The ATM/p53/p21 Pathway Influences Cell Fate Decision between Apoptosis and Senescence in Reoxygenated Hematopoietic Progenitor Cells*

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-gener-ated oxidative stress induced senescence, tested as staining for SA- (cid:1) -galactosidase (SA- (cid:1) -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 G 0 /G 1 accumulation without significant apoptosis after 24 h of treatments. Reoxygenated bone marrow progenitor cells expressed SA- (cid:1) -gal and senescence-associated proteins p53 and p21 WAF1 . Reoxygenated Fancc (cid:2) / (cid:2) progenitor cells, which underwent significant apoptosis and senescence, tested as staining for SA- (cid:1) -gal, also expressed p16 Suppression of apoptosis by the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone dramatically increased senescent Fancc (cid:2) / (cid:2) progenitor cells. Senescence induction, tested as staining

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) 1 (4). Furthermore, reoxygenation-induced DNA damage and p53 activation is dependent on the protein kinase ATM (ataxia-telangiectasia mutated; Refs. 4 and 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, p21 WAF1 , and p16 INK4A 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 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-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 reoxygenationgenerated oxidative stress. Our study demonstrates that oxidative stress generated by reoxygenation can induce premature senescence, tested as staining for SA-␤-galactosidase (SA-␤gal), of FanccϪ/Ϫ 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.

EXPERIMENTAL PROCEDURES
Mice, Isolation of BM Lin Ϫ Sca-1 ϩ c-kit ϩ (LSK) Cells, and Treatments-WT and Fancc Ϫ/Ϫ mice were generated by interbreeding the heterozygous Fancc ϩ/Ϫ mice (a gift from Dr. Manuel Buchwald, University of Toronto; Ref. 10). Lineage-negative (Lin Ϫ ) cells were isolated using a lineage cell depletion kit (Miltenyi Biotec Inc.) in accordance with manufacturer's instruction. BM Lin Ϫ cells were then stained with Sca-1-PE and c-kit-APC antibodies followed by cell sorting using a FACSCalibur (BD Biosciences). The resulting LSK cells were cultured in Iscove's modified Dulbecco's medium containing stem cell factor (100 ng/ml), interleukin-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°C in normoxia (humidified air with 21% O 2 , 5% CO 2 ); 2) the reoxygenated cultures were subjected to hypoxia (1% O 2 ) * 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.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
for 4 h then shifted to 21% O 2 ; 3) the reoxygenated-NAC cultures were the same as those in set 2 except that the medium contained the anti-oxidant NAC at a concentration of 1 mM. Pan-caspase inhibitor Z-VAD-FMK (Calbiochem) was added to cell cultures immediately after reoxygenation at 100 M. For 2-aminopurine (2-AP) treatment, cells were incubated with 10 mM 2-AP (Sigma) during hypoxia-reoxygenation treatments.
Apoptosis Assay-Aliquots of 1 ϫ 10 5 BM Lin Ϫ 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 negative controls. Apoptosis was therefore analyzed in different populations of Lin Ϫ 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 stem cell factor, 10 ng/ml interleukin-3, 100 ng/ml granulocyte-colony-stimulating factor, and 4 units/ml erythropoietin. Colonies (colony-forming cells (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 FanccϪ/Ϫ BM (2 ϫ 10 6 ) cells were transplanted by tail vein injection into sublethal irradiated (3.5 Gy) 8-week-old mice along with 5 ϫ 10 5 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 (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 TM sequence accession number NM007499; GGT-GACTATAAAATCATTTAA) 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 assessed by the single cell gel electrophoresis (comet)

FIG. 1.
Reoxygenation-generated oxidative stress induces DNA damage and growth inhibition in BM progenitor cells. KSL cells were incubated in 20% O 2 (control) or first subjected to hypoxia (1% O 2 ) for 4 h then reoxygenation (Reoxy) (20%). The cells were then incubated in 20% for 48 h. The NAC cultures (ReoxyϩNAC) were the same as the reoxygenated cells except that the medium contained NAC (1 mM). A, representative images of the comet assays used to analyze DNA strand breaks. Numbers below the 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 Ϯ S.D. of three independent experiments. B, untreated (Control) or reoxygenated WT and FanccϪ/Ϫ BM KSL cells were evaluated for CFC activity at day 7. Data represent the number (mean Ϯ S.D.) of total number of colonies from three independent experiments. *, statistical significance between paired samples at p Ͻ 0.05. C, untreated (Control) or reoxygenated WT and FanccϪ/Ϫ BM Lin Ϫ cells were stained with lineage maker antibodies (biotinconjugated) along with Sca-1-PE and ckit-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 FanccϪ/Ϫ BM Lin Ϫ 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 G 0 ϩ G 1 phases. E, untreated (Control) or reoxygenated WT and FanccϪ/Ϫ BM cells were transplanted into sublethal irradiated NOD/SCID mice along with 1 ϫ 10 6 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 the corners indicate percent of events in that quadrant.
assay (11), using a Fpg-FLARE (fragment length analysis using repair enzymes) comet assay kit in accordance with the manufacturer's instructions (Trevigen). SA-␤-gal activity was determined using a SA-␤gal staining kit (Cell Signaling Technology) according to the manufacturer's instruction.
Statistics-Data were analyzed statistically using a Student's t test. The level of statistical significance stated in the text was based on the p values. p Ͻ 0.05 was considered statistically significant.

