**2.3 Glutathione in cell proliferation**

272 Selected Topics in DNA Repair

view of the present knowledge of the role of oxidative stress in promoting cancer, its damaging effects to DNA, and its action on cell proliferation and apoptosis. Malignant cells produce more radical species and, although antioxidant defence could also be induced in these cells, they display a pro-oxidant state. However, apparently the oxidative stress generated in these high proliferative cells does not exceed the level where oxidative damage becomes so severe that cell function is impaired. This finding is in line with previously cited reports and many others that support the role of reactive oxidative species mediated

**2.2 The bridge between the oxidative stress and cell proliferation - Glutathione** 

Glutathione (GSH) is the most abundant non-protein thiol in mammalian cells (Meister & Anderson, 1983). It is considered essential for survival in mammalian cells (Viña, 1990) and yeast Meister & Anderson, 1983; Viña et al., 1978), but not in prokaryotic cells. The exact nature of this important difference has not been elucidated. Glutathione was discovered in 1888 by Rey Pailhade as "organic hydrogenate of sulphur" (Rey Paihade, 1988) and "rediscovered" and fully described by Sir Frederic Gowland Hopkins in the 1920s (Hopkins,

Glutathione has attracted the scientific interest with variable intensity along the century since its discovery and many important cellular functions of this tripeptide were revealed along the years. Glutathione shows a widespread localization within cells and considerably high concentration in cells and tissues (up to 10 mM) (Tateishi et al., 1974). Examples of normal physiological functions of glutathione known for a long time include regulation of the transport of certain amino acids (Viña & Viña, 1983) control of cytoskeleton assembly (Burchil et al. 1978) and regulation of enzymatic activity (Ernst et al., 1978; Ziegler, 1985). During 1960s, GSH was demonstrated to be a co-substrate for a number of important enzymatic reactions: GSH-S-transferase was described (Booth, 1961) and its role in a first– line defence against electrophilic insult, obviously dependent on glutathione, was suggested (Boyland, 1969). These pioneer works became the bases for many studies that lead to the development of concepts such as drug and foreign compound detoxification, and multidrug resistance (Smith, 1977) of crucial importance in the modern cancer therapy. Glutathione, as it lacks toxicity linked to cysteine (Viña et al., 1983), is considered perfect as a cellular thiol "redox buffer" with a purpose to maintain a given thiol/disulfide redox potential (Sies, 1999). Therefore, the redox properties and abundance that characterize this molecule grant it a major role in protecting the cell against oxidants and electrophiles, and during 1980s this

Association of redox regulation with toxicity events lead to the introduction of the concept of "oxidative stress" at biochemical and cellular level (Sies & Cadenas, 1985). Oxidative stress is generally defined as an imbalance between prooxidants and antioxidants with a considerable effect on other cellular components, including redox sensitive functional groups of proteins. Nowadays, with the increasing awareness of the importance of ROS and glutathione in cellular signalling, and the cellular redox environment in fundamental physiological processes, a new definition of oxidative stress is proposed. According to Jones, 2006, oxidative stress may be better defined as a disruption of redox signaling and control. Interestingly, more than 10 years ago, searching for a molecular link between oxidative stress and cell proliferation, Cotgrave IA and Gerdes RG recommended similar term:

signalling in the promotion of cell growth.

1929) and quoted by Sies several years later (Sies, 1999).

particular role of glutathione is central in many research efforts.

"oxidant mediated regulation" (Cortgreave and Gerdes, 1998).

Several studies from more than 20 years ago have suggested that changes in low molecular weight thiols (LMWT) are associated with regulation of cell growth. Harris and Patt published (Harris & Pat, 1969) that nonproliferating mouse tumour cells contained LMWT than proliferating cells and in early eighties various authors report similar results: human lung and ovarian tumour cells during the exponential growth demonstrate higher GSH levels than during nondividing state (Harris & Pat, 1969; Post, 1983). In accordance to these findings, Kosower and Kosower (Kosower & Kosower, 1978) have demonstrated that decrease of GSH biosynthesis in vivo inhibits tumour growth rate. Moreover, it was suggested that cellular GSH may have to reach certain critical levels before proliferation can be initiated and that variations in the protein sulphydryl redox status may directly relate to regulation of cell growth (Atzori et al. 1990).

Defining the intrinsic cellular redox environment by estimation of glutathione (GSH)/glutathione disulfide (GSSG) redox state, the group of Dean P. Jones (Nkabyo et al. 2002) concluded that each phase in the life of the cell is characterized by the certain redox state. Proliferating cells are in the most reduced state, with the values of Eh between -260mV and -230mV (Schafer & Buettner, 2001). Upon a growth arrest caused by differentiation (Nkabyo et al. 2002) or contact inhibition (Schafer & Buettner, 2001) cells are 40 mV more oxidised (-220mV to -190mV) while the apoptotic process is accompanied by further oxidation up to -165mV (Sun & Oberley, 1996).

Therefore, while the cell progresses from proliferation, through contact inhibition, differentiation, and finally apoptosis, there is an intrinsic and natural progression from more reduced to more oxidised cellular redox environment. The universality of this model which applies to various cells from different organisms (reviewed in Schafer & Buettner, 2001) inspired a daring hypothesis of Schafer and Buettner on the implication and function of thiols and disulfides as nano-switches. The GSSG/2GSH couple is imagined as a switchboard that move the cell from proliferation through differentiation towards programmed cell death, if the redox environment could not be maintained, or necrosis when the oxidative insult is to severe.

