Effects Of P66shcA Gene On Cell Cycle Regulation In PC12 Cells

p66ShcA protein is the longest isoform of ShcA proteins, which belong to a family including three members, known as ShcA, ShcB and ShcC. All Shc proteins share a unique highly conserved domain organization characterized by an N-terminal PTB domain and a C-terminal SH2 domain, but p66ShcA has an additional N-terminal CH domain, termed CH2. When cells are treated with UV& H₂O₂, p66ShcA is phosphorylated by p53 to promote apoptosis. Latest studies provide a clearly relationship between apoptosis and ROS. Thus, p66ShcA is suggested to be at the crossroad of oxidative stress and genetics of aging.To observe the effect of p66shcA on the cell cycle and to explore the role of p66shcA in the mechanism of neural development,the pEGFP-p66shcA plasmid was transiently transfected into PC12 with Lipofectamine. The effect of p66shcA on the cell cycle was observed by flow cytometer. The mRNA and protein expression p27 were detected by RT-PCR and immunohistochemistry respectively. There was an elevated expression of p66shcA in the cells transfected with pEGFP-p66shcA. Upon flow cytometry , there were more cells in G0/G1 phase and fewer cells in G2/M phase in pEGFP-p66shcA transfected cells(P< 0.01). The mRNA expression of p27 increased (P<0.01) . The results indicated that p66shcA might play an important role in the neural differentiation by inhibiting the cell cycle , which was related to the decreased cyclin E and increased p27 expression. When we add NGF to the pEGFP-p66shcA transfected cells for two days, we found the more cells are differentiated than the control cells, which suggested that p66shcA has some relationship with cell differentiation.