p21Cip1 expression is increased in ambient oxygen, compared to estimated physiological (5%) levels in rat muscle precursor cell culture

Objective: While it is common practice to culture cells in the presence of ambient oxygen (similar to 21% O-2), O-2 level observed in the physiological environment is often much lower. Previous efforts to culture a variety of different stem cells, including muscle precursor cells (MPC), under O-2 conditions that better mimic in vivo conditions have resulted in enhanced proliferation. In the present study, we hypothesized that 20% O-2 in culture represents a sufficient stimulus to cause increased expression of two key negative regulators of the cell-cycle Cip/Kip family of cyclin-dependent kinase inhibitors, p21(Cip1) and p27(Kip1), in MPCs. Materials and methods: MPCs were isolated from Fischer 344 x Brown Norway F-1 hybrid male rats and O-2 was adjusted in culture using a tri-gas incubator. Results: 5-Bromo-2'-deoxyuridine incorporation, cell number and nuclear proliferating cell nuclear antigen expression were all decreased after 48 h culture in 20% O-2, compared to 5% O-2. Twenty per cent O-2 had no effect on either p27(Kip1) promoter activity or protein expression. Although p21(Cip1) promoter activity remained unchanged between 5% and 20% O-2, there were significant increases in both p21(Cip1) mRNA and protein expression. Furthermore, 20% O-2 caused an increase in p21(Cip1) mRNA stability and p53 transcription factor activity. Conclusion: These findings are considered important because they reveal p21(Cip1) as a critical regulatory protein that needs to be considered when interpreting proliferation data from MPCs studied in culture. In addition, O-2-dependent regulation of MPC proliferation is relevant to conditions, including sarcopenia, heart failure, cancer and muscular dystrophy, where increased oxidative stress exists.