Mutagenesis vol. 24 no. 2 pp. 161–167, 2009
Advance Access Publication 8 December 2008
Histone H2AX phosphorylation in response to changes in chromatin structure induced
by altered osmolarity
Jennifer Baure, Atefeh Izadi1, Vannina Suarez1,
Erich Giedzinski1, James E. Cleaver2, John R. Fike and
Charles L. Limoli1,*
Department of Neurological Surgery, University of California, San Francisco,
CA 94110, USA,1Department of Radiation Oncology, University of
California, Irvine Medical Sciences I, Room B-149, Irvine, CA 92697-2695,
USA and2Auerback Melanoma Laboratory, UCSF Cancer Center, University
of California, San Francisco, CA 94143, USA
DNA strand breaks trigger marked phosphorylation of
histone H2AX (i.e. g-H2AX). While DNA double-strand
breaks (DSBs) provide a strong stimulus for this event, the
accompanying structural alterations in chromatin may
represent the actual signal that elicits g-H2AX. Our data
show that changes in chromatin structure are sufficient to
elicit extensive g-H2AX formation in the relative absence
of DNA strand breaks. Cells subjected to hypotonic (0.05
M) treatment exhibit g-H2AX levels that are equivalent to
those found after the induction of 80–200 DNA DSBs (i.e.
2–5 Gy). Despite this significant increase in phosphoryla-
tion, cell survival remains relatively unaffected (<10%
cytotoxicity), and there is no significant increase in
apoptosis. Nuclear staining profiles indicate that g-H2AX-
positive cells induced under altered tonicity exhibit vari-
able levels of staining, ranging from uniform pan staining
to discrete punctate foci more characteristic of DNA
strand breakage. The capability to induce significant
g-H2AX formation under altered tonicity in the relative
absence of DNA strand breaks suggests that this histone
modification evolved in response to changes in chromatin
The nuclear histones play a major role in defining chromatin
structure, and covalent modification of these proteins can
trigger a wide range of biological responses. One such
modification that has received a great deal of attention involves
the phosphorylation of histone H2AX to form c-H2AX. The
formation of c-H2AX was first found to be dependent on DNA
strand-breaking agents, particularly those that produce DNA
double-strand breaks (DSBs) (1,2). Since then a great deal of
effort has been focused on understanding the significance of
this phosphorylation event (3–7). While there is little doubt that
this histone modification can be stimulated by DNA strand
breaks, it is less clear whether phosphorylation of H2AX is
entirely dependent on DNA strand breakage.
Phosphorylation of H2AX has been reported in the relative
absence of DNA damage, but usually under circumstances
involving the remodeling and/or segregation of chromosomes
(8–10). Generally, studies analyzing c-H2AX formation utilize
agents or conditions that cause DNA strand breaks directly
(e.g. ionizing radiation) or indirectly (via replication arrest and
fork breakdown). Another inherent feature of these studies is
that regardless of the mode of strand break production, the
local structure of the chromatin is changed. The inability to
separate the inevitable changes in chromatin structure that
accompany overt DNA strand breaks may have obscured
a more fundamental role for H2AX phosphorylation, one that
evolved in response to structural changes in chromatin.
To address the nature of the c-H2AX-stimulating signal in
the present study, we relied on a strategy designed to produce
structural changes in chromatin in the relative absence of DNA
breaks. Considerable data exist showing that c-H2AX is
formed in response to DSBs (1–3), and numerous studies have
shown that cells subjected to irradiation (6,11,12), replication
arrest (4,13,14) or apoptosis (15) exhibit significant increases
in c-H2AX. Immunohistochemical detection of c-H2AX in
single cells is characterized by distinct intranuclear foci
(4,6,16). Presumably, these foci are first formed in close
proximity to the sites of DSBs, but can correspond to much
larger megabase regions of the chromatin, as local changes in
chromatin structure expand to impact larger chromatin domains
(1). The size and distribution of radiation-induced c-H2AX foci
can be altered in response to changing salt conditions (17,18),
implying that once c-H2AX foci are formed, their size and/or
distribution can be altered further in response to changing
chromatin conformations. In the current study, we adopted
a distinctly different approach, determining whether exposure
to hypotonic salt conditions designed to alter chromatin
structure were sufficient to induce c-H2AX alone, rather than
alter its immunohistochemical appearance once formed after
There has been a previous effort to induce c-H2AX in the
absence of DNA strand breaks using low salt, but that
investigation was not able to demonstrate any real change in
the status of c-H2AX (19). One reason for this finding might be
due to a salt concentration (100 mM) that was insufficiently low
to elicit the altered chromatin topology needed to trigger H2AX
phosphorylation. Thus, we contend that conditions able to alter
chromatin structure without causing significant DNA strand
breakage are in principal sufficient to elicit phosphorylation of
histone H2AX. Here we describe our studies showing that
incubation under relatively non-toxic conditions of hypotonic
salt is indeed sufficient to induce significant levels of c-H2AX.
