Karin Barnouin Marlene L. Dubuisson , René H. Medema ... · 1 H 2 O 2 induces a transient...

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H2O2 induces a transient multi-phase cell cycle arrest in mouse fibroblasts through

modulating cyclin D and p21Cip1 expression.

Karin Barnouin 3, 4, 7 Marlene L. Dubuisson2, 7, 8, Emma S. Child5, Silvia Fernandez

de Mattos1, 2 Janet Glassford1, 2, René H. Medema6, David J. Mann5, and

Eric W.-F. Lam 1, 2, 9

1 CRC labs and Section of Cancer Cell Biology, Imperial College School of Medicine at

Hammersmith Hospital, Du Cane Road, London W12 ONN, UK2 Ludwig Institute for Cancer Research and Section of Virology and Cell Biology,

Imperial College School of Medicine at St Mary's, Norfolk Place, London W2 1PG, UK.3 Department of Biochemistry and Molecular Biology, University College London,

Gower Street, London, WC1E 6BT, UK4 Ludwig Institute for Cancer Research, 91 Riding House Street, London, W1 7BS, UK.5Department of Biological Sciences, Imperial College of Science, Technology and

Medicine, London SW7 2AY, UK6Department of Molecular Biology H8, Netherlands Cancer Institute, Plesmanlaan 121,

1066 CX Amsterdam, The Netherlands.7Contributed equally to the study and should be considered joint first authors8Present address: Animal Biology Unit, Catholic University of Louvain, Place Croix

du Sud, 5, B-1348 Louvain-la-Neuve, Belgium9 Correspondence: Eric Lam, CRC labs and Section of Cancer Cell Biology, Imperial

College School of Medicine at Hammersmith Hospital, Du Cane Road, London W12

ONN, UK Telephone: 44-20-8383-5829 Fax: 44-20-8383-5830; e-mail:

[email protected]

Running title: H2O2 induces multi-phase transient cell cycle arrest

Keywords: H2O2/ p21Cip1/ cyclin D/fibroblasts/ cell cycle arrest

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on February 4, 2002 as Manuscript M111123200 by guest on A

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Abstract

To defend against the potential damages induced by reactive oxygen species(ROS),

proliferating cells enter a transient cell cycle arrest. To elucidate the mechanism by which

this cell cycle arrest is mediated, we treated mouse fibroblasts with H2O2. The result

showed that sublethal doses of H2O2 induced a transient multi-phase cell cycle arrest at

G1, S, G2, but not M, phases. Western blot analysis demonstrated that this transient cell

cycle arrest is associated with the down-regulation of cyclin D1 and -D3 and up-

regulation of the CKI p21Cip1 expression. We also demonstrate that the induction in

p21Cip1 expression by H2O2 is at least partially mediated at the transcriptional level and

can occur in the absence of p53 function. Further immunoprecipitation-kinase and

immunodepletion assays indicated that in response to H2O2 treatment, the down-

regulation of cyclin Ds expression are associated with repression of cyclin D-CDK4,

while the accumulation of p21Cip1 is responsible for the inhibition of cyclin E and A-

CDK2 activity and associated with the down-regulation of cyclin B-CDC2 activity. These

data could account for the cell cycle arrest at G1, S, and G2 phases following H2O2

stimulation. However, using p21 null MEFs and cyclin-inducible NIH 3T3 cell lines, we

showed that deletion of p21Cip1, restoration of cyclin D expression, or over-expression of

cyclin E alone is insufficient to effectively overcome the cell cycle arrest caused by sub-

lethal doses of H2O2. By contrast, over-expression of the Human Herpesvirus 8 K cyclin,

which can mimic the function of cyclin D and E, is enough to override this transient cell

cycle arrest. On the basis of our findings, we propose a model in which moderate levels

of H2O2 induce a transient multi-phase cell cycle arrest at least partially through up-

regulation of p21Cip1 and down-regulation of cyclin D expression.

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INTRODUCTION

Reactive oxygen species (ROS), including superoxide anion (O2.-), hydrogen

peroxide (H202), and hydroxyl radicals (HO.) are natural by-products generated by living

organisms as a consequence of aerobic metabolism. (1-3). Accumulation of excess ROS,

which are toxic to cells, can cause oxidative stress leading to damage to proteins, nucleic

acid, and cell membranes (4,5). Oxidative stress has also been implicated in a variety of

human diseases, including atherosclerosis, pulmonary fibrosis, cancer, neurodegenerative

diseases, and ageing (2,3,6). To protect against the potentially detrimental effects caused

by elevated levels of ROS, cells deploy antioxidant defences and activate damage

removal, repair and replacement systems. To counter oxidative stress, cells constitutively

express enzymes that neutralise ROS and repair and replace the damage caused by ROS

(4,5). In addition, cells also mount ‘adaptive responses’ to elevated levels of oxidative

stress. Mammalian cells respond to oxidative stress with an increase in expression of

antioxidant enzymes, including glutathione-S-transferases, peroxidases, superoxide

dismutases and activation of protective genes, including those encoding the heat shock

proteins. Stationary phase (non-cycling) cells are intrinsically resistant to high levels of

ROS, whereas actively dividing cells are prone to oxidative damage, because their DNA

is uncoiled and exposed while they engage in rapid DNA replication. To defend against

the potential damages induced by oxidative stress, proliferating cells enter a transient cell

cycle arrest, during which DNA is protected by histone proteins, energy is conserved

through reduced expression of non-essential genes, and the expression of shock and stress

proteins is increased. This transient growth arrest also allows time to repair and/or replace


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the damaged DNA so that the mutated DNA will not be replicated and/or transferred to

daughter cells. This also gives the cells extra time to mount an “adaptive response” to

counteract further oxidative damage. Thus, this transient growth arrest is a crucial

component of the cellular response to oxidative stress (7,8).

However, besides transient cell cycle arrest, the cell also exhibits a wide range of

adaptive cellular responses ranging from transient growth arrest, to permanent growth

arrest, to apoptosis and ultimately to necrosis, depending on the level of oxidative stress

experienced (7,8). Sub-lethal levels of ROS induce a temporary cell cycle arrest which is

believed to protect cells from DNA damage itself, consuming excess energy and

resources, and incorporating mutations following DNA damages. Following repair or

detoxication, the dividing cells reinitiate cell cycle progression. However, when the

oxidative stress is too severe and the cells do not have the ability to adapt or resist the

stress or to repair the damaged cellular components, the cells may respond by undergoing

permanent cell cycle arrest or apoptosis. At even higher levels of ROS, cells undergo cell

death by necrosis. Nevertheless, recent emerging evidence also demonstrates that ROS

are physiological mediators of cell signalling and functions and are produced by a variety

of cells after stimulation with cytokines, peptide growth factors, and agonists of receptors

(9-11). For example, cytosolic ROS produced in response to stimulation by growth

factors are involved mediating the proliferative response (11). Therefore, depending on

the level, ROS exerts two physiological effects: damage to various cell components and

activation of specific signalling pathways.


