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Hydrogen peroxide mediates higher order chromatin degradation
Neurochemistry International 42 (2003) 123–129
Hydrogen peroxide mediates higher order chromatin degradation H. Bai, G.W. Konat∗ Department of Neurobiology and Anatomy, West Virginia University School of Medicine, 4052 HSN, P.O. Box 9128, Morgantown, WV 26506-9128, USA Received 17 October 2001; accepted 26 April 2002
Abstract Although a large body of evidence supports a causative link between oxidative stress and neurodegeneration, the mechanisms are still elusive. We have recently demonstrated that hydrogen peroxide (H2 O2 ), the major mediator of oxidative stress triggers higher order chromatin degradation (HOCD), i.e. excision of chromatin loops at the matrix attachment regions (MARs). The present study was designed to determine the specificity of H2 O2 in respect to HOCD induction. Rat glioma C6 cells were exposed to H2 O2 and other oxidants, and the fragmentation of genomic DNA was assessed by field inversion gel electrophoresis (FIGE). S1 digestion before FIGE was used to detect single strand fragmentation. The exposure of C6 cells to H2 O2 induced a rapid and extensive HOCD. Thus, within 30 min, total chromatin was single strandedly digested into 50 kb fragments. Evident HOCD was elicited by H2 O2 at concentrations as low as 5 M. HOCD was mostly reversible during 4–8 h following the removal of H2 O2 from the medium indicating an efficient relegation of the chromatin fragments. No HOCD was induced by H2 O2 in isolated nuclei indicating that HOCD-endonuclease is activated indirectly by cytoplasmic signal pathways triggered by H2 O2 . The exposure of cells to a synthetic peroxide, i.e. tert-butyrylhydroperoxide (tBH) also induced HOCD, but to a lesser extent than H2 O2 . Contrary to the peroxides, the exposure of cells to equitoxic concentration of hypochlorite and spermine NONOate, a nitric oxide generator, failed to induce rapid HOCD. These results indicate that rapid HOCD is not a result of oxidative stress per se, but is rather triggered by signaling cascades initiated specifically by H2 O2 . Furthermore, the rapid and extensive HOCD was observed in several rat and human cell lines challenged with H2 O2 , indicating that the process is not restricted to glial cells, but rather represents a general response of cells to H2 O2 . © 2003 Elsevier Science Ltd. All rights reserved. Keywords: C6 cells; Peroxides; Hypochlorite; Nitric oxide; Oxidative stress
1. Introduction Oxidative stress resulting from an excessive generation of reactive oxygen species (ROS) is the key component in the etiopathology of both acute and chronic neurodegenerative conditions. Because ROS readily oxidize the DNA molecule (Halliwell and Aruoma, 1991; Wink et al., 1991; Breen and Murphy, 1995; Henle and Linn, 1997), the effects of oxidative stress are likely mediated by the accumulation of somatic mutations that in postmitotic neural cells inevitably result in the loss of important cell function, and ultimately, cell degeneration and death. We have recently shown that ROS can damage genomic DNA indirectly (Mouzannar et al., 2001; Konat et al., 2001). Thus, hydrogen peroxide (H2 O2 ), the major mediator of oxidative stress, rapidly induces higher order chromatin degradation (HOCD), i.e. enzymatic excision of chromatin loops and their oligomers from the chromosomes at matrix attachment ∗
Corresponding author. Tel.: +1-304-293-0594; fax: +1-304-293-8159. E-mail address: [email protected] (G.W. Konat).
regions (MARs). H2 O2 exerts the effect at concentrations featured during pathological conditions. For example, extensive HOCD is induced by a 15 min exposure of oligodendrocytes to 100 M H2 O2 , and such a concentration is generated and maintained for over an hour in the brain during ischemia/reperfusion (Hyslop et al., 1995). DNA strand fragmentation is the most disruptive form of DNA damage, and if unrepaired or improperly repaired, the excision of chromatin loops leads to the loss of genomic integrity, and consequently, to cell death. In fact, HOCD is an integral component of programmed cell death (PCD; Brown et al., 1993; Oberhammer et al., 1993; Sun et al., 1993; Cohen et al., 1994; Sun and Cohen, 1994; Zhivotovsky et al., 1994; Lagarkova et al., 1995; Beere et al., 1995; Walker and Sikorska, 1997). Even a partial HOCD induced by sublethal oxidative stress prevailing in chronic neurodegenerative conditions is a potent mutagenic event leading to the loss or truncation of genes. Moreover, the mis-religation of chromatin loops may dramatically change gene expression in surviving cells. Because free DNA ends can be rapidly trimmed by exonucleases before being religated, also a
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transient chromatin fragmentation is highly mutagenic. These mechanisms are consistent with the types of mutations previously found in cells exposed to H2 O2 (Turker et al., 1999). Studies of HOCD in cells undergoing PCD revealed that the DNA fragmentation results from the activation of an endonuclease located at MARs, proceeds through a single strand scission mechanism, and ultimately leads to bifilar fragmentation through the accumulation of single strand breaks (Walker et al., 1997). Our initial study (Mouzannar et al., 2001) showed that DNA scission during H2 O2 -induced HOCD is congruent with this mechanism. We also demonstrated that approximately half of oligodendrocyte chromatin undergoes single stranded digestion within 10 min of H2 O2 exposure, and that the rate of digestion is dependent on H2 O2 concentration in the range from 50 to 500 M. The DNA breaks are partly religated upon the removal of H2 O2 . The activation of HOCD-endonuclease by H2 O2 appears not to be mediated by proteolysis. Subsequent studies (Konat et al., 2001) revealed that HOCD is not directly mediated by H2 O2 -induced increase of intracellular calcium concentration. However, resting levels of intracellular calcium are required for the maintenance of HOCD-endonuclease in the active form. The endonuclease itself is calcium independent but magnesium dependent. The present study was undertaken to determine whether HOCD is specifically elicited by H2 O2 , or whether HOCD represents a general response of cells to oxidative stress. We compared the response of rat glioma C6 cells to equitoxic concentrations of H2 O2 , tert-butyrylhydroperoxide (tBH), hypochlorite and a nitric oxide generator, spermine NONOate. Subsequent chromatin analysis of oxidatively stressed cells showed that only the peroxides, and particularly H2 O2 induce rapid HOCD.
medium and grown overnight. Rat B104 neuroblastoma, human SH SY5Y neuroblastoma, human skin JB6 epithelial and human NGF7 breast cancer cells were cultured under the same conditions.
