Ros-Induced Histone Modifications and their Role in Cell Survival and Cell Death*

Drug Metabolism Reviews, 38: 755–767, 2006Copyright © Informa HealthcareISSN: 0360-2532 print / 1097-9883 onlineDOI: 10.1080/03602530600959649

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ROS-INDUCED HISTONE MODIFICATIONS AND THEIR ROLE IN CELL SURVIVAL AND CELL DEATH

Terrence J. Monks, Ruiyu Xie, Kulbhushan Tikoo, and Serrine S. LauDepartment of Pharmacology and Toxicology, College of Pharmacy, University ofArizona Health Sciences Center, Tucson, Arizona; Laboratory of Chromatin Biol-ogy, Department of Pharmacology and Toxicology, National Institute ofPharmaceutical Education and Research, Sec. 67, Mohali 160062, Punjab, India

Much is known about the distal DNA damage repair response. In particular, many of theenzymes and auxiliary proteins that participate in DNA repair have been characterized.In addition, knowledge of signaling pathways activated in response to DNA damage isincreasing. In contrast, comparatively less is known of DNA damage-sensing moleculesor of the specific alterations to chromatin structure recognized by such DNA damagesensors. Thus, precisely how chromatin structure is altered in response to DNA damageand how such alterations regulate DNA repair processes remain important unansweredquestions. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidlyafter double-strand break formation, extends over megabase chromatin domains, and isrequired for stable accumulation of repair proteins at damage foci. We have shown thatreactive oxygen species (ROS)-induced DNA single-strand breaks induce the incorpora-tion of 32P specifically into histone H3. ADP-Ribosylation of histones may stimulate localchromatin relaxation to facilitate the repair process, and, indeed, histone ribosylationpreceded DNA damage-induced histone H3 phosphorylation. However, H3 phosphoryla-tion occurred concomitant with overall chromatin condensation, as revealed by decreasedsensitivity of chromatin to digestion by micrococcal nuclease and by DAPI staining ofnuclei. Inhibitors of the ERK and p38MAPK pathways and inhibition of poly(ADP-ribose) polymerase all reduced ROS-induced H3 phosphorylation, chromatin condensa-tion, and cell death. Precisely how changes in the post-translational modification ofhistone H3 regulate the survival response remains unclear. Attempts to determine theprecise site of histone H3 phosphorylation, putative histone H3 kinases, and histone H3interacting proteins are underway.

Key Words: Cell death; Chromatin; DNA damage; Histones; Mitotic catastrophe; Oncoticcell death; Post-translational modification; Premature chromatin condensation; Reactiveoxygen species; Stress response signaling.

Presented at the Seventh International Symposium on Biological Reactive Intermediates, Tucson,Arizona, January 4–7, 2006.

Address correspondence to Terrence J. Monks, Ph.D., Department of Pharmacology and Toxicology,College of Pharmacy, 1703 E Mabel, P.O. Box 210207, Tucson, AZ 85721-0207, USA; Fax: 520-626-6944;E-mail: [email protected]

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756 T. J. MONKS ET AL.

