MINI-REVIEW

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Regulation of Reactive Oxygen Species in Stem Cells and Cancer Stem Cells
CHIHARU I. KOBAYASHI
Shinjuku-ku, Tokyo, Japan
AND

Cellular Physiology

TOSHIO SUDA*

Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, Keio University School of Medicine,

Stem cells are dened by their ability to self-renew and their multi-potent differentiation capacity. As such, stem cells maintain tissue homeostasis throughout the life of a multicellular organism. Aerobic metabolism, while enabling efcient energy production, also generates reactive oxygen species (ROS), which damage cellular components. Until recently, the focus in stem cell biology has been on the adverse effects of ROS, particularly the damaging effects of ROS accumulation on tissue aging and the development of cancer, and various anti-oxidative and anti-stress mechanisms of stem cells have been characterized. However, it has become increasingly clear that, in some cases, redox status plays an important role in stem cell maintenance, i.e., regulation of the cell cycle. An active area of current research is redox regulation in various cancer stem cells, the malignant counterparts of normal stem cells that are viewed as good targets of cancer therapy. In contrast to cancer cells, in which ROS levels are increased, some cancer stem cells maintain low ROS levels, exhibiting redox patterns that are similar to the corresponding normal stem cell. To fully elucidate the mechanisms involved in stem cell maintenance and to effectively target cancer stem cells, it is essential to understand ROS regulatory mechanisms in these different cell types. Here, the mechanisms of redox regulation in normal stem cells, cancer cells, and cancer stem cells are reviewed. J. Cell. Physiol. 227: 421430, 2012. 2011 Wiley Periodicals, Inc.

With the emergence of photosynthetic bacteria on the earth about 3 billion years ago, oxygen gradually came to prevail in the atmosphere. Eukaryotic organisms became aerobic about 2 billion years ago through incorporation of a proteobacterial ancestor, which enabled highly efcient ATP production and eventually led to the emergence of diverse multicellular organisms (Chan, 2006). Reactive oxygen species (ROS) are highly chemically reactive byproducts of aerobic metabolism. Aerobic organisms have developed systems to cope with these potentially toxic byproducts and defend against the damaging effects of ROS, primarily multiple scavenging mechanisms. On the other hand, ROS are sometimes used in normal physiological responses (Finkel and Holbrook, 2000). In multicellular organisms that are comprised of hundreds of tissues with distinct functions, each type of tissue is believed to arise from a rare progenitor cell, called a multipotent stem cell. These stem cells are dened by their ability to self-renew and their multipotent differentiation capacity. Currently, stem cells that give rise to several types of mammalian tissue have been well-characterized, including those that give rise to the cells and/or tissues of the hematopoietic system (Orkin and Zon, 2008), central nervous system (Zhao et al., 2008), epidermis (Blanpain and Fuchs, 2009), skeletal muscle (Wagers and Conboy, 2005), mammary gland (Visvader, 2009), testis (Yoshida, 2010), and digestive tract (Barker et al., 2010). Since stem cells are in theory sustained for long periods of time in our bodies, it is presumed that they are resistant to many stressors, including chemical compounds, ultraviolet light, ionizing radiation, and oxidative stress. The details of these stress defense mechanisms is a fundamental issue in stem cell biology (Fig. 1). In certain stem cells, such as hematopoietic stem cells (HSCs), the accumulation of DNA damage is prevented because cell-cycle progression is limited, and the cells exist primarily in a state of quiescence. The direct progeny of HSCs, termed short-term hematopoietic stem cells (ST-HSCs) or multipotent progenitors (MPPs), actively proliferate and generate all of the cells of the hematopoietic lineage, but because they do not have long-term (LT) self-renewal capacity, they eventually become exhausted (Orkin and Zon, 2008) (Fig. 2). In this way, gene mutations that may accumulate during proliferation are
2 0 1 1 W I L E Y P E R I O D I C A L S , I N C .

eliminated. Although this quiescent stem cell model is attractive, it is not clear whether it can be applied to other stem cell systems. For instance, murine stem cells of the intestine and stomach, which are characterized by Lgr5 expression, divide every 24 h (Snippert and Clevers, 2011), suggesting that quiescence is not the only stress defense mechanism. Distinct DNA damage responses and DNA repair mechanisms in stem cells that render them tolerant to stressors have recently been demonstrated (Blanpain et al., 2011). Furthermore, tissuespecic stem cells in the body respond differentially to DNA damage. Another feature of stem cells is high efux capacity through ATP-dependent transporters, which serves to prevent

Abbreviations: AMP, adenosine monophosphate; ATP, adenosine triphosphate; CAT, catalase; CML, chronic myeloid leukemia; e-, electron; ETC, electron transport chain; FAD, avin adenine dinucleotide; FADH2, reduced avin adenine dinucleotide; G6P, glucose-6-phosphate; Gclm, glutamate-cysteine ligase regulatory subunit; GPX, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSS, glutathione synthase; GSSG, glutathione disulde; H2O, water; H2O2, hydrogen peroxide; LT-HSC, longterm hematopoietic stem cell; MPP, multipotent progenitor; NAD, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adeninedinucleotide; NADP, nicotinamide adenine dinucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NOX, NADPH oxidase; O2, molecular oxygen; O , 2 superoxide radical; OH, hydroxyl radical; PRX, peroxiredoxin; ROS, reactive oxygen species; SOD, superoxide dismutase; STHSC, short-term hematopoietic stem cell; TCA, tri-carbonic acid; TR, thioredoxin reductase; TRX(S2), oxidized thioredoxin; TRX(SH2), reduced thioredoxin; UV, ultraviolet light. *Correspondence to: Toshio Suda, MD, PhD, Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: sudato@sc.itc.keio.ac.jp Received 19 March 2011; Accepted 21 March 2011 Published online in Wiley Online Library (wileyonlinelibrary.com), 29 March 2011. DOI: 10.1002/jcp.22764