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The mammalian cell division cycle is traditionally divided into G1(Gap-phase 1),

S(DNA synthesis), G2(Gap-phase 2), and M(Mitosis). Progression through each phase of

the cell cycle is controlled by co-operative activity of distinct cyclin-dependent kinases

(CDKs) and their regulatory subunits, cyclins (12). The cyclins have specificity for

different CDK subunits. The D-type cyclins (cyclin D1, D2 and D3) bind to and activate

CDK4 and CDK6 preferentially, while cyclin E interacts predominantly with CDK2,

cyclin A associates primarily with CDK2 and CDC2 (12-14) and cyclin B specifically

with CDC2 (also called CDK1) (12,15,16). The association of CDK4 or 6 with D-type

cyclins is important for G1 phase progression, while the CDK2-cyclin E complex is

essential for initiation of S phase. Progression through S phase is regulated by the CDK2-

cyclin A complex, while the transition from G2 to M is mediated by CDC2–cyclin B. The

principal cellular substrates of the cyclin-CDKs are members of the retinoblastoma

protein (pRB) family of pocket proteins (pRB, p107 and p130) (17-19). In their

hypophosphorylated forms, these pocket proteins bind to members of the E2F family of

transcription factors, thereby negatively regulating transcription of E2F-dependent genes

that are required for entry into and transition through S phase of the cell cycle (17,20-22).

During G1 to S transition, phosphorylation of pRB is initiated by cyclin D-dependent

kinases and is completed by cyclin E-CDK2 and cyclin A-CDK2 (12,13). CDKs are

negatively regulated by two classes of CDK-inhibitors (CKIs), the CIP/KIP and the INK4

families of proteins. The CIP/KIP proteins (p21Cip1, p27 Kip1 and p57 Kip2) target both

cyclin D-CDK4/6 and cyclin E/A-CDK2 by binding to the cyclin/CDK complexes, while

the INK4 family (p15 INK4b, p16INK4a, p18INK4c and p19INK4d) specifically inhibit cyclin D-

CDK4/6 complexes through direct association with the CDK components, thereby


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preventing their interaction with D-type cyclins (12,13). The CIP/KIP proteins are

inhibitors of cyclin E- and A-dependent CDK2, but p21Cip1 (more than p27Kip1) at low

stoichiometric levels act as positive regulators of cyclin D-dependent CDK4/6 kinases

(23,24).

An understanding of the cellular responses to oxidative stress will provide useful

insights into the mechanisms of ageing and transformation as well as the pathogenesis of

a variety of ageing-related diseases. The cellular responses to oxidative stress, in

particular those in response to sub-lethal doses of ROS, has not been thoroughly

characterised. Several recent studies using human diploid fibroblasts showed that H202

induced a permanent G1 growth arrest, which is phenotypically similar to replicative

senescence (25-27). In this study, we investigated the nature of the transient cell cycle

arrest induced by H202, as a source of ROS, in mouse fibroblasts and go on to explore the

mechanisms by which H202 induced this proliferation arrest.


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Experimental Procedures

Fibroblasts, MEF isolation and cell cultures

Wild-type mice and mice with deletion of p21Cip1 (28) genes were maintained at the

animal facilities of the Imperial College, UK. Primary mouse embryo fibroblasts (MEFs)

were isolated from day 13.5 embryos derived form the corresponding colonies of wild-

type or gene ‘knock-out’ mice as described previously (29). Briefly, each embryo was

dispersed and trypsinised for 20 min at 37°C and the resulting cells were grown for 1 day

in a 10 cm diameter tissue culture plate. After which, the cells were replated onto a 15 cm

dish and allowed to grow for 2 days. These cells, designated passage number 0 cells,

were stored in liquid nitrogen for later use. The MEFs were cultured and passaged as

described previously (30). Briefly, 106 cells were replated every three days onto 100 mm

plates. NIH 3T3, Swiss 3T3, and p21Cip1-null (31) fibroblasts were cultured in Dulbecco's

modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum and

penicillin/streptomycin. The NIH 3T3-D1, NIH 3T3-E and NIH 3T3-K cell lines have

been described previously (32), and exogenous cyclin expression was induced by

incubation with 3 mM isopropyl beta-D-thiogalactoside (IPTG; Sigma, UK) for 24 h.

Hydrogen peroxide (H202) was purchased from Sigma, UK (H-1009, 30% w/w solution)

and was administered to the cells as a 10X solution in growth medium. For H202

treatment, fibroblasts were grown to 60% confluence and the tissue culture medium

changed before addition of H202.


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Cell synchronisation

For synchronising cells at G0/G1 phase by serum deprivation (33), SWISS 3T3

fibroblasts were incubated in DMEM with 0.5% FCS for 48 h, before 10% FCS was

added to induce the cells to re-enter the cell cycle. For synchronisation at G1/S transition,

NIH 3T3 cells were first incubated with thymidine (25 mM, Sigma UK) for 16 h,

released into the cell cycle for 12 h after removal of thymidine, and then treated again

with thymidine for another 16 h. After the second block, thymidine was washed away and

replaced with culture medium. Typically, 65%-75% of cells re-entered the cell cycle and

progressed into S phase after the double thymidine block. To block cells at the

metaphase/anaphase of M, cells were treated with nocodazole (50ng/ml; Sigma, UK) for

16 h. The rounded-up cells were detached by ‘mechanical shake-off’, washed, and

resuspended in nocodazole-free culture medium for them to re-enter the cell cycle.

Cell cycle analysis

Cell cycle analysis was performed by combined propidium iodide (PI) and

bromodeoxyuridine (BrdU) staining. Subconfluent fibroblasts with or without H2O2

treatment were incubated for 30 min with 10 µM bromodeoxyuridine (BrdU; Sigma,

UK). Cells were trypsinised, collected by centrifugation, and resuspended in PBS, before

fixing in 90% ethanol. The fixed cells were incubated first with 2M HCl, then with 0.5%

Triton X-100 for 30 min at room temperature, and then with fluorescein isothiocyanate

(FITC)–conjugated anti-BrdU antibodies (BD Biosciences, UK) at 1:3 dilution for 30

min, with PBS washes between each treatments. The cells were incubated with 5µg/ml


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propidium iodide (Sigma, UK), 0.1 mg/ml RNAse A (Sigma, UK), 0.1% NP-40, and

0.1% trisodium citrate for 30 min prior to analysis using a Becton Dickinson FACSort

analyser. The cell cycle profile was analysed using the Cell Quest software.

Western blot analysis and antibodies

Western blot cell extracts were prepared by lysing cells with 3 times packed cell

volume of lysis buffer (20mM HEPES pH7.9, 150mM NaCl, 1mM MgCl2, 5mM EDTA

(pH.8.0), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50mM NaF, 5mM

sodium orthovanadate) on ice for 20 min. Protein yield was quantified by Bio-Rad Dc

protein assay kit (Bio-Rad). Samples corresponding to 50µg of lysates were separated by

SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and

recognised by appropriate antibodies. The antibodies against p21Cip1 (M-19), p27Kip1 (C-

19), CDK4 (C-22), CDK6 (C-21), CDK2 (M-2), CDC2 (C-19), cyclin D1 (HD11), cyclin

D1-3 (H-295), cyclin A (C-19), cyclin E (M-20), and cyclin B1 (M-433) were purchased

from Santa Cruz Biotechnology. Anti-p27Kip1 (K25020) and anti-p21Cip1 (F-5) monoclonal

antibodies were acquired from Transduction Laboratories and from Santa Cruz

Biotechnology, respectively. The exogenous cyclins in the inducible NIH 3T3-D1, NIH

3T3-E, and NIH 3T3-K cell lines were detected by antibodies against Flag (M2 from

Sigma, UK), human cyclin E (HE12 from Santa Cruz) and HA (12CA5 from Roche,

respectively. Cyclin D1 was Flag-tagged, the K cyclin was HA-tagged, and the human

cyclin E can be distinguished from the endogenous mouse protein by the use of an

antibody specific for human cyclin E. The anti-alpha tubulin, monoclonal TAT-1 has

been described previously (32). The primary antibodies were detected using horseradish


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peroxidase-linked goat anti-mouse or anti-rabbit IgG (Dako) and visualised by the

enhanced chemiluminescent (ECL) detection system (Amersham Pharmacia Biotech,

UK).

