The ATM/p53/p21 pathway influences cell fate decision

between apoptosis and senescence in reoxygenated

hematopoietic progenitor cells

Xiaoling Zhang1, June Li1, Daniel P. Sejas1, and Qishen Pang1,2* Form 1Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, and 2Department of Pediatrics University of Cincinnati College of Medicine. 3125 Eden Avenue, Cincinnati, Ohio 45267

Running title: Reoxygenation induces hematopoietic cell senescence

*To whom reprint requests should be addressed. Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229. Phone: (513) 636-1152. Fax: (513) 636-3768. E-mail: qishen.pang@cchmc.org.

1 The abbreviations used are: AA, aplastic anemia; 2-AP, 2-aminopurine; ATM,

Ataxia-Telangiectasia Mutated; HSC, hematopoietic stem cell; FA, Fanconi Anemia; IL, interleukin; NAC, N-acetyl-L-cysteine; SCF, stem cell factor; siRNA,

small interfering RNA.

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JBC Papers in Press. Published on March 7, 2005 as Manuscript M502262200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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ABSTRACT

Hematopoietic cells are often exposed to transient hypoxia as they

develop and migrate between blood and tissues. We tested the hypothesis that

hypoxia-then-reoxygenation represent a stress for hematopoietic progenitor cells.

Here we report that reoxygenation-generated oxidative stress induced

senescence, tested as staining for SA-β-gal, of bone marrow progenitor cells.

Reoxygenation induced significant DNA damage and inhibited colony formation

in lineage-depleted bone marrow cells enriched for progenitor cells. These

reoxygenated cells exhibited a prolonged G0/G1 accumulation without significant

apoptosis after 24 hours of treatments. Reoxygenated bone marrow progenitor

cells expressed SA-β-galactosidase and senescence-associated proteins p53

and p21WAF1. Reoxygenated Fancc-/- progenitor cells, which underwent

significant apoptosis and senescence, tested as staining for SA-β-gal, also

expressed p16INK4A. Suppression of apoptosis by the pan-caspase inhibitor Z-

VAD-FMK dramatically increased senescent Fancc-/- progenitor cells.

Senescence induction, tested as staining for SA-β-gal, in reoxygenated

progenitor cells was closely correlated with extent of DNA damage and

phosphorylation of ATM at Ser1981 and p53 at Ser15. Moreover, inhibition of

ATM signaling reduced SA-β-gal positivity but increased apoptosis of

reoxygenated progenitor cells. Thus, these results suggest that the ATM/p53/p21

pathway influences cell fate decision between apoptosis and senescence in

reoxygenated hematopoietic progenitor cells.

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INTRODUCTION

Hematopoietic cells are often exposed to transient hypoxia and

reoxygenation as they develop and migrate between blood and tissues.

Continuous cycles of hypoxia-then-reoxygenation has long been known to

increase in the production of oxidants (1), which could cause DNA damage,

protein oxidation, and lipid peroxidation (2, 3). In human colorectal cell line RKO

and lymphoblasts, hypoxia-then-reoxygenation induces DNA damage and

activates p53, which can be inhibited by the anti-oxidant N-acetyl-L-cysteine

(NAC) (4). Furthermore, reoxygenation-induced DNA damage and p53 activation

is dependent on the protein kinase ATM (Ataxia-Telangiectasia Mutated; 4, 5).

Oxidative stress-induced DNA damage is well known to cause loss of cell

replication and multiple molecular changes involved in premature senescence,

such as up-regulation of senescence-associated proteins p53, p21WAF1 and

p16INK4A, and permanent growth arrest (6). Little is known about the effects of

reoxygenation-generated oxidative stress on the survival and maintenance

hematopoietic progenitor and stem (HSC) cells.

Studies on pathophysiological mechanisms of oxidative stress responses

in stem cell diseases such as aplastic anemia (AA) has been very instructive and

provides insights into the function of normal hematopoietic stem cells and their

self-renewal capacity (7). One of the well-studied AA disease models is Fanconi

anemia (FA), a genetic disorder characterized by progressive bone marrow

failure and a predisposition to cancer (8, 9). We hypothesized that hypoxia-

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reoxygenation represents a physiological stress for HSC and progenitor cells,

particularly those from FA patients, and sought to determine the molecular

response of hematopoietic progenitor cells to reoxygenation-generated oxidative

stress. Our study demonstrates that oxidative stress generated by reoxygenation

can induce premature senescence, tested as staining for SA-β-gal, of Fancc-/-

hematopoietic progenitor cells through the ATM/p53/p21 pathway and suggests

that stress-induced senescence may be a novel mechanism underlying

hematopoietic cell depletion in bone marrow (BM) failure diseases including FA.