Materials and methods
The following cell lines were used: 1) immortalized neural precursor cells
isolated from the mouse cerebellum (20), 2) primary neural precursor cells
isolated from the mouse subventricular or hippocampal dentate subgranular
zones (21), 3) immortalized human fibroblasts (HCA) (22) and 4) primary
human fibroblasts (IMR90). Cerebellar precursor cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA)
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serum (Hyclone), 100 units/ml penicillin, 100 lg/ml streptomycin and 1.25 lg/ml
fungizone (Invitrogen). Subventricular precursor cells were grown in Dulbecco’s
Modified Eagle’s Medium: Nutrient Mixture F-12 (1:1) (DME/F12; Invitrogen),
0.4% bovine serum albumin (BSA; Sigma, St Louis, MO), 0.76 units/ml heparin
MA) and 40 ng/ml fibroblast growth factor (FGF; Peprotech, Rocky Hills, NJ).
Hippocampal precursor cells were grown in DME/F12 supplemented with
100 units/ml penicillin, 100 lg/ml streptomycin, 20 ng/ml FGF and N2
supplement (Invitrogen). HCA cells were grown in Minimal Essential
Medium (MEM; Invitrogen) supplemented with 10% FBS, 2 mM glutamine
(Invitrogen), 100 units/ml penicillin, 100 lg/ml streptomycin and MEM non-
essential amino acids (Invitrogen). IMR90 cells were grown in DMEM
supplemented with 15% FBS, 2 mM glutamine, 100 units/ml penicillin and
100 lg/ml streptomycin. All cell lines were grown at 37?C anchored to flasks,
except for the subventricular neural precursor cells that are cultured as
neurospheres in suspension (23). Under these conditions, all cell types exhibited
doubling times of 24–36 h. To analyze the impact of hypotonic treatments on
confluence-arrested cerebellar cultures, cells were maintained as confluent
monolayers for 5 days in the presence of 1% serum.
For hypotonic treatments, a 0.05 M salt solution was made using divalent
cation-free phosphate-buffered saline (PBS, pH 7.4) diluted in water. Cells
were then incubated in these solutions at 37?C for the specified period of time.
For cellular exposures to c-rays, a
Associates, San Fernando, CA; Mark I) was used at a dose rate of 1.17 Gy/min.
137Cs irradiator (J.L. Shepard and
Fluorescent-activated cell sorting analysis
Fluorescent-activated cell sorting (FACS) was used to quantify the levels of
H2AX phosphorylation following hypotonic treatments. Cells subjected to
altered tonicity (with or without recovery in isotonic medium) were counted
and then fixed and permeabilized in preparation for FACS analysis following
the instructions provided in the H2AX FACS kit (Upstate, NY). The
appropriate amount of anti-H2AX antibody (Millipore, Billerica, MA) was
added to each sample and incubated overnight at 4?C. The next day, cells were
stained with propidium iodide and left at 4?C for 4 h before FACS analysis.
Negative controls (untreated) and positive controls (c-irradiated) for c-H2AX
were routinely included for comparison. To compute the relative levels of
H2AX phosphorylation, the ratio of ungated fluorescent means (test cells/
untreated controls) was used.
Cell cycle analysis of cerebellar cells after hypotonic incubation and
recovery was accomplished as previously described (21). Cells were analysed
for DNA content by FACS analysis based on propidium iodide fluorescence
and data were analysed using the CellQuest? algorithm. Cell cycle
distributions were derived from a minimum of 3000 gated events under set
parameters that minimized reverse chi-square values.