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Fig. 1. Stem cell defense mechanisms and possible cell stress responses. Indicated are several stem cell properties that confer stem cell resistance to cytotoxic insult. Once injured, the fate of the cell is survival or cell death.

the accumulation of chemical compounds in the cell (Challen and Little, 2006).
ROS Generation, Elimination, and Toxicity

ROS is a collective term for oxygen species that are more reactive than free oxygen. Superoxide, hydrogen peroxide (H2O2), hydroxyl radical, and singlet oxygen comprise the main ROS. Broadly speaking, ROS also encompasses nitric oxide. ROS cause the peroxidation of nucleic acids, lipids, amino acids, and carbohydrates, and excessive amounts of ROS, from any source, lead to cellular senescence, apoptosis, or

carcinogenesis (Balaban et al., 2005). Overexpression of superoxide dismutase (SOD), an antioxidant enzyme, in either Drosophila ovarian germline stem cells (GSCs) or the GSC niche, can deter the aging of GSCs and increase GSC proliferation, suggesting that ROS-induced cellular damage may contribute to stem cell aging (Pan et al., 2007). Under physiological conditions, the principal source of ROS is mitochondria (Balaban et al., 2005) (Fig. 3). Exogenously supplied oxygen reaches cells through the bloodstream and enters the electron transport chain of the mitochondria. During this process, superoxide is generated as a consequence of incidental electron leakage, and is immediately converted

Fig. 2. Hierarchy of hematopoietic stem cells (HSCs). LT-HSCs show higher self-renewal activity than ST-HSCs. LT-HSCs are quiescent in cell cycle.

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Fig. 3. Schematic illustration of ROS generation and elimination pathways. O is generated mainly through the mitochondrial electron 2 transport chain, and also by membrane bound NOX, and quickly converted into H2O2 by SOD. H2O2 is then neutralized by several enzymes and proteins such as CAT, PRX, and GPX in processes coupled to GSH and TRX(SH2) oxidation. TRX(S2) and GSSG are reduced by TR and GR, respectively, in an NADPH-dependent reaction. Highly reactive OH is generated from H2O2, leading to oxidization of macromolecules in the cell.

into H2O2 by Mn-containing mitochondrial SOD (MnSOD, SOD2) or Cu/Zn-containing cytosolic SODs (SOD1 and SOD3) (Pervaiz et al., 2009). H2O2 can be converted into highly toxic hydroxyl radicals through acquisition of an electron, or eliminated by the action of glutathione peroxidase (Gpx) (Cohen and Hochstein, 1963), peroxiredoxin (Prx) (Hofmann et al., 2002), or catalase (Michiels et al., 1994). Cells also possess an active ROS-generating system in their NADPH oxidase complexes (NOXes) (Katsuyama, 2010), which will be described in detail in the following sections. Evidence to date has shown that aerobic organisms have not only developed passive defense mechanisms against ROS, but also actively use ROS in signal transduction pathways that regulate cell survival and proliferation (Clement and Stamenkovic, 1996; Sattler et al., 1999; Chen and Pervaiz, 2007). However, the signicance of ROS in stem cells is only starting to become clear. Some stem cells are more tolerant to ROS than others (Takubo et al., 2006, 2008). For instance, mammalian HSCs, the redox regulation of which is currently an intense area of investigation, are more vulnerable to ROS than their progenitors; exposure to ROS causes these cells to lose stemness and die. Using a uorescent ROS probe, DCF-DA, Jang and Sharkis (2007) were able to divide HSCs (lineage marker-negative, CD45-positive and annexin V-negative) into two fractions: DCF-DAhigh and DCF-DAlow. The investigators were then able to show that the DCF-DAlow fraction retained a higher reconstitution capacity than the DCF-DAhigh fraction (Jang and Sharkis, 2007). Interestingly, the DCF-DAhigh fraction had higher myeloid differentiation capacity than the DCF-DAlow fraction, which indicated that the DCF-DAhigh fraction was myeloid shifted, one of the characteristics of aged HSCs. Treatment with antioxidants or inhibitors of p38 mitogen
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activated protein kinase (MAPK), which protects HSCs against ROS damage, restored the colony-forming capacity of the DCF-DAhigh fraction in vitro. Although it is not clear whether the DCF-DAhigh fraction arises as a result of exposure to excessive ROS or during the normal differentiation process, these results show that ROS can compromise the maintenance of HSCs.
ROS-Regulating Mechanisms in HSCs

