ORIGINAL PAPER

Tumor suppressor Ing1b facilitates DNA repair and preventsoxidative stress induced cell death

Anand Rotte • Gang Li • Madhuri Bhandaru

Published online: 16 November 2013

� Springer Science+Business Media New York 2013

Abstract Inhibitor of growth (ING) family of proteins

are known to coordinate with histone acetyltransferases

and regulate the key events of cell cycle and DNA repair.

Previous work from our lab showed that Ing1b regulated

the nucleotide excision repair by facilitating histone acet-

ylation and subsequent chromatin relaxation. Further, it

was also shown that Ing1b protected the cells from geno-

mic instability induced cell death by promoting ubiquiti-

nation of proliferating cell nuclear antigen (PCNA). In the

present study we explored the role of Ing1b in the repair of

oxidized DNA and prevention of oxidative stress induced

genotoxic cell death. Using HCT116 cells we show that

Ing1b protein expression is induced by treatment with

H2O2. Ing1b lacking cells showed decreased ability to

repair the oxidized DNA. PCNA monoubiquitination, a

critical event of DNA repair was blunted in Ing1b knock

down cells and augmented in Ing1b over expressing cells.

Moreover, oxidative stress induced cell death was higher in

cells lacking Ing1b whereas it was lower in Ing1b over

expressing cells. Finally we show that inhibition of histone

deacetylases, rescued the Ing1b knock down cells from

cytotoxic effects of H2O2 treatment.

Keywords Ing1b � PCNA ubiquitination � DNA

repair � Histone acetylation � Cell survival

Introduction

Oxidative stress has been implicated in wide range of dis-

orders including cancer, diabetes and neurodegeneration [1–

5]. It is defined as a condition in which there is an uncon-

trolled increase in the cellular levels of reactive oxygen

species (ROS). The source of oxidative stress can be both

exogenous like chemotherapeutic agents or UV radiation

and endogenous, as a byproduct of mitochondrial energy

metabolism. The outcome of increased cellular ROS, is

oxidation of the cell contents which include lipids, proteins

and DNA. Among the various types of oxidative modifica-

tions to DNA, 7,8-dihydro-8-oxo-guanine (8-oxoG) repre-

sents the most abundant lesion [6]. DNA lesions due to

oxidation are promptly repaired by a specialized pathway

called as ‘Base Excision Repair’ where in the oxidized base

is cleaved from the DNA strand and replaced due to the

coordinated activity of DNA glycosylase, apurinic/apyrim-

idinic endonuclease (APE1), poly(ADP-ribose) polymerase-

1 (PARP), X-ray repair cross complementing protein1

(XRCC1), proliferating cell nuclear antigen (PCNA), DNA

polymerases, and other repair factors [3, 7, 8]. The regulation

of DNA damage check point activity by oxidative stress is

not clearly understood though recent evidences suggest the

involvement of proteins regulating the check point during

replication blockade [9, 10]. The inhibitor of growth (ING)

proteins are known to regulate various biological processes

including DNA repair, cell cycle, apoptosis, and senescence

[11–20]. ING proteins are known to be components of var-

ious histone acetyltransferases and are supposed to carry out

at least part of their functions through chromatin remodeling

[21–23]. Previous work from our group showed that Ing1b

facilitated nucleotide excision repair by promoting chro-

matin accessibility to xeroderma pigmentosum, comple-

mentation group A (XPA) [14], and our previous studies also

A. Rotte (&) � G. Li � M. Bhandaru

Department of Dermatology and Skin Science, University of

British Columbia, Research Pavilion, 828 West, 10th Avenue,

Vancouver, BC V5Z 1L8, Canada

e-mail: anand.rotte@ubc.ca

123

Apoptosis (2014) 19:518–526

DOI 10.1007/s10495-013-0940-5

showed the indispensable role of Ing1b in maintenance of

genomic stability after UV induced replication stress [23].

Cells lacking Ing1b were found to be sensitive to UV

induced stress with comparatively higher incidence of

chromatid breaks, and higher percentage of cell death [23].