Kinase assays, immunoprecipitation and immunodepletion

For kinase assays, cells collected were washed with PBS and lysed in lysis buffer

containing 50 mM Hepes/NaOH pH 7.4, 150 mM NaCl, 20 mM EDTA, 0.5 % Triton-X

100, 10 mM β-gycerophosphate, 1 mM PMSF, 27 TIU/ml aprotinin, 2.5 µg /ml

leupeptin, 2 mM DTT, 10 mM NaF, 2 mM Na3VO4. 200 µg protein lysate was

immunoprecipitated with 1 - 2 µg of antibody (CDC2, CDK4, cyclin E or cyclin A- for

CDK2 immunoprecipitation, 100 µg protein and 1 µg of antibody was used) and protein

sepharose G beads (Pharmacia) (50% v/v) overnight at 4 0C. The G beads were washed

twice with lysis buffer and once with assay buffer (50 mM Hepes/NaOH pH 7.4, 10 mM

MgCl2, 10 mM MnCl2, 1 mM DTT, 10 mM β-gycerophosphate, 0.1 µM PKA inhibitor).

The kinase assay was performed with 500 ng histone H1/assay or C-term GST-Rb (C-

Term 792-928) (34) as substrate, 20 µM ATP, and 0.1 µCi γ-[32P]ATP (3000 Ci/mmol;

Amersham) in 20 µl assay buffer. The samples were then incubated for 30 minutes at 37 0

C. The reaction was terminated with the addition of 50 µl sample buffer and boiling for 3

minutes. Samples were resolved on a 12% gel (15 µl per 15 well mini gel). The gel was

then fixed and coomassie stained, dried and exposed to a phosphoimager screen.

Immunoprecipitation was performed as described for the kinase assay. For

immunodepletion experiments, two extra immunoprecipitations were performed with


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either the anti-p21Cip1 or the anti-p27Kip1 monoclonal antibody cross-linked to protein G

beads with the dimethylpimelidate method (35). The subsequent immunodepleted

supernatants were then analysed by Western Blotting.

Immunofluorescence and Mitotic index assay

Cells cultured on coverslips were treated with or without 250 µM H2O2 for 4

hours. Cells were washed with PBS and fixed with 4 % formaldehyde, and then washed

and permeablised for 5 minutes with 0.5% v/v Triton/PBS. The cells were then incubated

for 1 h to overnight with monoclonal antibodies against p21Cip1 or p27Kip1, washed and

incubated with goat-anti-mouse secondary-FITC conjugated antibodies (Molecular

Bioprobes) to visualise the staining.

For mitotic index assay, exponentially growing fibroblasts were cultured on

coverslips and synchronised by double thymidine block and release. After entering G2/M

phase, these cells were treated with 100ng/ml nocodazole (to trap cells that had

progressed through G2 into mitosis) in the presence or absence of 250 µM H2O2. Cells

were fixed and stained as above except 20 µg/ml of 4’, 6-diamidino-2-phenylindole

(DAPI; Sigma, UK) was added with the secondary antibody for 1 hour to visualise the

DNA. The nuclear morphology of the cells was analysed by fluorescence microscopy.

The number of cells displaying condensed chromosome morphology was counted and

scored as mitotic and expressed as a percentage of the total.


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Northern Blot Analysis

Total RNA was isolated using the RNeasy Kit (Qiagen, UK) and quantified by

absorbance at 260nm. Twenty µg of RNA, prepared as above, was resolved on 1.5%

formaldehyde-agarose gels. Following electrophoresis, RNA were transferred to Hybond-

N membrane (Amersham Pharmacia Biotech, UK) and subjected to Northern blotting as

previously described (33). p21Cip1 mRNA was detected by hybridization with its full-

length 32P-labelled mouse cDNA probe (36) kindly provided by Dr. B. Vogelstein, Johns

Hopkins University School of Medicine, Baltimore, USA. Actin mRNA was detected by

a β-actin probe (37).

Transfections and gene reporter assays

Transfection of NIH3T3 cells were performed using the calcium phosphate co-

precipitation method as described previously (33). Briefly, calcium phosphate

precipitates containing 10 µg of the wild type mouse p21Cip1 promoter-luciferase reporter

plasmid (pGL3b-4542) (38) together with 2 µg of a ß-galactosidase transfection control

plasmid (pJ4Ωß-gal) (33) were incubated overnight with cycling 5 x 106 NIH3T3 cells in

a 10 cm dish. The transfected NIH3T3 were then washed, treated with H2O2 and

harvested for luciferase and ß-galactosidase assays, as described previously (33).


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RESULTS

Sub-lethal doses of H2O2 induce a transient cell cycle arrest in NIH 3T3 fibroblasts

Previous studies showed that sub-lethal dose of H2O2 induced senescence-like permanent

G1 cell cycle arrest in human fibroblasts 48 h following stimulation (26,27). To

investigate the more imminent and short term effects of H2O2 on cell cycle progression,

we treated NIH 3T3 fibroblasts with sub-lethal doses (100 µM to 500 µM) of H2O2 and

discovered that H2O2 caused cells to transiently arrest progression through the cell cycle

(data not shown). Initial examination of the propidium iodide staining alone indicated that

there was no significant change in cell cycle distribution after H2O2 treatment. However,

more detailed analysis of BrdU incorporation demonstrated a dramatic decrease in DNA

synthesis detectable as early as 2 h following the addition of 250 µM of H2O2 (Fig. 1).

DNA synthesis was almost completely abolished between 4-8 h following H2O2

treatment, after which the cells were temporarily delayed in the G2/M phases. At 48 h,

cells attained a more normal cell cycle profile although proportionately more cells were

found in G1 phase, perhaps reflecting the build up of senescent-like cells previously

reported (26). Notably, there was no apparent change in the proportion of cells in G1, S

and G2/M phases of the cell cycle accompanying the transient arrest in DNA synthesis

induced by the H2O2 treatment. This indicated that the H2O2 induced growth arrest was not

confined only to S phase and also involved other phases of the cell cycle. Taken together,

these findings suggested that sub-lethal doses of H2O2 induced a rapid but transient multi-

phase cell cycle arrest in mouse fibroblasts. To demonstrate that this effect was not only


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restricted to the NIH3T3 cell line, we also examined the effect of H2O2 on the

proliferation of low passage (<2 passages) mouse embryo fibroblasts (MEFs) and found

that H2O2 also induced temporary cell cycle arrest in normal primary cells (see Fig. 8).