Analyses of genetically manipulated mice in which the stem cells contain high levels of ROS have been important in understanding the regulatory mechanisms that govern the redox status of HSCs (Fig. 4). For instance, mice decient in Atm (Atm/), which in humans is the gene responsible for ataxia telangiectasia, have increased levels of ROS in various organs (Ito et al., 2004). The elevated ROS levels in Atm/ mice are due to dysregulation of the major anti-oxidative systems mediated by catalase, Gpx, SOD, and glutathione reductase (Barzilai et al., 2002). HSCs of Atm/ mice have a dramatically decreased reconstitution capacity compared to progenitors, and aged Atm/ mice develop myelodysplasia with elevated levels of ROS. Activation of p38MAPK is also observed in ATM-depleted HSCs. Elevated ROS or activation of p38MAPK resulted in depletion of normal HSCs after serial transplantation, and this decreased reconstitution capacity was normalized by treatment with an antioxidant, N-acetyl L-cysteine (NAC). These results point to a causative role of ROS in the impaired function of ATM-decient HSCs. Evidence has also accumulated showing that forkhead homeobox type O (FoxO) transcription factors play a pivotal role in the regulation of the redox status of HSCs (Storz, 2011).

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Fig. 4. Signaling pathways associated with ROS generation and elimination in HSCs. The PI3K/AKT pathway is activated under conditions of high oxygen or high nutrients, and is accompanied by enhanced protein synthesis, oxidative phosphorylation, and ROS generation. Over-activation of the AKT pathway results in exhaustion of HSCs through a ROS-independent manner. Quiescent HSCs maintain low ROS levels via several systems, including promotion of glycolysis and autophagy, and upregulation of FoxO proteins. The relationship between LKB1 and ROS is unclear, but it may facilitate HSC maintenance through distinct mechanisms.

The FoxO family includes FoxO1, FoxO3, FoxO4, and FoxO6 in mammals. FoxO proteins have been implicated in metabolism control, cell survival, cellular proliferation, DNA damage repair response, and stress resistance. FoxO3a knockout mice have higher levels of H2O2 due to reduced levels of antioxidant enzymes such as catalase or SOD2, both of which are downstream targets of FoxO3a (Miyamoto et al., 2007). Excessive H2O2-induced phosphorylation of p38MAPK in these mice results in expression of the cellular senescence-related genes p16Ink4a and p19Arf, and loss of HSC self-renewal capacity. This abnormity was restored by inhibition of p38MAPK or reduction of ROS levels through administration of antioxidants. Tothova et al. (2007), in analyzing HSCs from FoxO1/FoxO3a/ FoxO4 triple-knockout mice, reported similar results. In these triple-knockout mice, there were fewer HSCs, and those that were present exhibited accelerated cell-cycle progression and increased apoptosis. Again, the defects were rescued by administration of antioxidants. These results support the idea that FoxO family proteins are key mediators of ROS regulation in HSCs and contribute to the maintenance of self-renewal capacity. FoxO3a has also been shown to transactivate and directly interact with ATM, thereby inhibiting ROS generation and potentiating the DNA damage response (Tsai et al., 2008; Yalcin et al., 2008). FoxO proteins are regulated both negatively and positively through phosphorylation by several upstream signal transduction pathways. The phosphoinositide 3-kinase (PI3K)/ AKT pathway is the most well-recognized negative regulator of the FoxO transcription factors (Brunet et al., 1999). AKT is a
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kinase that is activated by growth factors such as insulin or conditions of abundant nutrients. Once activated, AKT phosphorylates FoxO at three consensus AKT phosphorylation sites (Naka et al., 2008). Phosphorylated FoxO is then exported from the nucleus to the cytoplasm by a member of the 14-3-3 family of proteins, resulting in loss of transcriptional activity (Brunet et al., 2002). In the hematopoietic system, the intracellular localization of FoxO3a varies according to differentiation stage. In HSCs, FoxO3a localizes to the nucleus, whereas in hematopoietic progenitors, it localizes to the cytoplasm. Correspondingly, the PI3K/AKT pathway is suppressed in HSCs and activated in hematopoietic progenitors (Yamazaki et al., 2006; Miyamoto et al., 2007). Constitutive activation of PI3K/AKT signaling through deletion of phosphatase and tensin homolog (PTEN), a PIP3 phosphatase, results in acceleration of the cell cycle, and eventually leads to exhaustion of HSCs (Yilmaz et al., 2006). However, the mechanism of depletion of HSCs in Pten-deleted mice cannot be explained by upregulation of ROS, in contrast to FoxO3a decient mice, since Pten-deleted mice do not have elevated ROS levels. Furthermore, the reconstitution capacity of HSCs in Pten-negative mice was restored by deletion of p53 or p16Ink4a, but not by administration of NAC (Lee et al., 2010). Moreover, Kharas et al. (2010) reported no elevation of ROS in HSCs transduced with constitutively active AKT. Thus, the relationship between FoxO and the PI3K/AKT pathway in HSCs awaits further clarication. FoxO is positively regulated by AMP-activated protein kinase (AMPK), an intracellular sensor of starvation that monitors

