The importance of the nuclear glutathione in the Cell Proliferation

• Main Author: Markovic, Jelena
• Other Authors: Viña Ribes, José
• Format: Doctoral Thesis
• Language: English
• Published: Universitat de València 2009
• Subjects: Facultat de Medicina i Odontologia; 612
• Online Access: http://hdl.handle.net/10803/9922; http://nbn-resolving.de/urn:isbn:9788437076362

The present thesis offers an insight in the importance of nuclear GSH in cell proliferation. The research was performed in three different cellular models of diverse proliferating activity: immortalized mouse embryonic fibroblasts 3T3, mammary adenocarcinoma cell line MCF7 and primary embryonic neuralonal culture. The results presented here provide evidence that suggest that the relationship between GSH level and telomerase activity, previously described by our group for 3T3 fibroblasts is a common phenomenon in mammalian cells. Reduced glutathione emerged as a driving force of cell cycle progression; high level of GSH coincided with the peak of telomerase activity and preceded the exponential phase of cell growth in all three cell types analyzed. Moreover, the comparative studies of three distinct proliferating models revealed that the level of GSH corresponded to the intensity of the proliferation and clearly correlated with the level of DNA synthesis. This finding inspired the focus on the nuclear compartmentalization of GSH. Interestingly, nuclear localization of GSH was observed before the exponential phase of cell growth in all three cell types, while the GSH distribution within the cell was uniform when the cells stopped proliferating. Comprehensive analysis of the nuclear GSH levels along the cell cycle, and the experiments with the depletion of nuclear GSH conducted in 3T3 fibroblasts, provided further evidence on crucial importance of nuclear GSH for the onset of cell proliferation. In the attempt to elucidate the possible mechanism of nuclear GSH compartmentalization, we have found no evidence either of active GSH transport or of its de novo synthesis in the nucleus. The implication of bcl2 in nuclear transport of GSH was evaluated in the study of MCF7 cells over-expressing this protein. In this model, bcl2 caused an early and striking increase of total cellular glutathione, nuclear GSH and telomerase activity; unexpectedly, these alterations induced no significant effect on cell proliferation.Based on these findings we have been able to draw a series of conclusions: The level of cellular glutathione in the particular cell line could define its proliferating capacity. Its variations along the cell culture dictate the rhythm of cell proliferation, possibly by the modulation of redox sensitive cell cycle regulatory proteins.The telomerase activity is highly sensible to the cellular redox state, regardless of the cell type, its inherent proliferation capacity or the origin of the cells. The dependence of the cell cycle progression on the level of glutathione could be attributed, at least in part, to its modulation of telomerase activity The nuclear compartmentalization of glutathione is a prerequisite for the onset of the exponential phase of the cell growth. The redox state of nuclear proteins, defined by the glutathiolation and oxidation, varies significantly with the cell proliferation activity.The depletion of nuclear glutathione severely impairs the cell cycle progressionDuring the changes in the nuclear membrane that precede the cell division, nuclear Bcl-2 could facilitate the translocation of glutathione to the cell nucleus. The overexpression of bcl-2 induced an early increase of the total glutathione level, telomerase activity and nuclear GSH compartmentalization.In summary, our results reveal the importance of nuclear GSH compartmentalization in early phases of cell proliferation. This study suggests that the control of the cell cycle progression from G1 to S phase is dependent on elevated nuclear GSH levels and originates from its influence on telomerase activity and DNA synthesis. === Integridad celular se defiende en el núcleo, en su "software" - el acido desoxiribonucleico (ADN) que controla los procesos celulares atreves de la regulación de la expresión génica. El papel protector del glutatión (GSH) sobre el ADN, en su síntesis de ADN y en el mantenimiento de organización del matriz nuclear ha sido claramente demostrado. La gran diversidad de procesos vitales dependientes del glutatión y realizados en diferentes orgánulos de la célula implica la existencia de mecanismos que controlarían su concentración y garantizarían el ambiente adecuado en cada compartimiento. El núcleo cambia dramáticamente durante las diferentes fases del ciclo celular, por lo tanto los estudios de la compartimentación nuclear del GSH tienen que tener en cuenta esta dinámica celular. El objetivo de la presente tesis era investigar la importancia del nivel de GSH y su distribución celular, especialmente la compartimentación nuclear, en la proliferación celular y posibles mecanismos involucrados en el control del dicho fenómeno. Los resultados obtenidos nos han permitido sacar una serie de conclusiones: El nivel de glutation celular puede determinar la capacidad proliferativa de una línea celular, posiblemente vía la modulación de las proteínas reguladoras del ciclo celular sensibles al estado redox. La actividad de la telomerasa, altamente sensible al estado redox, independientemente del tipo celular, su inherente capacidad proliferativa o la especie de la que procede el cultivo celular puede ser vínculo entre la progresión del ciclo celular y el nivel de glutation. La compartimentación nuclear del glutation es imprescindible en el principio de la fase exponencial del crecimiento celular. El estado redox de las proteínas nucleares, definido por su glutationilacion y oxidación, varía significativamente con la actividad proliferativa de las células. La depleción del glutation nuclear interfiere gravemente con la progresión del ciclo celular. La sobre expresión del Bcl-2 induce el incremento del nivel del glutation celular, de la actividad telomerasa y de la compartimentación nuclear del GSH. El nivel de GSH y su compartimentación nuclear son de crucial importancia para el inicio de la proliferación celular. El fenómeno parece universal, confirmado en cultivos celulares provenientes de diferentes especies y con distintos niveles de proliferación.