2. Materials and methods
3. Results
2.1. Materials
3.1. Kinetics of HOCD in C6 cells
DMEM/F-12 (1:1) medium, antibiotic–antimycotic mixture were from Gibco (Grand Island, NY). Fetal calf serum (FCS), H2 O2 , sodium hypochlorite and tBH were from Sigma (St Louis, MO). The nitric oxide generator, spermine NONOate was from Cayman (Ann Arbor, MI) and S1 nuclease was from Roche Diagnostic Co. (Indianapolis, IN).
In agreement with our previous study in rat oligodendrocytes (Mouzannar et al., 2001), the exposure of rat glioma C6 cells to H2 O2 induced a rapid and extensive chromatin digestion in a pattern consistent with HOCD (Fig. 1). Thus, the initial digestion generates fragments ≥400 kb that are likely to represent chromatin loop oligomers cut at AT-rich MARs (Bode et al., 1992). Subsequent digestion at MARs with a lower AT content (Dickinson et al., 1992) generates individual chromatin loops of approximately 50 kb. As shown in Fig. 1 untreated C6 cells contained an appreciable amount of single strand breaks that could be visualized as ≥400 kb ssDNA fragments (ssDNA gel). These fragments have been previously observed in oligodendrocytes (Mouzannar et al., 2001) and in other cells (Walker et al., 1997), and are likely to result from a dynamic equilibrium between scission and ligation at AT-rich MARs (Konat et al., 2001). A profound
2.2. Cell cultures Rat glioma C6 cells obtained from American Type Culture Collection (Manassas, VA; passage 42–45) were plated at a density of 2×106 cells in 30 mm petri dishes, and grown in DMEM/F-12 (1:1) medium supplemented with 1% of antibiotic–antimycotic mixture and 10% FCS at 37 ◦ C under 95% air/5% CO2 for 2–4 h to facilitate adhesion to the substratum. Subsequently, the cells were placed in serum-free
2.3. ROS exposure Cells were washed in Hank’s balanced salt solution (BSS) containing no phenol red. Three milliliters of BSS containing H2 O2 , ClO− , tBH or spermine NONOate were added, and the cells were incubated at 37 ◦ C for up to 30 min. Following ROS exposure, the cells were collected and the integrity of genomic DNA was analyzed (see below). In some cases the cells were allowed to recover in normal serum-free DMEM/F-12 for desired time. 2.4. Genomic DNA analysis Cells were embedded in agarose and the fragmentation of genomic DNA was analyzed by field inversion gel electrophoresis (FIGE) as described in Mouzannar et al. (2001). Nuclei were isolated as in Konat et al. (2001) and embedded in agarose as above. To detect fragments resulting from single stranded scission, the embedded DNA was digested with S1 nuclease prior to FIGE. Consequently, ssDNA gel bands represent a total of dsDNA and ssDNA fragments. Concatenated chromosome of lambda phage (Bio-Rad, Hercules, CA) was used as a DNA size marker. The DNA content in agarose plugs was determined spectrophotometrically (Mouzannar et al., 2001). Unless otherwise indicated all experiments were performed in quadruplicates, and the figures show representative gels. Because gels presented in the figures have been individually contrast adjusted, band intensities can only be compared within the same gel.
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Fig. 1. Time course of H2 O2 -induced higher order chromatin degradation in rat glioma C6 cells. The cells were exposed to 1 mM H2 O2 for various periods of time as indicated. The integrity of genomic DNA was analyzed by FIGE. The picture shows negatives of EtBr stained gels. The embedded DNA was subjected to FIGE before (dsDNA gel), or after (ssDNA gel) S1 digestion to detect and discriminate between double and single strand fragmentation, respectively.
increase in these fragments was observed 1 min after the exposure to H2 O2 . After 5 min of exposure, further digestion into 50 kb fragments was evident. By 10 min of exposure, practically the whole genomic DNA was digested as seen from the disappearance of DNA from the gel wells. By 30 min of exposure, the genomic DNA was fully digested into 50 kb fragments. Double stranded fragmentation resultant from the accumulation of single strand breaks (Walker et al., 1997) was first detectable 5 min after H2 O2 exposure (dsDNA gel). The amount of dsDNA ≥400 kb fragments increased up to 30 min of exposure. Chromatin fragmentation into 50 kb dsDNA was also evident. A discernible digestion into both ssDNA and dsDNA of ≥400 kb could be induced by 5 M H2 O2 (Fig. 2). Chromatin digestion increased with H2 O2 concentration up to 500 M, and plateaued thereafter. The rapid and extensive HOCD induced by H2 O2 validates C6 cells as a convenient model system to study this process. 3.2. H2 O2 -induced HOCD requires cytoplasmic signaling pathways
Fig. 3. The effect of H2 O2 on chromatin digestion in purified nuclei. Nuclei (106 ) isolated from C6 cells were suspended in 50 l of INC buffer (10 mM Tris–HCl, 15 mM NaCl, 60 mM KCl, 0.32 M sucrose, pH 7.5) and incubated for 60 min at 37 ◦ C in the presence of 1 mM H2 O2 and 5 mM MgCl2 , as indicated. The integrity of genomic DNA was analyzed by FIGE. The picture shows negatives of EtBr stained gels. The embedded DNA was subjected to FIGE before (dsDNA gel), or after (ssDNA gel) S1 digestion to detect and discriminate between double and single strand fragmentation, respectively.
