A Recombinant Adenovirus Expressing P27(KIP1) Induces Cell Cycle Arrest and Apoptosis In Human 786–0 Renal Carcinoma Cells

Abstract

We evaluated the effects of the over expression of p27Kip1, a cyclin dependent kinase inhibitor and tumor suppressor protein, on the 786-0 human renal carcinoma cell line. The recombinant adenovirus Adp27Kip1 was evaluated for the induction of p27 protein expression in 786-0 renal carcinoma cells. Expression time and optimal vector concentration were determined. Growth curve studies, cell cycle analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling were done to determine the effects of p27Kip1 on the cell cycle. Cyclin dependent protein kinase (Cdk) inhibitor (CDKI) activity assays were done to determine the expression/activities of Cdks and Western blot analysis was performed to determine the presence of CDKIs and other cell cycle regulator proteins. Nude mouse xenografts were established to demonstrate the in vivo efficacy of Adp27Kip1. p27Kip1 protein expression was detected within 12 hours after Adp27Kip1 infection and it remained stable for at least 48 hours. Growth studies demonstrated that Adp27Kip1 infection resulted in the inhibition of proliferation by 3 days after infection and cell death was detected by day 5. Cell cycle analysis of DNA content indicated an accumulation of cells in the G1 phase of Adp27Kip1 infected cells and a corresponding decrease in S phase cells within 48 hours after infection. Cdk activity was determined, and Cdk2, Cdk4 and Cdc2 kinase activities were inhibited, consistent with p27Kip1 over expression. The levels of the CDKIs p16 and p18 were elevated 24 hours after Adp27Kip1 infection, while p21 levels remained unchanged. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling revealed that Adp27Kip1 infection but not infection by control virus induced detectable apoptosis within 24 hours. Adp27Kip1 significantly caused the reduction in the size of tumors of the renal cell carcinoma xenografts. This study demonstrates the potential effectiveness of Adp27Kip1 as a vector for gene therapy studies of renal cell carcinoma.