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changes in the AMP/ATP ratio (Greer et al., 2007). Jun N-terminal kinase (JNK)-mediated phosphorylation is also a major mechanism of induction of FoxO activity in response to H2O2 (Essers et al., 2004). Phosphorylation of two threonine residues of FoxO by JNK leads to nuclear translocation (Kops et al., 2002). Additionally, post-translational modications such as acetylation by CBP/p300 are also necessary for FoxOmediated cellular detoxication, cell-cycle arrest, and DNA repair or apoptosis in responses to ROS stress (van der Horst and Burgering, 2007). Although acetylation of FoxO family members by CBP/p300 in HSCs has yet to be reported, posttranslational activation by CBP/p300 is an intriguing possibility, given the crucial role of CBP/p300 in both the maintenance and differentiation of HSCs (Rebel et al., 2002). Chuikov et al. (2010) demonstrated that PR domaincontaining16 (Prdm16), a zinc nger transcription factor that regulates leukemogenesis, palatogenesis and brown-fat development, is required for stem cell maintenance in the hematopoietic and nervous systems. Prdm16 deciency led to increased ROS levels and depletion of stem cells. In the central nervous systems, Prdm16 appeared to promote neural stem/ progenitor cell function, partly by promoting hepatocyte growth factor (HGF) expression. Administration of antioxidants to Prdm16-decient mice partially rescued the defects in neural stem/progenitor cell function and neural development, suggesting the role of Prdm16 as a modulator of oxidative stress in stem cell maintenance. It has been shown that p53 protects stem cells from ROS damage through the regulation of redox factors such as thioredoxin, metallothioneins, or the redox-repair enzyme APE1/ref1 (Hafsi and Hainaut, 2011). However, Abbas et al. (2010) demonstrated that activation of p53 results in the depletion of stem cells via ROS accumulation. In this study, the investigators focused on the role of Mdm2, an E3 ubiquitin ligase involved in the degradation of p53, in the maintenance of HSCs. The p53 variant p53515c (encoding p53R172P), a hypomorphic allele of p53 that lacks pro-apoptotic activity, rescued the embryonic lethality of Mdm2/mice. The group generated Mdm2/p53515c/515c mice, and found that expression of p53R172P induced the accumulation of ROS, resulting in cell-cycle arrest, senescence and cell death of HSCs through activation of p16Ink4a. This defect was rescued by administration of antioxidants, hypoxic culture, or p16Ink4a deletion, demonstrating the pivotal role of Mdm2 in down-regulation of the p53-mediated response to ROS. In fact, p53 activates several ROS-inducing genes, such as Pig1 (Galectin-7), Pig8, and Pig12 (Polyak et al., 1997; Li et al., 1999; Donald et al., 2001), and excess p53 may results in a dysregulated p53-ROS positive feedback loop. It seems therefore that an appropriate level of p53 is essential for the maintenance of HSCs in terms of ROS regulation.
Active Generation of ROS in Stem Cells

knockout mice were in a constitutively quiescent state and were not able to differentiate into MPPs due to MPP cell apoptosis (Juntilla et al., 2010). These AKT1//AKT2/ HSCs also had decreased levels of ROS. The colony-forming capacity of the cells was restored by raising the levels of ROS, indicating that the appropriate level of ROS is important for normal HSC function. Lewandowski et al. (2010) showed using in vivo imaging that homing of HSCs to the bone marrow after transplantation was mediated by ROS. A surprising nding in terms of redox regulation of stem and precursor cell function was recently reported by Le Belle et al. (2011). In contrast to what is observed with HSCs and oligodendrocyte/type-2 astrocyte progenitor cells (also known as oligodendrocyte precursor cells), both of which retain selfrenewal capacity under reduced conditions, increased oxidative status promoted stem cell activity of neuroepithelial stem cells (NSCs) in the central nervous system. Pharmacological inhibition of NOXes inhibited NSC selfrenewal, suggesting that NOXes play a critical role in maintaining adequate ROS levels in NSCs. Brain-derived neurotrophic factor (BDNF), which can promote the selfrenewal capacity of NSCs, increased the levels of ROS in these cells. Similarly, the PI3k/AKT pathway appeared to be essential for NSC self-renewal. Lending support to this nding was a report by Limoli et al. (2004) showing that oxidation promotes the self-renewal and division of hippocampal cells in the CNS, which generate neurons. In analyzing cardiac and embryonic stem cells, Li and Marban (2010) reported that physiological levels of intracellular ROS are required to maintain genomic stability in stem cells through activation of the DNA repair pathway. Taken together, in contrast to the conventional idea of ROS as the enemy of stem cells, the evidence suggests that ROS production is tightly controlled to promote proliferation and survival of both stem and progenitor cells, and that the appropriate levels may depend on cellular context.
Links Between ROS and Metabolism

Intracellular ROS are generated as a result of oxidation of NADPH by NOX family members (NOX 1-5, Duox1, and 2) (Katsuyama, 2010). Little is known about the function of these enzymes in HSCs. In human CD34 HSCs and progenitor cells, NOX1, 2, 4 are expressed. In these cells, half of the oxygen consumed is mitochondrial in origin, and the rest is derived from NADPH (Piccoli et al., 2005; Piccoli et al., 2007). Deletion of Nox2 causes reduced HSC mobilization from the bone marrow to peripheral blood after treatment with G-CSF, IL-8, cyclophosphamide (van Os et al., 2000), or ischemia (Urao et al., 2008). Constitutive activation of AKT accelerates HSC proliferation, resulting in depletion of HSCs. However, complete loss of this pathway also seems to impair normal hematopoiesis. HSCs isolated from AKT1/AKT2 doubleJOURNAL OF CELLULAR PHYSIOLOGY