INTRODUCTION

Molecular/Cellular Stress Responses to ROS-Induced DNA Damage

Although the cellular response to chemical-induced stress is relatively well charac-terized, particularly the response to DNA damage, factors that govern the outcome of thestress response (cell survival or cell death) are less clearly defined. In a model of reactiveoxygen species- (ROS) induced cytotoxicity, treatment of renal proximal tubular epithelial(LLC-PK1) cells with the ROS-generating nephrotoxicant and nephrocarcinogen2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ) results in cell death. Despite the factthat LLC-PK1 cells are capable of engaging the machinery necessary to induce apoptoticcell death, TGHQ-treated LLC-PK1 cells die via oncotic cell death (Jia et al., 2004). Afrequent response to ROS-induced cell stress that ultimately leads to oncotic cell death isthe premature engagement of chromosome condensation (PCC) and the ensuing mitoticcatastrophe. During the transition from the G2 phase into mitosis, relaxed interphase chro-matin must be converted into mitotic condensed chromatin, a process considered essentialfor nuclear division. Therefore, a critical event during the cell cycle is the timing of theinitiation of DNA replication (S-phase entry). Rigid controls function to prevent repeatedrounds of DNA replication without intervening mitoses or the initiation of mitosis beforeDNA replication is complete (“mitotic catastrophe”). Although some of the genetic inter-actions that participate in this process have recently been identified in yeast (Novak andTyson, 1997), little is known about their mammalian counterparts. Indeed, until recentlyrelatively little was known about the mechanisms and factors that regulate this transitionin chromatin structure. Because a variety of phosphatase inhibitors induce PCC (Coco-Martin and Begg, 1997), protein phosphorylation likely plays an important role in thisprocess. However, the targets for phosphorylation and the corresponding protein kinasesare poorly defined. Moreover, the signal transduction pathways activated during the“commitment” phase of oncotic cell death are insufficiently characterized.

ROS-mediated MAPK activation and cell death. Oxidative stress is knownto activate mitogen-activated protein kinases (MAPKs) (Cobb, 1999). The MAPK familyis comprised of three major subgroups: extracellular signal-regulated protein kinase(ERK), c-Jun N-terminal kinases/stress-activated protein kinase (JNK/SAPK), and p38MAPK (Cobb, 1999). ERKs behave mainly as mitogen-activated proliferation/differentia-tion factors, whereas JNK/SAPK and p38 MAPK are mainly stress-activated proteinsrelated to apoptotic cell death. The MAPKs are all rapidly activated by TGHQ in LLC-PK1 cells (Ramachandiran et al., 2002). Although in most situations the MAPK signalingpathway is associated with cell survival, the activation of MAPKs, especially the ERKsubfamily, appears to play a causal role in ROS-induced cell death of renal proximal tubularepithelial cells (Ramachandiran et al., 2002). Thus, inhibition of ERK1/2 activation withPD98059 or inhibition of p38 MAPK activation with SB202190 attenuates cell deathinduced by TGHQ (Ramachandiran et al., 2002). In contrast, the JNK inhibitor SP600125has no effect on TGHQ-induced cell death (Ramachandiran et al., 2002). ERK1/2 activa-tion may therefore contribute to both cell proliferation or cell death, dependent upon celltype and the specific context. In a few cases, activated ERK1/2 seems to behave as a celldeath-inducing factor. Thus, ERK activation is related to vanadate-induced oncotic celldeath of vascular smooth muscle cells (Daum et al., 1998), in H2O2-induced cell death ofoligodendrocytes (Bhatt and Zhang, 1999), in T cells (van den Brink et al., 1999), and inpleural mesothelial cells (Jiminez et al., 1997). Precisely how ERK activation is coupledto cell death in each of these models remains to be elucidated.

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ROS-INDUCED HISTONE MODIFICATIONS 757

ROS and chromatin. Although the histone proteins play a vital role in maintain-ing chromatin structure, they also participate in the dynamics of chromatin remodelingduring both gene activation and gene silencing. This is achieved by a variety of post-transla-tional modifications, including phosphorylation, acetylation, ubiquitination, methylation,and ADP ribosylation, varying combinations of which influence the interaction of histoneswith DNA within the nucleosome, resulting in changes in chromatin structure and function(Wolfe and Hayes, 1999; Cheung et al., 2000). In particular, phosphorylation of histones H1and H3 on Ser-10 and 28 within its basic amino terminal tail (Mahadevan et al., 1991; Sauveet al., 1999; Wei et al., 1999; Goto et al., 1999) has long been implicated in chromosomecondensation during mitosis (Koshland and Strunikov, 1996) and in response to variousmitogenic stimuli, such as growth factors or phorbol esters. These sites of phosphorylation inhistone H3 are highly conserved and are flanked by basic amino acid residues, which arealso susceptible to multiple post-translational modifications. Similarly, in histone H2B, twohighly conserved phosphorylation sites are located at serines 14 and 32. Interestingly, thesehighly conserved serine residues in histones H3 and H2B are both 17 amino acids apart andlocated within the N-terminal histone tail domain (Cheung et al., 2000).