Immunohistochemistry was performed to determine the nuclear distribution of
irradiation or hypotonic salt treatments. All treatments were carried out while
cells were still attached to slides, after which cells were fixed in 4%
paraformaldehyde and permeabilized in a 0.2% solution of Triton X-100 in
PBS. Subsequent detection was accomplished after blocking in 10% FBS/1%
BSA for 1 h, using a 1:1000 dilution of the flourescein isothiocyanate (FITC)-
labelled mouse monoclonal antibody against c-H2AX (Upstate) in the
background reducing antibody diluent (DAKO plus S3022; Upstate). Following
overnight incubation at 4?C, slides were counterstained with 4#,6-diamidino-2-
phenylindole (DAPI) in MP Prolong GOLD antifade (Invitrogen-Molecular
Probes, Carlsbad, CA). Nuclei staining positive for c-H2AX were scored double
blind using a subjective scale based on the intensity of the fluorescent nuclear
signal. Brightly and moderately staining nuclei visible against a DAPI
counterstain were scored as either strong or medium staining cells, respectively.
Cells that could be unambiguously identified as c-H2AX positive only after
eliminating the nuclear DAPI signal (via a FITC bandpass filter) were classified
as weakly staining cells.
Apoptosis was measured as described by us previously (18). Briefly, apoptosis
was assayed using the annexin V?FITC apoptosis kit (BD Biosiences, San
Jose, CA) following the manufacturer’s recommendations. Immediately
following specific hypotonic treatments (30?60 min) and recovery in isotonic
media (30?120 min), cells were re-suspended in binding buffer, incubated with
the annexin?FITC conjugate and analysed by FACS.
To determine how the salt treatments impacted survival, clonogenic assays
were performed using the two immortalized cell lines (cerebellar precursors,
HCA fibroblasts). Cells subjected to hypotonicity were diluted, plated in
triplicate and stained (25% crystal violet in ethanol) after visible colonies
developed after 10–14 days. Surviving fraction was calculated as the number of
colonies counted divided by the number seeded corrected for plating efficiency.
Plating efficiencies for the immortalized neural precursors and fibroblasts were
52 (?6) and 76 (?8)%, respectively.
To test the hypothesis that c-H2AX could be induced
independent of DNA strand breaks, cerebellar precursor cells
were subjected to a range of incubation times under hypotonic
conditions and allowed to recover in isotonic medium for
a fixed period of 30 min before processing for FACS analysis.
These initial studies showed that relatively short exposures (10
min) to low salt were sufficient to elicit significant increases in
c-H2AX levels over background (Figure 1). Hypotonic treat-
ments up to 1 h resulted in progressively higher levels of
c-H2AX, with the most rapid rise occurring during the initial 20
min (Figure 1). Hypotonic treatment of other cells suggested that
changes in chromatin structure provide a general signal for
triggering H2AX phosphorylation across different tissues (i.e.
neural precursor cells, fibroblasts) and species (i.e. rodent,
human). Analysis of primary neural precursors (hippocampal
and subventricular) and primary (IMR90) and immortalized
(HCA) human fibroblasts indicated that all cells analysed
responded to reduced tonicity (30 and 60 min) by increasing
c-H2AX levels (Table I). Overall, hypotonic treatments were
found to increase c-H2AX levels by 1.3–2.3 fold. For cells
subjected tohypotonic treatments, FACS histogramsshowedthat
increasing treatment times led to a progressive rightward shift
in a single fluorescent peak, representing increased levels of
c-H2AX (Figure 2). Extended hypotonic treatments were also
1020 304050 60
Hypotonic Incubation Time (min)
Relative γ −H2AX Levels
Fig. 1. H2AX phosphorylation induced by hypotonic exposure in cerebellar
precursor cells. Cells were subjected to hypotonic (0.05 M) treatments for
various lengths of time then allowed to recover in isotonic medium for 30 min
before processing for c-H2AX levels by FACS. Hypotonic treatments were
found to progressively increase c-H2AX levels over 60 min of incubation. The
levels of c-H2AX quantified in cells subjected to x-irradiation are indicated
along the ordinate for comparative purposes. Relative c-H2AX levels were all
normalized to untreated controls set to unity and represent the average of at
least three experiments (?standard deviation).
