**4. The depletion of nuclear glutathione hampers the cell cycle progression**

With the intention of providing further evidence of the importance of nuclear GSH in the initiation of cell proliferation, we have found ourselves in front of a challenge of depleting nuclear glutathione. A number of reports have focused on the consequences of the depletion of cellular glutathione levels on changes in cellular proliferation (Thomas et al., 1995; Hansen et al., 2006). However, all those reports were performed measuring cellular or total glutathione levels, but there is no information relating cellular proliferation with nuclear glutathione levels. A number of studies have indicated the existence of a nuclear GSH pool that resists depletion after exposure of cells to BSO (Thomas et al., 1995). BSO treatment resulted in the concentration dependent depletion of cytoplasmic GSH, while the depletion of mitochondrial and nuclear pool of GSH required concentrations higher than 100 µM, which induced DNA damage (Green et al., 2006). Spyrou and Holmgren (Spyrou & Holmgren, 1996) showed that inhibition of glutathione synthesis by 0.1 mM BSO was able to decrease GSH synthesis after treatment for 12 hours, but GSH-depleted cells grew as well as control 3T6 cells with no decrease in DNA synthesis. Thus, incubation of cells with low concentration of BSO, although decreases glutathione levels, does not change cell proliferation. On the other hand, Thomas *et al.* (Thomas et al., 1995) showed that non toxic concentrations of N-ethyl maleimide or DEM decreased the GSH level in the nucleus and cytoplasm to a similar extent, whereas the nuclear pool of GSH was much more resistant to BSO depletion.

Based on this findings, we have designed a model to study the effects on the cell proliferation parameters caused by GSH depletion both in the nucleus and the cytoplasm, using 100µM DEM, comparing to the administration of 10 µM BSO when nuclear GSH level is preserved.

The Nuclear Compartmentation of Glutathione: Effect on Cell Cycle Progression 283

levels by DEM strongly impairs cell proliferation. This difference could be due to the fact that DEM decreases both nuclear and cytosolic glutathione levels in opposition to BSO, which only decreases cytosolic glutathione. It is worth mentioning that the impairment of cell proliferation could not be attributed to the alkylating properties of DEM, since the simultaneous administration of GSHe completely prevented it, nor to the toxicity of the

In addition, we have observed the delay in the cell cycle progression caused by DEM, when both nuclear and cytoplasmic GSH was depleted, which is absent in the treatment with BSO when nuclear GSH pool was preserved. Interestingly, Esposito *et al*. (Esposito et al., 2002) showed that direct administration of DEM on the nuclear extracts of COS7 cells induces cell cycle arrest. So, it is daring to speculate that, despite the depletion of cytoplasmic GSH with DEM could not be overlooked, the effect on the cell cycle progression could be attributed to the depletion of the nuclear GSH. Moreover, as Esposito shows, the depletion of nuclear GSH strongly induces a p53-independent accumulation of p21, which causes a cell cycle arrest. In our study, the expression of cell cycle regulatory protein, suggested previously to be under redox control, Id2, was decreased when the level of nuclear GSH was depleted.

It has been known that cell proliferation is regulated by a variety of mechanisms working to allow the activation and repression of growth stimulatory genes, one of them being the transcription factors. Previous *in vitro* reports show that the activity of transcription factors is related to its redox environment. In addition, change in the redox potential could induce variations in the activity of those transcription factors. Alterations as small as + 15 mV in the redox potential can result in transcription factor translocation and activation or deactivation, depending on the direction of the redox shift (Hutter et al., 1997; Sen & Packer, 1996; Sun & Oberley, 1996). Recently Reddy *et al.* (Reddy et al., 2008) have shown that Nrf2 deficiency leads to oxidative stress and DNA lesions, accompanied by impairment of cell-cycle progression, mainly G(2)/M-phase arrest. Both N-acetylcysteine and glutathione (GSH) supplementation ablated the DNA lesions and DNA damage-response pathways in Nrf2 (-/-) cells; however only GSH could rescue the impaired co-localization of mitosispromoting factors and the growth arrest. Our results demonstrate for the first time that it is

treatment because the cell death was not significantly augmented.