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 Ϫ Sca-1 ϩ c-kit ϩ (KSL) BM cells compared with untreated counterparts (Fig. 1A). The Fancc-deficient mice have a 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 FanccϪ/Ϫ KSL cells induced significant (3.8-fold) more DNA strand breakage than reoxygenated WT KSL cells (Fig. 1A). Treatment of reoxygenated WT or FanccϪ/Ϫ 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 CFCs derived from both reoxygenated WT and FanccϪ/Ϫ BM progenitors was significantly decreased compared with their untreated counterparts, and NAC completely restored the 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 FanccϪ/Ϫ BM KSL cells than in WT KSL cells shortly after exposure to high oxygen, apo-ptotic cells decreased thereafter (Fig. 1C). In addition, the extent of the increase was not as significant as reoxygenationgenerated 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 G 0 ϩ G 1 accumulation (Fig. 1D), suggesting that there might exist an overactivated G 0 /G 1 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 sublethally irradiated NOD/ SCID recipients. Engraftment was evaluated 4 weeks after transplantation by flow cytometric determination of donorderived cells (H2kb ϩ ) in BM cell suspensions of the bone marrow harvested from recipient animals. The bone marrow of animals that received transplants of reoxygenated WT or FanccϪ/Ϫ cells showed 2-or 3-fold lower engraftment than the untreated counterparts, respectively (Fig. 1E). Consistent with the observations of others (10), Fancc deficiency impairs the repopulating ability of FanccϪ/Ϫ BM progenitors.

