A Recombinant Adenovirus Expressing P27(KIP1) Induces Cell Cycle Arrest and Apoptosis In Human 786–0 Renal Carcinoma Cells
Abstract
Purpose:
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.
Materials and Methods:
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.
Results:
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.
Conclusions:
This study demonstrates the potential effectiveness of Adp27Kip1 as a vector for gene therapy studies of renal cell carcinoma.
References
- 1 : Role of cyclin-dependent kinases and their inhibitors in cellular differentiation and development. Curr Top Microbiol Immunol1998; 227: 57. Google Scholar
- 2 : A recombinant adenovirus expressing p27Kip1 induces cell cycle arrest and loss of Cyclin-Cdk activity in human breast cancer cells. Oncogene1997; 14: 2283. Google Scholar
- 3 : Promoting apoptosis: a novel activity associated with the cyclin-dependent kinase inhibitor p27. Cancer Res1997; 57: 5441. Google Scholar
- 4 : Expression of cell cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients. Nat Med1997; 3: 222. Google Scholar
- 5 : Prognostic role of the cyclin-dependent kinase inhibitor p27Kip1 in non-small cell lung cancer. Cancer Res1997; 57: 3381. Google Scholar
- 6 : Overexpression of p27Kip1 inhibits the growth of both normal and transformed human mammary epithelial cells. Cancer Res1998; 58: 3448. Google Scholar
- 7 : Adenoviral expression of the cyclin-dependent kinase inhibitor p27Kip1: a strategy for breast cancer gene therapy. J Natl Cancer Inst1998; 90: 1836. Google Scholar
- 8 : Comparison of the effectiveness of adenovirus vectors expressing cyclin kinase inhibitors p16INK4A, p18INK4C, p19INK4D, p21(WAF1/CIP1) and p27KIP1 in inducing cell cycle arrest, apoptosis and inhibition of tumorigenicity. Oncogene1999; 18: 1663. Google Scholar
- 9 : Effects of adenovirus-mediated p16INK4A expression on cell cycle arrest are determined by endogenous p16 and Rb status in human cancer cells. Oncogene1998; 16: 265. Google Scholar
- 10 : Molecular genetics of renal carcinogenesis. Toxicol Pathol1998; 26: 113. Google Scholar
- 11 : The von-Hippel-Lindau tumor suppressor gene product forms a stable complex with human Cul-2, a member of the Cdc 53 family of proteins. Proc Natl Acad Sci USA1997; 94: 2156. Google Scholar
- 12 : Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase. Science1999; 284: 657. Crossref, Medline, Google Scholar
- 13 : Functions of von Hippel-Lindau tumor suppressor protein. J Intern Med1998; 243: 535. Google Scholar
- 14 : Recombinant adenovirus expressing von Hippel-Lindau mediated cell cycle arrest is associated with the induction of cyclin-dependent kinase inhibitor p27Kip1. Biochem Biophys Res Comm1998; 253: 672. Google Scholar
- 15 : Requirement for the von Hippel-Lindau tumor suppressor gene for functional epidermal growth factor receptor blockade by monoclonal antibody C225 in renal cell carcinoma. Clin Can Res2000; 6: 1518. Google Scholar
- 16 : Decreased expression of p27Kip1 is associated with decreased survival in patients with metastatic renal cell carcinoma. J Urol1999; 161: 196. abstract 748. Google Scholar
- 17 : Cytotoxic effects of adenovirus-mediated wild type p53 protein expression in normal and tumor mammary epithelial cells. Clin Cancer Res1995; 1: 889. Google Scholar
- 18 : Mutations of the VHL tumor suppressor gene in renal carcinoma. Nat Genet1994; 7: 85. Google Scholar
- 19 : Adenovirus-mediated gene transfer to human breast tumor cells: an approach for cancer gene therapy and bone marrow purging. Cancer Res1996; 56: 1346. Google Scholar
- 20 : Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27Kip1. J Clin Invest1999; 103: 597. Google Scholar
- 21 : BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature1994; 369: 321. Google Scholar
- 22 : Bcl-2 associates with the ras-related protein Ras p23. Nature1993; 366: 274. Google Scholar
- 23 : p27Kip1 overexpression causes apoptotic death of mammalian cells. Oncogene1997; 15: 2991. Google Scholar
- 24 : Efficacy of multiple administrations of a recombinant adenovirus expressing wild type p53 in an immune-competent mouse tumor model. Gene Ther1998; 5: 605. Google Scholar
- 25 : Efficacy of p53 adenovirus-medicated gene therapy against human breast cancer xenografts. Cancer Gene Ther1997; 4: 129. Google Scholar
- 26 : Gene therapy for cancer: what have we done and where are we going?. J Natl Cancer Inst1997; 89: 21. Google Scholar
From the Departments of Urology and Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana
