Expression of Aequorea green fluorescent protein produces excess intracellular reactive oxygen species, leading to apoptosis

The experimental studies described herein were initiated by observations of the enhanced green fluorescent protein (GFP) within the MCF7 cells. Observation of cells transfected with GFP, and expressing GFP, indicated that the cells were not dividing at a rate equal to those lacking visible expression. It was also observed that the cells appeared to experience apoptosis at an increased rate compared with those not expressing GFP. Therefore, the directed initiative of the experiments became analysis and evaluation of the effects of GFP on transfected cells. The working hypothesis was that excess intracellular reactive oxygen species (ROS) were being generated within this transfected mammalian cell line, and that the system could be utilized as a model to investigate the effects of increased ROS on mammalian cells as well as the experimental elimination of those effects.; A mammalian cell system was constructed, utilizing the green fluorescent protein, and analysis of the system indicated that cells expressing GFP have a much higher concentration of intracellular H₂O₂. This elevated intracellular H₂O₂ level stimulates proliferation in the cells exposed to media containing excess H₂O₂. Addition of antioxidants to the media demonstrated that supplementation with a potent antioxidant, such as reduced glutathione, serves to partially protect the cells from oxidative damage by decreasing intracellular concentrations of H₂O₂. Ambrotose® may be added to media, as well, to reduce the concentration of H₂O₂ in the media. Apoptosis was examined with ELISA and TUNEL assays, further verifying the beneficial effects of reduced glutathione supplementation and Ambrotose® supplementation.; Overall, the experiments demonstrated that this system may be used as a functional model for investigating obviation of ROS in mammalian cells, the effects of excess hydrogen peroxide production, as well as investigating the capacity of supplemental substances for intracellular hydrogen peroxide reduction. Since the effects of ROS are known to play a role in many pathologies such as Alzheimer's, Down's, ischemia, and numerous other diseases, this system could provide a tool for investigation into clinical treatments.