Amino Acids and mTOR Mediate Distinct Metabolic Checkpoints in Mammalian G 1 Cell Cycle

Objective: In multicellular organisms, cell division is regulated by growth factors (GFs). In the absence of GFs, cells exit the cell cycle at a site in G1 referred to as the restriction point (R) and enter a state of quiescence known as G0. Additionally, nutrient availability impacts on G1 cell cycle progression. While there is a vast literature on G1 cell cycle progression, confusion remains – especially with regard to the temporal location of R relative to nutrient-mediated checkpoints. In this report, we have investigated the relationship between R and a series of metabolic cell cycle checkpoints that regulate passage into S-phase. Methods: We used double-block experiments to order G1 checkpoints that monitor the presence of GFs, essential amino acids (EEAs), the conditionally essential amino acid glutamine, and inhibition of mTOR. Cell cycle progression was monitored by uptake of [3H]-thymidine and flow cytometry, and analysis of cell cycle regulatory proteins was by Western-blot. Results: We report here that the GF-mediated R can be temporally distinguished from a series of late G1 metabolic checkpoints mediated by EAAs, glutamine, and mTOR – the mammalian/mechanistic target of rapamycin. R is clearly upstream from an EAA checkpoint, which is upstream from a glutamine checkpoint. mTOR is downstream from both the amino acid checkpoints, close to S-phase. Significantly, in addition to GF autonomy, we find human cancer cells also have dysregulated metabolic checkpoints. Conclusion: The data provided here are consistent with a GF-dependent mid-G1 R where cells determine whether it is appropriate to divide, followed by a series of late-G1 metabolic checkpoints mediated by amino acids and mTOR where cells determine whether they have sufficient nutrients to accomplish the task. Since mTOR inhibition arrests cells the latest in G1, it is likely the final arbiter for nutrient sufficiency prior to committing to replicating the genome. Citation: Saqcena M, Menon D, Patel D, Mukhopadhyay S, Chow V, et al. (2013) Amino Acids and mTOR Mediate Distinct Metabolic Checkpoints in Mammalian G1 Cell Cycle. PLoS ONE 8(8): e74157. doi:10.1371/journal.pone. 0074157 Editor: Claude Prigent, Institut de Génétique et Développement de Rennes, France Received April 8, 2013; Accepted July 29, 2013; Published August 19, 2013 Copyright: © 2013 Saqcena et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Support by National Cancer Institute grant CA46677 is acknowledged. Research Centers in Minority Institutions award RR-03037 from the National Center for Research Resources of the National Institutes of Health, which supports infrastructure and instrumentation in the Biological Sciences Department at Hunter College, is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: foster@genectr.hunter. cuny.edu