Increases in histone H1 kinase activity during heat shock occur coincidentally withPCC and are associated with M-phase kinase complexes containing cyclin B1 (Mackey et al.,1996). Early studies demonstrated that increases in H1 phosphorylation occurred during mito-sis in a variety of eukaryotes (Roth and Allis, 1992). However, although H1 hyperphosphory-lation is temporally associated with entry into mitosis and requires Cdc2 kinase activity(Gurley et al., 1978; Davis et al., 1983; Langan et al., 1989), subsequent studies revealed thatchromatin condensation can occur in the absence of this modification (Guo et al., 1995) andeven without H1 itself (Shen et al., 1995). In contrast to the data on H1 phosphorylation,experimental evidence strongly implicates a functional role for H3 phosphorylation in chro-mosome condensation (Th'ng et al., 1994; Sauve et al., 1999), findings that appear to contra-dict the role of H3 phosphorylation in transcription-dependent chromatin decondensation.This apparent paradox has been addressed by Strahl and Allis (2000) and attributed to the“histone code” hypothesis, which states that multiple histone modifications act in concert tospecify a distinct functional response. Thus, multiple histone H3 phosphorylations, on resi-dues Ser10 and 28 (Hendzel et al., 1997; Van Hooser et al., 1998; Chadee et al., 1999; Weiet al., 1999; Goto et al., 1999) in the presence of other modifications on the same or multiplehistone tails, may be required to produce competent chromosome condensation during mitosis.DNA damage also results in the specific post-translational modification of histones. In partic-ular, phosphorylation of the histone variant H2A.X occurs rapidly after DNA double-strandbreak formation, extends over megabase chromatin domains, and is required for the efficientand stable recruitment of repair proteins to sites of DNA damage (Thiriet and Hayes, 2005).

MAPK signaling and ROS-induced cell death. A novel role for the MAPKpathway in progression from G2 into mitosis has been demonstrated (Wright et al., 1999).Thus, when MAPK activation was inhibited with PD98059, which selectively inhibitsMEK, an upstream regulator of ERKs, in S-phase synchronized NIH 3T3 cells, the cellsarrested in G2. Expression of a dominant-negative form of MAPKK1 was also found todelay the progression of cells through G2 (Wright et al., 1999). MAPKK activity wasrequired for the timely activation of Cdc2 and progression into mitosis. These findingsprovide a potential mechanistic explanation for our own findings that TGHQ-induced histoneH3 phosphorylation and chromatin condensation are inhibited by PD98059. More importantly,PD98059 protected against TGHQ-induced oncotic cell death, and cytoprotection corre-lated with decreases in H3 phosphorylation (Tikoo et al., 2001). Thus, ROS-dependent

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ERK activation may be coupled to LLC-PK1 cell death via changes in chromatin structure,mediated by increases in the phosphorylation of histone H3, a post-translational modifica-tion required for both chromosome condensation and segregation during mitosis, and PCCleading to cell death. Indeed, TGHQ-induced phosphorylation of histone H3 was accom-panied by increases in chromatin condensation, observed by DAPI-fluorescent staining, andby increases in the sensitivity of chromatin to digestion by micrococcal nuclease (Tikoo et al.,2001). Moreover, the changes in chromatin structure preceded cell death. Significantly,the biochemical and immunohistochemical findings in LLC-PK1 cells were consistentwith changes in chromatin structure that occur in vivo in renal proximal tubular epithelialcell nuclei following exposure of rats to a structurally related quinol-thioether (Rivera et al.,1994). Interestingly, more than 20 years ago, temperature sensitive (ts) mutants of babyhamster kidney cells (tsBN2 cells) (Kai et al., 1983) sustained histone H1 and H3 phos-phorylation at temperatures that also induced PCC (Ajiro et al., 1983). However, preventionof PCC only occurred concomitant with decreases in H3 phosphorylation, and not with H1phosphorylation (Ajiro et al., 1985). Our data therefore suggest that LLC-PK1 cells respondto TGHQ-induced oxidative stress by the aberrant stimulation chromosome condensationin the absence of the signals and machinery necessary to coordinate mitosis. Whether abrogationof histone H3 phosphorylation is required for protection against ROS-induced oncotic celldeath in LLC-PK1 cells is not known. However, mitotic histone H3 phosphorylation promotesthe disassociation of the histone H3 amino terminal tail from DNA (Sauve et al., 1999).This change in chromatin structure permits the association of additional factors withDNA. ROS-induced phosphorylation of histone H3 in LLC-PK1 cells might thus result inthe exposure of DNA to chromosome condensing factors, facilitating chromatin condensation.

