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Drug Metabolism Reviews, 38: 755767, 2006

Copyright Informa Healthcare

ISSN: 0360-2532 print / 1097-9883 online

DOI: 10.1080/03602530600959649

755

ROS-INDUCED HISTONE MODIFICATIONS AND THEIRROLE IN CELL SURVIVAL AND CELL DEATH

Terrence J. Monks, Ruiyu Xie, Kulbhushan Tikoo, andSerrine S. LauDepartment of Pharmacology and Toxicology, College of Pharmacy, University of

Arizona Health Sciences Center, Tucson, Arizona; Laboratory of Chromatin Biol-

ogy, Department of Pharmacology and Toxicology, National Institute of

Pharmaceutical Education and Research, Sec. 67, Mohali 160062, Punjab, India

Much is known about the distal DNA damage repair response. In particular, many of the

enzymes and auxiliary proteins that participate in DNA repair have been characterized.

In addition, knowledge of signaling pathways activated in response to DNA damage is

increasing. In contrast, comparatively less is known of DNA damage-sensing molecules

or of the specific alterations to chromatin structure recognized by such DNA damage

sensors. Thus, precisely how chromatin structure is altered in response to DNA damage

and how such alterations regulate DNA repair processes remain important unanswered

questions. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly

after double-strand break formation, extends over megabase chromatin domains, and is

required for stable accumulation of repair proteins at damage foci. We have shown that

reactive oxygen species (ROS)-induced DNA single-strand breaks induce the incorpora-tion of32P specifically into histone H3. ADP-Ribosylation of histones may stimulate local

chromatin relaxation to facilitate the repair process, and, indeed, histone ribosylation

preceded DNA damage-induced histone H3 phosphorylation. However, H3 phosphoryla-

tion occurred concomitant with overall chromatin condensation, as revealed by decreased

sensitivity of chromatin to digestion by micrococcal nuclease and by DAPI staining of

nuclei. 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 of

histone H3 regulate the survival response remains unclear. Attempts to determine the

precise site of histone H3 phosphorylation, putative histone H3 kinases, and histone H3

interacting proteins are underway.

Key Words: Cell death; Chromatin; DNA damage; Histones; Mitotic catastrophe; Oncoticcell death; Post-translational modification; Premature chromatin condensation; Reactive

oxygen species; Stress response signaling.

Presented at the Seventh International Symposium on Biological Reactive Intermediates, Tucson,

Arizona, January 47, 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 reactive

oxygen species- (ROS) induced cytotoxicity, treatment of renal proximal tubular epithelial

(LLC-PK1) cells with the ROS-generating nephrotoxicant and nephrocarcinogen

2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ) results in cell death. Despite the fact

that LLC-PK1 cells are capable of engaging the machinery necessary to induce apoptotic

cell death, TGHQ-treated LLC-PK1 cells die via oncotic cell death (Jia et al., 2004). A

frequent response to ROS-induced cell stress that ultimately leads to oncotic cell death is

the premature engagement of chromosome condensation (PCC) and the ensuing mitotic

catastrophe. 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 the

initiation of DNA replication (S-phase entry). Rigid controls function to prevent repeated

rounds of DNA replication without intervening mitoses or the initiation of mitosis before

DNA replication is complete (mitotic catastrophe). Although some of the genetic inter-

actions that participate in this process have recently been identified in yeast (Novak and

Tyson, 1997), little is known about their mammalian counterparts. Indeed, until recently

relatively little was known about the mechanisms and factors that regulate this transition

in chromatin structure. Because a variety of phosphatase inhibitors induce PCC (Coco-

Martin and Begg, 1997), protein phosphorylation likely plays an important role in this

process. However, the targets for phosphorylation and the corresponding protein kinases

are poorly defined. Moreover, the signal transduction pathways activated during thecommitment 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 family

is comprised of three major subgroups: extracellular signal-regulated protein kinase

(ERK), c-Jun N-terminal kinases/stress-activated protein kinase (JNK/SAPK), and p38

MAPK (Cobb, 1999). ERKs behave mainly as mitogen-activated proliferation/differentia-

tion factors, whereas JNK/SAPK and p38 MAPK are mainly stress-activated proteins

related 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 signaling

pathway 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 tubular

epithelial cells (Ramachandiran et al., 2002). Thus, inhibition of ERK1/2 activation with