J. Baure et al.
likely reflected an increased heterogeneity of H2AX phosphor-
ylation within the population of cells exposed to low salt.
To determine the persistence of salt-induced changes in
c-H2AX, cerebellar precursor cells were exposed (up to 1 h) in
hypotonic salt, before switching to isotonic medium, where
cells were thenallowed to recover over various times (up to 2h).
Under hypotonic conditions, cells remained competent to phos-
phorylate H2AX, where the level of c-H2AX increased (50%)
solid line). Upon return to isotonic media, little change in c-
H2AX levels were observed in these cells until 2 h, where c-
H2AX increased another 30% (Figure 3).
The formation c-H2AX after hypotonic exposure was
inhibited completely by wortmannin (20 lM, data not shown),
suggesting that these treatments were activating a member of
the phosphoinositide 3-kinases (PI3 kinase) family that could
phosphorylate the histone target. Wortmannin had a similar
inhibitory effect on c-H2AX formation in control cells kept
under isotonic conditions but irradiated with 10 Gy. Several
other experiments were run to determine whether DNA
intercalators could elicit c-H2AX formation, and despite
repeated attempts using ethidium bromide or chloroquine at
50 and 250 ng/ml, results were routinely negative (data not
shown). To determine whether hypotonic conditions might
perturb replication in S-phase cells and elicit c-H2AX and/or
interfere with cell cycle progression, cells were subjected to
hypotonic conditions and analysed by FACS for any changes
in the distribution of cells throughout the cell cycle. Cells
incubated under hypotonic conditions for 30 or 60 min and
allowed to recover for various lengths of time in isotonic
medium did not show any major changes in cell cycle
distribution (Table II). S-phase percentages were minimally
changed compared to untreated controls, suggesting that the
majority of H2AX phosphorylation resulting from hypotonic
exposure was not restricted to the fraction of S-phase cells. To
more conclusively rule out the possibility that c-H2AX
following hypotonic treatments was primarily due to an effect
on S-phase cells, cerebellar precursor cells subjected to
confluence arrest were analysed for changes in H2AX
phosphorylation. Hypotonic exposures of 30 and 60 min
Table I. H2AX phosphorylation induced in mammalian cells exposed to
1.5 ? 0.1
1.7 ? 0.05
2.0 ? 0.2
2.3 ? 0.3
1.2 ? 0.1
1.9 ? 0.1
1.3 ? 0.07
1.4 ? 0.1
aTime in isotonic medium before assay.
cNeural precursor cells from different regions of the brain.
Fig. 2. FACS histograms of cerebellar precursor cells. Fluorescent histograms
lead to progressively higher levels of c-H2AX (rightward shift along x-axis).
3060 90120150 180
Relative γ–H2AX Levels
Recovery time (min)
Isotonic media added
Fig. 3. Persistence of H2AX phosphorylation induced by hypotonic treatments
in cerebellar precursor cells. Cells were incubated under hypotonic conditions
for up to 60 min, then returned to isotonic media for various lengths of time.
Cells assayed before return to isotonic media showed that c-H2AX was formed
soon after hypotonic treatment. Upon return to isotonic media, cells previously
subjected to hypotonic treatment showed relatively little change in c-H2AX
levels, until 2 h where c-H2AX levels were found to increase.
Table II. Cell cycle distribution of cerebeiiar precursor cells subjected to
aTime in isotonic medium before assay.
bPercentages calculated using the ModFit? algorithm.
cControls kept in isotonic suspension for the minimum and maximum treatment
Response of H2AX to changes in osmolarity
followed by 30 min of isotonic recovery increased c-H2AX
levels by 1.4 ? 0.1-fold and 1.8 ? 0.04-fold, respectively, over
untreated controls. These values are in close agreement with
those for log-phase cells (Figure 1) and suggest that hypotonic-
induced changes in H2AX phosphorylation are not limited to
the yields of strong, moderate and weakly staining cells were
compared to negative (isotonic treatment) and positive (c-
irradiated) controls (Figure 4, Table III). Untreated controls
showed weak c-H2AX staining (4.2%) and were not found to
exhibit the more robust nuclear staining profiles (i.e. moderate or
strong staining) (Figure 4, lower left panel). In contrast, nearly all
cells (?99%) subjected to c-irradiation were found to exhibit
strong nuclear staining with numerous brightly staining c-H2AX
foci (Figure 4, lower center panel). Compared to irradiated
samples, cells subjected to hypotonic salt showed qualitative
different staining patterns for c-H2AX (Figure 4, upper and lower
right panels). In general, hypotonic-treated cells showed a more
however, exhibit a spectrum of different staining profiles varying
from uniform (pan staining) to more punctate (visible foci).
in the number of weakly staining cells with increasing incubation
was found to be less dependent on hypotonic incubation time.
