Cell cycle inhibition as a mode of abnormal development: The role of cell cycle checkpoint proteins and cyclin-dependent kinase inhibitors in neurodevelopmental toxicant defense.

摘要

Neurodevelopment is an intensively complex process involving the production and organization of over a trillion cells into regions with specialized functions. Patterning of these structures is orchestrated via signaling pathways that provide cues for immature precursor cells to continue proliferating or exit the cell cycle and differentiate into mature, functional neurons and glia. The transition from the proliferative to differentiated state is ultimately accomplished via cell cycle regulatory proteins that act to confer cell cycle exit. This same class of proteins also responds to stress signaling to prevent cellular growth in suboptimal conditions or to repair DNA. Little is known regarding the adverse effects of premature cell cycle exit, effected by these cell cycle regulators, following toxicant exposure during neurodevelopment. Using methylmercury (MeHg) as a model toxicant, this dissertation explores the involvement of cell cycle regulatory proteins in the balance between necrosis, apoptosis, cell cycle inhibition at specific cell cycle phases, and neuronal differentiation following MeHg exposure. Mouse embryonal fibroblasts wildtype and null for Tp53 (p53) are utilized to ascertain the role of p53 signaling pathways in MeHg's effects on cell cycle arrest and cell death. This experimentally derived data is then compared to a literature based mathematical model of the cell cycle that uses rates to describe p53 controlled cell cycle checkpoints and apoptotic pathways. The mathematical model accurately predicts cell cycle phase distribution and should be of utility to inform mechanism specific dose-response predictions of toxicant exposures that cause cell cycle inhibition. In order to examine the involvement of cell cycle regulatory proteins on cell cycle exit during differentiation and toxicant response, we established a novel embryonic mouse midbrain neural precursor cell (NPC) culture and demonstrated a role for both p27 and p53 proteins in effecting differentiation in vitro and in vivo. Midbrain NPC cultures were then used to test the hypothesis that sub-lethal exposure of cycling NPCs to MeHg would result in cell cycle arrest and premature neuronal differentiation in a p53 dependent manner. At a concentration of MeHg resulting in a 50% reduction of cells able to reach a new round of cycling (0.5 muM), we observed increases in cholinergic and GABAergic but not dopaminergic neurons in p53+/+ but not p53-/- NPCs. Taken as a whole, this body of work demonstrates a significant role for p53 in neurodevelopmental toxicant defense, and supports cell cycle inhibition and premature differentiation as possible modes of abnormal development following low dose toxicant exposures.