Activation of the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway by growth factors or nutrient signals such as glucose and amino acids promotes protein synthesis, resulting in cell growth and proliferation. Analysis of tissuespecic knockout of TSC (tuberous sclerosis complex)1, an inhibitor of mTOR, in the hematopoietic system of mice revealed that in HSCs, the mTOR pathway was over-activated, the HSCs were no longer quiescent and they had lost their capacity for reconstitution (Chen et al., 2008; Gan et al., 2008). In contrast to Pten-deleted mice, ROS were elevated in Tsc1-deleted HSCs, and the number of mitochondria was increased. Genes related to mitochondrial biosynthesis were also upregulated (Chen et al., 2008). Recently, another impressive mediator linking AKT/mTOR and FoxO proteins with metabolism in HSCs was identied. Three groups independently reported that Lkb1 (liver kinase B1) is indispensable for HSC maintenance (Gan et al., 2010; Gurumurthy et al., 2010; Nakada et al., 2010). In Lkb1 knockout mice, there was a gradual decline in hematopoiesis followed by a transient increase in hematopoietic progenitors; reconstitution capacity after bone marrow transplantation was also lost in these mice. LKB1 is the gene responsible for PeutzJeghers syndrome. Lkb1 directly activates AMPK in response to elevated AMP/ATP ratios, resulting in activation of downstream targets, including Tsc1 and FoxO, via phosphorylation (Kahn et al., 2005). Importantly, the loss of Lkb1 phenotype was not rescued by activation of AMPK or administration of ROS scavengers alone; thus, Lkb1 may maintain HSC quiescence through distinct mechanisms, perhaps involving modulation of mitochondrial function through the PPARg (peroxisome proliferator-activated receptor-g) co-activator Pgc1 (Gan et al.,

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2010), or enhanced genomic stability through as yet unknown mechanisms (Nakada et al., 2010). Autophagy is one of the most important cellular defense systems against starvation, and its role in HSCs is just now being unveiled. FIP (feline infectious peritonitis) 200, a regulator of autophagy, triggers intracellular energy generation under conditions of nutrient starvation. Fetal HSCs lacking FIP200 have more mitochondria and elevated levels of ROS, resulting in defects in stem cell function (Liu et al., 2010). These results indicate that metabolic signaling may suppress ROS production not only in adult HSCs but in fetal HSCs as well. As summarized above, various metabolic pathways are associated with ROS production and maintenance of HSCs. The mechanisms by which these pathways interact to maintain appropriate levels of ROS remain to be elucidated. Another intriguing issue that has yet to be fully explored is how intracellular metabolism cooperates with extracellular nutrients.
HSCs in Hypoxic Microenvironments

analysis of the murine bone marrow and demonstrated that LT-BrdU-labeled HSCs were in a hypoxic state (Kubota et al., 2008). Additionally, live real-time imaging of murine bone marrow showed that HSCs gradually moved away from the vessels in the bone marrow after transplantation (Lo Celso et al., 2009; Xie et al., 2009).
The Role of Hypoxia in Stem Cells