Fig. 5. Nuclear GSH depletion affects Id2 expression.

Fig. 3. Changes in cell proliferation caused by GSH depletion.

As reported previously by various authors (Britten et al., 1991; Green et al., 2006; Thomas et al., 1995) nuclear GSH pool was preserved. By contrast, depletion of glutathione levels by DEM induces a marked decrease in nuclear glutathione levels.

Fig. 4. The depletion of GSH in the nucleus.

The compartmentalization of glutathione depletion could explain the observed differences in the inhibition of cell proliferation. Indeed, our results show that inhibition of glutathione

As reported previously by various authors (Britten et al., 1991; Green et al., 2006; Thomas et al., 1995) nuclear GSH pool was preserved. By contrast, depletion of glutathione levels by

The compartmentalization of glutathione depletion could explain the observed differences in the inhibition of cell proliferation. Indeed, our results show that inhibition of glutathione

Fig. 3. Changes in cell proliferation caused by GSH depletion.

DEM induces a marked decrease in nuclear glutathione levels.

Fig. 4. The depletion of GSH in the nucleus.

levels by DEM strongly impairs cell proliferation. This difference could be due to the fact that DEM decreases both nuclear and cytosolic glutathione levels in opposition to BSO, which only decreases cytosolic glutathione. It is worth mentioning that the impairment of cell proliferation could not be attributed to the alkylating properties of DEM, since the simultaneous administration of GSHe completely prevented it, nor to the toxicity of the treatment because the cell death was not significantly augmented.

In addition, we have observed the delay in the cell cycle progression caused by DEM, when both nuclear and cytoplasmic GSH was depleted, which is absent in the treatment with BSO when nuclear GSH pool was preserved. Interestingly, Esposito *et al*. (Esposito et al., 2002) showed that direct administration of DEM on the nuclear extracts of COS7 cells induces cell cycle arrest. So, it is daring to speculate that, despite the depletion of cytoplasmic GSH with DEM could not be overlooked, the effect on the cell cycle progression could be attributed to the depletion of the nuclear GSH. Moreover, as Esposito shows, the depletion of nuclear GSH strongly induces a p53-independent accumulation of p21, which causes a cell cycle arrest. In our study, the expression of cell cycle regulatory protein, suggested previously to be under redox control, Id2, was decreased when the level of nuclear GSH was depleted.

Fig. 5. Nuclear GSH depletion affects Id2 expression.

It has been known that cell proliferation is regulated by a variety of mechanisms working to allow the activation and repression of growth stimulatory genes, one of them being the transcription factors. Previous *in vitro* reports show that the activity of transcription factors is related to its redox environment. In addition, change in the redox potential could induce variations in the activity of those transcription factors. Alterations as small as + 15 mV in the redox potential can result in transcription factor translocation and activation or deactivation, depending on the direction of the redox shift (Hutter et al., 1997; Sen & Packer, 1996; Sun & Oberley, 1996). Recently Reddy *et al.* (Reddy et al., 2008) have shown that Nrf2 deficiency leads to oxidative stress and DNA lesions, accompanied by impairment of cell-cycle progression, mainly G(2)/M-phase arrest. Both N-acetylcysteine and glutathione (GSH) supplementation ablated the DNA lesions and DNA damage-response pathways in Nrf2 (-/-) cells; however only GSH could rescue the impaired co-localization of mitosispromoting factors and the growth arrest. Our results demonstrate for the first time that it is

