UV-B Irradiated Cell Lines Execute Programmed Cell Death in Various Forms

We investigated changes typical for apoptosis in various cell lines after UV-B irradiation. Using established methods for detection of apoptosis we demonstrate changes of cellular morphology, phosphatidylserine (PS) exposure, ollgonucleosomal DNA fragmentation and generation of hypochrome nuclei. To isolated high-molecular-weight (hmwt) DNA fragments we engaged a new method avoiding pulse field gel electrophoresis. Most UV-B irradiated cell lines showed oligonucleosomal DNA fragmentation, hypochrome nuclei, morphological changes, annexin-V binding and positive TUNEL reaction. However, no oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. Whereas HaCaT cells displayed all other changes typical for apoptosis, Raji cells were TUNEL negative, formed low amounts of hmwt DNA and showed an 'atypically' low hypochrome shift. Nevertheless, UV-B irradiated Raji cells excluded propidium iodide (PI), bound annexin-V and stopped proliferation. This suggests that Raji cells underwent growth arrest with exposure of PS being the only feature of apoptosis. However, in the presence of phagocytes expressing the phosphatidylserine receptor these cells would share the removal pathway with apoptotic cells. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation or chromatin condensation is not suitable to exclude programmed (apoptotic?) cell death.     

Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA

The occurrence and spatial distribution of intracellular DNA fragmentation was investigated by in situ 3 end labelling of DNA breaks in K562 cells treated in such a way to cause either apoptotic or necrotic cell death. The localisation of DNA breaks was examined by confocal laser microscopy and compared with the electron-microscopic appearance of the cells. In addition, the number of cells with fragmented DNA was counted and compared with the number of dead cells, as determined by the nigrosin dye exclusion test. Apoptosis was induced by cultivation of the cells in the presence of actinomycin D. Cells undergoing apoptosis were characterised by massive intracellular DNA fragmentation that was highly ordered into successive steps. Cells in early stages of the apoptotic process had DNA breaks diffusely distributed in the entire nucleus, except the nucleolus, with crescent-like accumulations beyond the nuclear membrane. In the more advanced stages, the nucleus was transformed into many round bodies with intense labelling. Intracellular accumulations of fragmented DNA corresponded exactly to electron-dense chromatin seen in the electron microscope, whereas diffuse DNA breaks had no morphological correlate at the ultrastructural level. In necrosis induced by ionomycin, NaN₃, or rapid freezing combined with thawing, no DNA fragmentation occurred at the onset of cell death, but appeared 24 h later. This fragmentation was not characterised by a unique morphology, but represented the breakdown of the chromatin in the configuration remaining after cell death. Therefore, apoptosis is characterised by DNA fragmentation that proceeds in a regular orderly sequence at the beginning of cell death, and can be detected by in situ 3end labelling of DNA breaks.     

Analysis of apoptosis by cytometry using TUNEL assay

Activation of endonucleases that cleave chromosomal DNA preferentially at internucleosomal sections is a hallmark of apoptosis. DNA fragmentation revealed by the presence of a multitude of DNA strand breaks, therefore, is considered to be the gold standard for identification apoptotic cells. Several variants of the methodology that is based on fluorochrome-labeling of 3'-OH termini of DNA strand breaks in situ with the use of exogenous terminal deoxynucleotidyl transferase (TdT), commonly defined as the TUNEL assay, have been developed by us. This Chapter describes the variant based on strand breaks labeling with Br-dUTP that is subsequently detected immunocytochemically with Br-dUAb. Compared with other TUNEL variants the Br-dU-labeling assay offers the greatest sensitivity in detecting DNA breaks. Described also are modifications of the protocol that allow one to use other than Br-dUTP fluorochrome-tagged deoxynucleotides to label DNA breaks. Concurrent staining of DNA with propidium or 4',6-diamidino-2-phenylindole (DAPI) and multiparameter analysis of cells by flow- or laser scanning cytometry enables one to correlate induction of apoptosis with the cell cycle phase.     