The activation of HOCD-endonuclease can be mediated by direct oxidation of the enzyme by H2 O2 . Because
Fig. 2. Higher order chromatin degradation as a function of H2 O2 concentration. C6 cells were exposed for 30 min to various concentrations of H2 O2 as indicated. The integrity of genomic DNA was analyzed by FIGE. The picture shows negatives of EtBr stained gels. The embedded DNA was subjected to FIGE before (dsDNA gel), or after (ssDNA gel) S1 digestion to detect and discriminate between double and single strand fragmentation, respectively.
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H2 O2 also acts as a second messenger (Rhee, 1999), endonuclease activation can be achieved indirectly through H2 O2 -triggered signaling cascades. To resolve these mechanisms isolated nuclei were challenged with H2 O2 (Fig. 3). Contrary to intact cells (Figs. 1 and 2), the incubation of isolated nuclei with H2 O2 did not induce HOCD. As previously shown (Konat et al., 2001) HOCD-endonuclease can be activated by Mg2+ in isolated C6 cell nuclei. Also, there was no effect of H2 O2 on HOCD-endonuclease activity in the presence of Mg2+ . The inability of H2 O2 to activate HOCD-endonuclease in isolated nuclei, either in the presence or in the absence of Mg2+ , indicates that H2 O2 does not directly activate HOCD-endonuclease, but that the activation is rather mediated by cytoplasmic signaling cascades triggered by H2 O2 . 3.3. Induction of HOCD by other ROS To determine whether oxidants other than H2 O2 can elicit HOCD we exposed C6 cells to hypochlorite (ClO− ), tBH and nitric oxide generator, spermine NONOate. We had chosen ClO− , because the mechanisms of oxidation by ClO− are distinctly different from the mechanisms of oxidation by H2 O2 . ClO− oxidizes biomolecules through the generation of singlet oxygen, whereas H2 O2 acts through the generation of hydroxyl radicals. Because tBH is a peroxide, its mode of action should be similar to that of H2 O2 . Spermine NONOate spontaneously decays with a half-life of 39 min, and generates nitric oxide, which is a mild oxidant. A 30 min exposure to all the oxidants resulted in a delayed cell depletion that is indicative of PCD (Pittman et al., 1999). When assessed 24 h after the exposure, cell depletion was directly related to the concentration of the oxidants (Fig. 4). 1 mM H2 O2 , 1 mM ClO− , 2 mM tBH and 10 mM spermine NONOate were approximately equitoxic to C6 cells as seen from 40 to 60% cell death rate. The ability of the oxidants to induce rapid HOCD varied greatly as shown in Fig. 5. Thus, within 30 min of exposure,
Fig. 4. Toxicity of H2 O2 , hypochlorite (ClO− ), tert-butyrylhydroperoxide (tBH) and spermine NONOate (NO) to C6 cells. C6 cells were exposed to various concentrations of H2 O2 (filled circles), ClO− (open circles), tBH (filled triangles) or NO (open triangles) for 30 min. Subsequently, the cells were incubated in normal growth medium for 24 h. The cells were detached by trypsinization, and viable cells were counted in a hemocytometer. The results represent averages ± S.D. calculated from six independent experiments.
1 mM H2 O2 induced a total digestion of chromatin into 50 kb ssDNA fragments, and a profound digestion into ≥400 kb dsDNA fragments. Both single and double strand fragmentation decreased profoundly within the following 4 h of incubation in normal growth medium. Because no loss of DNA occurred in these cultures for up to 8 h (Fig. 6), the results prove the existence of efficient DNA relegation. However, at 8 h postexposure, the amount of ≥400 kb ssDNA and dsDNA fragments increased again. Also, 2 mM tBH induced HOCD within 30 min of exposure, but to a lesser extent than H2 O2 . Fig. 7 shows that even 10 mM tBH was by far less effective that 1 mM H2 O2 . On a par with cells exposed to H2 O2 , the
Fig. 5. Time course of higher order chromatin degradation induced by H2 O2 , tert-butyrylhydroperoxide (tBH), hypochlorite (ClO− ) and spermine NONOate (NO) in C6 cells. C6 cells were exposed for 30 min to 1 mM H2 O2 , 2 mM tBH, 1 mM ClO− or 10 mM NO, and then incubated in normal growth medium for various time periods, as indicated (Pex.). The integrity of genomic DNA was analyzed by FIGE. The picture shows negatives of EtBr stained gels. The embedded DNA was subjected to FIGE before (dsDNA gel), or after (ssDNA gel) S1 digestion to detect and discriminate between double and single strand fragmentation, respectively.