Recent work by Ceruti et al., showed the significance of

Ing1b in the repair of DNA damaged due to oxidation, using

cultured cell lines and mouse embryonic fibroblasts [12].

H2O2 treatment was shown to induce Ing1b mRNA levels

and cells lacking Ing1b were reported to be more sensitive to

H2O2 induced DNA damage as seen by higher levels of c-

H2AX [12]. However, the authors did not illustrate the

possible role of Ing1b in the oxidative stress induced PCNA-

monoubiquitination. Further, the authors also did not illus-

trate the effect of Ing1b on the genotoxic stress caused by

H2O2 treatment. The present study was therefore undertaken

to demonstrate the role of Ing1b in oxidative stress induced

PCNA-monoubiquitination and repair of oxidized DNA, and

in preventing the oxidative stress induced cell death.

Materials and methods

Cell culture, antibodies, chemicals and H2O2 treatment

HCT116 cells were cultured in Dulbecco’s modified Eagle

media (DMEM) (Invitrogen, Burlington, ON, Canada)

supplemented with 10 % fetal bovine serum (Invitrogen),

100 U/ml penicillin and 100 mg/ml streptomycin (Invit-

rogen) in 5 % CO2 humidified atmosphere at 37 �C. Anti-

actin and anti-Flag antibodies were purchased from Sigma-

Aldrich (St Louis, MO, USA); and anti-PCNA from Mil-

lipore (Billerica, MA, USA); ING1b, and ORC2 from

Santa Cruz Biotechnology (Santa Cruz, CA, USA) and

mouse anti-Flag from Applied Biological Materials

(Richmond, BC, Canada). To induce oxidative stress, H2O2

was added to the culture medium of the cells at indicated

concentrations. 30 min after addition of H2O2, medium

was removed and cells were washed once with 19 PBS,

and fresh ‘complete’ medium was added to the cells.

Expression plasmid, siRNA transfections

Expression plasmids were transfected into HCT116 cells by

Effectene Transfection Reagent (Qiagen, Mississauga, ON,

Canada) according to the manufacturer’s instruction. siRNAs

were synthesized by Qiagen. ING1b siRNA sequences as

follows: 50-acccacgtactgtctgtgcaa-30. siRNA was transfected

to cells by siLenFect Lipid reagent (Bio-Rad, Mississauga,

ON, Canada) according to manufacturer’s instruction. Assays

were performed 48 h after transfection.

Subcellular fractionation

Fractionation of chromatin bound and unbound fractions

were described previously [14, 24]. Briefly, cytoplasmic

and nucleoplasmic proteins were isolated by cytoskeletal

buffer (CSK) (100 mM NaCl, 300 mM sucrose, 3 mM

MgCl2, 10 mM PIPES pH 6.8, 1 mM EGTA, 0.2 % Triton

X-100) with protease inhibitors for 15 min on ice. After

centrifugation at 900g for 5 min at 4 �C, chromatin bound

proteins in the pellet were resuspended in modified RIPA

buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 1 mM

EDTA, 0.1 % NP-40, 0.25 % sodium dodecyl sulphate)

and sonicated.

Western blotting

Cells were harvested and washed with PBS thrice. Whole-

cell proteins were extracted and protein concentration was

determined by protein assay (Bio-Rad), western blot ana-

lysis was performed as described previously [25]. The

following antibodies were used for western blot: Ing1b,

ORC2, LAMP2 (1:250; Santa Cruz, CA, USA), PCNA

(1:2000 Billerica, MA, USA), Flag (1:1000, Applied Bio-

logical Materials, Richmond, BC, Canada), and actin

(1:5000; Immunechem Pharmaceuticals, Burnaby, BC,

Canada). Monoubiquitinated PCNA was detected by a

higher molecular weight band (*8–10 kDa) using PCNA

antibody. Infrared IR dye-labelled secondary antibody was

applied to the blot for 1 h at room temperature and then

signals were detected with Odyssey Infrared Imaging

System (LI-COR Biosciences, Lincoln, NE).