To assess the toxicity of the hypotonic treatments,
clonogenic assays were performed using immortalized neural
precursors and fibroblasts. Cells were also subjected to
irradiation to assess their response to an agent known to
produce DNA strand breaks. Both cell lines showed typical
responses to irradiation, having shouldered survival curves and
exponential killing with D0values of 1.56 (fibroblasts) and
1.91 Gy (neural precursors) (Figure 6). In comparison,
Fig. 4. Immunohistochemistry of c-H2AX in neural precursor cells subjected to hypotonic conditions. Neural precursor cells were subjected to hypotonic salt
(60 min) and allowed to recover (30 min) before fixation and immunohistochemical analysis of c-H2AX nuclear staining patterns. Upper panels (?40) show
examples of strong, medium and weakly staining cells. The dual bandpass image (left) shows the signals for both c-H2AX (FITC) and DAPI nuclear counterstains,
while a single bandpass (right) used to eliminate DAPI fluorescence reveals more weakly staining cells from the same field. Lower panels (?60) show examples of
the minimal c-H2AX staining found in isotonic controls (left), the maximal punctate staining in irradiated (5 Gy) cells fixed 20 min later (center) and the more
uniform c-H2AX staining found in cells subjected to hypotonic salt (right).
J. Baure et al.
hypotonic treatments lasting up to 60 min resulted in virtually
no cell kill (Figure 6). Analysis of non-clonogenic cells (i.e.
primary subventricular and hippocampal precursors) for viability
using the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl-
amino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay (func-
survival after hypotonic treatments (data not shown).
Cerebellar and subventricular neural precursor cells sub-
jected to hypotonic treatments were also evaluated for
apoptosis using annexin V staining. Following 30- or 60-min
treatments in hypotonic salt, cells were allowed to recover for
either 30 or 120 min in isotonic medium before annexin V
binding and FACS analysis. Both cell types gave qualitatively
similar results, showing that the percentage of apoptotic cells
actually decreased (although not significantly) after hypotonic
treatments (Table III).
The primary objective of these studies was to determine if we
could detect the formation of c-H2AX under conditions that did
not induce overt changes in chromatin structure associated with
DNA strand breaks. Here we present evidence that c-H2AX can
be formed in significant yield under minimally toxic conditions
of altered tonicity. Cells subjected to hypotonic conditions
rapidly form c-H2AX in a manner dependent on the time of
exposure (Figure 1). This effect appears to be general, at least,
for neural precursors and fibroblasts, cells that routinely showed
elevated c-H2AX levels following hypotonic treatments.
Cells retain the requisite kinase activity necessary to
phosphorylate histone H2AX under hypotonic conditions
(Figure 3). While not the focus of this investigation, the
capability of wortmannin to inhibit the formation of c-H2AX
suggests that the PI3 kinases (e.g. ataxia telangiectasia mutated,
ataxia telangiectasia and Rad3 related and/or DNA-dependent
Table III. Immunohistochemical staining of c-H2AX and analysis of apoptosis in cells exposed to hypotonic salt
Recovery time (min)Nuclear H2AX staininga,b
Fold change in annexinV
% Strong% Medium% Weak
14.7 ? 2.0
4.2 ? 1.1
29.6 ? 3.1
0.66 ? 0.12
0.86 ? 0.06
0.87 ? 0.24
1.0 ? 0.40
8.8 ? 1.6
607.5 ? 0.812.1 ? 3.329.01 ? 5.2
7.6 ? 1.4
3.75 ? 2.0
17.2 ? 2.1
12.3 ? 2.6
40.8 ? 5.6
48.1 ? 7.4
n/d, not deteremined.
a?1000 total cells were analysed after each treatment.
bTotal background staining was 4.2% (weak) from 0.5 to 2 h in isotonic medium.
cData averaged from cerebellar and subventricular neural precursor cells.
dFold change in the number of annexin V-positive cells compared to controls.
