Adrienne L. Katner, Praveen Gootam, Quoc Bao Ly Hoang, James R. Gnarra, and Walter Rayford
Departments of Urology (A.L.K., P.G., Q.H., W.R.) and Biochemistry and Molecular Biology (J.R.G.), Louisiana State University Health Sciences Center, New Orleans, LA., 70112
J. of Urology- TBA
ABSTRACT
Purpose: This study was undertaken to evaluate the effects of over-expression of p27Kip1, a cyclin-dependent kinase-inhibitor and tumor-suppressor protein, on the human renal carcinoma cell line, 786-0.
Materials and Methods: Adp27Kip1, a recombinant adenovirus was evaluated for induction of p27 protein expression in 786-0 renal carcinoma cells. Time of expression and optimal vector concentration was determined. Growth curve studies, cell cycle analysis and TUNEL analyses were conducted to determine effects of p27Kip1 on cell cycle. CdkI activity assays were conducted to determine expression/activities of cyclin-dependent kinases, and westerns were conducted to determine presence of CdkIs and other cell cycle regulator proteins. Nude mouse xenografts were established to demonstrate the in vivo efficacy of Adp27Kip1.
Results: Expression of p27Kip1 protein was detected within 12 hours after Adp27Kip1 infection and remained stable for at least 48 hours. Growth studies demonstrated that Adp27Kip1 infection resulted in inhibition of proliferation by three days post-infection, and cell death was detected by day five. Cell cycle analysis of DNA content indicated an accumulation of cells in G1-phase of Adp27Kip1-infected cells and a corresponding decrease in S-phase cells within 48 hours after infection. Cyclin dependent kinase activity was determined and Cdk2, Cdk4 and Cdc2 kinase activities were all inhibited, consistent with p27Kip1 over-expression. Levels of Cdk-inhibitors, p16 and p18, were elevated 24 hours after Adp27Kip1 infection, while p21 levels remained unchanged. TUNEL analysis demonstrated 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 RCC xenografts.
Conclusions: These studies demonstrate the potential effectiveness of Adp27Kip1 as a vector for gene therapy studies in renal