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EXPERIMENTAL PROCEDURES

Mice, Isolation of BM Lin Sca-1 c-kit (LSK) Cells, and - + + Treatments-- WT

and Fancc-/- mice were generated by interbreeding the heterozygous Fancc+/-

mice (a gift from Dr. Manuel Buchwald, University of Toronto; 10). Lineage-

negative (Lin )- cells were isolated using a lineage cell depletion kit (Miltenyi

Biotec Inc.) in accordance with Manufacturer’s instruction. BM Lin cells - were

then stained with Sca-1-PE and c-kit-APC antibodies followed by cell sorting

using a FACSCalibur (Becton Dickinson, San Jose, CA). The resulting LSK cells

were cultured in IMDM medium containing stem cell factor (SCF;100 ng/ml),

interleukin (IL)-6 (20 ng/ml) and Flt-3L (50 ng/ml) (R & D Systems). Three sets of

cells were incubated in parallel: (1) the control cultures were incubated at 37 oC

in normoxia (humidified air with 21% O2, 5% CO2); (2) the reoxygenated cultures

were subjected to hypoxia (1% O2) for 4 h then shifted to 21% O2; (3) the

reoxygenated-NAC cultures were the same as those in (2) except that the

medium contained the anti-oxidant N-acetyl-L-cysteine (NAC) at a concentration

of 1mM. Pan-caspase inhibitor Z-VAD-FMK (Calbiochem) was added to cell

cultures immediately after reoxygenation at 100 µM. For 2-aminopurine (2-AP)

treatment, cells were incubated with 10 mM 2-AP (Sigma) during hypoxia-

reoxygenation treatments.

Apoptosis Assay-- Aliquots of 1 × 105 BM Lin cells - were stained with Sca-

1-PE and c-kit-APC antibodies followed by annexin V staining. These

experiments also included PE and APC isotype controls, and FITC positive and

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negative controls. Apoptosis was therefore analyzed in different populations of

Lin- cells by flow cytometry.

Clonogenic Progenitor Cell Assays and BM Transplantation -- BM LSK

cells were subjected to hypoxia-reoxygenation with or without 2-AP, and cultured

in a 35 mm tissue culture dish in 4 ml of semisolid medium containing 3 ml of

MethoCult M 3134 (Stem Cell Technologies) and the following growth factors:

100 ng/mL SCF, 10 ng/mL IL-3, 100 ng/mL G-CSF, and 4 U/mL erythropoietin.

Colonies (CFCs) were counted on day 7. To evaluate the effect of reoxygenation

on the repopulation ability of the BM progenitor cells, we used a NOD/SCID

repopulation assay. NOD/SCID mice (Jackson Laboratories) were handled under

sterile conditions and maintained under microisolaters. WT or Fancc-/- BM (2 x

106) cells were transplanted by tail-vein injection into sublethal irradiated (3.5 Gy)

8-week-old mice along with 5 x 105 competitor cells. BM cells from the

transplanted mice were stained with H2kb-PE (for donor-derived cells) and H2kd-

FITC (for recipient-derived cells) antibodies (BD Pharmingen), and analyzed by

flow cytometry to detect donor-derived hematopoietic progenitors.

SiRNA and Assays for DNA Damage and Senescence – The siRNA

oligonucleotides targeting nucleotides 8111-8131 of mouse ATM mRNA

(GeneBank sequence accession number NM007499;

GGTGACTATAAAATCATTTAA) were cloned in the pSM2c retroviral vector

(Open Biosystems). Infected cells were selected for puromycin resistance. The

generation of DNA strand breaks in control and reoxygenated BM LSK cells was

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assessed by the single cell gel electrophoresis (comet) assay (11), using a Fpg-

FLARE (fragment length analysis using repair enzymes) comet assay kit in

accordance with the manufacturer's instructions (Trevigen). SA-β-gal activity was

determined using a SA-β-galactosidase (SA-β-gal) staining kit (Cell Signaling

Technology) according to the manufacturer’s instruction.