Cell cycle analysis

Cell cycle analysis was performed in HCT116 cells using a

protocol previously described [25]. Briefly, cells were

collected by trypsinization and pelleted by centrifugation at

5009g for 5. After overnight fixation, in 70 % ethanol at

4 �C, cell pellets were washed with 19 PBS and then

resuspended in 0.5 ml of FACS buffer (19 PBS with

0.5 mM EDTA and 0.5 % BSA) containing 25 lg/ml of

RNase A and 50 lg/ml of propidium iodide (PI) (Sigma).

After incubating the samples in the dark at room temper-

ature for 15 min, samples were analyzed by EPICS XL-

MCL flow cytometer (Beckman Coulter, Miami, FL) to

determine the percentage of subdiploid DNA. Cells in sub-

G1 phase were regarded as apoptotic cells.

Host-cell-reactivation (HCR) assay

The use of HCR assay to measure the DNA repair capacity

(DRC) has been previously described [26, 27]. The pGL3

control luciferase plasmid (Firefly) was oxidatively

Apoptosis (2014) 19:518–526 519

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damaged in vitro by dilution to 50 mg/ml and exposure to

the indicated concentration of H2O2 (v/v) at room tem-

perature for 1 h. Undamaged control plasmids were treated

with the vehicle solutions without exposure to the dam-

aging agents. After all treatments, the damaged or

undamaged DNA was purified by ethanol precipitation, and

resuspended in autoclaved water. pRL control luciferase

plasmid (Renilla) was used as an internal control for

transfection. To measure the DRC, HCT116 cells were first

transfected with control or Ing1b siRNA and then with

luciferase plasmids as per the standard transfection proce-

dures. 48 h after transfection, luciferase activity was

measured using dual-luciferase reporter assay kit (Pro-

mega, Madison, WI, USA). DRC (%) was calculated as the

ratio of the damaged plasmid luciferase activity to the

undamaged plasmid luciferase activity, multiplied by 100.

Statistics

All data were tested for significance using Student’s

unpaired two-tailed t test and only results with p \ 0.05

were considered statistically significant.

Results

Ing1b protein expression is induced by H2O2 treatment

Previously it was shown that DNA damage due to ultra-

violet radiation (UV-B) induced the expression of Ing1b at

the protein level [28, 29]. UV-B radiation is known to

generate reactive oxygen species (ROS) in the cell and

thereby cause oxidative damage to DNA [30]. H2O2

treatment was recently shown to induce Ing1b mRNA

levels in a variety of cell lines but the authors reported a

disconnection between mRNA and protein levels as there

was no change in Ing1b protein upon H2O2 treatment [12].

We hypothesized that induction of oxidative stress in the

cells with exogenous H2O2 treatment, could induce Ing1b

protein expression during the recovery phase. To test our

hypothesis, we treated the HCT116 cells with a previously

reported concentration of H2O2 (100 lM) for 30 min and

followed the Ing1b protein expression in recovering cells

over the period of 24 h [9]. As shown in Fig. 1a, the

induction in Ing1b protein expression was apparent at 4 h

of recovery time and increased as the time progressed.

Next we treated the cells with varying concentrations of

H2O2 (50–500 lM) for 30 min and measured the Ing1b

expression after 4 h. As seen in Fig. 1b, there was a dose

dependent increase in Ing1b expression from 50 lM to

200 lM followed by a slight decrease at 500 lM H2O2,

possibly because the concentration had cytotoxic effects on

HCT116 cells.

Repair of oxidized DNA is dependent on endogenous

Ing1b expression

ING family proteins have been shown to be involved in

DNA repair and Ing1b has been shown to be involved in

nucleotide excision repair and in the translesion synthesis

(TLS) pathway [14, 23]. Therefore, we studied the DNA

repair in Ing1b knock down HCT116 cells using HCR

assay. The assay is based on measuring the activity of

oxidized luciferase (firefly) plasmid in the cells. As seen in

Fig. 2, HCT116 cells transfected with control siRNA were

able to completely repair the firefly plasmid damaged with

Fig. 1 Oxidative stress induces the protein expression of Ing1b.

a WB showing the time response in HCT116 cells. HCT116 cells

were treated with 100 lM H2O2 for 30 min and then washed with

19 PBS and replaced with fresh medium. Cells were harvested at

indicated time points and the lysates were analyzed by western

blotting. b WB showing the dose response curve in HCT116 cells.