H. Bai, G.W. Konat / Neurochemistry International 42 (2003) 123–129
Fig. 6. DNA content of H2 O2 -treated C6 cell cultures. C6 cells were exposed to 1 mM H2 O2 for 30 min, and then allowed to recover in normal growth medium for various time periods, as indicated. Subsequently, the cells were embedded in agarose, and the DNA was quantitated spectrophotometrically (Mouzannar et al., 2001). The results represent averages±S.D. calculated from 4 to 6 independent experiments.
breaks were efficiently repaired within 4 h of incubation, and the fragmentation increased again at 8 h of incubation in normal growth medium. Contrary to these peroxides, 1 mM ClO− did not induce HOCD within 30 min of exposure, but rather decreased the amount of ≥400 kb ssDNA fragments present in untreated cell (Fig. 5). HOCD became evident only 8 h after the exposure. Spermine NONOate also failed to induce rapid HOCD. These results indicate that only peroxides have the ability to induce rapid HOCD. 3.4. HOCD in nonglial cells Subsequent experiments were performed to determine whether H2 O2 -induced HOCD is a special property of rat
Fig. 7. Higher order chromatin degradation as a function of tert-butyrylhydroperoxide (tBH) concentration. C6 cells were exposed for 30 min to either 1 mM H2 O2 , or to tBH at indicated concentrations. The integrity of genomic DNA was analyzed by FIGE. The picture shows negatives of EtBr stained gels. The embedded DNA was subjected to FIGE before (dsDNA gel), or after (ssDNA gel) S1 digestion to detect and discriminate between double and single strand fragmentation, respectively.
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Fig. 8. Higher order chromatin degradation induced by H2 O2 in various cells. The integrity of genomic DNA was analyzed in either untreated cells (H2 O2 , −) or in cells exposed to 1 mM H2 O2 (H2 O2 , +) for 30 min. The embedded DNA was digested with S1 nuclease, and single strand fragments were detected by FIGE. The picture shows negatives of EtBr stained gels. B104, rat B104 neuroblastoma cells; NGF7, human NGF7 breast cancer cells; JB6, human JB6 epithelial cells; SY5Y human SH SY5Y neuroblastoma cells.
glial cells, or is a general response of cells to H2 O2 . Rat B104 neuroblastoma, human SH SY5Y neuroblastoma, human skin JB6 epithelial and human NGF7 breast cancer cells were examined, and the results are presented in Fig. 8. HOCD was elicited to varying extend in all cells within 30 min exposure to 1 mM H2 O2 . The results demonstrate that H2 O2 -induced HOCD is operative in cells of different origin.
4. Discussion We have previously shown that H2 O2 induces a very rapid HOCD that involves approximately half of oligodendrocyte chromatin (Mouzannar et al., 2001). Glioma C6 cells used in this study confirmed the rapidity of HOCD. However, the extent of single strand digestion was greater than in oligodendrocytes encompassing practically the whole chromatin (Fig. 1). This difference may be related to the difference in proliferative status of these two cell types. While oligodendrocytes are postmitotic cells with firmly established hetero and euchromatin domains, C6 cells are actively proliferating cells and their chromatin may be more accessible to digestion. In addition, C6 cells are more responsive to H2 O2 than oligodendrocytes as conspicuous HOCD can be induced by 5 M H2 O2 in C6 cells (Fig. 2), whereas 50 M H2 O2 is required for oligodendrocytes (Mouzannar et al., 2001). Consequently, C6 cells provide a convenient model system to study H2 O2 -induced HOCD. The mechanism by which H2 O2 activates HOCD-endonuclease, and thus, induces HOCD, is not clear. However, the inability of ClO− and nitric oxide to induce rapid HOCD (Fig. 5) indicates that HOCD is not a result of oxidative stress per se. These results together with the induction of HOCD by tBH indicate that the process is specifically related to peroxides. A growing body of evidence indicates
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that H2 O2 , natural and relatively stable peroxide, is an intracellular signaling molecule (reviewed by Rhee, 1999). H2 O2 exerts its effect mainly through redox modification of proteins that alters their activity. The lack of H2 O2 effect on HOCD in isolated nuclei (Fig. 3) shows that the activation of the endonuclease is not related to its redox modification, but is rather mediated indirectly by upstream cytoplasmic event triggered by H2 O2 or tBH. Protein phosphorylation may provide the triggering signal as H2 O2 has been shown to activate protein kinases (Bae et al., 1997; Bhat and Zhang, 1999), and inactivate protein phosphatases (Lee et al., 1998; Robinson et al., 1999). We are currently testing the role of protein phosphorylation in H2 O2 -induced HOCD. PCD induced by various stimuli features both HOCD (Brown et al., 1993; Oberhammer et al., 1993; Sun et al., 1993; Cohen et al., 1994; Sun and Cohen, 1994; Zhivotovsky et al., 1994; Beere et al., 1995; Lagarkova et al., 1995; Walker and Sikorska, 1997) and oxidative stress (Quillet-Mary et al., 1995; Bredesen, 1995; Um et al., 1996; Kroemer et al., 1997). Oxidative stress is instigated by degenerating mitochondria through the generation of excessive amounts of superoxide radical that is rapidly converted into H2 O2 by omnipresent superoxide dismutase. The accumulation of H2 O2 may provide the signal that elicits HOCD in cells undergoing PCD. Such a signaling role of H2 O2 could explain the delayed HOCD in oxidatively stressed cells (Fig. 5). Accordingly, H2 O2 exposure induces rapid HOCD that is mostly reversible when H2 O2 is removed. However, the brief H2 O2 exposure leads to delayed mitochondrial dysfunction resulting in the generation of endogenous H2 O2 that induces the second wave of HOCD. Because ClO− is not an adequate stimulus to trigger HOCD, no rapid HOCD is observed within 30 min of exposure. However, oxidative stress instigated by this strong oxidant leads to delayed mitochondrial dysfunction, the generation of endogenous H2 O2 , and consequently, a substantial HOCD 8 h later. The induction of rapid HOCD by H2 O2 is not restricted to glial cells, i.e. oligodendrocytes and C6 cells, but can be elicited in cells of neuronal origin, i.e. neuroblastoma cells as well (Fig. 8). Furthermore, also human cells of neural and nonneural origin show a similar induction. Consequently, the rapid HOCD represents a general response of cells to H 2 O2 .