Immunocytochemistry – BM LSK cells were stained with primary

antibodies (monoclonal anti-ATMser1981, Rockland Immunochem. Res.;

monoclonal anti-p53ser15, and polyclonal anti-p21WAF1, Cell Signaling; monoclonal

anti-p16, Santa Cruz Biotechnologies), and then with Rhodamine Red-X -

conjugated Goat Anti-Rabbit IgG or Rhodamine Red-X -conjugated Goat Anti-

mouse IgG (Jackson Immuno Research). Nuclei were counter-stained with DAPI

(Sigma).

Statistics--Data was analyzed statistically using a Student’s t test. The

level of statistical significance stated in the text was based on the p values.

P < 0.05 was considered statistically significant.

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RESULTS AND DISCUSSION

Reoxygenation-generated Oxidative Stress Induces DNA Damage and

Inhibits Colony Formation in BM Progenitor Cells – Because reoxygenation

represents oxidative stress to the cell and oxidative stress induces DNA damage

(2, 4, 6), we first sought to determine whether hypoxia-then-reoxygenation

caused DNA damage in BM progenitor cells. Analysis of DNA strand breaks by

comet assay revealed that there was increased accumulation of DNA damage in

reoxygenated Lin-Sca-1+c-kit+ (KSL) BM cells compared to untreated

counterparts (Fig. 1A). The Fancc-deficient mice have profound defect in the

hematopoietic stem and progenitor cell compartment and FA HSCs and

progenitors have been shown to be hypersensitive to a variety of stresses

including oxidative stress (8, 12, 13). Consistent with these observations,

reoxygenated Fancc-/- KSL cells induced significant (3.8-fold) more DNA strand

breakage than reoxygenated WT KSL cells (Fig. 1A). Treatment of reoxygenated

WT or Fancc-/- KSL cells with the anti-oxidant NAC completely abrogated the

effect (Fig. 1A), suggesting that the DNA damage was generated by oxidative

stress. The number of colony-forming cells (CFC) derived from both

reoxygenated WT and Fancc-/- BM progenitors was significantly decreased

compared to their untreated counterparts and NAC completely restored

progenitor activity of these reoxygenated KSL cells (Fig. 1B).

Reoxygenated BM Progenitor Cells Undergo Growth Arrest and Have

Reduced BM Repopulating Ability – While reoxygenation induced more apoptosis

in Fancc-/- BM KSL cells than in WT KSL cells shortly after exposure to high

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oxygen, apoptotic cells decreased thereafter (Fig. 1C). In addition, the extent of

the increase was not as significant as reoxygenation-generated DNA damage at

24 h post-reoxygenation (compare Fig. 1A). Actually, apoptosis decreased to

basal level 48 h post-reoxygenation (Fig. 1C). Thus, apoptosis is not the major

consequence of the DNA damage. We therefore evaluated the cell cycle profile

of these BM cells. Reoxygenated BM cells clearly exhibited a prolonged G0+G1

accumulation (Fig. 1D), suggesting that there might exist an overactivated G0/G1

checkpoint in these BM progenitor cells. The marrow repopulating ability of

reoxygenated progenitors was assessed by transplanting equal numbers of either

untreated (control) or reoxygenated progenitors into sub-lethally irradiated

NOD/SCID recipients. Engraftment was evaluated 4 weeks after transplantation

by flow cytometric determination of donor-derived cells (H2kb+) in BM cell

suspensions of the bone marrow harvested from recipient animals. The bone

marrow of animals that received transplants of reoxygenated WT or Fancc-/- cells

showed 2- or 3-fold lower engraftment than the untreated counterparts,

respectively (Fig. 1E). Consistent with others’ observation (10), Fancc deficiency

impairs the repopulating ability of Fancc-/- BM progenitors.

Cell Fate Choice between Senescence, Test as Staining for SA-β-gal, and

Apoptosis in Reoxygenated BM Progenitor Cells - Because reoxygenated BM

progenitor cells underwent G0/G1 arrest, we reasoned that stress-induced

senescence might be one fate of these cells. To examine this possibility, we

stained WT and Fancc-/- BM KSL cells for SA-β-galactosidase, a biomarker for

senescence (14). Nearly 20% of the reoxygenated WT KSL cells and more than

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40% of the reoxygenated Fancc-/- KSL cells stained positive for SA-β-

galactosidase activity after 48 h of reoxygenation (Fig. 2A).