ROS-Induced Poly(ADP-ribose) Polymerase Activation. The generation ofROS has been implicated in the pathogenesis of renal ischemia/reperfusion injury andmany other pathological conditions. DNA strand breaks caused by ROS lead to the activa-tion of poly(ADP-ribose)polymerase (PARP), the excessive activation of which results inthe depletion of both NAD+ and ATP (Pieper et al., 1999). It has been suggested thatdepletions in NAD+ and ATP in response to DNA damage contribute to cell death as aconsequence of deficits in energy stores. For example, Chatterjee et al. (1999) showed thatincubation of primary cultures of rat proximal tubule epithelial cells with 1 mM H2O2inhibited mitochondrial respiration and increased LDH release, with concomitant increasesin PARP activity. Moreover, inhibitors of PARP protected against H2O2-mediated celldeath (Cristovao and Rueff, 1996). Deletion of PARP also protects against NMDA-receptor-activated neurotoxicity (Eliasson et al., 1997; Endres et al., 1997), myocardialischemia (Zingarelli et al., 1998), inflammation elicited by a variety of mediators(Zingarelli et al., 1999; Szabo et al., 1997; Oliver et al., 1999), and streptozocin-induceddiabetes (Matsutani et al., 1999; Burkart et al., 1999; Pieper et al., 1999). In all these mod-els of cell death, the experimental evidence indicates that cell death occurs by oncosis(Kerr et al., 1972; Wylie et al., 1980; Ankacrona et al., 1995; Nicotera et al., 1997).

PARP inhibitors not only block oncotic cell death (Ha and Snyder, 1999; Filipovicet al., 1999), but also appear to shift the mode of cell death from oncosis to apoptosis in oxi-dant-stressed endothelial cells (Walisser and Theis, 1999). In addition, using fibroblastsobtained from mice with a targeted deletion of PARP (PARP−/−) DNA damage induced byeither MNNG or H2O2 failed to deplete intracellular concentrations of ATP, and the cellswere protected against oncotic cell death (Ha and Snyder, 1999) despite exhibiting exten-sive DNA damage. However, the PARP−/− cells still underwent apoptotic cell death. In con-trast, PARP+/+ cells treated with either MNNG or H2O2 died by oncosis, suggesting that

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PARP activation may regulate the mode of cell death, perhaps by modulating ATP (andNAD+ ?) concentrations. Because inhibition of PARP activity or PARP gene deletion canprevent both ATP depletion and the induction of oncosis, it has been suggested that PARPoveractivation-induced oncosis is an “active” rather than a passive process (Ha and Snyder,1999). This interpretation raises the question of whether the machinery exists with whichthe cell can switch the mode of cell death. The answer to this question has profound clinicalimplications. For example, in many clinical situations, such as inflammation, vascularstroke, and myocardial infarction, the predominant mechanism of cell death appears to beoncotic. By extension, it has been predicted that PARP inhibitors may have therapeuticbenefit (Ha and Snyder, 1999). Consistent with this hypothesis, PARP inhibition or genedeletion attenuates tissue injury associated with stroke, myocardial infarct, and diabeticpancreatic damage (Eliasson et al., 1997; Endres et al., 1997; Zingarelli et al., 1998).