Although HSCs are vulnerable to oxidative stress, hematopoiesis continues throughout the lifetime of an animal. It has been hypothesized that in addition to cell-intrinsic mediators such as FoxO that help maintain low ROS levels in HCSs, the microenvironment around the HSC also helps restrict ROS production. It is known that long-term HSCs (LT-HSCs) that retain a high capacity for reconstitution reside mainly in the endosteum of the bone marrow (Oh and Kwon, 2010). Since the endosteum is enriched with capillaries, one cannot simply postulate that the endosteum is a low ROS-generating hypoxic region. Given that the oxygen consumption of a hematopoietic cell is relatively high, simulation models of oxygen diffusion in the bone marrow have shown that the partial pressure of oxygen in a region that is only 100 mm away from a capillary could be one-tenth of the partial pressure of the capillary (Chow et al., 2001). The partial pressure of oxygen in the human bone marrow is approximately 50 mmHg, and oxygen saturation is 80% (Harrison et al., 2002). Based on this simulation, the niche of quiescent HSCs that are distant from sinusoidal vessels may be a more hypoxic environment. Using FACS analysis of a tracer administered to mice, Parmar et al. (2007) showed that undifferentiated HSCs were relatively enriched in low-reux regions. When the mice were given pimonidazole, a marker of hypoxia, beforehand, the group found that cells in the low-reux region were also positive for pimonidazole. Furthermore, administration of tirapazamine, which damages hypoxic cells, resulted in the loss of HSCs. Human cord blood HSCs that were transplanted into NOD/ Shi-scid/IL-2Rgnull (NOG) mice were positive for pimonidazole (Shima et al., 2009). These results suggest that HSCs occupy a hypoxic niche in the body. On the other hand, Kiel et al. (2005) reported that 60% of HSCs reside along the vascular endothelium, based on a twodimensional analysis of a resected segment of the bone marrow (Kiel et al., 2005). The question then is how HSCs are maintained in a hypoxic state while residing near the endothelium. First, HSCs may maintain hypoxia due to malfunctioning of the capillaries in the endosteum. Second, the intracellular partial pressure of oxygen, which is determined by the balance of oxygen supply and consumption, is important for the ability of HSCs to detect hypoxia. If oxygen consumption in HSCs is relatively high, HSCs could induce hypoxia-related gene expression even though they reside near capillaries. Lastly, twodimensional analysis of a resected segment of the bone marrow may not fully illuminate the complex relationship between HSCs and their surrounding microenvironment. To address this issue, we performed a whole-mount immunocytochemical
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As described above, HSCs may preferentially maintain low oxygen levels through both cell-autonomous and nonautonomous mechanisms. What then is the signicance of low levels of oxygen in HSCs? Characteristics of undifferentiated HSCs such as colony-forming ability or reconstitution capacity are retained after hypoxic culture compared with normoxic culture (20% oxygen) (Cipolleschi et al., 1993; Shima et al., 2009; Ivanovic et al., 2000; Danet et al., 2003). Undifferentiated HSCs are also described as side population (SP) cells, in that they constitute a cell fraction with higher efux activity when stained with the DNA-labeling dye Hoechst 33342 (Goodell et al., 1996). Krishnamurthy et al. (2004) showed that hypoxia promotes the maintenance of HSC SP characteristics (Krishnamurthy et al., 2004). The efux of Hoechst 33342 by HSCs is mediated by Bcrp (Breast cancer resistance protein)-1, which is an ATP-dependent transporter. When HSCs were cultured under hypoxia, the expression of Bcrp-1 was elevated and the SP population was increased compared with HSCs cultured under normoxia. Bcrp-1 transports heme and porphyrin out of HSCs, components that otherwise easily accumulate in HSCs in hypoxic environments like the bone marrow, resulting in cell damage. It seems that the hypoxic niche of the bone marrow may protect undifferentiated HSCs via the induction of Bcrp-1 expression. However, Brcp-1 knockout mice do not exhibit defects in hematopoiesis (Zhou et al., 2002). Clearly, there is much to be elucidated about the relationship between HSCs and the hypoxic microenvironment of the bone marrow in vivo. Recently, it was shown that HIF-1a, a key regulator of the intracellular response to hypoxia, is highly expressed in LT-HSCs at both the transcriptional and protein levels (Simsek et al., 2010; Takubo et al., 2010). HSCs derived from conditional HIF-1a knockout mice (HIF-1aD/D) exhibited impaired reconstitution capacity with high expression of p16Ink4a and p19Arf. This phenotype was rescued by overexpression of B lymphoma Mo-MLV insertion region 1 homolog (Bmi1), a polycomb gene that inhibits the expression of p16Ink4a and p19Arf (Oguro et al., 2006). Injection of polyinosinic polycytidylic acid (pIpC) into HSCs from Mx1-Cre:HIF-1aox/ox mice resulted in acceleration of the cell cycle, reduced tolerance against stresses such as 5-FU or aging, and loss of serial reconstitution capacity, suggesting a pivotal role for HIF-1a in HSCs. However, while depletion of HIF-1a appears to decrease HSC capacity via expression of p16Ink4a and p19Arf, the role of HIF-1a seems to be somewhat complex. Conditional deletion of VHL in HSCs revealed that HSC functions were differentially inuenced by HIF-1a in a dose-dependent manner. VHL in humans is the gene responsible for von Hippel Lindau disease. VHL is an E3 ubiquitin ligase specic for HIF-1a. HIF-1a protein was increased in both homozygous (VHLD/D) and heterozygous (VHL/D) conditional deletion mutants of VHL. Interestingly, although there were few normal hematopoietic progenitors (CD34 LSK fraction) in G0 phase, the percentage of VHL/Dhematopoietic progenitors in G0 phase was dramatically increased. Since HIF-1a is not usually expressed in hematopoietic progenitors, these results suggested that stabilization of HIF-1a through VHL heterozygous deletion enabled VHL/Dhematopoietic progenitors to enter a quiescent state of the cell cycle. Moreover, VHLD/DHSCs, in which HIF-1a levels were maximally upregulated, were in a quiescent state

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but had completely lost their reconstitution capacity. This loss of reconstitution capacity could be due to a reduced homing capacity of VHLD/DHSCs. This suppression of the cell cycle and reconstitution capacity was restored in HIF-1aD/DVHLD/Ddouble-knockout HSCs, indicating that the defect in VHLD/DHSCs was HIF-1a-dependent. These results demonstrate that a moderate increase in HIF-1a is benecial for HSCs. Simsek et al. (2010) showed that HSCs utilize glycolysis rather than mitochondrial oxidative phosphorylation to meet the energy requirements of the cell, leading the authors to speculate that HIF-1a was a possible regulator of this distinct metabolic prole. The same group also showed that Meis1, a TALE family member associated with denitive hematopoiesis, transcriptionally activates HIF-1a in HSCs, highlighting the importance of the Meis1/HIF-1a/glycolysis axis. In neural and embryonic stem cells under hypoxic conditions, HIF-1a mediates the activation of Wnt/b-catenin signaling through upregulation of b-catenin and induction of expression of the downstream effectors, lymphoid enhancer binding factor (LEF)-1 and T-cell-specic transcription factor (TCF)-1 (Mazumdar et al., 2010). Wnt/b-catenin activity was closely correlated with low oxygen regions in the subgranular zone of the hippocampus, a key NSC niche. Loss of HIF-1a decreased Wnt-dependent hippocampal neurogenic niche capacity, including NSC proliferation, differentiation, and neural maturation. In light of a contradictory report showing that HIF-1a inhibited Wnt/b-catenin activity in HCT116 colon cancer cells, it seems safe to say that the interaction between HIF-1a and Wnt/b-catenin in stem/progenitor cells may differ from more differentiated cell types, including cancer cells (Kaidi et al., 2007). Nevertheless, it appears that oxygen concentration may have a direct role in the regulation of different types of stem cells.
Cancer, ROS, and Hypoxia

