Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Transferase-1 (CPT-1). We will measure the levels of fatty acid oxidation to determine which ofthe inhibitors is more efficient in our cell line, or whether we need to use both for completeinhibition.AMPK will be up-regulated by inducing CA-AMPK expression, and the same experiments as in 1.1 will beperformed. Additionally, cycle progression into S-phase will be determined in samples of the population bythe cell phase determination kit at regular intervals of 30 minutes.2: AMPK influence on cell cycle arrest and apoptosisHypothesis 2: AMPK induces cell cycle arrest in response to glucose depletion at multiple points in G1.Prolonged AMPK activation induces apoptosis in early G1 but not in late G1.2.1 Determining the restriction point (R) in G1.It has been established that there is a growth factor restriction point R in mammalian cells (Cooper, 2007).To determine whether the AMPK checkpoint corresponds to R, we will firstly determine where exactly R isin the cells used.Experiment 2.1In order to determine R, synchronized cells are deprived of growth factors at various times after M:Synchronized cells are seeded in a number of wells in complete BrdU/uridine containing medium.Subsequently, individual wells are successively washed (at time intervals of about 1/10 th of the length ofG1) and provided with serum-free medium containing BrdU/uridine. Incubation continues for the previouslydetermined time required for non serum-starved cells to completely reach S-phase (as in Schorl et al.,2003). A 50% rise in BrdU positive cells indicates the time point when cells are no more affected bygrowth factor deprivation, and thus the restriction point, R.2.2 Does AMPK induce quiescence in G1 in response to decreased glucose levels?Here we aim to confirm that AMPK is able to induce quiescence in G1 in response to glucose deprivationin the cell lines under investigation, as has been demonstrated previously for other cell lines (seebackground).Experiment 2.2.1Quiescence will be determined by measuring the differences in levels of Cyclin A, B and D1, p130and phosphorylated Rb (see methods experiment 0.2 and 0.3). If levels of these “quiescent markers” aresignificantly different than control levels, they will serve as a reference to determine quiescence in laterexperiments. Furthermore, we will measure whether G1 is prolonged after AMPK activation. We expect toobserve cell cycle re-entry after increased levels of ATP and decreased AMPK activity.Experiment 2.2.2 Is cell cycle arrest in response to glucose deprivation AMPK dependent?Synchronized cells will be glucose starved while AMPK is inhibited by Compound C. If under theseconditions the cells are arrested, an alternative, AMPK independent pathway must be responsible forhalting the cell cycle in response to low glucose levels.2.3 Where in G1 can AMPK induce cell cycle arrest?The following experiment will be conducted to determine the effect of selective activation of AMPKthroughout G1.Experiment 2.3.1Glucose starved cells treated with Compound C will be used to determine whether AMPK is able to inducecell cycle arrest at different points in G1. The synchronized cells will be divided over several wells with alow-glucose and Compound C containing medium. As in experiment 2.1, individual wells are washed atsuccessive points in time (at time intervals of about 1/10 th of the length of G1) and afterwards CompoundC-free medium containing AICAR with the same low glucose concentration is added. This will lead toSCI 332 Advanced Molecular Cell Biology Research Proposal 47