PD98059 or inhibition of p38 MAPK activation with SB202190 attenuates cell death

induced by TGHQ (Ramachandiran et al., 2002). In contrast, the JNK inhibitor SP600125

has 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 cell

type and the specific context. In a few cases, activated ERK1/2 seems to behave as a cell

death-inducing factor. Thus, ERK activation is related to vanadate-induced oncotic cell

death of vascular smooth muscle cells (Daum et al., 1998), in H2O2-induced cell death of

oligodendrocytes (Bhatt and Zhang, 1999), in T cells (van den Brink et al., 1999), and in

pleural 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 remodeling

during 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 H1

and H3 on Ser-10 and 28 within its basic amino terminal tail (Mahadevan et al., 1991; Sauve

et al., 1999; Wei et al., 1999; Goto et al., 1999) has long been implicated in chromosome

condensation during mitosis (Koshland and Strunikov, 1996) and in response to various

mitogenic stimuli, such as growth factors or phorbol esters. These sites of phosphorylation in

histone H3 are highly conserved and are flanked by basic amino acid residues, which are

also susceptible to multiple post-translational modifications. Similarly, in histone H2B, two

highly conserved phosphorylation sites are located at serines 14 and 32. Interestingly, these

highly 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 with

PCC 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 that

chromatin condensation can occur in the absence of this modification (Guo et al., 1995) and

even 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 to

specify 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; Wei

et al., 1999; Goto et al., 1999) in the presence of other modifications on the same or multiple

histone 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-strand

break 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 MAPK

pathway in progression from G2 into mitosis has been demonstrated (Wright et al., 1999).

Thus, when MAPK activation was inhibited with PD98059, which selectively inhibits

MEK, an upstream regulator of ERKs, in S-phase synchronized NIH 3T3 cells, the cells

arrested in G2. Expression of a dominant-negative form of MAPKK1 was also found to

delay the progression of cells through G2 (Wright et al., 1999). MAPKK activity was

required for the timely activation of Cdc2 and progression into mitosis. These findings

provide a potential mechanistic explanation for our own findings that TGHQ-induced histone

H3 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 PCC

leading 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 consistent

with changes in chromatin structure that occur in vivo in renal proximal tubular epithelial

cell 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 baby

hamster 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, prevention

of PCC only occurred concomitant with decreases in H3 phosphorylation, and not with H1

phosphorylation (Ajiro et al., 1985). Our data therefore suggest that LLC-PK1 cells respondto TGHQ-induced oxidative stress by the aberrant stimulation chromosome condensation

in the absence of the signals and machinery necessary to coordinate mitosis. Whether abrogation

of histone H3 phosphorylation is required for protection against ROS-induced oncotic cell

death in LLC-PK1 cells is not known. However, mitotic histone H3 phosphorylation promotes

the 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 with

DNA. ROS-induced phosphorylation of histone H3 in LLC-PK1 cells might thus result in

the exposure of DNA to chromosome condensing factors, facilitating chromatin condensation.

ROS-Induced Poly(ADP-ribose) Polymerase Activation. The generation of

ROS has been implicated in the pathogenesis of renal ischemia/reperfusion injury and

many 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 in

the depletion of both NAD+ and ATP (Pieper et al., 1999). It has been suggested that

depletions in NAD+ and ATP in response to DNA damage contribute to cell death as a

consequence of deficits in energy stores. For example, Chatterjee et al. (1999) showed that

incubation of primary cultures of rat proximal tubule epithelial cells with 1 mM H2O2

inhibited mitochondrial respiration and increased LDH release, with concomitant increases

in PARP activity. Moreover, inhibitors of PARP protected against H2O2-mediated cell

death (Cristovao and Rueff, 1996). Deletion of PARP also protects against NMDA-

receptor-activated neurotoxicity (Eliasson et al., 1997; Endres et al., 1997), myocardial

ischemia (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-induced

diabetes (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; Filipovic

et 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 fibroblasts

obtained from mice with a targeted deletion of PARP (PARP/) DNA damage induced by

either MNNG or H2O2 failed to deplete intracellular concentrations of ATP, and the cells

were 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 (and

NAD+ ?) concentrations. Because inhibition of PARP activity or PARP gene deletion can

prevent both ATP depletion and the induction of oncosis, it has been suggested that PARP

overactivation-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 clinical

implications. For example, in many clinical situations, such as inflammation, vascular

stroke, and myocardial infarction, the predominant mechanism of cell death appears to be

oncotic. By extension, it has been predicted that PARP inhibitors may have therapeutic

benefit (Ha and Snyder, 1999). Consistent with this hypothesis, PARP inhibition or gene

deletion attenuates tissue injury associated with stroke, myocardial infarct, and diabetic

pancreatic damage (Eliasson et al., 1997; Endres et al., 1997; Zingarelli et al., 1998).