H2O2 triggers a cell cycle arrest at G1, S and early G2/M phases in SWISS 3T3

fibroblasts released from a G0 block

To confirm this hypothesis and investigate the nature of this multi-phase cell cycle arrest,

we tested the ability of H2O2 to arrest fibroblasts at different phases of the cell cycle. In

order to synchronise cells at different cell cycle phases, Swiss 3T3 fibroblasts were first

arrested at G0 by serum starvation and then stimulated to re-enter the cell cycle by

reintroduction of serum (Fig. 2). After serum-stimulation, the fibroblasts traversed late

G1, early S, G2/M phases of the cell cycle at 12, 16, 24 h, respectively. At 28 h following

serum stimulation, the majority of the cells re-entered G1 from G2/M (Fig. 2A). To study

the effects of H2O2 on different cell cycle phases, these serum-stimulated cells were either

untreated or treated with 250 µM H2O2 and harvested 4 h later for cell cycle analysis.

H2O2 prevented the fibroblasts from progressing further through the cell cycle when

added to cells at G1 and S phases (12, 16, and 20h post-serum stimulation) (Fig. 2A). The

results showed that when H2O2 was added to the serum stimulated cells at 24 h, a

substantial number of the cells continued to progress into G1 from G2/M (Fig. 2A). This

indicated that most G2/M cells were insensitive to H2O2 induced cell cycle arrest. In a

second independent experiment (Fig. 2B), the number of cells entering G1 following

treatment with H2O2 at 24 h post-serum stimulation was significantly smaller than their

untreated counterparts. This is likely to reflect the fact that the majority of the cells used


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in Fig. 1A were slightly further advanced in the cell cycle than those in Fig. 2B and were

in M phase rather than G2 (see below). Since some of the cells in G2/M did progress into

G1 as illustrated in Fig. 2A, this cell cycle block was unlikely to be late in M. These

observations indicated that H2O2 induced growth arrest in cells at G1, S and early part of

G2/M phases.

H2O2 triggers NIH 3T3 cells to arrest at G1, S and early G2/M, but not M, phases

We next tested the effects of H2O2 on NIH 3T3 fibroblasts released from a nocodazole

induced M phase block (Fig. 3). The results showed that pre-treatment with 250 µM H2O2

failed to prevent the fibroblasts from progressing from M to G1 phase as both the

untreated and H2O2 treated cells escaped into G1 at similar kinetics after released from M.

It was also found that H2O2 only became effective in imposing growth arrest once the

cells progressed from M into G1 and S phases of the cell cycle. Consistent with earlier

results, these results confirmed that sub-lethal doses of H2O2 induced a rapid but transient

growth arrest at G1, S and early G2/M phases.

H2O2 blocks cells at G2 but not M phase

To determine precisely where in G2/M phase H2O2 induces this transient cell cycle arrest,

we examined whether H2O2 treatment could block cells from progressing from G2 into M

phase. To achieve this, we first arrested NIH 3T3 cells at the G1/S boundary using a

double thymidine cell cycle block and then released them to progress through the cell

cycle (Fig. 4). The majority of these synchronised cells acquired a 4N DNA content and

thus reached G2/M at 6 h following release from the G1/S block. It was notable that a


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proportion of the cells did not leave G1/S after release from the second thymidine block.

However, these cells never re-entered the cell cycle and consequently, did not interfere

with the G2/M synchronised population of cells. The cells traversing G2/M, at 6 h after

release from G1/S, were subjected to nocodazole treatment in the presence or absence of

250 µM H2O2. Nocodazole (50ng/ml) was added to trap cells at metaphase to allow the

analysis of the percentage of cells that had progressed from G2 into mitosis. At 4 h after

addition of nocodazole in the presence or absence of H2O2, cells were fixed and the

percentage of mitotic cells was determined following immunofluorescence staining using

the DNA-interchelating fluorochrome DAPI (4',6-diamidino-2-phenylindole) (Fig. 4B).

Propidium iodide staining showed that the proportion of cells arrested at G2/M (with 4N

DNA content) is similar with or without H2O2 treatment (Fig. 4). It was also found that

the percentage of mitotic cells was significantly lower in the H2O2 treated fibroblasts than

their untreated counterparts (Fig. 4B), even though the total number of cells arrested at

G2/M with and without H2O2 were comparable, as revealed by the propidium iodide

staining (Fig. 4A). The results indicated that almost all H2O2 treated cells with 4N DNA

content resulted from a cell cycle block at G2 but not M. This finding, therefore, suggests

that H2O2 inhibits cell proliferation at G2, but not M, phase of the cell cycle.

The H2O2 induced cell cycle arrest is associated with down-regulation of cyclin Ds and

up-regulation of p21Cip1 expression

We next proceeded to investigate the mechanism involved in this multi-phase cell cycle

arrest. To achieve this, we analysed the expression of different components of the cyclin-

dependent kinase complexes, which are important for regulating cell cycle progression,


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following treatment with 250 µM H2O2 (Fig. 5). The Western blot results showed that

while there was no obvious change in the expression levels of cyclin E and-A, CDK2,

and -4, and CDC2 in response to H2O2 stimulation, there was a significant reduction in

cyclin D1 and -D3 expression. Following H2O2 treatment, cyclin D1 and -D3 expression

reached a nadir at 4 h, before recovering to higher levels. Moreover, it was found that the

CKI p21Cip1 was up-regulated by H2O2 treatment and its expression peaked at 4 h after

H2O2 treatment. Notably, cyclin D2 and p57Kip2 expression is not detectable in these

mouse fibroblasts (data not shown). Interestingly, the kinetics for down-regulation of

cyclin D1 and -D3 and up-regulation of p21Cip1 coincided with that of the H2O2 induced

growth arrest, indicating that the cyclin D1 and D3 and p21Cip1 could have a role in

mediating this transient cell cycle arrest. It was notable that the expression level of

another CKI, p27Kip1, decreased after H2O2 stimulation and was inversely correlated with

cell cycle arrest, suggesting that p27 Kip1 is therefore unlikely to be involved in the cell

cycle arrest induced by H2O2.

The induction of p21Cip1 by H2O2 occurs at transcriptional level but does not require p53

function

To investigate the mechanism for p21Cip1 regulation by H2O2, we performed Northern blot

analysis on NIH3T3 treated with H2O2 (Fig 5). The results showed that H2O2 increased

p21Cip1 mRNA levels with kinetics similar to that of the p21Cip1 protein, indicating that

p21 transcription increases is a component of the H2O2 response. To test if this induction

of p21Cip1 expression by H2O2 was also mediated at gene promoter level, we transiently

transfected NIH 3T3 cells with a p21Cip1 promoter/luciferase reporter construct and


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monitored the luciferase activity following treatment with 250 µM H2O2 (Fig. 5). p21Cip1

promoter activity essentially paralleled the changes in p21Cip1 mRNA levels, indicating

that the induction of p21Cip1 in response to H2O2 treatment can be largely accounted for

through alterations in transcription rate, although other mechanisms have not been

eliminated.

The CKI p21Cip1 is one of the primary transcriptional targets of the tumour

suppressor p53. p53 has previously been shown to mediate G1 arrest induced by DNA

damage through activating p21Cip1 gene transcription and it is possible that the induction

of p21Cip1 expression by H2O2 was mediated through p53. Indeed, immunoblot analysis

(Fig. 5) indicated that p53 levels did increase in response to H2O2 treatment. However,

p53 expression subsided before the accumulation of p21Cip1 at either protein or mRNA

levels, indicating that the induction of p21Cip1 expression was not a direct consequence of

p53 accumulation. To further confirm this idea, we treated MEFs derived from p53-/-

mice with 250 µM H2O2 and followed the expression patterns of p21Cip1, p27 Kip1 and

cyclin Ds. Western blot results showed that the induction of p21Cip1 expression by H2O2 in

p53-/- MEFs occurred at similar kinetics as in the NIH 3T3 fibroblasts, indicating that p53

is not essential for the induction of p21Cip1 expression by H2O2. Likewise, the down-

regulation of p27 Kip1 and cyclin D1 and -D3 expression also occurred in the absence of

p53 following H2O2 stimulation.