Because we observed reoxygenation-induced apoptosis, especially in

Fancc-/- BM progenitor cells, we asked whether blockage of apoptosis would

increase senescence, tested as staining for SA-β-gal, in these reoxygenated BM

cells. Indeed, when these reoxygenated BM KSL cells were incubated in the

presence of the pan-caspase inhibitor Z-VAD-FMK, more than 60% of the Fancc-

/- cells entered senescence, tested as staining for SA-β-gal, compared to ~ 40%

without the apoptotic inhibitor (Fig. 2B). Therefore, reoxygenated Fancc-/- BM

progenitor cells blocked for apoptosis may be prone to developing senescence.

Reoxygenation-induced Senescence, Test as Staining for SA-β-gal, in BM

Progenitor Cells Involves the ATM/p53/p21WAF1 Pathway - Because

reoxygenation induces DNA damage and subsequent p53 activation, which is

dependent on the ATM kinase (4, 5), we asked whether reoxygenation-induced

senescence, tested as staining for SA-β-gal, in BM progenitor cells involves the

ATM/p53/p21WAF1 pathway. We examined the reoxygenation-induced

phosphorylation of ATM (Ser1981) and p53 (Ser15), and expression of p21 in

BM KSL cells. ATM autophosphorylation at Ser1981 activates the kinase and is

largely responsible for phosphorylating p53 at Ser15 in response to DNA damage

(15, 16). We found approximately 20% each of ATMSer1981- and p53Ser15-

positive (Fig. 3A-B) in reoxygenated WT BM KSL cells. In reoxygenated Fancc-/-

BM KSL cells, the intensity and percentages of cells stained positive for

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ATMSer1981 and p53Ser15 increased significantly (Fig. 3A-B). We also found

that higher levels of p21-positive (22%) stained cells were present in

reoxygenated WT BM KSL cells compared to untreated WT cells. The

percentage of p21-positive cells increased to 66% in those FA BM KSL cells (Fig.

3A-B).

To provide evidence that there is a link between activation of the

ATM/p53/p21 pathway and senescence, we wanted to know whether blockage of

ATM signaling inhibited reoxygenation-induced senescence, tested as staining

for SA-β-gal, in BM progenitor cells. It is known that inhibition of ATM can relieve

senescent cell cycle arrest (17). We used both siRNA and the kinase inhibitor 2-

AP, which has been shown to suppress ATM activation (18, 19). BM KSL cells

expressing the ATM siRNA or treated with 2-AP effectively reduced ATMSer1981

and p53Ser15 in reoxygenated WT and Fancc-/- BM KSL cells (Fig. 3C-D). We

then determined progenitor activity of these reoxygenated BM KSL cells in a

clonogenic assay (Fig. 3E). Our prediction was that if inhibition of ATM reversed

senescence, tested as staining for SA-β-gal, then the senescence-reversed cells

would be able to proliferate as reflected by the enhanced progenitor activity.

Interestingly, reoxygenated WT BM KSL cells expressing ATM siRNA or treated

with 2-AP exhibited increased clonogenic ability (nearly restored colony formation

to the level of untreated WT BM KSL cells; Fig. 3E). However, both siATM and 2-

AP treatments had little affect on progenitor activity of the reoxygenated Fancc-/-

BM KSL cells (Fig. 3E).

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Recent reports show that the ability of the cell to relieve senescent cell

cycle arrest or reverse senescence depends on the levels of p16INK4A expression

(19, 20). Our observations that inhibition of ATM signaling increased clonogenic

ability (Fig. 3E) of WT but not Fancc-/- progenitor cells led us to examine the

relationship between p16INK4A expression and senescence, tested as staining for

SA-β-gal. Reoxygenation hardly induced p16INK4A expression in WT BM KSL

cells, but did so strongly in Fancc-/- BM KSL cells (Fig. 3F). Expression of siATM

or 2-AP treatment reduced p16 expression in reoxygenated WT cells but did not

have detectable effect on Fancc-/- BM KSL cells (Fig. 3F), indicating that

reoxygenation-induced p16INK4A expression in Fancc-/- BM KSL cells was

independent of ATM activity. However, when we correlated ATM inhibition and

p16INK4A expression with SA-β-gal activity, we found that reoxygenated WT BM

KSL cells treated with 2-AP and siATM were almost devoid of SA-β-gal-positive

cells (decreased from 14.6% to 2.5 and 3.3%, respectively; Fig. 3D). In contrast,

inhibition of ATM activity did not significantly reduced SA-β-gal staining in

reoxygenated Fancc-/- BM KSL cells, which expressed high levels of p16INK4A

(Fig. 3F). Interestingly, inhibition of ATM activity increased apoptosis in

reoxygenated WT and Fancc-/- BM KSL cells (Fig. 3F). These results indicate

that inhibition of ATM can reverse senescence, tested as staining for SA-β-gal, of

BM progenitor cells that do not or express low level of p16, and suggest that the

ATM/p53/p21 pathway influences cell fate decision between apoptosis and

senescence in reoxygenated hematopoietic progenitor cells.