Control 30 - 3060 - 30 90 - 30120 - 30
gamma-H2AX staining (%)
Fig. 5. Immunohistochemical analyses of c-H2AX-positive cells. Bar charts
derived from the data shown in Table III, plot the percentage of c-H2AX-
positive cells scored as strong, moderate or weakly staining as described (see
Materials and methods). Control cells were incubated in isotonic medium (30–
120 min), while test cells were incubated in hypotonic salt (30–120 min)
followed by recovery (30 min) in isotonic media. Numbers along x-axis refer
to ‘hypotonic–isotonic’ treatment times (min).
Radiation Dose (Gy)
Hypotonic Incubation Time (min x 1/6)
Fig. 6. Cell survival after hypotonic exposure or c-irradiation. Gamma-
irradiated cerebellar precursor cells (NPC, circles) and immortalized human
fibroblasts (HCA, squares) show typical shouldered survival curves over
a dose range of 1–10 Gy (solid lines). Hypotonic incubation of these same cells
over the course of 60 min leads to relatively little toxicity (dashed lines). For
comparison, hypotonic data are plotted at 1/6 scale (i.e. 60 min 5 10 Gy).
Experiments repeated in triplicate (?standard error).
Response of H2AX to changes in osmolarity
protein kinase) are likely to be involved in mediating H2AX
phosphorylation after hypotonic exposure (18,24). This idea is
also supported by a number of past studies analyzing the
substrate specificities of this kinase family after different types
of cellular stress (25–27). The formation of c-H2AX in
response to hypotonic treatment persists well after return to
isotonic medium (Figure 3), suggesting that certain disruptions
to chromatin structure persist, and/or the ability of phospha-
tases to return H2AX to the unphosphorylated state are com-
promised. It may also be possible that hypotonic conditions
compromise cellular metabolism and energy pools, potentially
impacting H2AX phosphorylation by altering the response of
DNA damage and chromatin sensing pathways. While different
salt levels are likely to have many effects in cells, the impact of
the resultant structural alterations to chromatin induced under
hypotonic conditions are still likely to play a contributory if not
causal role in eliciting c-H2AX.
Past studies have tried to quantify the cellular response to
DNA damage through immunohistochemical approaches
aimed at analyzingthenuclear
(4,6,28,29). Such efforts have clearly demonstrated that agents
and/or conditions capable of causing DNA DSBs directly (e.g.
ionizing radiation) or indirectly (e.g. via replication fork
breakdown) lead to the formation of brightly staining c-H2AX
foci of widely varying size. However, efforts to quantify
fluorescent c-H2AX foci within nuclei are often hampered by
the marked heterogeneity in the level of background c-H2AX
staining. If, as we suggest, c-H2AX levels are sensitive to chro-
matin change, then much of the heterogeneity in nuclear c-
H2AX staining profiles may simply reflect the multiple
structural conformations of DNA actively undergoing replica-
tion and/or repair. In support of this possibility, past studies
from us and others have found that background H2AX staining
is highest during S-phase (4,30,31).
For the reasons alluded to above, we chose to focus on the
quantification of c-H2AX levels via FACS analysis. Immuno-
histochemical analyses undertaken for comparative purposes
demonstrated unequivocally the capability of hypotonic treat-
ments to elicit c-H2AX nuclear staining. The different nuclear
staining profiles evident after various treatments did, however,
reveal marked variations in c-H2AX signal intensity and
distribution in the nucleus. Overall, hypotonic treatments led to
more uniform pan-nuclear staining while c-irradiated cells all
showed very discrete punctate patterns of nuclear staining
(Figure 4). The number of weakly staining nuclei increased
from 30 to 50% as hypotonic incubations increased from 30 to
120 min (Table III, Figure 5). Similar hypotonic treatments had
a smaller impact on the fraction of cells staining more
intensely, where the yield of positive cells scored as strong
or moderate fluctuated around mean values of 6.9 ? 2% and
14 ? 2%, respectively (Table III, Figure 5).
