Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007IntroductionThe discovery of the cell cycle and its key regulators, cyclins and cyclin-dependent kinases, has had anenormous impact on cell biology. Many diseases, most notably cancer, are involved with improperregulation of the cell cycle, which has therefore attracted much scientific attention.The cell cycle is not the only cycle. In yeast, there is evidence for a metabolic cycle, where periodsof relatively high rates of (mitochondrial) respiration and periods with more non-respiratory modes ofenergy generation cyclically alternate (Reinke and Gatfield, 2006).Interestingly, this metabolic cycle seems to be coordinated with the cell cycle (Klevecz et al.,2003), and is associated with temporal patterns of gene expression (Chen et al., 2007). This suggests thatthe metabolic cycle might play a role in cell cycle regulation.The yeast metabolic cycle also seemed to be associated with cyclic changes in the cellular redoxstate of the cell (Reinke and Gatfield, 2006). Periods with highly levels of respiration were relativelyoxidized, whereas a reduced state was characterized by more non-respiratory metabolism.The existence of other cycles than the cell cycle has attracted much attention. It has even beensuggested that this redox cycle could underlie and regulate nearly all cyclic processes in the cell, varyingfrom the metabolic cycle to day-night rhythms (Lloyd and Murray, 2007). Since this could possibly providealternative ways of manipulating many cellular processes and new treatments for cancer, the study ofthese cycles is highly relevant.In mammalian cells, there are indications for a metabolic cycle and a redox cycle controlling cell cycleprogression as well. A recent study showed that cyclin D1, an important protein in the regulation of G1/Sphase progression, can modulate NRF-1 levels, a transcription factor controlling expression of manymitochondrial proteins (Wang et al., 2006). This suggests that the cell cycle and metabolism might becoordinated through control of mitochondrial activity.Some of the most interesting oxidants, with high potential for control of other processes arereactive oxygen species. It is quite widely known that high levels of ROS can cause cellular apoptosis(Boonstra and Post, 2004), but more recent studies have also shed light on more subtle effects of ROS onthe cell cycle (reviewed in Menon and Goswami, 2007), such as the finding that ROS are necessary forG 1 /S progression (Havens et al., 2006).Since mitochondria are a major source of ROS, the redox cycle might function as an intermediatebetween the cell cycle and a potential metabolic cycle.Several pieces of the puzzle are missing for the interplay between a metabolic cycle, a redox cycle andthe cell cycle in mammalian cells. In this research, these pieces will be added. Three parts can bedistinguished in this research proposal:1. Metabolic cycleIt is not known whether mitochondrial activity fluctuates in coordination with the mammalian cell cycle.This research will measure mitochondrial activity throughout the cell cycle, and compare it to mitochondrialactivity in quiescent cells. This will allow the characterization of a metabolic cycle in mammalian cells.2. Links between a metabolic cycle and a redox cycleEven though some studies have measured ROS levels throughout the cell cycle, there are contradictionsin the literature (Conour et al., 2004; Havens et al., 2006). In this part, first ROS levels throughout the cellcycle will be measured. Then, it is of interest whether mitochondria are responsible for these changes inROS levels, since mitochondrial contribute for 85% to the cellular superoxide levels (Foster et al., 2006,citing Dröge et al., 2002 and Boveris and Chance, 1973) By using a combination of specific probes, for thefirst time statements can be made about fluctuations in ROS levels mediated by mitochondria.3. Links between a redox cycle and the cell cyclePrevious studies have mainly focused on the effects of ROS on the cell cycle when ROS levels beyondthe physiological range were used. Here, it will be investigated whether the hypothesized more subtlechange in ROS levels throughout the cell cycle is essential for cell cycle progression. It has been foundSCI 332 Advanced Molecular Cell Biology Research Proposal 10
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007that ROS are necessary for G 1 /S transition (Havens et al., 2006). To identify the influence of a redox cycleon the entire cell cycle, this research will focus on the G 2 /M transition.Background InformationThis section will present relevant background information. Three main topics will be discussed. First,information on the metabolic cycle will be presented, which will be followed by a discussion of ROS andthe redox cycle. Lastly, the effects of ROS on the cell cycle will be elaborated on.Existence of a metabolic cycleIt has been shown that there is a cyclic change in metabolic activity in yeast that is coordinated with thecell cycle (Tu et al., 2005; Reinke and Gatfield 2006). A period where metabolism is mainly respiratory isalternated with a period where anaerobic metabolism is prevalent. The entire cycle can be divided intothree parts: an oxidative phase, a reductive/building phase and a reductive/charging phase. In the first,oxidative, phase, respiration, oxidative phosphorylation and ATP production are high, meaning that themitochondria are metabolically active. In the reductive/building phase DNA is replicated and the celldivides, while respiration is greatly reduced. Lastly, in the reductive/charging phase, the cell prepares forthe next respiratory burst through non-respiratory modes of energy and protein degradation (Reinke andGatfield, 2006). This cycle is accompanied by a highly organized transcriptional cycle of genes thatencode for proteins associated with energy production, metabolism and protein synthesis (Tu et al., 2006).The transcription of these nuclear genes involved in mitochondrial metabolism starts right at the end of theoxidative phase (Reinke and Gatfield 2006). This indicates that after the oxidative phase in whichmitochondria are very active, the mitochondria switch to a resting state to rebuild their resources. Theexistence of a metabolic cycle and the accompanied transcriptional cycle in yeast indicates thatmetabolism and cell cycle progression can be closely linked through gene expressionIn mammalian cells, such a metabolic cycle has never been found. However, there are indications thatmitochondria can alternate between metabolic states depending on availability of ADP, substrates foroxidative phosphorylation and oxygen. Such a change in mitochondrial activity can occur in nonproliferatingcells, but there might also be transcriptional regulation of metabolic activity, potentially relatedto the cell cycle. It has been shown that when cyclin D1 is knocked-out there is an increase inmitochondrial activity. This is accompanied by increased activity of NRF-1, a transcription factor controllingmany mitochondrial proteins (Wang et al., 2006; Sakamaki et al., 2006). As cyclin D1 is involved in theG1/S transition, this is another indication that mitochondrial metabolic activity and the cell cycle caninteract.An accurate measure of mitochondrial metabolic activity is oxygen consumption. Mitochondria account forapproximately 90% of the cellular oxygen consumption, meaning that oxygen consumption can be directlyrelated to mitochondrial activity (Boveris et al., 2006; Gnaiger, 2007). In a study in yeast, oxygenmeasurements were used to measure mitochondrial activity throughout the cell cycle (Tu et al., 2006),which led to the identification of the yeast metabolic cycle.Another measure of mitochondrial metabolic activity is the mitochondrial membrane potential. Thispotential is formed by protons that are pumped into the mitochondrial intermembrane space by theelectron transport chain. Subsequently, when protons flow back through its pore, the ATPase uses thisgradient to generate ATP. It has been reported that mitochondrial membrane potential decreases whenthe mitochondria are in a resting state, while it increases in active mitochondria (Boveris et al. 2006).Existence of a redox cycleIn mammalian cells, fluctuations in the intracellular redox state have been proposed to work as a growthregulator during the cell cycle. The cell contains many electron donors and electron receptors, orreductants and oxidants, respectively. Two examples are the NAD(P)H/NAD(P)+ balance, and reactiveoxygen species and their antioxidants. The most accurate description of a redox cycle would be afluctuation in cellular reduction potential, as this takes into account all cellular reductants and oxidants.SCI 332 Advanced Molecular Cell Biology Research Proposal 11
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