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In summary, we have shown that (1) reoxygenation-generated oxidative

stress induced DNA damage and G0/G1 arrest in BM progenitor cells without

significant apoptosis, (2) reoxygenation induced senescence, test as staining for

SA-β-gal, in reoxygenated BM progenitor cells, (3) induction of senescence,

tested as staining for SA-β-gal, in reoxygenated BM progenitor cells closely

correlated with extent of DNA damage and phosphorylation of ATM at Ser1981

and p53 at Ser15, and (4) inhibition of ATM signaling reversed reoxygenation-

induced senescence, tested as staining for SA-β-gal, but increased apoptosis in

BM progenitor cells. Thus, these results suggest that reoxygenation induces

senescence in hematopoietic progenitor cells through the ATM/p53/p21 pathway.

These findings are especially relevant to the survival and maintenance of

hematopoietic progenitor cells that are often exposed to transient hypoxia in vivo

and, since hematopoietic stem/progenitor cell depletion is the major cause of BM

failure occurred in aplastic anemia including FA, to the molecular etiology of BM

diseases.

We demonstrated that the ATM kinase played a major role in transducing

the reoxygenation-induced DNA damage signal in BM progenitor cells.

Reoxygenation can generate oxidative stress, which can damage DNA. It is

known that ATM transmits the signal of DNA damage induced by oxidative

stress. For instance, oncogenic insults promote the accumulation of reactive

oxygen species, resulting in DNA damage and apoptosis by a p53-dependent

pathway (21-23). More recently, ATM has been shown to play an essential role in

transmitting DNA damage signals generated by reoxygenation, through

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phosphorylation of p53Ser15 (4, 5). We used siRNA targeting ATM and the

protein kinase inhibitor 2-AP to investigate the involvement of ATM in DNA

damage response of reoxygenated BM progenitor cells. Inhibition of ATM

signaling resulted in reduction of ATM-Ser1981, p53Ser15 and p21 expression

and reversal of senescence, tested as staining for SA-β-gal, in reoxygenated BM

progenitor cells but sensitized these BM cells to apoptosis (Fig. 3). Our results

thus indicate for the first time that senescence, tested as staining for SA-β-gal,

induction in reoxygenated BM progenitor cells is regulated by the ATM/p53/p21

pathway. We propose the existence of distinct mechanisms for WT and Fancc-/-

BM cells with regard to cell cycle arrest in response to DNA damage induced by

reoxygenation-generated oxidative stress. Upon experiencing DNA damage, WT

BM cells initially arrest cell cycle progression by ATM-dependent activation of the

G1 checkpoint to gain time for DNA repair. The major pathway responsible for

triggering the G1 checkpoint involves the activation of p53 by ATM, the p53-

mediated induction of p21, and a reduction in the level of RB phosphorylation. If

the DNA damage cannot be repaired timely, the cells undergo reversal

senescence to prevent accumulation of genetic mutations until the damage has

been repair. In Fancc-/- BM progenitor cells, however, excessive DNA damage

causes overactivation of the ATM kinase, resulting in a hyperactive G1

checkpoint. The arrested Fancc-/- BM cells enter senescence while the DNA

damage remains unrepaired. Inhibition of ATM in these FA cells thus likely

results in bypass of G1 checkpoint and undergoing apoptosis.

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Acknowledgements--We thank Dr. Manuel Buchwald (Hospital for Sick

Children, University of Toronto) for the Fancc+/- mice. Q.P. thanks Dr. Grover

Bagby (Oregon Health Science University) for continued support.

This work was supported by an American Cancer Society (Ohio Division)

Support grant, a Fanconi Anemia Research Fund grant, and a Trustee grant from

the Cincinnati Children’s Hospital Medical Center to Q.P.

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Figure legends

Figure 1. Reoxygenation-generated Oxidative Stress Induces DNA Damage

and Growth Inhibition in BM Progenitor Cells. KSL cells were incubated in

20% O2 (control) or first subjected to hypoxia (1% O2) for 4 h then reoxygenation

(20%; Reoxy: reoxygenation). The cells were then incubated in 20% for 48 h.