Cleavage of Poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a target of caspases during apoptosis: its cleavage onto 89- and 24-kDa fragments is considered to be a hallmark of the apoptotic mode of cell death. Another hallmark is the activation of endonuclease which targets internucleosomal DNA. The aim of the present study was to reveal cell cycle phase specificity as well as the temporal and sequence relationships of PARP cleavage vis-à-vis DNA fragmentation in two model systems of apoptosis, one induced by DNA damage via cell treatment with camptothecin (CPT) (mitochondria-induced pathway) and another by the cytotoxic ligand tumor necrosis factor alpha (TNF-alpha) (cell surface death receptor pathway). PARP cleavage was detected immunocytochemically using antibody which recognizes its 89-kDa fragment (PARP p89) while DNA fragmentation was assayed by in situ labeling of DNA strand breaks. The frequency and extent of PARP cleavage as well as DNA fragmentation were measured by mutiparameter flow and laser scanning cytometry. PARP cleavage, selective to S phase cells, was detected 90 min after administration of CPT. PARP cleavage in the cells treated with TNF-alpha was not selective to any cell cycle phase and was seen already after 30 min. DNA fragmentation trailed PARP cleavage by about 30 min and showed a similar pattern of cell cycle specificity. PARP p89 was present in nuclear chromatin but at least in the early phase of apoptosis it did not colocalize with DNA strand breaks. The rate of cleavage of PARP molecules in individual cells whether induced by CPT or TNF-alpha was rapid as reflected by the paucity of cells with a mixture of cleaved and noncleaved PARP molecules. In contrast, DNA fragmentation proceeded stepwise before reaching the maximal number of DNA strand breaks. Although time windows for PARP cleavage vs DNA fragmentation were different at early stages of apoptosis, a good overall correlation between the cytometric assays of apoptotic cells identification based on these events was observed in both CPT- and TNF-alpha-treated cultures.     

Early Detection of DNA Strand Breaks in the Brain After Transient Focal Ischemia: Implications for the Role of DNA Damage in Apoptosis and Neuronal Cell Death

Abstract: Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16–72 h of reperfusion, many SSB- and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.     

Contribution of apoptosis to responses in the comet assay

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.     

Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis

Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named gammaH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of gammaH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.     

Intact Cell Evidence for the Early Synthesis, and Subsequent Late Apopain-Mediated Suppression, of Poly(ADP-ribose) during Apoptosis

Poly(ADP-ribose) polymerase (PARP), which is catalytically activated by DNA strand breaks, has been implicated in apoptosis, or programmed cell death. A protease (CPP32) responsible for the cleavage of PARP and necessary for apoptosis was recently purified and characterized. The coordinated sequence of events related to PARP activation and cleavage in apoptosis has now been examined in individual cells. Apoptosis was studied in a human osteosarcoma cell line that undergoes a slow (8 to 10 days), spontaneous, and reproducible death program in culture. Changes in the abundance of intact PARP, poly(ADP-ribose) (PAR), and a proteolytic cleavage product of PARP that contains the DNA-binding domain were examined during apoptosis in the context of individual, whole cells by immunofluorescence with specific antibodies. The synthesis of PAR from NAD increased early, within 2 days of cell plating for apoptosis, prior to the appearance of internucleosomal DNA cleavage and before the cells become irreversibly committed to apoptosis, since replating yields viable, nonapoptotic cells. Strong expression of full-length PARP was also detected, by immunofluorescence as well as by Western analysis, during this same time period. However, after ∼4 days in culture, the abundance of both full-length PARP and PAR decreased markedly. After 6 days, a proteolytic cleavage product containing the DNA-binding domain of PARP was detected immunocytochemically and confirmed by Western analysis, both in the nuclei and in the cytoplasm of cells. A recombinant peptide spanning the DNA-binding domain of PARP was expressed, purified, and biotinylated, and was then used as a probe for DNA strand breaks. Fluorescence microscopy with this probe revealed extensive DNA fragmentation during the later stages of apoptosis. This is the first report, using individual,intact cells,demonstrating that poly(ADP-ribosyl)ation of nuclear proteins occurs prior to the commitment to apoptosis, that inactivation and cleavage of PARP begin shortly thereafter, and that very little PAR per se is present during the later stages of apoptosis, despite the presence of a very large number of DNA strand breaks. These results suggest a negative regulatory role for PARP during apoptosis, which in turn may reflect the requirement for adequate NAD and ATP during the later stages of programmed cell death.     

DNA Damage Induced by DNA Topoisomerase I- and Topoisomerase II- Inhibitors Detected by Histone H2AXphosphorylation in Relation to the Cell Cycle Phase and Apoptosis

Histone H2AX is phosphorylated on Ser-139 by ATM kinase in response to damage that induces dsDNA breaks. Immunocytochemical detection of phosphorylated H2AX (gH2AX), thus, reveals the presence of dsDNA breaks in chromatin. Multiparameter cytometry was presently used to correlate the appearance of gH2AX with:

a. cell cycle phase;

b. caspase-3 activation; and

c. apoptosis-associated DNA fragmentation in individual human leukemic HL-60 cells treated with the DNA topoisomerase I (topo1) inhibitors topotecan (TPT) and camptothecin (CPT) or with the topo2 inhibitor mitoxantrone (MTX).

