Cell cycle regulation by post-translational and post-transcriptional mechanisms in an anaerobic extremist – The anoxic tolerant turtle, Trachemys scripta elegans
As a model for vertebrate long-term survival in oxygen restricted environments, the red-eared slider turtle (T. s. elegans) can adapt at the biochemical level to deal with hibernation occurring in oxygen-free (anoxic) cold water (<10°C). In this thesis I hypothesized that the mechanisms which suppress ATP-expensive cell cycle activity, would contribute to establishing an hypometabolic state. To explore this possibility, this thesis studied the post-transcriptional and post-translational mechanisms of cell cycle arrest during anoxic stress in the freshwater turtle.
Results indicated a general regulation of critical cell cycle components, in addition to the possible regulation by signaling cascades (Akt/GSK-3β and ATR/Chk2) that are known to regulate G1/G0 phases of the cell cycle. Importantly, there is extensive regulation of Cyclin D1 protein by (1) Akt/GSK-3β signaling, (2) post-translational modification, (3) an AU-rich region, and (4) microRNA-induced translational suppression. This study also identified a phase-specific cell cycle arrest mechanism involving the Rb/E2F DNA-binding complex in both anoxic liver and kidney tissues. A novel DNA-binding complex ELISA technique was able to identify that both kidney and liver establish an Rb/E2F1 mediated G1 arrest complex by 5 and 20 h anoxia, repectively. By 20 h anoxia, kidney tissue established a reversible state of G0, characterized by the prescence of a p130/E2F4 DNA-bound complex.
Overall, results from this thesis indicate that both kidney and liver enter into a G1 arrest during anoxia. By contrast, the cell cycle in white skeletal muscle was found to be minimally regulated during anoxia and this finding is likely a reflection of its overall post-mitotic nature. Interestingly, kidney established a state of G1 arrest within 5 h anoxia and subsequently transitioned to a sustainable G0 arrest by 20 h anoxia. However, it appears that liver G1 arrest was not established until 20 h anoxia. Future studies will need to explore the regulation of the cell cycle in liver after longer periods of anaerobiosis to determine whether hepatocytes are also able to transition into G0 arrest in a manner similar to kidney tissue.