Clearly, there are caveats associated with this type of
subjective immunohistochemical analyses, which limits the
types of conclusions that can be drawn. We reiterate that these
types of studies were included with the intent of highlighting
qualitative differences between c-H2AX staining patterns
found after irradiation and those found after hypotonic salt. It
is possible that the most brightly staining cells were those
destined to die (possibly via apoptosis), but at the time at which
our H2AX measurements were performed, annexin V staining
was minimal (Table III). If in fact pan-nuclear staining was
a marker for early apoptosis, then it also did not impact
clonogenic kill. Based on the increased H2AX phosphorylation
observed after hypotonic treatments, it is unlikely that
apoptosis could account for the majority of the increased
H2AX phosphorylation we report in this study. Despite
inherent differences between the quantification of c-H2AX-
positive cells by FACS or immunohistochemistry, each
technique did show that exposure to hypotonic conditions
increased the number of cells positive for c-H2AX.
Further support that c-H2AX is a response sensitive to
chromatin disruptions comes in a recent report showing that
UV light induces significant increases in c-H2AX levels
(30,32), a response that was muted in cells deficient for
nucleotide excision repair (NER). UVC light does not produce
DNA DSBs, but at the fluences used, hundreds of thousands of
thymine dimers and 6–4 photoproducts are formed throughout
the genome (33,34). These UV-induced lesions are excised
during NER, a repair process that involves the formation of
intermediate D-loop structures and gap-filling synthesis in the
DNA. If c-H2AX acts to respond to structural change in
chromatin, then activation of NER would be predicted to
increase c-H2AX levels. Repair competent cells exposed to
UVC light not only exhibited marked increases in c-H2AX but
also showed pan-nuclear staining patterns for c-H2AX (i.e.
similar to that shown in Figure 4) (30). These results lend
support to the idea that more subtle changes in chromatin
structure can elicit c-H2AX formation.
One of the critical issues these investigations sought to
clarify was if c-H2AX formation was not entirely (if at all)
dependent on DNA strand breakage. Because measuring low-
level yields of DNA DSBs in mammalian cells (i.e. 1 Gy
equivalent, ?40/cell) presents a considerable technical chal-
lenge (35,36), it becomes difficult to conclusively determine
whether a cell at any given time contains only a few or no
DSBs. Consequently, to unequivocally substantiate that the
hypotonic treatments used here do not create any DNA DSBs
would be difficult. However, the survival curves shown (Figure
6) do provide an unambiguous assessment of survival and
allow one to estimate relative DSB yields. Thus, at a dose of 5
Gy, ?200 DSBs elicit 80% cell kill and a 1.8-fold increase in
c-H2AX levels (Figure 1) in neural precursor cells. In
comparison, a 60-min hypotonic treatment elicits equivalent
c-H2AX levels (?1.7-fold over background) but only results in
13% cell kill, survival levels typically found at doses ,1 Gy
(i.e. ,40 DSBs). Therefore, based on the survival data shown,
the level of c-H2AX induced under hypotonic conditions
cannot be explained solely by the induction of DNA DSBs,
even if such treatments were in fact found to induce low yields
(?1 Gy) of these lesions.
In summary, our investigation was initiated not to dispute
the role of the DSB-dependent formation of c-H2AX but rather
to provide evidence that the phosphorylation of this histone is
responsive to alterations in chromatin structure that are not
strictly dependent upon strand break formation. Changes in
tonicity elicit predictable changes governing the interactions
between macromolecules in cells, and based on these expect-
ations, it is difficult to reconcile how the hypotonic conditions
used here would not elicit at least minimal changes in
chromatin structure. While we did not attempt to measure
such structural alterations, salt-induced changes were in fact
sufficient to elicit the phosphorylation of histone H2AX. We
maintain that c-H2AX formation is principally responsive to
changes in chromatin structure and that such changes likely
provided the selective pressure for the adaptation of c-H2AX
into a DNA damage-responsive niche.
J. Baure et al.
National Institutes of Health/National Institute of Neurological
Disorders and Stroke (1R01NS052781 to J.E.C.); American
Cancer Society (RSG-00-036-04-CNE to C.L.L.).
We would also like to express our gratitude to Katherine Tran for the
immunohistochemical staining and scoring of c-H2AX-positive cells. Conflict
of interest statement: None declared.
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Received on November 8, 2007; revised on October 29, 2008;
accepted on October 31, 2008
Response of H2AX to changes in osmolarity