Expression of Cell Cycle Checkpoints Regulator Proteins p27~(Kip1) and Its Mechanism of Regulation in DNA Damage

In the expression of p27 Kip1 and its mechanism of regulation in the DNA damage, the level of p27 Kip1 expression was found to decrease from 3 to 24 hours after 10 Gy γ-irradiation, and lose its inhibition function to cyclin-dependent kinase. Northern blotting results indicated that the mRNA level of p27 Kip1 did not change during ionizing radiation(IR) responses, which showed that the amount of p27 Kip1 was predominantly regulated by posttranslational mechanism, but the precise mechanism of p27 Kip1 regulation was unclear. It was well known that the degradation of p27 Kip1 mainly depends on cyclin E/Cdk2 kinase which could phosphorylate p27 Kip1, and then the phosphorylated p27 Kip1 degraded through ubiquitin-proteasome pathyway. Enzyme kinetics showed that the activity of Cyclin E/Cdk2 kinase complex was upregulated after irradiation, reached to maximum at 12 hours. When Cyclin E/Cdk2 inhibitor Olomoucine inhibited the activity of Cyclin E/Cdk2 kinase before IR, the level of p27 Kip1 increased. The level of ubiquitin-p27 Kip1 conjugates in non-irradiated and irradiated HeLa cells were detected and the level of ubiquitinated p27 Kip1 was found to increase following 10 Gy-irradiation. MG-132 (proteasome inhibitor) inhibited the degradation of p27 Kip1 which was induced by IR in HeLa cells. The data suggested that ubiquitin -proteasome pathway might involve in the regulation of p27 Kip1 during IR responses.