Proceedings of The Physiological Society

Kings College London (2005) J Physiol 565P, PC160


Cellular Distribution of Reduced Glutathione During Cell Cycle in 3T3 Fibroblasts

Markovic, Jelena ; Borras Blasco, Consuelo ; Ortega, Angel ; Pallardo, Federico ; Vina Ribes, Jose ;

1. Physiology, Medical School of the University of Valencia, Valencia, Valencia, Spain.

Reduced glutathione (GSH) is a low molecular weight thiol and the most abundant no enzymatic antioxidant in the cell. Its property to maintain cellular thiol/disulfide redox state gives it a central place in the control of vital cell processes of great diversity including cell proliferation (Jones et al. 2002). Our group has recently demonstrated that the peak of total GSH in 3T3 fibroblasts coincides with the peak of telomerase activity at 24h in culture and precedes the exponential phase of cell growth (Borras et al. 2004). Considering the growing evidence that shows the importance of GSH compartimentation (Voehringer et al. 1998), and its role in numerous processes that occur in the nucleus, we have focused our attention in following the changes in the GSH distribution throughout the cell cycle. To visualize GSH localization, cells were observed with a Leica confocal microscope connected to a PC with software for microscope control and image analysis. 3T3 fibroblasts were maintained in culture as described previously (Borras et al. 2004) and plated in 2cm2 chamber slides 5 days, 72h, 48h, 24h, 12h and 6h before the experiment, so cells in all phases of the cell cycle were dyed and analyzed on the same day. Triple staining was applied: propidium iodide 2μg/ml (PI) to identify dead cells, Hoechst 2μg/ml to localize nucleus and CellTracker green 5-chloromethylfluorescein diacetate 5 μM (CMFDA), which marks GSH (specificity 95%). We captured the emission of red (excitation 543nm), blue (excitation 364nm) and green (excitation 488nm) fluorescence to identify IP, Hoechst and CMFDA staining, respectively. Z-series obtained were converted to 2-D images of maximal fluorescence and analyzed by drawing regions around the cell and its nucleus. We calculated the nucleus/cell ratio (n/c) of the mean of CMFDA fluorescence in 3 separate experiments (100 cells per condition). Our results show that in 3T3 fibroblasts GSH concentrates in the nucleus during the first 24h (n/c=2.40±0.46) of culture (Borras et al. 2004). This phenomenon, to a lesser extent, is maintained until 48h (n/c=1.94±0.09), which coincides with exponential phase of cell growth (Borras et al. 2004). At 72h the majority of cells are characterized by evenly distributed GSH (n/c=1.33±0.10, p≤0.05 vs. 24h). At 5 days, as cells reach confluence and enter quiescence, the intensity of green fluorescence is very low and distribution is homogeneous (n/c=1.17±0.09, p≤0.05 vs. 24h). We believe that the start of active proliferation of 3T3 fibroblasts requires a reduced environment in the nucleus and that the level of GSH compartimentation regulates the rhythm of the cell cycle.

Where applicable, experiments conform with Society ethical requirements