**2.1 Reactive oxidative species in cell proliferation**

Classical work of Kelvin Davies (Davies, 1999), more than ten years ago, showed that the cells show a whole range of responses to oxidative stress that depends on the intensity of the stress. Low level of hydrogen peroxide induced mitogenic responses and stimulation of proliferation; this observation was firstly reported by Oberly (Oberly et al., 1981) who have described that oxidative stimuli, such as superoxide and hydrogen peroxide, could activate signalling pathways that lead to proliferation. Davies et al. further assert that considerable increase in the oxidant concentrations caused temporary growth arrest which became permanent with a progressive increase. When high H2O2 concentrations were used, apoptosis took place and at very high oxidant levels the cells were killed by necrosis. A year later, (Pani et al., 2000) demonstrate a causal link between redox changes and growth control by cell density: they show that low level of oxygen species in the environment of proliferating cells was not only stimulating but necessary for the correct mitogenic signaling. This study was immediately followed by the work of Menon et al., 2003 who suggested that an oxidation event early in G1 phase may be a critical regulatory step in the progression of the cells into S phase. This lead to the development of the model of the "redox cycle within a cell cycle" proposed by the same group several years later (Menon & Goswami 2007).

According to this model, the transient change in ROS could modify the redox state of cell cycle regulatory proteins, at their critical cysteine residues, and thus determine progression or arrest in the proliferation. Antioxidant mechanism could scavenge ROS and reverse the process. In accordance to these reports, Barry Halliwell (Halliwell, 2007) draw a complete

The Nuclear Compartmentation of Glutathione: Effect on Cell Cycle Progression 273

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

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

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

**2.4 Glutathiolation of regulatory proteins as a link between a stimulating oxidative** 

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

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

**2.3 Glutathione in cell proliferation** 

regulation of cell growth (Atzori et al. 1990).

oxidation up to -165mV (Sun & Oberley, 1996).

**event and reduced cell environment in cell proliferation** 

the oxidative insult is to severe.

cell proliferation came into sight.

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 signalling in the promotion of cell growth.

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

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 particular role of glutathione is central in many research efforts.

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