elevated ROS levels, as functional p53 works not only as a guardian of the genome against DNA damage, but also as a transcription factor that regulates the expression of many genes involved in the redox system. Mitochondrial DNA (mtDNA) mutations associated with increased ROS levels due to increased leakage of electrons have been reported in both solid tumors and leukemia (Carew et al., 2003; Indo et al., 2007; Ishikawa et al., 2008). Extrinsic factors such as inammatory cytokines (i.e., TNFa), nutrient imbalance, and a hypoxic environment can also result in dysregulation of ROS production (Cook et al., 2004; Azad et al., 2008). ROS facilitate the proliferation of cancer cells through the activation of downstream targets. For example, constitutive activation of the Keap1/Nrf2 pathway, which responds to elevated ROS levels, renders cancer cells resistant to oxidative stress through upregulation of various cytoprotective genes, thereby facilitating cancer progression (Taguchi et al., 2011). The AKT/mTOR pathway ultimately activates nuclear factor kB (NF-kB) to induce pro-survival signals, and ROS are required for NF-kB activation in HeLa cells in response to inammatory cytokines (Sulciner et al., 1996). ROS also have an adverse effect on cancer cells. High levels of ROS cause senescence-induced tumor suppression through activation of p16Ink4a (Takahashi et al., 2006). In addition, excess ROS may induce cancer cell death through non-specic oxidation of cellular components or by triggering cell death mechanisms (Trachootham et al., 2009). These results raise the question, how do cancer cells adapt to ROS? Several reports suggest that cancer cells may acquire adaptive mechanisms, including the activation of ROS-scavenging systems such as glutathione (GSH), as well as the inhibition of apoptosis (Trachootham et al., 2009), which may lead to malignant transformation, metastasis and chemo-resistance.
Cancer Stem Cells

Research on oxygen metabolism in cancer cells dates back to the early 20th century. Warburg found that cancer cells exhibit abnormal oxygen metabolism, in that they gain energy from glycolysis even under aerobic conditions, a phenomenon termed the Warburg effect (Warburg, 1956). There is a close relationship between cancer cells and hypoxia. Due to the chaotic vascular architecture of solid cancers, oxygen distribution is quite unequal, with some areas showing as low as 5 mmHg (0.7% O2) oxygen partial pressure (Hockel and Vaupel, 2001). Additionally, there is evidence that moderate hypoxia might be benecial for the survival of some types of cancer stem cells (Heddleston et al., 2010). Cancer cells also have abnormal redox systems. It has been reported that many types of cancer cells have increased levels of ROS compared with their normal counterparts. Changes in redox status may facilitate cancer progression in a Darwinian manner via acceleration of DNA damage. In using ROS as a signal transducer, cancer cells may acquire proliferative, invasive, and metastatic properties as well. In fact, increased ROS levels in cancer cells have been shown to correlate with the aggressiveness of tumors and poor prognosis (Patel et al., 2007; Kumar et al., 2008). The mechanisms of ROS generation in cancer are diverse, and the association of ROS with cancer metabolism has been reviewed in detail elsewhere (Cairns et al., 2011). Upregulation of ROS-producing enzymes by activated oncogenes is one of the major sources of ROS in cancer cells. For example, oncogenic K-ras transcriptionally activates NOX1, and ROS are essential for the cytoskeletal changes associated with cellular transformation and for tumorigenicity (Mitsushita et al., 2004; Shinohara et al., 2007). Proto-oncogenes such as SRC (Diaz et al., 2009) and c-MYC (Vafa et al., 2002), and overexpression of receptor tyrosine kinases (Benhar et al., 2001) induce ROS production as well. Loss of functional p53 also leads to redox imbalance and
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Cancer stem cells or cancer-initiating cells are a group of cells with self-renewal and differentiation capacity, and in this sense at least, resemble normal stem cells. Cancer stem cells are believed to be the cause of chemo- and radio-resistance and disease recurrence in many, if not all, types of cancer (Reya et al., 2001). The key questions for this review are whether cancer stem cells have similar redox properties as their non-tumorigenic offspring, or if the redox status in cancer stem cells is similar to that of their normal stem cell counterparts (Fig. 5). Although the redox status of cancer stem cells is not well-characterized, it has been suggested that cancer stem cells share some characteristics of normal stem cells. For instance, CD24/low/CD44 breast cancer stem cells are more tumorigenic and are relatively resistant to radiation at the DNA and cellular levels, due perhaps to signicantly lower levels of basal and radiation-induced ROS in these cells, indicative of higher levels of ROS scavengers (Phillips et al., 2006). Diehn et al. (2009) reported that ROS levels are lower in human and murine breast cancer stem cells compared to non-stem breast cancer cells (Diehn et al., 2009). The mechanism whereby ROS levels are kept low in breast cancer stem cells appears to involve upregulation of ROS-scavenging molecules, thereby contributing to tumor radio-resistance. Human gastrointestinal cancer stem cells with a high level of CD44 expression showed a enhanced capacity of GSH synthesis and defense against ROS by a cystineglutamate exchange transporter (Ishimoto et al., 2011). For glioblastoma, it has been reported that CD133 glioblastoma stem cells (GSCs) exhibit increased survival rates in response to ionizing radiation compared to non-stem glioblastoma cells due to a more efcient DNA repair response (Gilbertson and Rich, 2007). Additionally, evidence is accumulating on the importance of HIFs in GSCs (Li et al., 2009;