In response to TPT or CPT maximal increase of gH2AX immunofluorescence was seen in S-phase cells by 90 min. In contrast, following MTX treatment the maximal rise of gH2AX was detected at 2 h in G1 cells and the cell cycle phase specificity was much less apparent. A linear relationship between the drug concentration and increase of gH2AX immunofluorescence was seen only up to 200 nM TPT; a decline in gH2AX was apparent at a concentration range between 0.4 and 1.6 mM TPT. Thus, the intensity of gH2AX immunofluorescence, as a marker of cell survival following TPT treatment, can be used only within a limited range of drug concentration. Following treatment with TPT, CPT or MTX the peak of H2AX phosphorylation preceded caspase-3 activation and the appearance of apoptosis-associated DNA fragmentation, both selective to S-phase cells. Progression of apoptosis was paralleled by a decrease in gH2AX immunofluorescence. The data also indicate that regardless whether treated with inhibitors of topo1 or topo2, at comparable levels of dsDNA breaks, the cells replicating DNA have a higher proclivity to undergo apoptosis compared to G1 or G2/M cells.     

Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis

Chromosomal DNA and mitochondrial dysfunctions play a role on mammalian cell death induced by oxidative stress. The major biochemical dysfunction of chromosome is the presence of an ordered cleavage of the DNA backborn, which is separated and visualized as an electrophoretic pattern of fragments. Oxidative stress provides chromatin dysfunction such as single strand and double strand DNA fragmentation leading to cell death. More than 1 Mb of giant DNA, 200-800 kb or 50-300 kb high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced during apoptosis or necrosis induced by oxidative stress such as glutathione (GSH) depletion in several types of mammalian cells. Reactive oxygen species (ROS)-mediated DNA fragmentation is enhanced by polyunsaturated fatty acids including arachidonic acid or their hydroperoxides, leading to necrosis. Mitochondrial dysfunction on decrease of trans membrane potential, accumulation of ROS, membrane permeability transition and release of apoptotic factors during apoptosis or necrosis has been implicated. This review refers to the correlation of chromosomal DNA fragmentation and apoptosis or necrosis induced by GSH depletion, and the possible mechanisms of oxidative stress-induced cell death.     

Application of Biotin, Digoxigenin or Fluorescein Conjugated Deoxynucleotides to Label DNA Strand Breaks for Analysis of Cell Proliferation and Apoptosis Using Flow Cytometry

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deozynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20-30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluo-resceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.     

Application of Biotin,Digoxigenin or Fluorescein Conjugated Deoxynucleotides to Label DNA Strand Breaks for Analysis of Cell Proliferation and Apoptosis Using Flow Cytometry

A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deozynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20–30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluo-resceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.     

DNA single-strand breakage in mammalian cells induced by redox cycling quinones in the absence of oxidative stress

Quinone-induced cell death is often attributed to oxidative stress during which the formation of DNA strand breaks is thought to play an important role. In this study, extensive DNA damage was observed in human chronic myelogenous leukemic cells (K562) exposed for 15 minutes to low concentrations (15–100 μM) of the redox cycling quinones 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) and menadione. However, DNA strand breakage and cell death could not be attributed to oxidative stress as the intracellular level and redox status of the reducing equivalents NADP(H) and GSH were unaffected. The intracellular level of NAD⁺ was found to correlate well with the extent of DNA repair (r = 0.93, P < 0.02) and cell proliferation (r = 0.96, P < 0.01) in cells exposed to the quinones. In contrast, a significant decrease in the level of intracellular ATP was only observed in cells exposed to menadione (50–100 μM). These results suggest that redox cycling quinones are capable of inducing DNA damage in mammalian cells by a mechanism that does not involve oxidative stress. Following DNA damage, cell death is dependent on the availability of NAD⁺, which may be key to the rapid repair of strand breaks. © 1995 John Wiley & Sons, Inc.     

DNA damage induced by DNA topoisomerase I- and topoisomerase II-inhibitors detected by histone H2AX phosphorylation in relation to the cell cycle phase and apoptosis

Histone H2AX is phosphorylated on Ser-139 by ATM kinase in response to damage that induces dsDNA breaks. Immunocytochemical detection of phosphorylated H2AX (gammaH2AX), thus, reveals the presence of dsDNA breaks in chromatin. Multiparameter cytometry was presently used to correlate the appearance of gammaH2AX with: a. cell cycle phase; b. caspase-3 activation; and c. apoptosis-associated DNA fragmentation in individual human leukemic HL-60 cells treated with the DNA topoisomerase I (topo1) inhibitors topotecan (TPT) and camptothecin (CPT) or with the topo2 inhibitor mitoxantrone (MTX). In response to TPT or CPT maximal increase of gammaH2AX immunofluorescence was seen in S-phase cells by 90 min. In contrast, following MTX treatment the maximal rise of gammaH2AX was detected at 2 h in G1 cells and the cell cycle phase specificity was much less apparent. A linear relationship between the drug concentration and increase of gammaH2AX immunofluorescence was seen only up to 200 nM TPT; a decline in gammaH2AX was apparent at a concentration range between 0.4 and 1.6 microM TPT. Thus, the intensity of gammaH2AX immunofluorescence, as a marker of cell survival following TPT treatment, can be used only within a limited range of drug concentration. Following treatment with TPT, CPT or MTX the peak of H2AX phosphorylation preceded caspase-3 activation and the appearance of apoptosis-associated DNA fragmentation, both selective to S-phase cells. Progression of apoptosis was paralleled by a decrease in gammaH2AX immunofluorescence. The data also indicate that regardless whether treated with inhibitors of topo1 or topo2, at comparable levels of dsDNA breaks, the cells replicating DNA have a higher proclivity to undergo apoptosis compared to G1 or G2/M cells.     

