Effect of P15INK4b expression on the cell cycle and G1 phase related regulatory genes in human melanoma cells

Using the transfection technique, P15(INK4b) was introduced into P15(INK4b) gene deleted human melanoma A375 tells, and a cell model MLIK6 overexpressing P15(INK4b) was constructed. Comparing with the control cells MLC2, MLIK6 cells in G(t) phase increased by 11%, but those in S phase decreased by 15% by FCM. By the method of thymidine (TdR) and N2O arresting, the proportions of synchronized M phase cells of MLIK6 and MLC2 were measured and found to be 89.1% and 76.8%, respectively, and the tells in G(1) phase were 74.3% for MLIK6 and 76. 4% for MLC2. The result of H-3-TdR incorporation indicated that the transition of G(1)/S of MLIK6 cell was delayed 2 h as compared with that of MLC2 cells, and incorporation rate also decreased. The observation on expressions of some G(1)/S-related relatory regulating genes showed that in MLIK6 cells the protein level of P27(KIP1) increased with the decreasing expressions of cycllinD1, cyclinE and c-myc, especially cyclinD1 in late G(1) phase. The expression of cyclinE obviously decreased at G(1)/S transition, and c-myc was inhibited throughout all the process of G(1)-->S phase. All the results suggest that P15(INK4b) call delay G(1)/S transition of MLIK6 cells by inhibiting the cell cycle engine molecule, and by increasing the expression of Cdk inhibitor P27(KIP1) in different stages of G(1) phase.