The H2O2 induced cell cycle arrest is associated with repression of cyclin D-CDK4/6,

cyclin E-CDK2, cyclin A-CDK2, and cyclin B-CDC2 activity


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Progression through the cell cycle from G1 to M requires the sequential activation of

cyclin D-CDK4/6, cyclin E-CDK2, cyclin A-CDK2, cyclin B-CDC2 activity, which are

important for transition through G1, initiation of S, advance through S, and passage from

G2 to M, respectively (12,39-42). Our data showed that H2O2 induces a multi-phase cell

cycle arrest at G1, S and G2. Consequently, we next examined the effects of H2O2 on the

kinase activity of the cyclin D-CDK4, cyclin E-CDK2, cyclin A-CDK2 and cyclin B-

CDC2 complexes. CDK complexes were immunoprecipitated using specific anti-cyclin

and CDK antibodies and the levels of cyclin and CDK-associated kinase activity

measured against a bacterially synthesised pRB fragment (Rb 792-973) or histone H1 as

substrates (Fig. 6A). The immunoprecipitation-kinase assays showed that whereas the

activity of cyclin A, -E, -B, CDK4, CDK2 and CDC2 containing kinase complexes

remained at high levels in untreated fibroblasts, these cyclin-CDK complexes were

significantly inactivated in the H2O2-treated cells (Fig. 6A). Collectively, the kinase

assays showed that the H2O2-induced multi-phase growth arrest was correlated with

down-regulation of cyclin D-CDK4, cyclin A and E-CDK2, and Cyclin B-CDC2

associated kinase activity. Given that the level of CDK4 expression remained unchanged

before and after H2O2-stimulation, the decrease in cyclin D-CDK4 activity is likely to be

primarily the result of the down-regulation of cyclin D1, -2, and –3 expression induced

by H2O2, though the involvement of other mechanisms could not be excluded.

The WAF/KIP family of CKIs, including p21Cip1 and p27Kip1, have been shown to

trigger cell cycle arrest through specifically binding to and inhibiting CDK2 kinase

complexes. Therefore, the reduction of cyclin A and E-CDK2 activity could be due to the


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induction of p21Cip1 by H2O2. To explore further the roles of p21Cip1 and p27Kip1 in the

H2O2-induced cell cycle arrest, we used anti-p21Cip1 or anti-p27Kip1 antibodies cross-linked

to sepharose G beads to immunodeplete cyclin-dependent kinase complexes from NIH

3T3 fibroblasts before and 4 h after H2O2 treatment. The subsequent immunodepleted

lysates were then Western blotted for components of cyclin associated kinase complexes

(Fig. 6B). The result showed that significant levels of cyclin A, E, and CDK2 remained in

the untreated cell lysates after p21Cip1 and p27Kip1 immunodepletion, indicating that in

untreated cells the majority of the cyclin E-CDK2 and cyclin A-CDK2 complexes are

‘free’ of these two CKIs. It also demonstrated that although the anti-p21Cip1 antibodies

failed to deplete any of the cyclin A/E-CDK2 complexes in the untreated cells, they

removed the majority of the cyclin A and -E, and CDK2 proteins from the H2O2-treated

cell lysates. This indicates that the majority of the cyclin A or E-CDK2 complexes are

‘free’ in the untreated cells, but are associated with p21Cip1 following H2O2 stimulation. In

contrast, although the anti-p27Kip1 antibodies removed effectively all p27Kip1 protein from

the untreated and H2O2-treated cell lysates, the antibodies failed to eliminate the cyclins

and dependent kinases, including cyclin Ds, -A, -E, -B, CDK2, -4, and CDC2, before and

after H2O2-treatment. These results not only indicated that H2O2-treatment increased the

amount of cyclin A or E-CDK2 complexes associated with p21Cip1, but also suggested

that the majority of the cyclin A or E-CDK2 complexes were bound to p21Cip1 after H2O2-

stimulation. It was also found that the anti-p21Cip1 antibodies depleted cyclin D1 and

CDK4 in cell lysates with and without H2O2 treatment. Conversely, little or no cyclin B

and CDC2 was eliminated by the anti-p21Cip1 antibodies with or without H2O2 treatment.

Although our result showed that there was no binding of p21 to the cyclin B-CDC2


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complex, previous studies have shown that increased levels of p21 can induce a G2 arrest

by inhibiting the CAK-mediated Thr 161 phosphorylation of CDC2. Thus, the G2 arrest

could also be attributable to the induction of p21 expression by H2O2.

p21Cip1 localised exclusively in the nucleus and p27 Kip1 largely in the cytoplasm

Interestingly, although p27Kip1 was present abundantly in the cells both before and

after H2O 2 treatment, they failed to associate with the cyclin-CDK complexes. The

subcellular localisation of p21Cip1 and p27Kip1 plays a part in regulating their activity and

it is possible that the absence of p27Kip1 binding to cyclin A and E-CDK2 could be due to

the fact that p27Kip1 and the cyclin dependent kinases are not present in the same

subcellular compartments. Consistent with this, it has been documented previously that

p27Kip1 is localised in the cytoplasm of proliferating Swiss 3T3 cells (43). To investigate

this possibility, we studied the subcellular localisation of p21Cip1 and p27Kip1 before and 4

h after H2O2 treatment by immunofluorescence staining using antibodies specific for

p21Cip1 and p27Kip1 (Fig. 7). Immunofluorescence experiments showed that p21Cip1

localised primarily within the nucleus and p27Kip1 in the cytoplasm, and that H2O2

treatment did not affect the subcellular localisation of either p21Cip1 or p27Kip1 (Fig. 7). In

accordance with the Western blotting results, p21Cip1 expression was higher in the H2O2

treated cells compared to the untreated, where p21Cip1 levels were very low. As the

cyclin-CDK complexes are located predominantly in the nucleus, the cytoplasmic

localisation of p27Kip1 could help explain the lack of binding of p27Kip1 to the cyclin A/E-


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CDK2 complexes. Studies are currently underway to address the significance of this

observation.

Ectopic expression of K cyclin, but not cyclin D1 or cyclin E, can rescue fibroblasts from

the H2O2 induced cell cycle block

On the basis of our findings, we propose a model in which moderate levels of H2O2

induce a transient multi-phase cell cycle arrest through up-regulation of p21Cip1 and

down-regulation of cyclin D expression. To investigate the contribution of p21Cip1 in this

H2O2-induced cell cycle arrest, MEFs from wild-type and p21Cip1-/- mice were treated with

250 µM H2O2 (Fig. 8). The results showed that after H2O2 treatment, normal MEFs and

MEFs lacking p21Cip1 underwent cell cycle arrest with similar kinetics. This result

indicated that deletion of p21Cip1 function alone is not sufficient to overcome the cell

cycle arrest mediated by H2O2 and highlighted the fact that the H2O2-induced cell cycle

arrest also involved down-regulation of cyclin D-CDK4/6 activity, which cannot be

restored by deletion of p21Cip1. The result also indicated that another mechanism other

than or in addition to cyclin D down-regulation and p21Cip1 up-regulation must also be

involved. As an alternative approach to test the hypothesis, we examined if over-

expression of cyclin D, cyclin E or a viral K cyclin is enough to overcome the cell cycle

arrest induced by H2O2. K cyclin is a human herpesvirus 8 (HHV8) viral cyclin that can

mimic the function of cyclin Ds and is resistant to CKIs, including p21Cip1 and p27Kip1.

