Tour-de-Force

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Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Proposed ResearchWe will be investigating the involvement of PRC and PGC1 in the cell cycle as important regulators ofmitochondrial biogenesis, in cells transitioning from quiescent to proliferating directly via the metabolicburst.To investigate our hypotheses we chose two cell lines to do this in, the BALB/3T3 cells and U-2 OS cellswhich will be supplied to us by the company LGC Promochem – ATCC. The U-2 OS cells originate fromthe bone tissue of a 15 year old female Caucasian and are osteosarcoma cells. The cells will be culturedin McCoy’s 5A Medium with addition of Fetal Bovine Serum. The BALB/3T3 cells are fibroblast cells whichoriginate from 14- to 17- day old mouse embryos. They are very sensitive to contact inhibition and will becultured in the ATCC formulated Dulbecco’s Modified Eagle’s base Medium. To establish a complete andsuitable growth medium, we will add Calf Bovine Serum to the media, which will also be supplied by LGCPromochem – ATCC. The cells will be shipped to us frozen, therefore we will use the protocol supplied byATCC to culture the cells. The cell lines can be used as non-proliferating (quiescent), continuouslyproliferating and induced from quiescence upon serum stimulation. We chose this cell line because theyhave been successfully used in previous experiments in this topic of research (Vercauteren et al., 2006).In the experiments the induction of the metabolic burst will be carried out by adding specificconcentrations of serum stimulating media such as the bovine calf serum and the fetal bovine serum forthe U-2 OS cells and the BALB/3T3 cells respectively.For a detailed description of the methods and specific materials used in this research please refer toAppendix B. General protocols for the methods are provided in Appendix A.Subtopic 1To establish a solid basis for the proposed research program on mitochondrial biogenesis, experimentsfrom previous research will be replicated in the aforementioned cell lines to suit the experimental context.These experiments provide a value on their own and are needed as a basis for the experiments insubsequent subtopics.Mitochondrial DNA to nuclear DNA ratioFirstly, the ratio of mitochondrial DNA to nuclear DNA will be established during the cell cycle inproliferating cells and for the same amount of time in non-proliferating cells. To distinguish between effectsfrom the metabolic burst and the actual cell cycle, the experiment will be performed on continuouslyproliferating cells and cells in which the cell cycle is induced and thus experience a metabolic burst.Cell cycle synchronization is obtained via a double thymidine block, as the cell lines are not suited forcentral elutriation and will not suffer from disadvantages as inhibition of cell growth and apoptosis whenusing serum deprivation. Nocodazole is used to synchronize cells in M-phase, in order to have cellsynchronized for a full cell cycle. Quantitative real-time PCR will be used to measure the mitochondrialDNA to nuclear DNA ratio.Measuring mitochondrial mass during the cell cycleFluorescent-activated cell sorting (FACS) will be used to measure the relative changes in mitochondrialmass during the cell cycle. This provides information on the rate of mitochondrial biogenesis and indicatesperiods in which the rate of mitochondrial biogenesis changes. Such information is needed to focus onparticular periods or phases in the cell cycle.As the target(s) of a single probe for mitochondria may not be representative of the changes inmitochondrial mass, different probes will be used. The fluorescent probes need to be membrane potentialSCI 332 Advanced Molecular Cell Biology Research Proposal 64
Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007independent and mitochondrial uptake of the fluorescent probe should also be membrane potentialindependent.After flow cytometry, the relative changes in intensity will be used to indicate changes in mitochondrialmass during the cell cycle over 30 minute intervals.Subtopic 2The general picture of upstream regulation of mitochondrial biogenesis via the PPAR family is comingtogether piece by piece. Some steps have been determined, while others have merely been indicated.This part of the research will focus on clarifying some of the unknown but suggested steps in regulation ofmitochondrial biogenesis pathways through two main MAPK pathways linked to cell proliferation: Erk andp38.MAPK-p38 has been established to activate PGC-1α. Nevertheless, there is no experimental data whichlinks the proliferative role of p38 MAPK by means of PGC-1α activation. It has also been demonstratedthat p38 MAPK pathway results in the stimulation of CREB (Delghandi et al., 2005). This may be thestarting point of the signaling cascade that leads to the up regulation of PRC because CREB, like NRF-1is able to bind to PRC and induce mitochondrial transcription.MAPK-Erk has been suggested to reduce PGC-1α expression, as well as been shown to increase musclefibre percentage during injury repair in vivo. It has also been suggested that Erk travels into the nucleusand activates CREB. CREB, as already discussed has been indicated in phosphorylation of PGC-1α invitro as well as modelled in a complex with PRC and NRF. Due to some grey areas that remain in currentresearch this research wishes to determine if MAPK-Erk is able to upregulate as well as downregulatePPAR family expression by dephosphorylation and phosphorylation respectively.This section will first try to ascertain the involvement of these regulatory pathways, through identifyingkinase involvement. Once that is established the mechanisms of regulation will be examined more closelyby determining phosphorylative actions.In carrying out this proposal, we hope to establish some differences between p38 and Erk pathways andunderstand their precise role in this vital but complex process. Another goal is to establish moreknowledge about the extent of functional similarity between PPAR family proteins in these pathways. Todo this, we will examine each PPAR member individually and use RNAi to determine the effect of onePPAR member in the absence of another.Presence of PPAR proteins and mRNA levels of PPAR family in cell linesIt is important to establish the timing of PRC and PGC-1α and PGC-1β mRNA transcription so that timingcan be compared to the corresponding stage of the cell cycle. This will allow determination of anytemporal coordination of such pathways with cell cycle progression. To determine when mRNAtranscription occurs, quantitative Real-Time PCR will be used. The amount of product present will bemeasured by densitometry of the intensities of bands after electrophoresis. These results will becompared with results from the subtopic 1.Establishing Regulatory Pathways for PGC family / cell proliferationWe then wish to establish that there is in fact involvement of the considered pathways in our cell lines aswell as the importance of each. We wish to perform RT-PCR on MAPK-ERK and MAPK-p38 to determinepresence and levels thereof throughout the cell cycle.We will investigate kinase involvement by inhibiting the MAPK-ERK and MAPK-p38 pathways. Followingthis, we will measure the level of PPAR family members upon such inhibition through RNAi. In doing so,SCI 332 Advanced Molecular Cell Biology Research Proposal 65
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