria as was observed in coleoptile. The vacuolar vesicles are specific for the apoptotic plant cells. Thus, apoptosis in various tissues is an obligatory element of plant (wheat) growth and development even in the early stages of ontogenesis. Contrary to strong geroprotecting action in coleoptile, the known antioxidant BHT (ionol, 2.27·10^–4 M) does not prevent in the leaf cells the apoptotic internucleosomal DNA fragmentation and appearance of specific vacuolar vesicles containing subcellular organelles. Therefore, the antioxidant action on apoptosis in plants is tissue specific. Peroxides (H₂O₂, cumene hydroperoxide) stimulated apoptosis (internucleosomal DNA fragmentation) in coleoptile and induced it in an initial leaf when apoptosis in a control seedling leaf was not yet detected. Thus, apoptosis that is programmed in plant ontogenesis and controlled by reactive oxygen species (ROS) can be modulated by anti- and prooxidants.     

Simulated microgravity decreases DNA repair capacity and induces DNA damage in human lymphocytes

The effect of simulated microgravity on DNA damage and apoptosis is still controversial. The objective of this study was to test whether simulated microgravity conditions affect the expression of genes for DNA repair and apoptosis. To achieve this objective, human lymphocyte cells were grown in a NASA‐developed rotating wall vessel (RWV) bioreactor that simulates microgravity. The same cell line was grown in parallel under normal gravitational conditions in culture flasks. The effect of microgravity on the expression of genes was measured by quantitative real‐time PCR while DNA damage was examined by comet assay. The result of this study revealed that exposure to simulated microgravity condition decreases the expression of DNA repair genes. Mismatch repair (MMR) class of DNA repair pathway were more susceptible to microgravity condition‐induced gene expression changes than base excision repair (BER) and nucleotide excision repair (NER) class of DNA repair genes. Downregulation of genes involved in cell proliferation (CyclinD1 and PCNA) and apoptosis (Bax) was also observed. Microgravity‐induced changes in the expression of some of these genes were further verified at the protein level by Western blot analysis. The findings of this study suggest that microgravity may induce alterations in the expression of these DNA repair genes resulting in accumulation of DNA damage. Reduced expression of cell‐cycle genes suggests that microgravity may cause a reduction in cell growth. Downregulation of pro‐apoptotic genes further suggests that extended exposure to microgravity may result in a reduction in the cells' ability to undergo apoptosis. Any resistance to apoptosis seen in cells with damaged DNA may eventually lead to malignant transformation of those cells. J. Cell. Biochem. 107: 723–731, 2009. © 2009 Wiley‐Liss, Inc.     

Peptidoglycan from Staphylococcus aureus has an anti‐apoptotic effect in HaCaT keratinocytes mediated by the production of the cellular inhibitor of apoptosis protein‐2

Colonization of epithelium by microorganisms leads to inflammatory responses. In some cases an anti‐apoptotic response involving the cellular inhibitor of apoptosis protein‐2 (cIAP‐2) also occurs. Although strong expression of cIAP‐2 has been observed in lesional skin from psoriatic patients and in HaCaT keratinocytes treated with peptidoglycan (PGN) from Staphylococcus aureus, anti‐apoptotic responses induced in the skin by cIAP‐2 have seldom been studied. In this study, the effect of PGN on TNF‐α‐induced apoptotic HaCaT keratinocytes was assessed. Morphological analysis, quantification of cells with DNA fragmentation and active caspase‐3 detection was performed to assess apoptotic cell death. Greater LL‐37 and cIAP‐2 production was found in keratinocytes stimulated with PGN than in non‐treated cells (P < 0.05). In comparison with cells treated with TNF‐α only, a significant reduction in apoptotic cell death was observed when HaCaT were pretreated with PGN before inducing apoptosis with TNF‐α (P < 0.05). In addition, an inhibitor of cIAP‐2 activity (LCL161) stopped the PGN effect. These findings show that PGN from S. aureus has an anti‐apoptotic effect in keratinocytes mediated by cIAP‐2 production, suggesting that this anti‐apoptotic activity could favor proliferation of keratinocytes in psoriasis.     

The antiapoptotic protein AAC‐11 interacts with and regulates Acinus‐mediated DNA fragmentation

The nuclear factor Acinus has been suggested to mediate apoptotic chromatin condensation after caspase cleavage. However, this role has been challenged by recent observations suggesting a contribution of Acinus in apoptotic internucleosomal DNA cleavage. We report here that AAC‐11, a survival protein whose expression prevents apoptosis that occurs on deprivation of growth factors, physiologically binds to Acinus and prevents Acinus‐mediated DNA fragmentation. AAC‐11 was able to protect Acinus from caspase‐3 cleavage in vivo and in vitro, thus interfering with its biological function. Interestingly, AAC‐11 depletion markedly increased cellular sensitivity to anticancer drugs, whereas its expression interfered with drug‐induced cell death. AAC‐11 possesses a leucine‐zipper domain that dictates, upon oligomerization, its interaction with Acinus as well as the antiapoptotic effect of AAC‐11 on drug‐induced cell death. A cell permeable peptide that mimics the leucine‐zipper subdomain of AAC‐11, thus preventing its oligomerization, inhibited the AAC‐11–Acinus complex formation and potentiated drug‐mediated apoptosis in cancer cells. Our results, therefore, show that targeting AAC‐11 might be a potent strategy for cancer treatment by sensitization of tumour cells to chemotherapeutic drugs.     