Plasma membrane phospholipid asymmetry precedes DNA fragmentation in different apoptotic cell models

 Biochemical alterations occurring in many cell types during apoptosis include the loss of plasma membrane phospholipid asymmetry and nuclear DNA fragmentation. Annexin V staining detects phosphatidylserine translocation into the outer plasma membrane layer occurring during cell death, while the in situ tailing (IST or TUNEL) reaction labels the DNA strand breaks typical of apoptosis. To compare the time course of these processes we investigated methylprednisolone-induced apoptosis of rat thymocytes, topoisomerase inhibitor-induced apoptosis in the human histiocytic lymphoma cell line U937, and serum deprivation-induced apoptosis in the rat pheochromocytoma cell line, PC12. At all time points, FACS analysis and quantitative fluorescence light microscopy showed a higher proportion of annexin V-positive than IST-positive cells, with significantly different time courses in the apoptotic cell models investigated (Anova test). Results were confirmed by confocal microscopy. Our data indicate that the exposure of phosphatidylserine, a potential phagocyte recognition signal on the cell surface of apoptotic cells in vivo, precedes DNA strand breaks during apoptosis in different cell types. Accepted: 29 June 1998     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

Programmed Myocyte Cell Death Affects the Viable Myocardium after Infarction in Rats

To determine whether apoptotic and necrotic myocyte cell death occur acutely and chronically after infarction, the formation of DNA strand breaks and the localization of myosin monoclonal antibody labeling were analyzed in the surviving myocardium from 20 min to 1 month. DNA strand breaks in myocyte nuclei were detected as early as 3 h following coronary artery occlusion and were still present at 1 month. This cellular process was characterized biochemically by internucleosomal DNA fragmentation which produced DNA laddering on agarose gel electrophoresis. Quantitatively, 155 myocyte nuclei per 10⁶cells exhibited DNA strand breaks in the portion adjacent to the infarcted tissue at 3–12 h. This parameter increased to 704 at 1–2 days and subsequently decreased to 364 at 7 days, 188 at 14 days, and 204 at 1 month. In the remote myocardium, the number of myocyte nuclei with DNA strand breaks was 84 per 10⁶at 3–12 h and remained essentially constant up to 1 month. Programmed myocyte cell death was accompanied by a decrease in the expression of bcl-2 and an increase in the expression of bax. The changes in the expression of these genes were present at 1 and 7 days after coronary artery occlusion. In conclusion, the mechanical load produced by myocardial infarction and ventricular failure may affect the regulation of bcl-2 and bax in the viable myocytes, triggering programmed cell death and the remodeling of the ventricular wall.     

Activation of Xenopus eggs by the kinase inhibitor 6-DMAP suggests a differential regulation of cyclin B and p39(mos) proteolysis

The molecular mode of action of arsenic, a therapeutic agent employed in the treatment of acute promyelocytic leukemia, has been elusive. Here we provide evidence that arsenic compounds may act on mitochondria to induce apoptosis. Arsenite induces apoptosis accompanied by a loss of the mitochondrial transmembrane potential (Delta Psim). Inhibition of caspases prevents the arsenite-induced nuclear DNA loss, but has no effect on the Delta Psim dissipation and cytolysis induced by arsenite. In contrast, Bcl-2 expression induced by gene transfer prevents all hallmarks of arsenite-induced cell death, including the Delta Psim collapse. PK11195, a ligand of the mitochondrial benzodiazepine receptor, neutralizes this Bcl-2 effect. Mitochondria are required in a cell-free system to mediate arsenite-induced nuclear apoptosis. Arsenite causes the release of an apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space. This effect is prevented by the permeability transition (PT) pore inhibitor cyclosporin A, as well as by Bcl-2, which is known to function as an endogenous PT pore antagonist. Arsenite also opens the purified, reconstituted PT pore in vitro in a cyclosporin A- and Bcl-2-inhibitible fashion. Altogether these data suggest that arsenite can induce apoptosis via a direct effect on the mitochondrial PT pore.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage.The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2 h (fresh) or 5 days at 4 °C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.