Cell Fate Choice between Senescence, Tested as Staining for SA-␤-gal, and Apoptosis in Reoxygenated BM Progenitor
Cells-Because reoxygenated BM progenitor cells underwent G 0 /G 1 arrest, we reasoned that stress-induced senescence might be one fate of these cells. To examine this possibility, we stained WT and FanccϪ/Ϫ BM KSL cells for SA-␤-galactosidase, a biomarker for senescence (14). Nearly 20% of the reoxygenated WT KSL cells and more than 40% of the reoxygenated FanccϪ/Ϫ KSL cells stained positive for SA-␤-galactosidase activity after 48 h of reoxygenation ( Fig. 2A).
Because we observed reoxygenation-induced apoptosis, especially in FanccϪ/Ϫ BM progenitor cells, we asked whether blockage of apoptosis would increase senescence, tested as staining for SA-␤-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 FanccϪ/Ϫ cells entered senescence, tested as staining for SA-␤-gal, compared with ϳ 40% without the apoptotic inhibitor (Fig. 2B). Therefore, reoxygenated FanccϪ/Ϫ BM progenitor cells blocked for apoptosis may be prone to developing senescence. Reoxygenation-induced Senescence, Tested as Staining for SA-␤-gal, in BM Progenitor Cells Involves the ATM/p53/ p21 WAF1 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-␤-gal, in BM progenitor cells involves the ATM/p53/p21 WAF1 pathway. We examined the reoxygenation-induced phosphorylation of ATM (Ser-1981) FIG. 3. Reoxygenation-induced senescence in BM progenitor cells involves the ATM/p53/p21 WAF1 pathway. A, control and reoxygenated BM KSL cells were stained with the antibodies against ATM Ser-1981 (magnification, ϫ40), p53 Ser-15 (ϫ20), and p21 Waf1 (ϫ20) and then counterstained with 4Ј,6-diamidino-2-phenylindole. B, quantification of ATM Ser-1981 -, p53 Ser-15 -, and p21 Waf1 -positive cells. Ͼ100 cells were scored in each case. C, BM KSL cells were untreated (Control), reoxygenated, or reoxygenated in the presence of 2-AP (10 mM; Reoxyϩ2-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 Ser-1981 (magnification, ϫ40) and p53 Ser-15 (ϫ20). D, quantification of ATM Ser-1981 -and p53 Ser-15 -positive cells. Ͼ100 cells were scored in each case. E, BM KSL cells described in C were plated in semisolid, cytokine-containing medium. The CFC activity of WT and FanccϪ/Ϫ BM KSL cells was evaluated at day 7. Data represent the number (mean Ϯ S.D.) of total number of colonies from three independent experiments. *, statistical significance between paired samples at p Ͻ 0.05. F, BM KSL cells described in C were stained with the antibodies against p16 Ink4a (magnification, ϫ20). The numbers below the images are percentages of the cells stained positive for SA-␤-gal or having undergone apoptosis. Data represent mean Ϯ S.D. of three experiments. Reoxy, reoxygenated. and p53 (Ser-15) and expression of p21 in BM KSL cells. ATM autophosphorylation at Ser-1981 activates the kinase and is largely responsible for phosphorylating p53 at Ser-15 in response to DNA damage (15,16). We found ϳ20% each of ATM Ser-1981 -and p53 Ser-15 -positive (Fig. 3, A and B) in reoxygenated WT BM KSL cells. In reoxygenated FanccϪ/Ϫ BM KSL cells, the intensity and percentages of cells stained positive for ATM Ser-1981 and p53 Ser-15 increased significantly (Fig.  3, A and B). We also found that higher levels of p21-positive (22%) stained cells were present in reoxygenated WT BM KSL cells compared with untreated WT cells. The percentage of p21-positive cells increased to 66% in those FA BM KSL cells (Fig. 3, A and 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 reoxygenationinduced senescence, tested as staining for SA-␤-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 ATM Ser-1981 and p53  in reoxygenated WT and FanccϪ/Ϫ BM KSL cells (Fig. 3, C and  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-␤-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 FanccϪ/Ϫ BM KSL cells (Fig. 3E).
Recent reports show that the ability of the cell to relieve senescent cell cycle arrest or reverse senescence depends on the levels of p16 INK4A expression (19,20). Our observations that inhibition of ATM signaling increased clonogenic ability (Fig.  3E) of WT but not FanccϪ/Ϫ progenitor cells led us to examine the relationship between p16 INK4A expression and senescence, tested as staining for SA-␤-gal. Reoxygenation hardly induced p16 INK4A expression in WT BM KSL cells but did so strongly in FanccϪ/Ϫ 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 FanccϪ/Ϫ BM KSL cells (Fig. 3F), indicating that reoxygenation-induced p16 INK4A expression in FanccϪ/Ϫ BM KSL cells was independent of ATM activity. However, when we correlated ATM inhibition and p16 INK4A expression with SA-␤-gal activity, we found that reoxygenated WT BM KSL cells treated with 2-AP and siATM were almost devoid of SA-␤-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-␤gal staining in reoxygenated FanccϪ/Ϫ BM KSL cells, which expressed high levels of p16 INK4A (Fig. 3F). Interestingly, inhibition of ATM activity increased apoptosis in reoxygenated WT and FanccϪ/Ϫ BM KSL cells (Fig. 3F). These results indicate that inhibition of ATM can reverse senescence, tested as staining for SA-␤-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.
In summary, we have shown that 1) reoxygenation-generated oxidative stress induced DNA damage and G 0 /G 1 arrest in BM progenitor cells without significant apoptosis; 2) reoxygenation induced senescence, tested as staining for SA-␤-gal, in reoxygenated BM progenitor cells; 3) induction of senescence, tested as staining for SA-␤-gal, in reoxygenated BM progenitor cells closely correlated with the extent of DNA damage and phosphorylation of ATM at Ser-1981 and p53 at Ser-15; and 4) inhibition of ATM signaling reversed reoxygenation-induced senescence, tested as staining for SA-␤-gal, but increased apoptosis in BM progenitor cells. Thus, these results suggest that reoxygenation induces senescence in hematopoietic progenitor FIG. 3-continued 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)(22)(23). More recently, ATM has been shown to play an essential role in transmitting DNA damage signals generated by reoxygenation, through phosphorylation of p53 Ser-15 (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 Ser-1981 , p53 Ser-15 , and p21 expression and reversal of senescence, tested as staining for SA-␤-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-␤-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 FanccϪ/Ϫ 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 G 1 checkpoint to gain time for DNA repair. The major pathway responsible for triggering the G 1 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 in a timely manner, the cells undergo reversal senescence to prevent accumulation of genetic mutations until the damage has been repair. In FanccϪ/Ϫ BM progenitor cells, however, excessive DNA damage causes overactivation of the ATM kinase, result-ing in a hyperactive G 1 checkpoint. The arrested FanccϪ/Ϫ BM cells enter senescence, while the DNA damage remains unrepaired. Inhibition of ATM in these FA cells thus likely results in bypass of G 1 checkpoint and undergoing apoptosis.