The NAC cultures (Reoxy+NAC) were the same as the reoxygenated cells

except that the medium contained NAC (1mM). (A) Representative images of the

comet assays used to analyze DNA strand breaks. Numbers below images are

DNA damage quantified by determining the comet tail movement (increasing

values represent increasing amounts of DNA damage). The mean tail moment of

the WT cells without treatment (Control) is expressed as 100%. For each

treatment, 30 cells were scored for tail moment from random sampling. Data

reflect means ± SD of three independent experiments. (B) Untreated (Control) or

reoxygenated WT and Fancc-/- BM KSL cells were evaluated for colony-forming

cell (CFC) activity at day 7. Data represent the number (mean ± SD) of total

number of colonies from three independent experiments. *Statistical significance

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between paired samples at P < 0.05. (C) Untreated (Control) or reoxygenated

WT and Fancc-/- BM lin- cells were stained with lineage maker antibodies (biotin-

conjugated) along with Sca-1-PE and c-kit-APC antibodies, and then with

annexin V. Percentages of apoptosis in the KSL population were analyzed by

flow cytometry. (D) Untreated (Control) or reoxygenated WT and Fancc-/- BM lin-

cells were analyzed for cell cycle distribution at 24 h after reoxygenation. Shown

are representative flow cytometric presentations of three independent

experiments. Numbers in plots indicate percent of cells in G0+G1 phases. (E)

Untreated (Control) or reoxygenated WT and Fancc-/- BM cells were

transplanted into sublethal irradiated NOD/SCID mice along with 1 x 106

irradiated carrier cells per mouse. At 4 weeks after transplantation the bone

marrow was harvested and stained with H2kb-PE and H2kd-FITC (for donor and

recipient markers, respectively) antibodies and analyzed by flow cytometric

analysis. Numbers in corners indicate percent of events in that quadrant.

Figure 2. Reoxygenation-induced senescence, test as staining for SA-β-gal,

in BM Progenitor Cells. (A) WT and Fancc-/- BM KSL cells were subjected to

reoxygenation for 48 h, and stained for SA-β-gal. The numbers below the images

are percentages of the cells stained positive for SA-β-gal quantified by counting a

total of 1000 cells in random fields on a slide. The data represent the mean ±SD.

(B) Reoxygenated BM KSL cells were incubated in the presence of the pan-

caspase inhibitor Z-VAD-FMK (100 µM), and stained for SA-β-gal. Values

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represent mean ± SD of three experiments. *Statistical significance between

paired samples at P < 0.05.

Figure 3. Reoxygenation-induced Senescence in BM Progenitor Cells

Involves the ATM/p53/p21WAF1 Pathway. (A) Control and reoxygenated BM

KSL cells were stained with the antibodies against ATM-Ser1981 (magnification,

×40), p53-Ser15 (×20), and p21Waf1 (×20) and then counterstained with DAPI. (B)

Quantification of ATM-Ser1981, p53-Ser15 and p21Waf1-positive cells. >100 cells

were scored in each case. (C) BM KSL cells were untreated (Control),

reoxygenated, or reoxygenated in the presence of 2-AP (10 mM; Reoxy+2-AP).

siATM group represents cells that had been infected with siATM retroviruses and

selected for puromycin resistance for 48 h before reoxygenation treatment. Cells

were stained with the antibodies against ATM-Ser1981 (magnification, ×40) and

p53-Ser15 (×20). (D) Quantification of ATM-Ser1981 and p53-Ser15-positive

cells. >100 cells were scored in each case. (E) BM KSL cells described in (C)

were plated in semisolid, cytokine-containing medium. The colony-forming cell

(CFC) activity of WT and Fancc-/- BM KSL cells was evaluated at day 7. Data

represent the number (mean ± SD) of total number of colonies from three

independent experiments. *Statistical significance between paired samples at

P < 0.05. (F) BM KSL cells described in (C) were stained with the antibodies

against p16Ink4a (magnification, ×20). The numbers below the images are

percentages of the cells stained positive for SA-β-gal or undergone apoptosis.

Data represent mean ± SD of three experiments.

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Figure 1

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Figure 2

Figure 3

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Xiaoling Zhang, June Li, Daniel P. Sejas and Qishen Pangsenescence in reoxygenated hematopoietic progenitor cells

The ATM/p53/p21 pathway influences cell fate decision between apoptosis and

published online March 7, 2005J. Biol. Chem. 

  10.1074/jbc.M502262200Access the most updated version of this article at doi:

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