HCT116 cells were treated with indicated concentrations of H2O2 as

described above and harvested after 4 h of recovery and the lysates

were analyzed using western blot

Fig. 2 Ing1b regulates the DNA repair after oxidative stress.

HCT116 cells were transfected with control (open columns) or Ing1b

siRNA (closed columns), and after 8 h, transfected with damaged (1

or 3 % H2O2) or undamaged pGL3 luciferase (firefly) plasmid

together with pRL (renilla) plasmid at 10:1 ratio. 48 h after

transfection, luciferase activity was measured as described in methods

section. *p \ 0.01

520 Apoptosis (2014) 19:518–526

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1 % H2O2 and partly repair the plasmid damaged with 3 %

H2O2, where as the repair in cells transfected with Ing1b

siRNA was significantly blunted.

Ing1b regulates PCNA-ubiquitination upon oxidative

stress

Monoubiquitination of PCNA at K164, a known hallmark

for lesion bypass after UV induced replicative blockade,

has been recently shown to be also involved in DNA repair

after oxidative stress [9, 24]. Previous studies on PCNA-

ubiquitination upon replicative stress, have demonstrated a

regulatory role of Ing1b in the process [23]. We asked if

Ing1b had a similar regulatory role in PCNA ubiquitination

upon oxidative DNA damage. Zlatanou et al., in their

report on ‘oxidative stress induced PCNA monoubiquiti-

nation and its significance’, showed that treatment of cells

with H2O2 for 20 min followed by washing once with PBS

and replacement of medium induced a transient ubiquiti-

nation of PCNA. We reproduced the results from the study

by Zlatanou et al., in our cell line and in our laboratory

conditions [9]. As shown in Fig. 3a, H2O2 treatment led to

a transient ubiquitination of PCNA, a result consistent with

Fig. 3 a Time course of PCNA monoubiquitination. HCT116 cells were

treated with 100 lM H2O2 for 30 min and medium was replaced after

washing thecellswith19 PBS.Cellswere harvestedat indicated timepoints

and the soluble and chromatin fractions were separated as described in

‘‘methods’’ section. The proteins were analyzed using western blotting.

b Concentration response of PCNA ubiquitination. HCT116 cells were

treated as described above with indicated concentrations of H2O2 for 30 min

and cells were immediately harvested, and the proteins in chromatin fraction

were analyzed using western blotting. We do not know the origin of the

bands slightly below the monoubiquitinated PCNA band, which were of

relatively low intensity (indicated by asterisk), but we consider them related

to PCNA

Fig. 4 Oxidative stress induced ubiquitination of PCNA is dependent on

Ing1b. a Decreased PCNA ubiquitination in Ing1b knock down cells.

HCT116 cells were transfected with control or Ing1b siRNA. 48 h after

transfection, the cells were treated with 100 lM H2O2 for 30 min and the

cells were harvested at indicated time points. The whole cell extract (WCE),

soluble and chromatin fractions were isolated and analyzed by western

blotting. Asterisk-occasionally (but not always) we observed a band below

the actual Ing1b band, which we think was an unknown protein which cross

reacts with the Ing1b antibody we used. b PCNA fractionation is augmented

in Ing1b over expressing cells. HCT116 cells were transfected with empty

vector or 3XFlag Ing1b plasmid. 24 h after transfection, the cells were

treated with H2O2 as described above and analyzed by western blotting

Apoptosis (2014) 19:518–526 521

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the published report. We also found that the effect was