#### **2.4 Glutathiolation of regulatory proteins as a link between a stimulating oxidative event and reduced cell environment in cell proliferation**

During the last two decades the increasing body of evidence reveals that several transcription factors undergo oxidant modification necessary for their activation. For instance, the property of binding DNA and thus regulate gene expression of AP1, NfkB, p53, and SP1 depends on the redox status of cysteinyl thiols in their structures (Sun & Oberley, 1996). Thus, the idea of protein glutathiolation as a regulatory mechanism of importance in cell proliferation came into sight.

Glutathiolation is a protein modification which consists in the covalent union of the tripeptide glutathione to the SH group of the cysteine residue. For a long time this reaction was considered to be a consequence of the equilibrium between protein thiols and GSSG inevitably related to oxidative stress. From this point of view, glutathiolation fulfills two important functions. Firstly, its reversibility enables the preservation of glutathione in the cell and serves as a buffer for the reduction potential; otherwise, GSSG efflux would cause the loss of GSH from the cell, decreasing the reducing capacity which could be recovered only by the synthesis of new GSH (Schafer & Buettner, 2001). Secondly, it provides

The Nuclear Compartmentation of Glutathione: Effect on Cell Cycle Progression 275

essential (Rodriguez-Manzaneque et al., 1999) presumably their vital importance may be interdependent. Then GSH seems to be important in S phase. During the process of DNA replication, errors, such as double-strand breaks (DSBs) that arise from stalled replication forks, require attention by the DNA damage response proteins. Thus, the correct control of DNA synthesis and probably essential molecules, such as GSH, are necessary for the correct

One of the most important proteins involved in DNA damage signaling pathway is the ataxia-telangiectasa mutated protein (ATM). This central signaling protein, mainly for DSBs, is involved in the repairing DNA process necessary after replication stress. Thus cells lacking ATM fail to execute many of the cellular responses to DNA damage (Zhou & Elledge, 2000). In addition, control of ATM responses after DNA replication may be necessary for the correct cell cycle control. In that way, ATM is a central component in the cell cycle regulation. Therefore, patients with ataxia telangiectasia have reduction in DNA synthesis (Painter & Young, 1980). Furthermore, a recent work published by Guo Z. and coworkers describes using a series of elegant experiments how ATM sense the redox changes to modulate their activity (Guo et al, 2010). Interestingly, these authors propose that ATM may regulate global cellular responses to oxidative stre*s*s, remarking the essential link between redox control and DNA interacting, remodeling or repairing proteins. In Fanconi anemia for instance, Castillo and coworkers have shown that ATM dependent phosphorylation of FANCD2, one of the main proteins in the Fanconi anemia pathway of DNA repair, is necessary for normal S-phase checkpoint activation after oxidative stress

The eukaryotic chromosomes are capped by telomeres, which consist of TTAGGG DNA sequences repeated in tandem, associated with several proteins, which protect the final regions of chromosomes. These structures play an important role in the stability and the complete replication of the chromosomes. Conventional DNA polymerases cannot fully replicate the 3'-end of the lagging strand of linear molecules, and therefore in every cell division telomeric sequences are lost (Komberg, 1969). Telomerase is an important enzyme that ensures the maintenance of normal telomere length. This activity is high in human cancers (Kim et al., 1994), but virtually absent in normal human tissues, except germinal cells (Harley et al., 1990). Telomerase regulation is not completely understood, but its changes are related to both cancer and aging (Sharpless & Depinho, 2004). Studies carried out by Jady et al. show that human telomeres are more accessible during the S-phase (Jady et al., 2006) and that the telomerase assembly with telomeres takes place at this specific moment of the cell cycle (Jady et al., 2006; Tomlison et al., 2006). Telomerase plays a key role in cellular homeostasis, because it maintains the length of the telomeres. This especially important in germinal cells in which it is necessary to keep a normal telomeric length after many cellular divisions. Important contributions about the epigenetic control of telomeres have been reported recently (Koziel et al., 2011). In that way, Maria Blasco has suggested that telomeres are under epigenetic control (García-Cao et al., 2004). Mammalian telomeres and subtelomeric regions are enriched in epigenetic marks that are characteristic of heterochromatin. In addition, histone deacetylase enzymes, such as Sirt6, regulate the telomeric chromatin conformation in order to allow the interaction of WRN protein with

DNA processing.

(Castillo et al, 2011).

**2.4.2 Regulation of telomerase activity by glutathione**

these chromosomal regions (Michishita et al., 2008).

protection for protein-SH against irreversible modifications and protein damage in response to higher levels of oxidative stress (Dalle-Donne et al., 2007). Interestingly, it was demonstrated that glutathiolation as a posttranslational modification occurs not only during oxidative stress, but also under basal conditions and is involved in regulating distinct transcription factors, such as NF-kB (Pineda-Molina et al., 2001), its inhibitor factor IKK (Reynaert et al., 2006) and c-Jun (Canela et al. 2007). Apparently, the binding capacity of these proteins to DNA or other proteins is modulated by glutathiolation. This relatively recent focus on the implication of glutathiolation modulatory effects on protein function yielded important breakthrough in elucidation of the implication of this modification in various physiological and pathological situations (Giustarini et al. 2004) and raises interesting questions about its possible implications in cell proliferation.
