Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 20075. Measuring fluorescenceThe same procedure as in experiments 2.1.2 and 2.1.4 will be used.Experiment 2.1.6: NADPH oxidase ROS production throughout the cell cycleApart from the mitochondria and peroxisomes, the NADPH oxidase is also a producer of ROS, namelysuperoxide. (Boveris and Chance, 1973; Dröge, 2002). Therefore, in order to establish the relativecontribution of this enzyme to overall ROS production, NADPH oxidase will be inhibited by means of preactivatedapocynin, a specific NADPH oxidase inhibitor, and the effects on the cellular ROS levels will bedetermined.It is necessary to pre-activate apocynin, since the activation reaction of apocynin, which occurs in cellsafter incubation, can increase intracellular ROS levels (Vejrazka et al., 2005). When this activationreaction has been done before administering apocynin to the cells, this increase in ROS levels is notobserved (Vejrazka et al., 2005).1. NADPH oxdidase inhibition & O 2 - levelsTo measure the effect of NADPH oxidase inhibition on superoxide levels, the experiment 2.1.1 will berepeated. However, in this case, after cells are incubated with PF-1, they are washed with MEM (minimalessential medium) and suspended in pre-activated apocynin (for exact protocol, see Vejrazka et al., 2005).As a control, only medium (without apocynin) will be added to the cells. The fluorescence emitted by theprobe will be measured with a confocal scanning laser microscope. Intensity will then be semi-quantifiedusing the appropriate software.2. NADPH oxidase inhibition & H 2 O 2 levelsSince the NADPH oxidase itself produces superoxide, which is readily converted to hydrogen peroxide inthe cytosol, H 2 O 2 levels will also need to be established. Thus, experiment 2.1.2 will be repeated on cellsthat are transfected with the HyPer gene and are suspended in pre-activated apocynin (for protocol, seeVejrazka et al., 2005).However, it has been shown that endothelial cells can go into cell cycle arrest after inhibition ofNADPH oxidase. If the cell cycle arrest is also observed in this experiment, it will not be able to get animpression of how much NADPH oxidase contributes to ROS fluctuations. On the other hand, it will showthat the NADPH oxidase is important for cell cycle progression, probably due to its function in differentredox-signaling pathways, which have not been clearly identified yet.Experiment 2.1.7 Control for ROS production by laser excitationUsing confocal laser scanning microscopy for the detection of fluorescence allows for the focus andquantification of fluorescence in a single cell. However, the laser used to excite the probes might alsocontribute to the ROS levels observed. This has been observed in ROS-induced ROS release by certainspecific probes (Zorov et al, 2000). As a control, cells of every cell line will be taken and forced intoquiescence by using serum starvation. After that the following control experiments will be conducted:1. Measuring superoxide levels in quiescent cellsThese quiescent cells will be incubated with a PF-1 probe dissolved in DMSO and will then be excited byconfocal laser scanning microscopy and the fluorescence levels of 5 different cells will be quantified usingthe appropriate software.2. Measuring hydrogen-peroxide levels in quiescent cellsAdditionally quiescent cells will be transfected with HyPer and will then be excited by confocal laserscanning microscopy and the fluorescence levels of five different cells will be quantified using theappropriate software.3. Measuring superoxide levels in laser exposed quiescent cellsThe same experimental procedure as in 2.1.6.1 will be used, however before the incubation with PF-1 thecell will first be exposed to a laser excitation dose normally used for the detection of PF-1.4. Measuring hydrogen-peroxide levels in laser exposed quiescent cellsSCI 332 Advanced Molecular Cell Biology Research Proposal 21