ROS-induced histone modifications: 1) histone ribsoylation facilitateshistone H3 phosphorylation. As previously noted, current dogma suggests that

depletions 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, causes

DNA damage, lipid peroxidation, and oncotic cell death in LLC-PK1 cells, inhibiting lipid

peroxidation with lazeroids or Trolox prevented oncotic cell death without affecting DNA

damage 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 modification

of 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

(DAmours 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 least

understood. Poly(ADP-ribosylation) participates in histone shuttling and nucleosomal unfold-

ing, facilitating DNA excision from chromatin (Realini and Althaus, 1992). Histone H1 at

glutamate 2 and 116 can undergo poly ADP-ribosylation (van Holde, 1989). ADP ribosylation

is relatively rare in unperturbed cells. However, when DNA is damaged, the almost immediate

ribosylation 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 the

repair process. The end result of PARP inhibition will therefore depend upon the relative

effects 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 can

be 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 (Realini

and Althaus, 1992) it may also facilitate additional post-translational modification on the

subsequently exposed proteins. Consistent with this view, TGHQ-induced rapid (


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suggesting that ADP-ribosylation and histone H3 phosphorylation are coupled in this

model 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 previously

demonstrated and suggests that PARP-mediated ADP-ribosylation of histones facilitates

histone H3 phosphorylation and that these post-translational modifications contribute to

PCC and mitotic catastrophe. There is precedence for the coupling of various histone post-

translational modifications. For example, Imai et al. (2000) described a NAD-dependent

histone deacetylase, Sir2, and Sir 2 proteins exhibit NAD-dependent mono-ADP-ribosyl-

transferase activity (Frye, 1999). The coordination of multiple histone modifications

appears 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 to

play 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 were

labeled with 100 Ci/ml [2,8- 3H] adenosine for 4 h and then treated with 400 M TGHQ for increasing periods of

time. Histones were extracted from these cells, and 55 g 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 the

corresponding 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 then

co-treated with 400 M 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 H3phosphorylation 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 the

histone 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 is

modulated by competing activities of various histone acetyltransferases and histone

deacetylases, which behave as transcriptional activators and repressors, respectively. To

elucidate the relationship between ROS-induced histone H3 phosphorylation and histone

acetylation, 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 hyperacetylated

histones (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 M TGHQ for increasing periods of time. Histones

were extracted from these cells, and 35 g 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 M

TGHQ for 30 min; and lane h, only 3-aminobenzamide-treated cells for 30 min. Panel A shows the Coomassie

Blue-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 population

of acetylated histones are phosphorylated and that these can be distinguished from the

bulk histones by butyrate treatment. Interestingly, butyrate treatment shifted the target of

TGHQ-induced histone phosphorylation exclusively to histone H3. Hyperacteylated

(n=3/4) histone H4 was not phosphorylated in the combination of butyrate/TGHQ treated

cells (Fig. 3B, compare lane c [TGHQ only] with lane d [butyrate plus TGHQ]). The data

suggest 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 of

histone 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 400M of TGHQ. Histones were extracted from these cells, and 70 g of

protein were electrophoretically resolved on a Triton-acid urea gel. Proteins were overloaded on Triton-acid urea gel to

see acetylated histone subtypes on Coomassie Blue-stained gel. Lane a, untreated control cells; lane b, butyrate-treated

control cells; lane c, TGHQ-treated cells for 30 min; and lane d, cells pretreated with butyrate and co-treated with

TGHQ 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, 36

and in H4 lysine 20 (Jenuwein, 2006). Histone H3 can be trimethylated at each lysine

residue, 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 complexity

to 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 preferentially

acetylated. To gain insight into the possible role of histone methylation in modifying

chromatin structure in response to ROS-induced DNA damage, LLC-PK1 cells were

labeled by 14C-methyl-methionine and exposed to 400 M of TGHQ for 30 min. No sig-

nificant changes in the level of histone H3 and H4 methylation were observed in

response 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 M of TGHQ. Histones were extracted from these cells, and 45 g of protein

were 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 of

histones are not random in nature. Rather, the changes likely represent the concerted

establishment 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 be

manipulated, at the genetic and pharmacological level, to produce a potentially favorable

(survivable) tissue response. Basic knowledge of the mechanisms by which ROS induce

cell death may yield strategies for clinical interventions in the many pathologies in which

ROS play a prominent role.

ABBREVIATIONS

3-AB 3-aminobenzamide

ERK extracellular signal regulated protein kinase

MAPK mitogen-activated protein kinase

PARP of poly(ADP-ribose)polymerase

PCC premature chromatin condensation

ROS reactive oxygen species

TGHQ 2,3,5-tris-(glutathion-S-yl)hydroquinone.

ACKNOWLEDGMENTS

The work conducted in the authors laboratory was supported by awards from the

National Institutes of Health (DK 59491 and P30 ES 06694).

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