ROS-induced histone modifications: 1) histone ribsoylation facilitates

histone H3 phosphorylation. As previously noted, current dogma suggests thatdepletions in NAD and ATP in response to DNA damage contribute to cell death as a con-sequence of deficits in energy stores. However, although H2O2 depletes ATP, causesDNA damage, lipid peroxidation, and oncotic cell death in LLC-PK1 cells, inhibiting lipidperoxidation with lazeroids or Trolox prevented oncotic cell death without affecting DNAdamage or depletion in ATP (Andreoli et al., 1997). Thus, DNA damage-induced deple-tions in cellular ATP concentrations can be dissociated from oncotic cell death.

Although the biological functions of PARP are unclear, post-translational modificationof several nuclear proteins by PARP has been implicated in chromatin structure and function,in surveillance of the genome, and in the regulation of proteins that participate in DNA repair(D’Amours et al., 1999). However, under conditions where PARP is either inhibited pharma-cologically or deleted genetically, the potential consequences on PARP targets and their corre-sponding influence on cell survival have not been considered. Histones are also substrates forADP ribosylation, although the significance of this particular modification is the one leastunderstood. Poly(ADP-ribosylation) participates in histone shuttling and nucleosomal unfold-ing, facilitating DNA excision from chromatin (Realini and Althaus, 1992). Histone H1 atglutamate 2 and 116 can undergo poly ADP-ribosylation (van Holde, 1989). ADP ribosylationis relatively rare in unperturbed cells. However, when DNA is damaged, the almost immediateribosylation of histones is observed (Adamietz and Rudolph, 1984), and immunoaffinity-purified ADP-ribosylated oligonucleosomes contain many DNA nicks (Malik et al., 1983).Therefore poly ADP-ribosylation of histones may provide local relaxation to facilitate therepair process. The end result of PARP inhibition will therefore depend upon the relativeeffects of inhibiting DNA repair at the expense of conserving energy supplies (ATP). As pre-viously noted, however, DNA damage-induced depletions in cellular ATP concentrations canbe dissociated from oncotic cell death. Decreased PARP activity might therefore be cytopro-tective against oncotic cell death by interfering with its ability to regulate chromatin structure.

Because PARP participates in histone shuttling and nucleosomal unfolding (Realiniand Althaus, 1992) it may also facilitate additional post-translational modification on thesubsequently exposed proteins. Consistent with this view, TGHQ-induced rapid (<5 min)histone ribsoylation on histone H3, H4, H1, and H2B (Fig.1), and this histone ribosylationnot only precedes histone H3 phosphorylation (Fig.2), but also appeared to be a prerequi-site for histone H3 phosphorylation. Thus, inhibition of PARP with 3-aminobenzamide(3AB) at concentrations that produce few other effects (D'Amours et al., 1999) diminishedhistone H3 ribosylation (Fig. 1B, compare lane e [w/o 3AB] with lane g [w 3AB]) andprevented H3 phosphorylation (Fig. 2B, compare lane e [w/o 3AB] with lane g [w 3AB]),

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suggesting that ADP-ribosylation and histone H3 phosphorylation are coupled in thismodel of ROS-induced DNA damage and cell death. The prevention of both these post-translational modifications was accompanied by an increase in cell survival (Tikoo et al.,2001). The coupling of histone phosphorylation to ribosylation has not been previouslydemonstrated and suggests that PARP-mediated ADP-ribosylation of histones facilitateshistone H3 phosphorylation and that these post-translational modifications contribute toPCC and mitotic catastrophe. There is precedence for the coupling of various histone post-translational modifications. For example, Imai et al. (2000) described a NAD-dependenthistone deacetylase, Sir2, and Sir 2 proteins exhibit NAD-dependent mono-ADP-ribosyl-transferase activity (Frye, 1999). The coordination of multiple histone modificationsappears to be involved in the regulation of immediate early gene expression (Clayton et al.,2000). In particular, the coupling of histone H3 phosphorylation and acetylation appears toplay an important role in transcriptional regulation, particularly in response to factors thatengage the epidermal growth factor/MAPK signaling pathway (Clayton et al., 2000).

