**5. Oxidative stress response in FA hematopoietic cells: a FOXO3a connection**

Forkhead transcription factors of the FOXO class O including FOXO1, FOXO3a, FOXO4 and FOXO6, are implicated in the regulation of diverse physiologic processes, including cell cycle arrest, apoptosis, DNA repair, stress resistance, and metabolism (Brunet *et al.,* 2004; Huang *et al.,* 2007). It has been established previously that members of the FOXO family are negatively regulated by PKB/c-Akt in response to insulin/IGF signaling, and are involved in regulating cell cycle progression and cell death (Geert *et al.,* 2002; Essers *et al.,* 2004). Among these FOXO proteins, FOXO3a functions as a master regulator of oxidative stress (Huang *et al.,* 2007; Tsai *et al.,* 2008). Several recent studies demonstrate that FOXO3a protects quiescent HSCs from oxidative stress (Tothova *et al.,* 2002, 2007; Miyamoto *et al.,* 2005). Some other studies also indicatethat Foxo3a is involved in inflammatory responses, such as inflammatory arthritis, intestinal inflammation, rheumatoid blood and synovial tissue, angiogenesis and postnatal neovascularization etc. (Turrel-Davin *et al.,* 2009; Potente *et al.,* 2005; Jonsson *et al.,* 2005; Walbert *et al.,* 2004).

While strong evidence indicates that FA cells, including hematopoietic cells from FA patients, are intolerant to oxidative stress (Cohen-Haguenauer *et al.,* 2006; Cumming *et al.,* 2001; Du *et al.,* 2008; Futaki *et al.,* 2002; Hadjur *et al.,* 2001; Kruyt *et al.,* 1998; Paganno *et al.,*

demonstrated a defective hematopoiesis (Hadjur *et al.,* 2001). *Fancc-/-* cells exhibit hyperactivation of ASK1, a serine-threonine kinase that plays an important role in redox apoptotic signaling (Saadatzadeh *et al.,* 2004). Another FA protein, FANCG, interacts with cytochrome P450 2E1, which is associated with the production of reactive oxygen intermediates, and mitochondrial anti-oxidant enzyme peroxiredoxin-3 (Futaki *et al.,* 2002, Mukhopadhyay *et al.,* 2006), which suggested a possible role of FANCG in protection against oxidative DNA damage. Furthermore, FANCA and FANCG interact upon oxidative stress (Park *et al.,* 2004). These findings indicate a crucial role of FA proteins in oxidative stress signaling. We recently found that FANCD2 associated with FOXO3a, a master regulator in response to oxidative stress (Huang *et al.,* 2007; Li *et al.,* 2010; Tsai *et al.,* 2008). While these observations point to the involvement of FA proteins in oxidative stress response, the molecular pathways in which FA proteins function to modulate physiologic

oxidative stress have not been defined.

Table 2. Fanconi anemia proteins in redox signaling.

*et al.,* 2005; Jonsson *et al.,* 2005; Walbert *et al.,* 2004).

**connection** 

**5. Oxidative stress response in FA hematopoietic cells: a FOXO3a** 

Forkhead transcription factors of the FOXO class O including FOXO1, FOXO3a, FOXO4 and FOXO6, are implicated in the regulation of diverse physiologic processes, including cell cycle arrest, apoptosis, DNA repair, stress resistance, and metabolism (Brunet *et al.,* 2004; Huang *et al.,* 2007). It has been established previously that members of the FOXO family are negatively regulated by PKB/c-Akt in response to insulin/IGF signaling, and are involved in regulating cell cycle progression and cell death (Geert *et al.,* 2002; Essers *et al.,* 2004). Among these FOXO proteins, FOXO3a functions as a master regulator of oxidative stress (Huang *et al.,* 2007; Tsai *et al.,* 2008). Several recent studies demonstrate that FOXO3a protects quiescent HSCs from oxidative stress (Tothova *et al.,* 2002, 2007; Miyamoto *et al.,* 2005). Some other studies also indicatethat Foxo3a is involved in inflammatory responses, such as inflammatory arthritis, intestinal inflammation, rheumatoid blood and synovial tissue, angiogenesis and postnatal neovascularization etc. (Turrel-Davin *et al.,* 2009; Potente

While strong evidence indicates that FA cells, including hematopoietic cells from FA patients, are intolerant to oxidative stress (Cohen-Haguenauer *et al.,* 2006; Cumming *et al.,* 2001; Du *et al.,* 2008; Futaki *et al.,* 2002; Hadjur *et al.,* 2001; Kruyt *et al.,* 1998; Paganno *et al.,*

2005; Park *et al.,* 2004; Saadatzadeh *et al.,* 2004; Schindler *et al.,* 1988; Zhang *et al.,* 2005) and certain FA proteins interact with cellular factors involved in redox metabolism (Aggarwal *et al.,* 2003; Ames *et al.,* 1995; Bagby *et al.,* 2003), the molecular pathways in which FA proteins function to modulate physiologic oxidative stress have not been defined. Our recent identification of the FANCD2-FOXO3a complex (Li *et al.,* 2010) and preliminary characterization of impaired anti-oxidant defense in primary BM cells from FA patients opened new research opportunities to extend the functional study on the roles of FA proteins in the context of oxidative stress. We envision a model (Fig 2) in which the FA proteins regulate oxidative stress response through mechanisms involving functional interplay with the major oxidative stress-responsive transcription factor FOXO3a and protection of anti-oxidant genes from oxidative damage. Loss of these FA protein functions leads to elevated levels of ROS. As a consequence, FA HSC/P cells accumulate excessive DNA damage and increased genomic instability. However, further studies remains to be done in this context.

Fig. 2. A model for the role of FA proteins in oxidative stress signaling. In WT cells, the FA pathway helps keep cellular levels of ROS in check through functional interaction with the FOXO3a oxidative stress responsive pathway and safeguarding cellular anti-oxidant genes. In FA cells, both the FOXO3a pathway and the anti-oxidant defense are impaired due to loss of the FA protein functions. As a result, FA cells accumulate high levels of ROS, which damages DNA leading to genomic instability.