To this end, we used NIH 3T3 fibroblast cell lines which expressed cyclin D1, cyclin E,

or K cyclin, upon the addition of IPTG (32). The inducible expression of cyclin D1,


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cyclin E, and K cyclin in these three NIH 3T3 fibroblast cell lines by IPTG stimulation

was demonstrated by Western blot analysis of serum deprived quiescent cells in the

presence or absence of IPTG stimulation (Fig. 9). Cells were incubated in the absence or

presence of 250 µM or 500 µM H2O2 for 4 h, with and without prior induction of ectopic

cyclin expression. After treatment, cells were harvested for cell cycle analysis (Fig. 10).

Cyclin E was unable to effectively override the cell cycle arrest induced by H2O2. Over-

expression of cyclin D1 appeared to be able to partially revert the cell cycle arrest

imposed by low concentrations of H2O2 (250 µM), as revealed by the low levels of BrdU

incorporation in the presence cyclin D1 expression following H2O2 treatment. This ability

of cyclin D1 to partially overcome the H2O2 induced cell cycle arrest could be due to the

fact that cyclin D1 can overcome the p21Cip1-induced cell cycle arrest which has been

documented previously (44). The results also demonstrated that the H2O2-induced cell

cycle arrest can be effectively overridden by expression of K cyclin, consistent with our

theory that the multi-phase cell cycle arrest triggered by H2O2 is associated with down-

regulation of cyclin D and up-regulation of p21Cip1 expression.


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DISCUSSION

Hydrogen peroxide (H2O2) is an active oxygen species, which can diffuse freely

into cells and is relatively more stable compared with superoxide anions or hydroxyl

radicals (7). Hydrogen peroxide is first converted to a hydroxyl radical, which is a very

strong oxidant, before being converted to water. Using H2O2 as a source of oxidant, we

studied the cellular response to oxidative stress in proliferating immortalised and primary

mouse fibroblasts. The cellular response to oxidative stress involves activation of

checkpoints that delay cell cycle progression in order to provide time for repair of

damaged cellular components (eg. DNA, proteins and lipids) and also for mounting an

anti-oxidant defence. Our data shows that sub-lethal doses of H2O2 induce a rapid and

transient growth arrest in both primary and immortalised mouse fibroblasts. This cell

cycle arrest can easily be over-looked, especially if only propidium iodide staining is

used to study the cell cycle distribution of cells in response to H2O2 treatment. Besides

the cell cycle arrest, the complete absence of BrdU labelling in cells after H2O2 treatment

also indicates that there is a cessation of DNA synthesis. This cell cycle arrest was

apparent 2 h following exposure to H2O2 and lasted for about 6 h.

To characterise further the nature of this cell cycle arrest, we examined the effects

of H2O2 on fibroblasts optimally synchronised at different phases of the cell cycle using

three different methods: serum deprivation, nocodazole and thymidine blocks. Our results

suggested that H2O2 activates cell cycle checkpoints at G1 and S, and somewhere within

G2/M phases of the cell cycle. The observation that some, although not all, G2/M cells


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entered G1 after H2O2 stimulation suggests that H2O2 arrests cells somewhere in the early

part of G2/M. Consistent with this are the subsequent findings that H2O2 failed to delay

cells released from a nocodazole induced M phase block but prevented cells entering M

from G2, thus confirming that H2O2 arrests cells at G2 but not at M phase. Together,

these synchronised cell cycle phase experiments show that H2O2 activates cell cycle

checkpoints in G1, S, and G2 phases, but not in M phase of the cell cycle.

The molecular mechanisms involved in the H2O2 response appear complex. Our

initial experiments indicated the involvement of the D-type cyclins and the CKI p21Cip1.

D-type cyclin expression was markedly down-regulated in response to H2O2 and resulted

in a reduction in cyclin D-CDK4 activity. Since the progression of cells from G1 to S is

initiated by expression of D-type cyclins and their assembly with CDK4, the down-

regulation of cyclin D1 and D3 expression and their associated CDK4 activity by H2O2

can explain the G1 phase arrest induced by H2O2. Likewise, the down-regulation of cyclin

E-CDK2 and cyclin A-CDK2 activity by H2O2 could account for the block of DNA

synthesis caused by H2O2. Interestingly, the down-regulation of cyclin E-CDK2 and

cyclin A-CDK2 activity by H2O2 is not accompanied by significant changes in cyclin A,

E and CDK2 levels. This reduction in cyclin E/A-CDK2 after H2O2 is predominantly a

result of increase in p21Cip1 binding to cyclin E/A-CDK2. The increase in binding of

p21Cip1 to cyclin E/A-CDK2 is primarily the result of increased transcription of the p21Cip1

gene following H2O2 treatment. p21Cip1 expression can be activated by p53-dependent and

independent mechanisms in response to cellular stresses, such as DNA damage, to

mediate G1 cell cycle arrest (45-47). In the present study, we showed that H2O2 can


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induce transient cell cycle arrest and up-regulation of p21Cip1 in the absence of p53

function. Nevertheless, we still cannot exclude the possibility that p53 contributes to the

H2O2-induced p21Cip1 expression and cell cycle arrest in normal cells.

Cell cycle progression from G2 into M phase is regulated by cyclin A/B-CDC2

activity (14,42,48); therefore, the G2 arrest following H2O2treatment can be attributable

to the decrease in cyclin A/B-CDC2 associated kinase activity observed after H2O2

treatment. However, this decrease in cyclin A/B-CDC2 activity is not accompanied by

any detectable change in cyclin A/B and CDC2 expression. Previous studies have shown

that increased levels of p21Cip1 can induce a G2 arrest by inhibiting the CAK (CDK-

activating kinase)-mediated Thr 161 phosphorylation of CDC2 (49). As a result, the

H2O2-induced accumulation of p21Cip1 could also be, at least in part, responsible for

down-regulation of cyclin A/B-CDC2 dependent kinase activity and the consequent G2

arrest induced by H2O2. Consistent with our finding, this inhibitory function of p21Cip1

does not require its physical association with cyclin B or CDC2 (49). Indeed, over-

expression of p21Cip1 has been demonstrated to be able to enforce a G1 as well as G2 cell

cycle arrest through inhibiting the cyclin A, E, and B-associated kinase activity (49,50).

