Association of Gpx1 fluctuation in cell cycle progression.

This research demonstrates fluctuation of glutathione peroxidase1 (Gpx1) throughout cell cycle progression with significant decreased expression at mitosis of HeLa cell. This was achieved with western blot (WB) analysis of target proteins from each phase of synchronized cells. The synchronizations were performed with double thymidine (T/T) for G1/S arrest and thymidine followed by nocodazole (T/N) for G2/M arrest. The G1/S arrested cells were released in fresh medium for 3, 6, 9, 10, and 15h to obtain cell at each phase such as gap1 (G1), synthesis (S), gap2 (G2), mitosis (M), and gap1 (G1) phase, respectively, for investigating Gpx1 expression throughout a complete cycle. The synchronizations were confirmed using fluorescence activated cell sorting (FACS) and WB analysis of phase-specific markers. The fluctuations of Gpx1 expression were verified with universal protein actin and peroxiredoxin1 (Prx1) which are stable throughout the cell cycle. Intriguingly, immunoblots showed the level of Gpx1 decreases at mitosis phase and increased during mitotic exit to G1 phase in HeLa cells, while Prx1 protein level remained constant. The fractionation experiments reveal that only the cytosolic Gpx1 was decreased while their levels at mitochondria remain constant. The highest levels of mitochondrial ROS were measured in mitosis phase with FACS analysis using Mito sox indicating that antioxidant activity of Gpx1 for detoxifying excessive induced endogenous reactive oxygen species (ROS) in the mitosis phase could be the reason for such decreasing level. For unfolding the molecular mechanism of such decreased expression, the Gpx1 was investigated at transcriptional, translational, and proteosomal level. The results revealed that translational mechanism is involve in the decreased expression rather than transcriptional or proteosomal degradation at mitosis phase. This finding supports that Gpx1 is involved in the cell cycle progression through regulation of endogenous ROS. Based on this observation, further research could uncover their possible association with the infinitive division of a cancer cell.