Figure 1 Changes in histone ribosylation precede cell death in TGHQ-treated LLC-PK1 cells. LLC-PK1 cells werelabeled with 100 μCi/ml [2,8- 3H] adenosine for 4 h and then treated with 400 μM TGHQ for increasing periods oftime. Histones were extracted from these cells, and 55 μg protein were electrophoretically resolved on a 13.5% SDS-polyacrylamide gel. Gel transferred to a PVDF membrane and stained with Ponceau S; Panel A. Panel B shows thecorresponding autoradiograph. Lane a, untreated control cells; lane b, 5 min; lane c, 10 min; lane d, 20 min; lane e,30 min; lane f, 30 min, control untreated cells; lane g, pretreated with 1mM 3-aminobenzamide for 15 min and thenco-treated with 400 μM TGHQ for 30 min; and lane h, only 3-aminobenzamide-treated cells for 30 min.

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ROS-induced histone modifications: 2) ROS-induced histone H3

phosphorylation is preferentially associated with hyperacetylated

histones. The short stretches of basic amino acids that tend to flank phosphoryla-tion sites within the histone tails (see previous discussion) can undergo additional post-translational modifications, including acetylation and methylation. Acetylation sites in thehistone N terminal domain of H3 and H4 are lysine 9, 14, 18, 23, and 5, 8, 12, 16, respec-tively. The dynamics of histone acetylation on transcriptionally active chromatin ismodulated by competing activities of various histone acetyltransferases and histonedeacetylases, which behave as transcriptional activators and repressors, respectively. Toelucidate the relationship between ROS-induced histone H3 phosphorylation and histoneacetylation, quiescent LLC-PK1 cells were exposed to 5mM sodium butyrate for 12h and32P-labeled for the final 4h. Treatment of butyrate-exposed LLC-PK1 cells with TGHQ(Fig. 3, lanes c and d) for 30 min induced histone H3 phosphorylation. However, in butyrate-treated cells, phosphorylation of histone H3 occurred preferentially on hyperacetylatedhistones (Fig. 3, lane d). Furthermore, the Triton-acid urea gel revealed the ability ofTGHQ to induce the phosphorylation of constitutively hyperacetylated histone H4 (Fig, 3B,

Figure 2 Time course of TGHQ-induced histone H3 phosphorylation in LLC-PK1 cells that precedes celldeath. [32P]-Labeled LLC-PK1 cells were treated with 400 μM TGHQ for increasing periods of time. Histoneswere extracted from these cells, and 35 μg protein were electrophoretically resolved on a 13.5% SDS-polyacrylamide.Lane a, untreated control cells; lane b, 5 min; lane c, 10 min; lane d, 20 min; lane e, 30 min; lane f, 30 min,control untreated cells; lane g, pretreated with 1mM 3-aminobenzamide for 15 min and then co-treated with 400 μMTGHQ for 30 min; and lane h, only 3-aminobenzamide-treated cells for 30 min. Panel A shows the CoomassieBlue-stained gel. Panel B shows the corresponding autoradiograph.

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lane c) despite the fact that no hyperacetylated H4 protein is visible on the CoomassieBlue-stained gel (Fig. 3A, lane c). The results suggests that only a very small populationof acetylated histones are phosphorylated and that these can be distinguished from thebulk histones by butyrate treatment. Interestingly, butyrate treatment shifted the target ofTGHQ-induced histone phosphorylation exclusively to histone H3. Hyperacteylated(n=3/4) histone H4 was not phosphorylated in the combination of butyrate/TGHQ treatedcells (Fig. 3B, compare lane c [TGHQ only] with lane d [butyrate plus TGHQ]). The datasuggest that ROS- induced histone H3 kinases target butyrate sensitive histone H3 acety-lation sites (or vice versa), which, in combination, may be sufficient to disrupt nucleo-somes to facilitate access of the DNA repair machinery.

