Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007G2, the opposite will be done. Subsequently, entrance into mitosis will be monitored. This experiment willbe carried out in human fibroblast and human retinal epithelial cells.Experiment 3.1.1: Are ROS in general necessary for the G 2 /M transition?1. Experimental and control groupsTo decrease ROS levels in G2, antioxidants will be used. A complete elimination of ROS will not benecessary; levels significantly lower than the physiological range of ROS levels measured in question2 will be aimed at.• Experimental group: antioxidants• Control group: vehicleMany different antioxidants exist, and they possibly have different preferences for certain kinds ofROS. To cover a range of antioxidants as wide as possible, an ‘antioxidant cocktail’ will be used,containing various antioxidants in PBS that both mimic enzymatic antioxidants and affect redoxbuffers (Appendix B3.1.1). Control cells will only receive PBS.2. Determination of dose of antioxidants neededTo determine the amount of antioxidants required, 1 ml of the antioxidant cocktail in various dilutions(Appendix B3.1.1) will be added to synchronized cells in G2. The concentration necessary to lowerROS levels significantly below the physiological range will be determined using the methods used inquestion 2.3. Cell synchronization and treatmentCells will be synchronized using the methods described in Appendix B1.1 and cultured as described inAppendix B1.4. Addition of antioxidantsFor synchronized cells, the expected start of mitosis can be calculated from the length of the cell cyclephases. Two hours before the expected onset of mitosis, antioxidants or vehicle will be added to themedium.5. Mitotic arrestCells will be arrested in early mitosis to determine whether cells were able to pass the G2/M transitionpoint. Two hours before mitosis, nocodazole (20 µg/ml) will be added to the medium to arrest cells inprometaphase.6. Analysis of entry into mitosisWhen cells enter mitosis, histone H3 becomes phosphorylated (Hendzel et al., 1997). To determinewhether cells entered mitosis, an application of FACS adapted from the description by Manke andcolleagues (2005) will be used (Appendix B3.1.3). Cells will be arrested in prometaphase andincubated with anti-phospho-histone H3 antibodies. FACS will be used to measure the amount of cellsthat contain the phosphorylated form of histone H3.It is possible that cells were already past G2 at the time antioxidants will have been added, due to lossof synchrony or cells proliferating faster than expected. If cells were already past prometaphase,nocodazole will not arrest them anymore. In that case, decreased phosphorylation of histone H3 canmean either that cells could not enter mitosis, or that they were already past mitosis when antioxidantswere added. This can be controlled for by FACS using propdium iodide to stain for DNA content(Appendix B3.1.3). If FACS shows 2n DNA content, cells are properly arrested in mitosis and were inG2 when antioxidants were added. If FACS shows (some) 1n DNA content, some cells were alreadypast prometaphase when nocodazole and antioxidants were added, and have progressed to G1. Inthat case, the experiment should be repeated, but antioxidants and nocodazole should be addedearlier.Experiment 3.1.2: Is a change in ROS levels throughout the cell cycle necessary for the G 2 /M transition?1. Experimental and control groupsSCI 332 Advanced Molecular Cell Biology Research Proposal 24