Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007However, in this research the focus will lie on the balance between ROS and antioxidants. Therefore, inthis proposal, the definition of the redox cycle is the cyclic fluctuation in the “balance between the levels ofreactive oxygen species (ROS) produced during metabolism and the antioxidant system that scavengesthem” (Menon and Goswami, 2007).ROS are reactive radicals and hydrogen peroxide.Hydrogen peroxide itself is not a radical, but when reactingwith a metal it will form the very reactive and destructivehydroxyl radical. ROS are produced as a side effect ofseveral metabolic pathways. They can extract an electronfrom other compounds, destabilizing them and makingthem inactive or incapable of performing their normalfunction. (Foster et al., 2006, citing Dröge et al., 2002 andBoveris and Chance, 1973) There are several types ofROS, which are described in Table 1.1. However, as thisresearch concerns mitochondrial ROS production, it willfocus on the two ROS produced by mitochondria,hydrogen peroxide and superoxide.Subtypes of ROSStructureHydrogen peroxide H 2 O 2Hydroxyl radical HO .Hypochlorous acidHOCLNitric oxideNOPeroxyl radical ROO .Peroxynitrite anionSuperoxide anionSinglet oxigenTable 1.1: Different types of ROSONOO-. O 2 -1 O 2A redox cycle in mammalian cellsROS levels in different phases of the cell cycle have been measured. Havens and colleagues (2006)found that ROS levels were lowest in G1, increased during S and were highest in G2/M. However, Conourand colleagues (2004) measured ROS levels throughout the cell cycle and did not find significantdifferences between phases. They also measured the balance between the reduced and oxidized form ofglutathione (GSH/GSSG balance). Glutathione (GSH) is a tripeptide, consisting of cysteine, glycine andglutamic acid. It is an important player in the antioxidant system, because it is involved in a reductionreaction with hydrogen peroxide: one hydrogen peroxide react with two GSH molecules to yield two watermolecules and two oxidized glutathiones (GSSGs). This reaction is catalyzed by glutathione peroxidase(GPx). GSSG can be converted back into GSH by glutathione reductase (GR). The ratio betweenGSH/GSSG is an important and often used indicator of the oxidative/reductive state of a cell (Schafer andBuettner, 2001; Conour et al., 2004). According to Conour and colleagues (2004), reduced glutathione(GSH) levels were higher in the G2/M phase compared to the G1 phase, and cells in the S phase showedintermediate GSH levels. This suggests that in the progression from G1 to S to G2/M, less oxidants areproduced, and that therefore the state of the GSH/GSSG buffer shifts to the reduced side. Thiscontradiction needs to be resolved before final conclusions can be drawn.Various producers of ROSAn interesting question is to what extent this redox cycle is linked to mitochondrial function. If ROS levelsdirectly correlate to mitochondrial activity, these observations strongly suggest that mitochondrial activityfluctuates throughout the cell cycle. However, even though mitochondria are the main source of ROS,there are also other contributors, including the NADPH oxidase complex, peroxisomes (through fatty acidmetabolism), cytochrome P450 reductase, xanthine oxidase and myeloperoxidase (Menon and Goswami,2007). Furthermore, a change in redox state might also be regulated independently of ROS production, forexample by a change in the pro-oxidant/anti-oxidant balance.Not all of these are relevant for this research, mainly because some ROS producers are very celltypespecific. Myeloperoxidase, for example, is only present in leukocytes (Daugherty et al., 1994).Xanthine oxidase is only present intracellularly in the endothelial cells of the capillaries in the mammarygland, liver, intestine, heart and lung. (Adachi et al., 1993). For other cell types it is only localizedextracellularly. For superoxide and hydrogen peroxide production within the cell there are just threepossible generators: peroxisomes (Schrader and Fahimi, 2004), mitochondria (Boonstra and Post, 2004)and NADPH oxidase (Dröge, 2002). Furthermore, a change in antioxidant levels or activity can alsoinfluence cellular ROS levels. An overview of the various contributors to ROS levels can be seen in Figure1.1.SCI 332 Advanced Molecular Cell Biology Research Proposal 12