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

Using the transfeetion teehnique. P15INK4b was introduced into P15INk4b gene deleted human melanoma A375 cells,and a cell model MLED6 overexpressing P15INK4b WAS CONSTRUCTED.Comparing with the control cells MLC2,MLEK6cells in G1phase increased by 11%,but those in Sphase decreased by 15%by FCM.By the method of thymidine(TdR)and N2O arresting,the proportions of synchronized Mphase cells of MLEK6 ana MLC23 were measured and found to be 89.1% and 76.8%respectively ,and the cells in G1phase were 74.3% for MLID6 AND 76. 4% forMLC2.The result of3 H-TdR incorporation indicated that the transition of G1/Sof MLEK6 cell was delayed 2h as compared with that of MLC2 cells,and incorporation rate also decreased.The observation on exprissions of some G1/ S-resates relatory rigusating genes showed that in MLIK6 cells the protein leves of P27KIPI increased with the decreasing expressions of cyclinD1,cyclinE and c-myc,especially cyclinD1 in late G1phade.The expression of cyclinE obviously decreased at G1/S transition ,and c-myc wad inhibited throughout all the process of G1 S phase.All the risults suggest that P15INK4b can delayG1/S transition of MLEK6 cells by inhibiting the cell cycle engine ,and by increasing the expression of Cdk ingibitor P27KIPI in different stages of G1 phase.