visibly clear at a dose of 100 lM (Fig. 3b). Next we con-

tinued our studies on the oxidative stress response in

control and Ing1b knock down cells. Weak LAMP2 bands

and strong ORC2 bands in the chromatin fraction were

taken as positive controls for the separation of cellular

chromatin and soluble fraction, and as seen in Fig. 4, our

fractionation experiment was indeed of good quality. As

illustrated in Fig. 4a, knock down of Ing1b caused an

obvious reduction of oxidative stress induced PCNA

monoubiquitination. Ubiquitinated PCNA is expected to be

concentrated in the chromatin fraction and accordingly we

did not see any bands in the soluble fraction (Fig. 4). Then

we confirmed the role of Ing1b by performing the experi-

ments in Ing1b over expressing cells. As shown in Fig. 4b,

ectopic over expression of 3Xflag Ing1b in HCT116 cells

lead to an enhanced PCNA monoubiquitination upon oxi-

dative stress. Interestingly our experiments did not identify

any apparent change in XRCC1 expression or chromatin

loading upon oxidative stress (Figs. 3a, 4).

Ing1b reduces the cytotoxic effects of oxidative stress

Failure to repair the damaged DNA is known to cause cell

death and therefore we tested cell survival in Ing1b knock

down HCT116 cells in order to see if defective DNA repair

in Ing1b knock down cells would lead to increase in cel-

lular apoptosis. Accordingly, cells lacking Ing1b were

more sensitive to H2O2 induced death as evidenced by

higher percentage of sub-G1 cells. As seen in Fig. 5, cells

transfected with siControl seemed to tolerate H2O2 (100

and 200 lM) and did not show any increase in sub-G1

population after treatment, while Ing1b knock down cells

showed a significant increase in sub-G1 percentage after

treatment with H2O2 .

Next we tested if over expression of Ing1b would protect

the cells from the cytotoxic effects of oxidative stress. As

shown in Fig. 6, Ing1b over expressing cells had signifi-

cantly lesser cell death at both 100 and 200 lM concen-

trations of H2O2 indicating the protective effect of Ing1b.

However, the cell death was similar at 500 lM H2O2

probably because the concentration was too toxic and

activated alternate cytotoxic pathways. Interestingly, cells

in control group transfected with empty vector had appar-

ently higher cell death as compared to control group

transfected with control siRNA (Figs. 5b, 6b). We think

that the difference was due to the reagents used for trans-

fection and in our experience ‘silentfect’ (used for siRNA)

Fig. 5 Ing1b knock down augments the cell death after oxidative

stress. HCT116 cells were transfected with control or Ing1b siRNA.

48 h after transfection, the cells were treated with H2O2 (0–500 lM)

for 30 min and medium was replaced after washing the cells with

19 PBS. The cells were trypsinized and collected, 24 h after

treatment and cells were fixed in 70 % ethanol. Cell cycle was then

analyzed using flow cytometry. a Representative histograms showing

the propidium iodide fluorescence (PI-fluorescence) and b mean of 3

independent experiments

b

522 Apoptosis (2014) 19:518–526

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was comparatively mild, where as ‘effectene’, which was

used for over expression, was relatively strong. There was,

however, very little variation within the experiments, and

the differences between the respective groups reached

statistical significance (p \ 0.05, Students t test).

Histone deacetylase (HDAC) inhibitor pretreatment

rescues the effects of Ing1b knock down

Inhibition of histone deacetylation has been shown to res-

cue the UV induced cell death in Ing1b knock down cells

and Ing1b was reported to act by facilitating histone acet-

ylation [14, 23]. To check if a similar mechanism was

involved in oxidative stress response, we treated the Ing1b

knock down cells with Trichostatic acid (TSA, 5 ng/ml)

[23], a known HDAC inhibitor one hour prior to, during

and following the exposure to H2O2 (200 lM). We choose

200 micromolar concentration of H2O2 because the dif-

ference between siControl and siIng1b was apparent at this

concentration and more importantly the concentration was

not too toxic for the cells. As illustrated in Fig. 7, treatment

with HDAC inhibitor abrogated the cell death in Ing1b

knock down cells and dissipated the differences in per-

centage of cells arrested in G2/M phase after H2O2

treatment.