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Hydrogen peroxide mediates higher order chromatin degradation H. Bai, G.W. Konat∗ Department of Neurobiology and Anatomy, West Virginia University School of Medicine, 4052 HSN, P.O. Box 9128, Morgantown, WV 26506-9128, USA Received 17 October 2001; accepted 26 April 2002
Abstract Although a large body of evidence supports a causative link between oxidative stress and neurodegeneration, the mechanisms are still elusive. We have recently demonstrated that hydrogen peroxide (H2 O2 ), the major mediator of oxidative stress triggers higher order chromatin degradation (HOCD), i.e. excision of chromatin loops at the matrix attachment regions (MARs). The present study was designed to determine the specificity of H2 O2 in respect to HOCD induction. Rat glioma C6 cells were exposed to H2 O2 and other oxidants, and the fragmentation of genomic DNA was assessed by field inversion gel electrophoresis (FIGE). S1 digestion before FIGE was used to detect single strand fragmentation. The exposure of C6 cells to H2 O2 induced a rapid and extensive HOCD. Thus, within 30 min, total chromatin was single strandedly digested into 50 kb fragments. Evident HOCD was elicited by H2 O2 at concentrations as low as 5 M. HOCD was mostly reversible during 4–8 h following the removal of H2 O2 from the medium indicating an efficient relegation of the chromatin fragments. No HOCD was induced by H2 O2 in isolated nuclei indicating that HOCD-endonuclease is activated indirectly by cytoplasmic signal pathways triggered by H2 O2 . The exposure of cells to a synthetic peroxide, i.e. tert-butyrylhydroperoxide (tBH) also induced HOCD, but to a lesser extent than H2 O2 . Contrary to the peroxides, the exposure of cells to equitoxic concentration of hypochlorite and spermine NONOate, a nitric oxide generator, failed to induce rapid HOCD. These results indicate that rapid HOCD is not a result of oxidative stress per se, but is rather triggered by signaling cascades initiated specifically by H2 O2 . Furthermore, the rapid and extensive HOCD was observed in several rat and human cell lines challenged with H2 O2 , indicating that the process is not restricted to glial cells, but rather represents a general response of cells to H2 O2 . © 2003 Elsevier Science Ltd. All rights reserved. Keywords: C6 cells; Peroxides; Hypochlorite; Nitric oxide; Oxidative stress
1. Introduction Oxidative stress resulting from an excessive generation of reactive oxygen species (ROS) is the key component in the etiopathology of both acute and chronic neurodegenerative conditions. Because ROS readily oxidize the DNA molecule (Halliwell and Aruoma, 1991; Wink et al., 1991; Breen and Murphy, 1995; Henle and Linn, 1997), the effects of oxidative stress are likely mediated by the accumulation of somatic mutations that in postmitotic neural cells inevitably result in the loss of important cell function, and ultimately, cell degeneration and death. We have recently shown that ROS can damage genomic DNA indirectly (Mouzannar et al., 2001; Konat et al., 2001). Thus, hydrogen peroxide (H2 O2 ), the major mediator of oxidative stress, rapidly induces higher order chromatin degradation (HOCD), i.e. enzymatic excision of chromatin loops and their oligomers from the chromosomes at matrix attachment ∗
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regions (MARs). H2 O2 exerts the effect at concentrations featured during pathological conditions. For example, extensive HOCD is induced by a 15 min exposure of oligodendrocytes to 100 M H2 O2 , and such a concentration is generated and maintained for over an hour in the brain during ischemia/reperfusion (Hyslop et al., 1995). DNA strand fragmentation is the most disruptive form of DNA damage, and if unrepaired or improperly repaired, the excision of chromatin loops leads to the loss of genomic integrity, and consequently, to cell death. In fact, HOCD is an integral component of programmed cell death (PCD; Brown et al., 1993; Oberhammer et al., 1993; Sun et al., 1993; Cohen et al., 1994; Sun and Cohen, 1994; Zhivotovsky et al., 1994; Lagarkova et al., 1995; Beere et al., 1995; Walker and Sikorska, 1997). Even a partial HOCD induced by sublethal oxidative stress prevailing in chronic neurodegenerative conditions is a potent mutagenic event leading to the loss or truncation of genes. Moreover, the mis-religation of chromatin loops may dramatically change gene expression in surviving cells. Because free DNA ends can be rapidly trimmed by exonucleases before being religated, also a
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transient chromatin fragmentation is highly mutagenic. These mechanisms are consistent with the types of mutations previously found in cells exposed to H2 O2 (Turker et al., 1999). Studies of HOCD in cells undergoing PCD revealed that the DNA fragmentation results from the activation of an endonuclease located at MARs, proceeds through a single strand scission mechanism, and ultimately leads to bifilar fragmentation through the accumulation of single strand breaks (Walker et al., 1997). Our initial study (Mouzannar et al., 2001) showed that DNA scission during H2 O2 -induced HOCD is congruent with this mechanism. We also demonstrated that approximately half of oligodendrocyte chromatin undergoes single stranded digestion within 10 min of H2 O2 exposure, and that the rate of digestion is dependent on H2 O2 concentration in the range from 50 to 500 M. The DNA breaks are partly religated upon the removal of H2 O2 . The activation of HOCD-endonuclease by H2 O2 appears not to be mediated by proteolysis. Subsequent studies (Konat et al., 2001) revealed that HOCD is not directly mediated by H2 O2 -induced increase of intracellular calcium concentration. However, resting levels of intracellular calcium are required for the maintenance of HOCD-endonuclease in the active form. The endonuclease itself is calcium independent but magnesium dependent. The present study was undertaken to determine whether HOCD is specifically elicited by H2 O2 , or whether HOCD represents a general response of cells to oxidative stress. We compared the response of rat glioma C6 cells to equitoxic concentrations of H2 O2 , tert-butyrylhydroperoxide (tBH), hypochlorite and a nitric oxide generator, spermine NONOate. Subsequent chromatin analysis of oxidatively stressed cells showed that only the peroxides, and particularly H2 O2 induce rapid HOCD.
medium and grown overnight. Rat B104 neuroblastoma, human SH SY5Y neuroblastoma, human skin JB6 epithelial and human NGF7 breast cancer cells were cultured under the same conditions.