Dephosphorylation of two inhibitory residues, Thr14 and Tyr15, is required for the G2 to

M transition in mammalian cells and the cyclin B-CDC2 complex is inactive when CDC2

is phoshorylated at the Thr14 and Tyr15 sites (49). The Thr14 and Tyr15 residues of

CDC2 have also been shown to play an important role in mediating the DNA damage-

induced G2 cell cycle check point. Expression of a CDC2 mutant that cannot be

phosphorylated at Thr14 and Tyr15 (CDC2AF) has been demonstrated to reduce


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significantly the G2 delay induced by DNA damages in HeLa cells (51). Moreover,

nuclear localization of cyclin B1 has also been proved to control mitotic entry after DNA

damage (52). It is therefore possible that CDC2 phosphorylation at the residues Thr14

and Tyr15 and/or the subcellular localisation of cyclin B1 can also contribute to the

transient G2 arrest observed after H2O2 treatment. Similarly, the inhibitory

phosphorylation of Tyr 15 on CDK2 could have a role in the H2O2-induced S phase arrest

(45). Besides binding to cyclin-CDK complexes, p21Cip1 also associates with the

proliferating nuclear antigen (PCNA), a subunit of DNA polymerase δ and through this

interaction, inhibits DNA replication directly (53-55). This specific interaction of p21Cip1

with PCNA may also contribute to the rapid cessation of DNA synthesis following H2O2

treatment. Interestingly, a recent report also suggested that p21Cip1 and PCNA co-operate

to maintain cell cycle arrest at G2 after DNA damage (56). Moreover, another study also

demonstrated that p21Cip1 can cause G1 and G2 arrest in p53 null cells through binding to

PCNA (57). These reports all point to a potential role of PCNA in this H2O2-induced

transient cell cycle arrest, which warrants further examination.

Although significant levels of p27Kip1 were detected by Western blot analysis

before and after H2O2 stimulation, the immunodepletion assays demonstrate that little or

no cyclin A/E-CDK2 complexes were associated with p27Kip1. This finding could be

explained by the notable difference in subcellular localisation of p21Cip1 and p27Kip1.

Immunofluorescence staining reveals that p21Cip1 locates predominantly in the nucleus

while p27Kip1 accumulates mainly in the cytoplasm regardless of H2O2 treatment.

Collectively, these data show that the down-regulation of D-type cyclin and up-regulation

of p21Cip1 expression contributes to the multi-phase cell cycle arrest induced by H2O2. To


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establish further the roles of p21Cip1 and D-type cyclins in this transient cell cycle arrest

induced by H2O2 treatment, we tested if deletion of p21Cip1 or ectopic expression of cyclin

D1 or E can override the cell cycle arrest induced by H2O2. However, using p21Cip1 null

MEFs and NIH 3T3 cells that express cyclin D1 or E under the control of an inducible

promoter, we showed that deletion of p21Cip1, restoration of cyclin D expression, or over-

expression of cyclin E alone is insufficient to effectively overcome the cell cycle arrest

caused by sub-lethal doses of H2O2. By contrast, over-expression of a HHV8 K cyclin is

enough to override this transient cell cycle arrest. Although it has been shown that the

cyclin K/CDK6 is resistant to p21Cip1 inhibition and can mimic cyclin E/CDK2 activity

with respect to DNA synthesis initiation (32,58), it is conceivable that K cyclin can also

interfere with other cell cycle regulators to overcome this transient multi-phase cell cycle

arrest. The observation that H2O2 can still cause S phase arrest in p21Cip1 null MEFs

suggests that H2O2 can also inhibit CDK2 activity by a p21Cip1-independent mechanism.

Interestingly, a previous study has demonstrated that in response to DNA damage, c-abl

can contribute to the growth arrest by down-regulating the activity of CDK2 in a p21Cip1-

independent mechanism (59). Moreover, DNA damage has also been shown to inhibit

CDK activity and cell cycle progression in a p21Cip1 independent manner through

inactivating the CDC25 family of phosphatases, including CDC25 A, B and C. Genotoxic

stress has been demonstrated to inhibit CDKs through inactivating CDC25 family of

phosphatases that normally activate CDKs by dephosphorylating CDKs at negative

regulatory sites, including threonine 14 and tyrosine 15 on CDC2, the tyrosine 15 on

CDK2, and the tyrosine 17 on CDK4 (42,60,61). Further studies are required to explore

the part played by these p21-independent mechanisms in the multi-phase cell cycle arrest


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induced by H2O2 exposure. Collectively, our results show that sub-lethal doses of H2O2

induce a rapid and transient growth arrest at G1, S and G2 phases of cell cycle in mouse

fibroblasts. Our data also demonstrate that this multi-phase cell cycle arrest is, at least in

part, mediated by the down-regulation of D-type cyclin and induction of p21Cip1

expression, culminating in the inhibition of CDK4, CDK2 and CDC2-associated kinase

activities.

A previous study on human diploid fibroblasts showed that sub-lethal levels of

H2O2 induce a long-term senescence related cell cycle arrest at G1 phase of the cell cycle

(26). This report documents the long-term more permanent responses of human

fibroblasts to H2O2, whereas our present study concentrates on the more immediate and

temporary effects of H2O2. While some similarities are shared between the two studies, it

is evident that many differences exist. Most noticeably, in these H2O2-stimulated human

diploid fibroblasts, p53 expression appears to be essential for the induction of p21Cip1

expression and the cell cycle arrest by H2O2. By contrast, our results show that H2O2 can

induce p21Cip1 expression in the absence of functional p53. It is possible the majority of

the differences between two studies may reside wholly in the fact that the present study

was performed on mouse cells, while the earlier study was carried out on cells of human

origin (26). Although the oxidative stress induced transient cell cycle arrest is beginning

to attract increasing attention in recent years, the nature of this cell cycle arrest and

molecular mechanisms by which this growth arrest is mediated remained very much

undefined. To our knowledge, the present study represents the first detailed investigation

on the nature and molecular basis of the H2O2-induced transient cell cycle arrest.


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ACKNOWLEDGEMENTS

We acknowledge the generosity of Dr. B. Vogelstein for the mouse p21Cip1

cDNAs and Dr. M. Serrano for the p21Cip1-/- mice and Dr. W. Kaelin for GST-Rb792

plasmid. We thank Dr. Shaun Thomas for critical reading of the manuscript. The work of

Eric Lam and Janet Glassford are also supported by the Leukaemia Research Fund. Eric

Lam and David Mann are also funded by the Cancer Research Campaign. Karin

Barnouin, Marlene Dubuisson and Silvia Fernandez de Mattos are fellows of the

Kay Kendall Leukaemia Fund, FNRS (Belgium) and IARC, respectively.

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FIGURE LEGENDS

Figure 1. Cell cycle analysis of NIH 3T3 fibroblasts after H2O2 treatment.Subconfluent cycling NIH3T3 were treated with 250 µM H2O2, labelled with BrdU, andcollected at times indicated. The cells were permeablised and stained with propidiumiodide and FITC conjugated anti-BrdU antibodies, before being used for fluorescent-activated cell sorting (FACS) analysis to determine DNA content (upper) and replicative(S-phase) cells (lower), respectively. The percentages of cells in each cell cycle phase(G1, S and G2/M) determined by DNA contents and stained positive for BrdU wereindicated.

Figure 2. Cell cycle analysis of Swiss 3T3 fibroblasts synchronised by serumdeprivation after H2O2 treatment.Swiss 3T3 fibroblasts arrested at G0 by serum deprivation were stimulated to re-enter thecell cycle with 10% FCS. Cells were labelled with BrdU, fixed, stained with propidiumiodide and FITC conjugated anti-BrdU antibodies, and processed for FACS analysis as inFigure 1. (A) The upper panel shows the cell cycle profile of fibroblasts at 0, 12, 16, 20,24, and 28h after serum-stimulation. Swiss 3T3 fibroblasts at 12, 16, 20, 24, and 28h afterserum-stimulation were treated with 250 µM H2O2 and harvested 4 h later for cell cycleanalysis. The lower panel reveals the cell cycle status of cells 4h after H2O2 treatment. Asthe serum-stimulated cells were collected at 4 h intervals and the H2O2 treated cells wereharvested after 4 h, the cells at 16, 20, 24, and 28h after serum stimulation served as theuntreated control of the cells treated with H2O2 at 12, 16, 20, and 24 h, respectively. (B)A duplicated experiment as above. However, only the cell cycle profile of fibroblasts 24h after serum-stimulation and 4h afterwards (28 h after serum-stimulation) with orwithout H2O2 treatment is shown.