The Nuclear Compartmentation of Glutathione: Effect on Cell Cycle Progression 285

molecules, like glutathione, are considered to move by free and fast diffusion across the nuclear pore (Ribbeck & Gorlich, 2001); nevertheless ion gradients and transnuclear ATPdependent membrane potential have also been reported (Nigg, 1997). In a series of creative experiments published in early 1990ies, Feldherr CM and Akin D (Feldherr & Akin, 1990; Feldherr & Akin, 1993), shown that permeability of nuclear envelope and nuclear transport were higher in proliferating than in quiescent cells. Reported seven fold reduce in the nuclear transport capacity was induced by the alterations in the characteristics of the pores and not by the changes within the cytoplasm, specifically, the decrease in ATP concentration. One pore forming protein that has been brought into the connection to nuclear glutathione content is Bcl-2. Voehringer and colleagues (Voehringer et al., 1998) showed that over-expression of Bcl-2 recruits GSH to the nucleus. The presence of this protein at the nuclear envelope was demonstrated (Krajewski et al., 1993) and the association with the nuclear pore complexes was suggested. Moreover, Zimmermann et al. (Zimmermann et al., 2007) demonstrated that GSH binds to Bcl-2 in mitochondria,

A clear picture emerges showing that the presence of a reduced nuclear environment, probably provided by glutathione, glutaredoxin and thioredoxin mainly, is of paramount importance in the physiology of cell cycle, underscoring the role of oxidative stress in cell

Atzori, L.; Dypbukt, J.M.; Sundqvist, K.; Cotgreave, I.; Edman, C.C.; Moldéus, P.

Barbie, D.A.; Kudlow, B.A.; Frock, R.; Zhao, J.; Johnson, B.R.; Dyson, N.; Harlow, E. &

Benlloch, M.; Ortega, A.; Ferrer, P.; Segarra, R.; Obrador, E.; Asensi, M.; Carretero, J.

Bellomo, G.; Palladini, G. & Vairetti, M. (1997). Intranuclear distribution, function and fate of

Biaglow, J.E.; Varnes, M.E.; Clark, E.P. Epp, E.R. (1983). The role of thiols in cellular

Blasco, M.A. (2002). Telomerase beyond telomeres. *Nature Reviews Cancer*, 2, 8, (August

Borrás, C.; Esteve, J.M.; Viña, J.R.; Sastre, J.; Viña, J. Pallardó, F.V. (2004). Glutathione

*Physiology*, 143, 1, (April 1990), pp. 165-171, ISSN 0021-9541.

Grafström, R.C. (1990). Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts. *Journal of Cellular* 

Kennedy, B.K. (2004). Nuclear reorganization of mammalian DNA synthesis prior to cell cycle exit. *Molecular and cellular biology.* 24, 2, (January, 2004), pp. 595-607,

Estrela, J.M. (2005). Acceleration of glutathione efflux and inhibition of gammaglutamyltranspeptidase sensitize metastatic B16 melanoma cells to endotheliuminduced cytotoxicity. *Journal of Biological Chemistry*, 280, 8, (February 2005), pp.

glutathione and glutathione-S-conjugate in living rat hepatocytes studied by fluorescence microscopy. *Microscopy Research and Technique.* 36, 4, (February 1997),

response to radiation and drugs. *Radiation Research,* 95, 3, (September 1983), pp.

regulates telomerase activity in 3T3 fibroblasts. *Journal of Biological Chemistry*, 279,

providing a molecular basis for its antioxidant function.

proliferation.

**6. References** 

ISSN 0270-7306.

6950-6959, ISSN 0021-9258.

pp. 243-52, ISSN 1059-910X.

437-455, ISSN 0449-3060.

2002), pp. 627-33, ISSN 1474-175X.

33, (August 2004), pp. 34332-34335, ISSN 0021-9258.

nuclear GSH levels, and not total cellular glutathione levels, that specifically correlate with cellular proliferation. Glutathione is considered essential for survival in mammary cells and other eukaryotic cells, but not prokaryotic cells. However, although a number of important functions have been attributed to GSH, its outstanding role in nucleated, but not in prokaryotic cells, remains unknown. Our results underscore the important role of nuclear glutathione in cell physiology and suggest that manipulation of nuclear GSH levels could be of paramount importance during development and cancer.