Analysis of apoptosis and DNA damage in bovine cumulus cells after exposure in vitro to different zinc concentrations

The purpose of this study was to investigate the effect of Zn (zinc) concentration on CCs (cumulus cells) during in vitro maturation. For this purpose, DNA integrity of CCs by addition of different Zn concentrations [0 (control); 0.7 μg/ml (Zn1); 1.1 μg/ml (Zn2) and 1.5 μg/ml (Zn3)] to the culture medium was evaluated by comet assay. In addition, early apoptosis was analysed by annexin staining assay. CCs treated with Zn showed a significant decrease in the DNA damage in a dose‐dependent manner. Comet assay analysed for TM (tail moment) was significantly higher in cells cultured without Zn (control, P<0.01) with respect to cells treated with Zn (control: 5.24±16.05; Zn1: 1.13±5.31; Zn2: 0.10±0.36; Zn3: 0.017±0.06). All treatments were statistically different from the control (P=0.014 for Zn1; P<0.01 for Zn2 and Zn3). The frequency of apoptotic cells was higher in the control group (control: 0.142±0.07; Zn1: 0.109±0.0328; Zn2:0.102±0.013; Zn3: 0.0577±0.019). Statistical differences were found between control and Zn1 (P=0.0308), control and Zn2 (P=0.0077), control and Zn3 (P<0.0001), Zn1 and Zn3 (P<0.001) and Zn2 and Zn3 (P=0.0004). No differences were found between Zn1 and Zn2. In conclusion, low Zn concentrations increase DNA damage and apoptosis in CCs cultured in vitro. However, adequate Zn concentrations ‘protect’ the integrity of DNA molecule and diminish the percentage of apoptotic CC.     

Isocyanates induces DNA damage,apoptosis, oxidative stress,and inflammation in cultured human lymphocytes

Isocyanates, a group of low molecular weight aromatic and aliphatic compounds containing the isocyanate group (?NCO), are important raw materials with diverse industrial applications; however, pathophysiological implications resulting from occupational and accidental exposures of these compounds are hitherto unknown. Although preliminary evidence available in the literature suggests that isocyanates and their derivatives may have deleterious health effects including immunotoxicity, but molecular mechanisms underlying such an effect have never been addressed. The present study was carried out to assess the immunotoxic response of methyl isocyanate (MIC) on cultured human lymphocytes isolated from healthy human volunteers. Studies were conducted to evaluate both dose‐dependent and time‐course response (n = 3), using N‐succinimidyl N‐methylcarbamate, a surrogate chemical substitute to MIC. Evaluation of DNA damage by ataxia telangiectasia mutated (ATM) and γ H2AX protein phosphorylation states; measure of apoptotic index through annexin‐V/PI assay, apoptotic DNA ladder assay, and mitochondrial depolarization; induction of oxidative stress by CM‐H2DCFDA and formation of 8‐hydroxy‐2′ deoxy guanosine; levels of antioxidant defense system enzyme glutathione reductase; and multiplex cytometric bead array analysis to quantify the secreted levels of inflammatory cytokines, interleukin‐8, interleukin‐1β, interleukin‐6, interleukin‐10, tumor necrosis factor, and interleukin‐12p70 parameters were carried out. The results of the study showed a dose‐ and time‐dependent response, providing evidence to hitherto unknown molecular mechanisms of immunotoxic consequences of isocyanate exposure at a genomic level. We anticipate these data along with other studies reported in the literature would help to design better approaches in risk assessment of occupational and accidental exposure to isocyanates. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:429–440, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20260     

The diverse origins of circulating cell‐free DNA in the human body: a critical re‐evaluation of the literature

Since the detection of cell‐free DNA (cfDNA) in human plasma in 1948, it has been investigated as a non‐invasive screening tool for many diseases, especially solid tumours and foetal genetic abnormalities. However, to date our lack of knowledge regarding the origin and purpose of cfDNA in a physiological environment has limited its use to more obvious diagnostics, neglecting, for example, its potential utility in the identification of predisposition to disease, earlier detection of cancers, and lifestyle‐induced epigenetic changes. Moreover, the concept or mechanism of cfDNA could also have potential therapeutic uses such as in immuno‐ or gene therapy. This review presents an extensive compilation of the putative origins of cfDNA and then contrasts the contributions of cellular breakdown processes with active mechanisms for the release of cfDNA into the extracellular environment. The involvement of cfDNA derived from both cellular breakdown and active release in lateral information transfer is also discussed. We hope to encourage researchers to adopt a more holistic view of cfDNA research, taking into account all the biological pathways in which cfDNA is involved, and to give serious consideration to the integration of in vitro and in vivo research. We also wish to encourage researchers not to limit their focus to the apoptotic or necrotic fraction of cfDNA, but to investigate the intercellular messaging capabilities of the actively released fraction of cfDNA and to study the role of cfDNA in pathogenesis.