2. Materials and methods
3. Results
2.1. Materials
3.1. Kinetics of HOCD in C6 cells
DMEM/F-12 (1:1) medium, antibiotic–antimycotic mixture were from Gibco (Grand Island, NY). Fetal calf serum (FCS), H2 O2 , sodium hypochlorite and tBH were from Sigma (St Louis, MO). The nitric oxide generator, spermine NONOate was from Cayman (Ann Arbor, MI) and S1 nuclease was from Roche Diagnostic Co. (Indianapolis, IN).
In agreement with our previous study in rat oligodendrocytes (Mouzannar et al., 2001), the exposure of rat glioma C6 cells to H2 O2 induced a rapid and extensive chromatin digestion in a pattern consistent with HOCD (Fig. 1). Thus, the initial digestion generates fragments ≥400 kb that are likely to represent chromatin loop oligomers cut at AT-rich MARs (Bode et al., 1992). Subsequent digestion at MARs with a lower AT content (Dickinson et al., 1992) generates individual chromatin loops of approximately 50 kb. As shown in Fig. 1 untreated C6 cells contained an appreciable amount of single strand breaks that could be visualized as ≥400 kb ssDNA fragments (ssDNA gel). These fragments have been previously observed in oligodendrocytes (Mouzannar et al., 2001) and in other cells (Walker et al., 1997), and are likely to result from a dynamic equilibrium between scission and ligation at AT-rich MARs (Konat et al., 2001). A profound
2.2. Cell cultures Rat glioma C6 cells obtained from American Type Culture Collection (Manassas, VA; passage 42–45) were plated at a density of 2×106 cells in 30 mm petri dishes, and grown in DMEM/F-12 (1:1) medium supplemented with 1% of antibiotic–antimycotic mixture and 10% FCS at 37 ◦ C under 95% air/5% CO2 for 2–4 h to facilitate adhesion to the substratum. Subsequently, the cells were placed in serum-free
2.3. ROS exposure Cells were washed in Hank’s balanced salt solution (BSS) containing no phenol red. Three milliliters of BSS containing H2 O2 , ClO− , tBH or spermine NONOate were added, and the cells were incubated at 37 ◦ C for up to 30 min. Following ROS exposure, the cells were collected and the integrity of genomic DNA was analyzed (see below). In some cases the cells were allowed to recover in normal serum-free DMEM/F-12 for desired time. 2.4. Genomic DNA analysis Cells were embedded in agarose and the fragmentation of genomic DNA was analyzed by field inversion gel electrophoresis (FIGE) as described in Mouzannar et al. (2001). Nuclei were isolated as in Konat et al. (2001) and embedded in agarose as above. To detect fragments resulting from single stranded scission, the embedded DNA was digested with S1 nuclease prior to FIGE. Consequently, ssDNA gel bands represent a total of dsDNA and ssDNA fragments. Concatenated chromosome of lambda phage (Bio-Rad, Hercules, CA) was used as a DNA size marker. The DNA content in agarose plugs was determined spectrophotometrically (Mouzannar et al., 2001). Unless otherwise indicated all experiments were performed in quadruplicates, and the figures show representative gels. Because gels presented in the figures have been individually contrast adjusted, band intensities can only be compared within the same gel.
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Fig. 1. Time course of H2 O2 -induced higher order chromatin degradation in rat glioma C6 cells. The cells were exposed to 1 mM H2 O2 for various periods of time as indicated. The integrity of genomic DNA was analyzed by FIGE. The picture shows negatives of EtBr stained gels. The embedded DNA was subjected to FIGE before (dsDNA gel), or after (ssDNA gel) S1 digestion to detect and discriminate between double and single strand fragmentation, respectively.
increase in these fragments was observed 1 min after the exposure to H2 O2 . After 5 min of exposure, further digestion into 50 kb fragments was evident. By 10 min of exposure, practically the whole genomic DNA was digested as seen from the disappearance of DNA from the gel wells. By 30 min of exposure, the genomic DNA was fully digested into 50 kb fragments. Double stranded fragmentation resultant from the accumulation of single strand breaks (Walker et al., 1997) was first detectable 5 min after H2 O2 exposure (dsDNA gel). The amount of dsDNA ≥400 kb fragments increased up to 30 min of exposure. Chromatin fragmentation into 50 kb dsDNA was also evident. A discernible digestion into both ssDNA and dsDNA of ≥400 kb could be induced by 5 M H2 O2 (Fig. 2). Chromatin digestion increased with H2 O2 concentration up to 500 M, and plateaued thereafter. The rapid and extensive HOCD induced by H2 O2 validates C6 cells as a convenient model system to study this process. 3.2. H2 O2 -induced HOCD requires cytoplasmic signaling pathways
Fig. 3. The effect of H2 O2 on chromatin digestion in purified nuclei. Nuclei (106 ) isolated from C6 cells were suspended in 50 l of INC buffer (10 mM Tris–HCl, 15 mM NaCl, 60 mM KCl, 0.32 M sucrose, pH 7.5) and incubated for 60 min at 37 ◦ C in the presence of 1 mM H2 O2 and 5 mM MgCl2 , as indicated. The integrity of genomic DNA was analyzed by FIGE. The picture shows negatives of EtBr stained gels. The embedded DNA was subjected to FIGE before (dsDNA gel), or after (ssDNA gel) S1 digestion to detect and discriminate between double and single strand fragmentation, respectively.
