A Primary Study Of The Effect On The Cell Cycle Of Exogenous Wild Type P16 Gene Transfected Bladder Cancer Cell

Cell cycle control plays a key role in the growth of normal mammalian cells. One of the fundamental abnormalities in human tumors is dysregulated cell cycle control. It is generally accepted that active Cdk4 - Cyclin D1 complexes help cells to pass through the R point, a point of no return, after which cells become committed to a new round of replication. Accumulated evidences indicate that Cdk4 is the 'primary sensor' for driving cells through the R point and it is widely known that P16INK4a can arrest cells in G1.But how can the expression of exogenous P16INK4 gene affect the activity of Cdk4-Cyclin D1 remains unclear. In this study, the effect of exogenous wild type P16 gene on cancer cell growth was observed and we also examined the expression of P16 gene and the expression changes of cell cycle regulatory genes-Cdk4, Cyclin D1 and Rb in human bladder cancer cells. All these elucidate the inhibition effect of P16 gene on the cancer cell cycle control.Methods:The plasmid pcDNA3 containing the wild type p16 gene and neo gene was used in the transfection experiment. Afterward the transfection ,normal culture medium with additional 800μg/ml G418 was used for 12 days to select the transfected cells. Flow cytometry (FCM) was performed to analyze the cell cycle distribution. The expression of several proteins involved in cell cycle control including P16, Cdk4, Cyclin D1 and Rb was detected with immunocytochemistry (ICC).And the transcription of the four genes was analyzed with RT-PCR method.Results:The cell growth analysis revealed that the proliferation of P16 gene transfected cancer cells was inhibited after the transfection of exogenous wild P16 gene. The immunocytochemical results indicated that Cdk4 expressed intensively in the nuclei of EJ, and the expression of Cyclin D1 and Rb was positive while the stain for P16 antibody was faint; after the transfection of exogenous wild type P16 gene, the expression of Cdk4,Cyclin D1 and Rb were negative in the nuclei while the expression of P16 significantly increased in the nuclei and the cytoplasm. And the RT-PCR experiments suggested that the gene transcription of P16 significantly increased, meanwhile the transcription of the other three genes: Cdk4, Cyclin D1 and Rb remarkably reduced. Conclusion:Our results suggest that the transfection of exogenous wild P16 gene induces the inhibition of proliferation of the bladder cancer cells and the increasing expression P16 inhibits the expression of Cdk4, Cyclin D1 and Rb in nuclei, which results in the cell cycle being inhibited at G0/G1 phase. As a consequence, exogenous P16 has negative effects on the malignant proliferation of bladder cancer cells and it may be considered as target for potential anti-cancer drugs.