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Fig. 5. ROS levels in cancer and cancer stem cells. Some cancer stem cells, such as breast cancer stem cells, glioblastoma stem cells, and CML stem cells, have low ROS levels. These types of cancer stem cells are assumed to constitute a chemo- and radio-resistant fraction. Conversely, cancerous tissue derived from cancer stem cells tends to have higher ROS levels.

Seidel et al., 2010). HIF-2a has been implicated in the tumorigenic capacity of GSCs (Li et al., 2009). HIF-1a and HIF-2a are highly homologous and bind to similar hypoxia response element (HRE) sequences (Bertout et al., 2008). However, HIF-1a and HIF-2a have non-redundant biological roles due to their unique target genes and differential oxygen requirement for activation. Only HIF-2a is stable under conditions of 7% oxygen (McCord et al., 2009). Li et al. (2009) showed that HIF-2a was signicantly expressed only in GSCs, while HIF-1a was expressed in both stem and non-stem glioma cells under more severe hypoxic conditions. Several genes associated with the hypoxic response in normal cells, such as Glut1, Serpin B9, and VEGF, have been shown to be upregulated in GSCs (Bao et al., 2006; Li et al., 2009). Furthermore, hypoxia induces the expression of genes related to stem cell function such as Sox2 and Oct4 (McCord et al., 2009). Sox2, together with Sox4, was recently shown to play a pivotal role in the maintenance of GSCs (Ikushima et al., 2009). HIF-1a interacts with Notch under hypoxic conditions to promote a stem cell phenotype (Gustafsson et al., 2005). Chen et al. (2007) demonstrated that hypoxia greatly enhances Notch signaling (especially NOTCH-1) in lung adenocarcinoma (Chen et al., 2007). In looking specically at leukemic stem cells (LSCs), Guzman et al. (2005) showed that parthenolide (PTL) induces apoptosis of LSCs in AML and blast crisis CML (Guzman et al., 2005). The molecular mechanism of PTL-induced apoptosis appeared to involve inhibition of NF-kB, proapoptotic activation of p53, and increased ROS levels. Ito et al. (2008) demonstrated the critical role of the promyelocytic leukemia protein (PML) tumor suppressor in the maintenance of quiescent CML stem cells, introducing the possibility of eradicating CML stem cells with arsenic trioxide (As2O3) (Ito et al., 2008), a ROS generator that inhibits PML. It has been reported that loss of FoxO3 causes a myeloproliferative syndrome due to increased ROS
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accumulation in mutated hematopoietic progenitors, leading to the activation of the AKT/mTOR signaling pathway (Yalcin et al., 2010). In contrast, Naka et al. (2010) demonstrated that TGF-b/FoxO signaling is involved in maintaining the serial repopulating capacity of CML stem cells (Naka et al., 2010); inhibition of AKT by TGF-b resulted in the nuclear localization of FoxO3 in CML stem cells. Bernardi et al. (2006) showed that PML represses mTOR and negatively regulates the rate of synthesis of HIF-1a under hypoxic conditions, providing further evidence that these molecules might be key targets in LSCs (Bernardi et al., 2006). Characterization of the redox systems of cancer stem cells is now an active area of research, as it is critical that we begin to develop strategies to eradicate these specic cell populations.
Concluding Remarks

Oxidative stress caused by the cellular accumulation of ROS is intrinsically detrimental to stem cells. While stem cells have evolved to contain redox systems that protect against ROS, it has become increasingly clear that stem cells regulate cell-cycle phase via subtle changes and ne-tuning of their redox status. One such mechanism is the intrinsic redox control mediated by transcription factors such as the FoxO family of proteins. Extrinsic factors, typied by the hypoxic niche, have also been shown to play a pivotal role. The redox status of a few types of stem cells has been characterized, leaving the details of the mechanisms of regulation of stem cell function by redox state to be addressed by further study. Given the importance of several factors such as HIF-1a and Lkb1, which bridge redox regulation and cellular metabolism, the signicance of ROS in stem cell biology should be reframed by comprehensive approaches, including intra- and extracellular metabolomics. As for the relationship between cancer cells and ROS, the redox status of cancer cells differs from that of their normal

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counterparts. That said, recently, the existence of cancer stem cells that exhibit redox patterns that are similar to normal stem cells has been demonstrated for several cancers. To develop rational therapies that specically target cancer stem cells, it is essential that we clarify the differences between normal and cancer stem cells in terms of redox regulation and the response to ROS.
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