The activation of HOCD-endonuclease can be mediated by direct oxidation of the enzyme by H2 O2 . Because
Fig. 2. Higher order chromatin degradation as a function of H2 O2 concentration. C6 cells were exposed for 30 min to various concentrations of H2 O2 as indicated. The integrity of genomic DNA was analyzed by FIGE. The picture shows negatives of EtBr stained gels. The embedded DNA was subjected to FIGE before (dsDNA gel), or after (ssDNA gel) S1 digestion to detect and discriminate between double and single strand fragmentation, respectively.
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H2 O2 also acts as a second messenger (Rhee, 1999), endonuclease activation can be achieved indirectly through H2 O2 -triggered signaling cascades. To resolve these mechanisms isolated nuclei were challenged with H2 O2 (Fig. 3). Contrary to intact cells (Figs. 1 and 2), the incubation of isolated nuclei with H2 O2 did not induce HOCD. As previously shown (Konat et al., 2001) HOCD-endonuclease can be activated by Mg2+ in isolated C6 cell nuclei. Also, there was no effect of H2 O2 on HOCD-endonuclease activity in the presence of Mg2+ . The inability of H2 O2 to activate HOCD-endonuclease in isolated nuclei, either in the presence or in the absence of Mg2+ , indicates that H2 O2 does not directly activate HOCD-endonuclease, but that the activation is rather mediated by cytoplasmic signaling cascades triggered by H2 O2 . 3.3. Induction of HOCD by other ROS To determine whether oxidants other than H2 O2 can elicit HOCD we exposed C6 cells to hypochlorite (ClO− ), tBH and nitric oxide generator, spermine NONOate. We had chosen ClO− , because the mechanisms of oxidation by ClO− are distinctly different from the mechanisms of oxidation by H2 O2 . ClO− oxidizes biomolecules through the generation of singlet oxygen, whereas H2 O2 acts through the generation of hydroxyl radicals. Because tBH is a peroxide, its mode of action should be similar to that of H2 O2 . Spermine NONOate spontaneously decays with a half-life of 39 min, and generates nitric oxide, which is a mild oxidant. A 30 min exposure to all the oxidants resulted in a delayed cell depletion that is indicative of PCD (Pittman et al., 1999). When assessed 24 h after the exposure, cell depletion was directly related to the concentration of the oxidants (Fig. 4). 1 mM H2 O2 , 1 mM ClO− , 2 mM tBH and 10 mM spermine NONOate were approximately equitoxic to C6 cells as seen from 40 to 60% cell death rate. The ability of the oxidants to induce rapid HOCD varied greatly as shown in Fig. 5. Thus, within 30 min of exposure,
Fig. 4. Toxicity of H2 O2 , hypochlorite (ClO− ), tert-butyrylhydroperoxide (tBH) and spermine NONOate (NO) to C6 cells. C6 cells were exposed to various concentrations of H2 O2 (filled circles), ClO− (open circles), tBH (filled triangles) or NO (open triangles) for 30 min. Subsequently, the cells were incubated in normal growth medium for 24 h. The cells were detached by trypsinization, and viable cells were counted in a hemocytometer. The results represent averages ± S.D. calculated from six independent experiments.
1 mM H2 O2 induced a total digestion of chromatin into 50 kb ssDNA fragments, and a profound digestion into ≥400 kb dsDNA fragments. Both single and double strand fragmentation decreased profoundly within the following 4 h of incubation in normal growth medium. Because no loss of DNA occurred in these cultures for up to 8 h (Fig. 6), the results prove the existence of efficient DNA relegation. However, at 8 h postexposure, the amount of ≥400 kb ssDNA and dsDNA fragments increased again. Also, 2 mM tBH induced HOCD within 30 min of exposure, but to a lesser extent than H2 O2 . Fig. 7 shows that even 10 mM tBH was by far less effective that 1 mM H2 O2 . On a par with cells exposed to H2 O2 , the
Fig. 5. Time course of higher order chromatin degradation induced by H2 O2 , tert-butyrylhydroperoxide (tBH), hypochlorite (ClO− ) and spermine NONOate (NO) in C6 cells. C6 cells were exposed for 30 min to 1 mM H2 O2 , 2 mM tBH, 1 mM ClO− or 10 mM NO, and then incubated in normal growth medium for various time periods, as indicated (Pex.). The integrity of genomic DNA was analyzed by FIGE. The picture shows negatives of EtBr stained gels. The embedded DNA was subjected to FIGE before (dsDNA gel), or after (ssDNA gel) S1 digestion to detect and discriminate between double and single strand fragmentation, respectively.