ROS-induced histone modifications: histone methylation. The role ofhistone methylation is one of the least understood post-translational modifications affect-ing histones. Histone methylation is a relatively stable modification, with a slow turnover

Figure 3 TGHQ-induced histone H3 phosphorylation occurs in hyperacetylated histones. LLC-PK1 cells were pre-treated with a hiostone deacetylase inhibitor, sodium butyrate (5mM) for 12 h, and then labeled with [32P]-orthophos-phoric acid. Labeled cells were treated with 400μM of TGHQ. Histones were extracted from these cells, and 70 μg ofprotein were electrophoretically resolved on a Triton-acid urea gel. Proteins were overloaded on Triton-acid urea gel tosee acetylated histone subtypes on Coomassie Blue-stained gel. Lane a, untreated control cells; lane b, butyrate-treatedcontrol cells; lane c, TGHQ-treated cells for 30 min; and lane d, cells pretreated with butyrate and co-treated withTGHQ for 30 min. Panel A shows the Coomassie Blue-stained gel. Panel B shows the corresponding autoradiograph.

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rate. The primary sites modified by methylation in histone H3 are lysines 4, 9, 27, 36and in H4 lysine 20 (Jenuwein, 2006). Histone H3 can be trimethylated at each lysineresidue, whereas lysine 20 in H4 can only be dimethylated (Jenuwein, 2006). This fur-ther mono-, di- or trimethylation of lysine residues provides another level of complexityto this post-translational modification. Histone H4, which is slowly acetylated anddeacetylated, is also methylated in HeLa cells (Annunziato et al., 1995). Although his-tone methylation and dynamic acetylation are not directly coupled, methylation of his-tone H3 at lysine 4 occurs preferentially in a subpopulation that is preferentiallyacetylated. To gain insight into the possible role of histone methylation in modifyingchromatin structure in response to ROS-induced DNA damage, LLC-PK1 cells werelabeled by 14C-methyl-methionine and exposed to 400 μM of TGHQ for 30 min. No sig-nificant changes in the level of histone H3 and H4 methylation were observed inresponse to TGHQ (Fig. 4).

Figure 4 TGHQ-induced histone methylation in LLC-PK1 cells. LLC-PK1 cells were labeled with L-[Methyl-14C]methionine and treated with 400 μM of TGHQ. Histones were extracted from these cells, and 45 μg of proteinwere electrophoretically resolved on a 13.5% SDS-polyacrylamide gel. Lane a, untreated control cells; lane b,TGHQ-treated cells for 30 min; lane c, TGHQ-treated cells for 60 min; and lane c, control untreated cells after 60 min.Panel A shows the Coomassie Blue-stained gel. Panel B shows the corresponding autoradiograph.

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CONCLUSION

In conclusion, ROS-induced changes in the post-translational modification ofhistones are not random in nature. Rather, the changes likely represent the concertedestablishment of a template conducive to the recruitment and retention of the DNA repairmachinery. Moreover, our data, and that of others, indicate that responses to stress, includ-ing oxidative stress, that usually results in oncotic cell death (and tissue necrosis) can bemanipulated, at the genetic and pharmacological level, to produce a potentially favorable(survivable) tissue response. Basic knowledge of the mechanisms by which ROS inducecell death may yield strategies for clinical interventions in the many pathologies in whichROS play a prominent role.

ABBREVIATIONS

3-AB 3-aminobenzamideERK extracellular signal regulated protein kinaseMAPK mitogen-activated protein kinasePARP of poly(ADP-ribose)polymerasePCC premature chromatin condensationROS reactive oxygen speciesTGHQ 2,3,5-tris-(glutathion-S-yl)hydroquinone.

ACKNOWLEDGMENTS

The work conducted in the author’s laboratory was supported by awards from theNational Institutes of Health (DK 59491 and P30 ES 06694).

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Ros-Induced Histone Modifications and their Role in Cell Survival and Cell Death*