Figure 3. Cell cycle analysis of NIH 3T3 fibroblasts released from a nocodazoleblock with and without H 2O2 treatment.NIH 3T3 fibroblasts were arrested at M by nocodazole and released back into the cellcycle after nocodazole was washed away. Cell were labelled with BrdU, fixed, andprocessed for FACS analysis as in Figure 1.The upper panel shows the cell cycle profileof fibroblasts at 0, 4, 8, 12, and 16h after release from the nocodazole block. At 0, 4, 8,12, and 16h after release from the nocodazole block, NIH 3T3 fibroblasts were treatedwith 250 µM H2O2 and harvested 4 h later for cell cycle analysis. The lower panel revealsthe cell cycle status of cells after H2O2 treatment.

Figure 4. Cell cycle analysis of NIH 3T3 fibroblasts released from a doublethymidine block in the presence or absence of H2O2 treatment.NIH 3T3 fibroblasts were arrested at G1/S by a double thymidine block and releasedback into the cell cycle. These cells were labelled with BrdU, fixed, and processed forFACS analysis as in Figure 1. (A) The upper panel shows the cell cycle profile of


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fibroblasts at 0, 2, 4, 6, 8 h after release from the thymidine block. At 6h after releasefrom the G1/S block, NIH 3T3 fibroblasts were treated with 100µM nocodazole in thepresence and absence of 250 µM H2O2, harvested 4 h later, and processed for cell cycleanalysis (lower panel). (B) Fluorescence photomicrograph of the cells treated withnocodazole in the presence or absence of H2O2 after DAPI staining as in (A). The arrowswithin the photomicrograph indicate condensed nuclei. The mitotic index was calculatedas the number of condensed nuclei against the total, and expressed as mean±SD. Theresult represents the average of 3 independent studies, and 200 nuclei were analysed ineach study.

Figure 5. Expression of cell cycle regulators in NIH 3T3 fibroblasts following H2O2

treatment. (A) Cell lysates were prepared from NIH 3T3 fibroblasts at times indicatedfollowing treatment with 250 µM H2O2. The expression of cyclin -D1, -D3, -E, -A and -B, CDK2, -4, CDC2, p21Cip1, p27Kip1, p53 were analysed by Western blotting. Total RNAwas isolated in parallel and the expression of p21Cip1 and actin mRNA was determined byNorthern blotting. (B) A wild-type p21Cip1 promoter-luciferase construct was transfectedinto cycling NIH 3T3 cells. The transfected cells were treated with 250 µM H2O2,collected 4 h later, and lysed for luciferase assay. The luciferase activity was normalisedwith β-galactosidase activity specified by co-transfected pJ4Ωß-gal plasmid andexpressed as the mean±SD of at least three independent experiments each with duplicatedtranfection. (C) Lysates were prepared from p53-/- MEFs at times indicated followingtreatment with 250 µM H2O2, and the expression of cyclin -D1, -D3, p21Cip1and p27Kip1

were analysed by Western blotting.

Figure 6. Analysis of cyclin-dependent kinase complexes and their association withCKIs in NIH 3T3 fibroblasts following H 2O2 treatment.(A) Cell extracts prepared from untreated NIH 3T3 and NIH 3T3 cells 4 h after 250 µMH2O2 treatment were immunoprecipitated with antibodies against CDK2, CDC2, CDK4,cyclin E, cyclin A and cyclin B1. The precipitated complexes were examined for kinaseactivity using a pRB fragment or Histone H1 as substrates.(B) Cell extracts from untreated and H2O2-treated NIH 3T3 cells, as described above,were immunodepleted of p21Cip1 or p27Kip1 by incubation with excess amounts of mousemonoclonal p21Cip1 or p27Kip1-specific antibodies coupled to protein G-Sepharose.Supernatant immunodepleted of p21Cip1, and p27Kip1 were Western blotted for p21Cip1,p27Kip1, cyclin A, cyclin E, CDK2, cyclin B, CDC2, cyclin D1, CDK4 and CDK6expression. As controls, a mock immunodepletion was performed without antibodies.

Figure 7. Subcellular localisation of p21Cip1 and p27Kip1 in NIH 3T3 fibroblasts afterH2O2 treatment.(A) Subconfluent NIH 3T3 fibroblasts plated on coverslips were either untreated ortreated with of 250 µM H2O2. After 4 h, cells with or without H2O2 treatment were fixedand stained with anti-p21Cip1 or anti-p27Kip1 antibody. (B) H2O2-treated NIH 3T3


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fibroblasts (4 h) were stained with either anti-p21Cip1 or anti-p27Kip1 antibody. The samecells were counterstained with DAPI to identify the nuclei.

Figure 8. Cell cycle analysis of wild type, and p21 null mouse embryo fibroblastsafter H2O2 treatment.Low passage (≤2) wild-type or p21-/-, MEFs were treated with 250 µM H2O2, labelledwith BrdU for 30 min, and collected at times indicated. Cells were permeablised andstained with propidium iodide and FITC conjugated anti-BrdU antibodies, prior to FACSanalysis. The percentages of cells in each cell cycle phase (G1, S and G2/M) determinedby DNA contents and stained positive for BrdU were shown.

Figure 9. Expression of cyclins D1, E and K after IPTG induction in the NIH 3T3cell lines.NIH 3T3-D1, NIH 3T3-E, or NIH 3T3-K cell lines were either untreated or treated with 3mM IPTG for 24 h. Lysates derived from these IPTG induced cells were Western blottedfor ectopic cyclin D1, cyclin E and K cyclin expression. The blots were also probed fortubulin expression, which was used as loading control. The inducible cyclins D1, E and Kwere detected with specific antibodies against the Flag-tag, human cyclin E, and HA,respectively.

Figure 10. Analysis of the ability of cyclins D1, E and K to overcome the cell cyclearrest induced by H2O2 treatment.NIH 3T3-D1, NIH 3T3-E, or NIH 3T3-K cell lines were either untreated (-) or treated (+)with 3 mM IPTG for 24 h to induce cyclin expression. These IPTG pre-treated cells aswell as the untreated cells were then treated with either 0, 250 or 500 µM H2O2, prior tocollection for cell cycle analysis 4 h afterwards. BrdU was added to the cells for the final30 min of the experiment. The harvested cells were fixed and stained with propidiumiodide and FITC conjugated anti-BrdU antibodies (lower panel) prior to FACS analysis tomeasure the DNA contents and to determine the cells in DNA synthesis, respectively.The percentages of cells in each cell cycle phase (G1, S and G2/M) determined by DNAcontents and stained positive for BrdU were indicated.


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Janet Glassford, René H. Medema, David J. Mann and Eric W. LamKarin Barnouin, Marlene L. Dubuisson, Emma S. Child, Silvia Fernandez de Mattos,

modulating cyclin D and p21Cip1 expressionH2O2 induces a transient multi-phase cell cycle arrest in mouse fibroblasts through

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