Discussion

The role of Ing1b in the maintenance of genomic stability

upon DNA lesions caused by ultra violet radiation is well

documented [11–13, 16, 23], but its role in regulation of

oxidative stress induced DNA damage has not been well

characterized. Our paper demonstrates the novel role of

Ing1b in the base excision repair pathway. Ultra violet

radiation was shown to cause phosphorylation of Ing1b and

thereby lead to increased protein levels of Ing1b through

Ataxia Telangiectasia and Rad3 related (ATR) kinase and

Chk1 signaling [28, 29]. Recently it was shown that H2O2

treatment triggered the activation of Chk1 in an ATR

dependent but ATM independent manner [10]. It is possi-

ble that activation of Chk1 during oxidative stress could

cause a similar increase in cellular Ing1b protein levels as

seen with the UV-induced stress [29]. Our results on

induction of Ing1b expression at protein level are contrary

to the findings in a previous study, which reported an

increase in Ing1b mRNA levels but no change in protein

levels [12]. However, we think that there is a conspicuous

difference between our study and previous study by Ceruti

et al., regarding the protocol used to induce oxidative

stress. While in the previous study, the authors induced

oxidative stress in the cells by adding H2O2 to the medium,

Fig. 6 Ing1b over expression abrogates the oxidative stress induced

cell death. HCT116 cells were transfected with empty vector or

3XFlag Ing1b plasmid. 24 h after transfection, the cells were treated

with H2O2 (0–500 lM) for 30 min and medium was replaced after

washing the cells with 19 PBS. 24 h after treatment, cells were

collected and fixed as described in Fig. 5, and analyzed using flow

cytometry. a Representative histograms showing the propidium

iodide fluorescence (PI-fluorescence) and b mean of 3 independent

experiments

Apoptosis (2014) 19:518–526 523

123

which suggests that the cells were treated continuously, we

treated the cells with H2O2 only for 30 min and replaced

the medium [12]. We think that treatment protocol used by

Ceruti et al. was a bit strong and was the reason for the

observed difference in findings. We did not proceed with

elucidating the mechanisms involved in the induction of

Ing1b protein expression as the objective of this study was

primarily to explore the role of Ing1b in repair of DNA

damaged due to oxidative stress. Therefore we aimed to

study the cells upon knock down of endogenous Ing1b or

upon ectopic expression of Ing1b.

Monoubiquitination of PCNA, which is a critical event in

the signaling of translesion synthesis of DNA, was recently

shown to have equal importance in base excision repair [9,

24]. We were able to reproduce the events demonstrated

previously by Zlatanou et al., and were further able to elu-

cidate the significance of Ing1b in the PCNA ubiquitintion.

Interestingly, we did not see any apparent changes in

XRCC1 chromatin loading after treatment with H2O2. We

think that our experimental conditions possibly required the

‘‘long patch pathway’’ which involves PCNA and therefore

we did not see any changes in XRCC1, which regulates

‘‘short patch pathway’’ [8]. Our results show that cells

lacking Ing1b had decreased PCNA ubiqutination and on

other hand, over expression of Ing1b lead to increased levels

of ubiquinated PCNA, clearly demonstrating the role of

Ing1b. Insufficient PCNA ubiquitination could lead to

defective recruitment of repair factors on the sites of lesions

and thereby could lead to incomplete repair of the damaged

DNA. The immediate effect of persistent DNA lesions in the

cell would be genomic instability and activation of apoptotic

cascade [23]. Along those lines, Ing1b knock down cells

showed defective DNA repair as well as increased cell death.

Inhibitor of Growth (ING) family proteins are known mainly

to act by facilitating histone acetylation by histone acetyl-

transferases [11, 12, 23]. Acetylation leads to neutralization

of the positive charges on histones, which are required to

hold the negatively charged DNA base pairs [31–33]. His-

tone acetylation would thus lead to unwinding of DNA and

cause chromatin relaxation, which is a pre-requisite for the

repair factors to gain access to DNA lesions and to take part

in DNA repair [32, 33]. The system is restored to normal by

the activity of histone deacetylases (HDAC), which remove

the acetyl groups from histone molecules [34]. Inhibition of

HDAC activity would lead to continued presence of acety-

lated histones and to a relaxed and more accessible DNA.