H. Bai, G.W. Konat / Neurochemistry International 42 (2003) 123–129
Fig. 6. DNA content of H2 O2 -treated C6 cell cultures. C6 cells were exposed to 1 mM H2 O2 for 30 min, and then allowed to recover in normal growth medium for various time periods, as indicated. Subsequently, the cells were embedded in agarose, and the DNA was quantitated spectrophotometrically (Mouzannar et al., 2001). The results represent averages±S.D. calculated from 4 to 6 independent experiments.
breaks were efficiently repaired within 4 h of incubation, and the fragmentation increased again at 8 h of incubation in normal growth medium. Contrary to these peroxides, 1 mM ClO− did not induce HOCD within 30 min of exposure, but rather decreased the amount of ≥400 kb ssDNA fragments present in untreated cell (Fig. 5). HOCD became evident only 8 h after the exposure. Spermine NONOate also failed to induce rapid HOCD. These results indicate that only peroxides have the ability to induce rapid HOCD. 3.4. HOCD in nonglial cells Subsequent experiments were performed to determine whether H2 O2 -induced HOCD is a special property of rat
Fig. 7. Higher order chromatin degradation as a function of tert-butyrylhydroperoxide (tBH) concentration. C6 cells were exposed for 30 min to either 1 mM H2 O2 , or to tBH at indicated concentrations. The integrity of genomic DNA was analyzed by FIGE. The picture shows negatives of EtBr stained gels. The embedded DNA was subjected to FIGE before (dsDNA gel), or after (ssDNA gel) S1 digestion to detect and discriminate between double and single strand fragmentation, respectively.
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Fig. 8. Higher order chromatin degradation induced by H2 O2 in various cells. The integrity of genomic DNA was analyzed in either untreated cells (H2 O2 , −) or in cells exposed to 1 mM H2 O2 (H2 O2 , +) for 30 min. The embedded DNA was digested with S1 nuclease, and single strand fragments were detected by FIGE. The picture shows negatives of EtBr stained gels. B104, rat B104 neuroblastoma cells; NGF7, human NGF7 breast cancer cells; JB6, human JB6 epithelial cells; SY5Y human SH SY5Y neuroblastoma cells.
glial cells, or is a general response of cells to H2 O2 . Rat B104 neuroblastoma, human SH SY5Y neuroblastoma, human skin JB6 epithelial and human NGF7 breast cancer cells were examined, and the results are presented in Fig. 8. HOCD was elicited to varying extend in all cells within 30 min exposure to 1 mM H2 O2 . The results demonstrate that H2 O2 -induced HOCD is operative in cells of different origin.
4. Discussion We have previously shown that H2 O2 induces a very rapid HOCD that involves approximately half of oligodendrocyte chromatin (Mouzannar et al., 2001). Glioma C6 cells used in this study confirmed the rapidity of HOCD. However, the extent of single strand digestion was greater than in oligodendrocytes encompassing practically the whole chromatin (Fig. 1). This difference may be related to the difference in proliferative status of these two cell types. While oligodendrocytes are postmitotic cells with firmly established hetero and euchromatin domains, C6 cells are actively proliferating cells and their chromatin may be more accessible to digestion. In addition, C6 cells are more responsive to H2 O2 than oligodendrocytes as conspicuous HOCD can be induced by 5 M H2 O2 in C6 cells (Fig. 2), whereas 50 M H2 O2 is required for oligodendrocytes (Mouzannar et al., 2001). Consequently, C6 cells provide a convenient model system to study H2 O2 -induced HOCD. The mechanism by which H2 O2 activates HOCD-endonuclease, and thus, induces HOCD, is not clear. However, the inability of ClO− and nitric oxide to induce rapid HOCD (Fig. 5) indicates that HOCD is not a result of oxidative stress per se. These results together with the induction of HOCD by tBH indicate that the process is specifically related to peroxides. A growing body of evidence indicates
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that H2 O2 , natural and relatively stable peroxide, is an intracellular signaling molecule (reviewed by Rhee, 1999). H2 O2 exerts its effect mainly through redox modification of proteins that alters their activity. The lack of H2 O2 effect on HOCD in isolated nuclei (Fig. 3) shows that the activation of the endonuclease is not related to its redox modification, but is rather mediated indirectly by upstream cytoplasmic event triggered by H2 O2 or tBH. Protein phosphorylation may provide the triggering signal as H2 O2 has been shown to activate protein kinases (Bae et al., 1997; Bhat and Zhang, 1999), and inactivate protein phosphatases (Lee et al., 1998; Robinson et al., 1999). We are currently testing the role of protein phosphorylation in H2 O2 -induced HOCD. PCD induced by various stimuli features both HOCD (Brown et al., 1993; Oberhammer et al., 1993; Sun et al., 1993; Cohen et al., 1994; Sun and Cohen, 1994; Zhivotovsky et al., 1994; Beere et al., 1995; Lagarkova et al., 1995; Walker and Sikorska, 1997) and oxidative stress (Quillet-Mary et al., 1995; Bredesen, 1995; Um et al., 1996; Kroemer et al., 1997). Oxidative stress is instigated by degenerating mitochondria through the generation of excessive amounts of superoxide radical that is rapidly converted into H2 O2 by omnipresent superoxide dismutase. The accumulation of H2 O2 may provide the signal that elicits HOCD in cells undergoing PCD. Such a signaling role of H2 O2 could explain the delayed HOCD in oxidatively stressed cells (Fig. 5). Accordingly, H2 O2 exposure induces rapid HOCD that is mostly reversible when H2 O2 is removed. However, the brief H2 O2 exposure leads to delayed mitochondrial dysfunction resulting in the generation of endogenous H2 O2 that induces the second wave of HOCD. Because ClO− is not an adequate stimulus to trigger HOCD, no rapid HOCD is observed within 30 min of exposure. However, oxidative stress instigated by this strong oxidant leads to delayed mitochondrial dysfunction, the generation of endogenous H2 O2 , and consequently, a substantial HOCD 8 h later. The induction of rapid HOCD by H2 O2 is not restricted to glial cells, i.e. oligodendrocytes and C6 cells, but can be elicited in cells of neuronal origin, i.e. neuroblastoma cells as well (Fig. 8). Furthermore, also human cells of neural and nonneural origin show a similar induction. Consequently, the rapid HOCD represents a general response of cells to H 2 O2 .
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