HDAC inhibitor treatment of the Ing1b knock down cells

might have lead to chromatin relaxation and allowed the

Fig. 7 Pretreatment with HDAC inhibitors rescues Ing1b KD cells

from oxidative stress induced cell death. HCT116 cells were

transfected with control or Ing1b siRNA. 48 h after transfection, the

cells were treated with trichostatic acid (TSA, 5 ng/ml) 1 h prior to

and following treatment with H2O2 (200 lM) for 30 min and medium

was replaced after washing the cells with 19 PBS. 24 h after

treatment, cells were collected and fixed as described in Fig. 5, and

analyzed using flow cytometry. a Representative histograms showing

the propidium iodide fluorescence (PI-fluorescence) and b mean of 3

independent experiments

b

524 Apoptosis (2014) 19:518–526

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activation of DNA repair pathways similar to the Ing1b

positive cells. Understandably, HDAC inhibitor treatment

lead to comparable cell death in control and Ing1b knock

down cells. Oxidative stress is known to cause cell cycle

arrest in G2/M phase [35] and our results showed that there

was a clear increase in G2/M population of cells at a dose of

200 lM and above. Interestingly, treatment with HDAC

inhibitors had more pronounced effects on the G2/M arrest

as seen by slight increase in G2/M percentage in control cells

and more importantly, by the virtual dissipation of difference

in percentage of cells in G2/M phase after H2O2 treatment.

Possibly, HDAC inhibition lead to a more complete activa-

tion of DNA repair pathways and enhanced the G2/M arrest

after oxidative stress. Our results are in agreement with the

previous study which reported that TSA treatment rescued

the Ing1b lacking cells from UV-C induced cytotoxicity

[23], but seem to contradict the results from the study by

Ceruti et al. which showed that TSA treatment exacerbated

the H2O2 induced DNA damage [12]. We think that our

experimental design differed from the study by Ceruti et al.,

and the dose (100 nM TSA to *16.5 nM)) and pretreatment

time (5 vs 1 h) used by them were relatively high [12]. We

speculate that treatment with higher concentration of TSA,

and for longer time led to greater degree of chromatin

relaxation in the cells prior to and during treatment with

H2O2, allowing for a greater degree of DNA damage. As

pointed earlier, we treated the cells only for 30 min, whereas

the report by Ceruti et al. indicates a continuous treatment

with H2O2, which could be another reason for difference in

the findings.

We think that the upregulation of Ing1b protein levels is

interesting feature of our findings as we see the effect at 4 h

of recovery after exposure to H2O2. The kinetics of the repair

mechanisms shown by previous reports and our studies,

showed that the presence of Ing1b was essential at very initial

stages (at time T = 0 h) [9]. The protein upregulation was

thus not part of the DNA repair mechanism and was sort of an

additional effect of the pathway. However, its significance

need not be underestimated because Ing1b is known to cause

cell cycle arrest and induce cellular senescence [13, 17].

Oxidative stress is also known to induce senescence in cells

and it would be interesting to study the role Ing1b in this

process [2, 26]. We speculate that cells lacking Ing1b would

have more initial cell death under stress conditions, but

would have more oncogenic mutations and lesser activation

of senescence which is considered as a delayed effect of

oxidative stress. Further, we believe that the mechanisms

regulating the cellular handling of intracellular reactive

oxygen species (ROS) are particularly important for cells

where intracellular ROS acts as a second messenger [36–38].

We think it would be interesting to study role of Ing1b in

survival of immune cells, where activated cells need to tol-

erate the intracellular ROS.

To summarize our paper describes an upregulation of

Ing1b in cells exposed to oxidative stress, elucidates the

critical role played by Ing1b in the repair of oxidized DNA

and demonstrates the decreased survival in cells lacking

Ing1b.

Acknowledgments We thank Dr Ronald P C Wong for the valuable

discussions during the course of the project. This project was sup-

ported by grants from Canadian Institute of Health Research (MOP-

93810).

Conflict of interest The authors declared that they have no conflict

of interest.

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