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The Mitochondrial Apoptosis-Inducing Factor Plays a Role in E2F-1–Induced Apoptosis in Human Colon Cancer Cells

From the Departments of Surgical Oncology (SAV, YL, XL, KKH) and Thoracic and Cardiovascular Surgery (AP, SGS), University of Texas, M. D. Anderson Cancer Center, Houston, Texas; and the Department of Surgery (KY), Jikei University School of Medicine, Tokyo, Japan.

Correspondence: Address correspondence and reprint requests to: Kelly K. Hunt, MD, FACS, Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 444, Houston, TX 77030; Fax: 713-792-4689; E-mail: khunt{at}mdanderson.org

Background: Overexpression of the transcription factor E2F-1 provokes apoptosis in cancer cells; the mechanism, however, is not completely understood. We sought to evaluate E2F-1 gene therapy in human colon cancer and to investigate the apoptotic pathway involved.

Methods: Adenoviral vectors were used to transfect the E2F-1 gene (Ad5E2F-1) or the control gene luciferase (Ad5Luc) into four human colon carcinoma cell lines. Apoptosis was confirmed by flow cytometry and poly (ADP-ribose) polymerase cleavage. Expression of apoptotic factors was determined with Western blot analysis. Inhibitory assays were used to determine the involvement of caspases in the apoptotic pathway.

Results: Overexpression of E2F-1 was evident in all cells treated with Ad5E2F-1; upregulation of Bcl-2, and activation of caspases were noted. The apoptosis-inducing factor in the cytosolic fraction was markedly upregulated after Ad5E2F-1 treatment. E2F-1 overexpression inhibited proliferation and induced significant apoptosis in all cell lines (P < .005). This apoptotic response could be only partially blocked by caspase inhibitors.

Conclusions: These findings demonstrate that E2F-1 induces apoptosis and inhibits proliferation in human colon cancer cell lines. The marked upregulation of apoptosis-inducing factor and the fact that E2F-1–induced apoptosis is incompletely blocked by caspase inhibitors suggest a caspase-independent pathway of E2F-1–mediated apoptosis, reported here for the first time.

Key Words: Apoptosis • E2F-1 • Colon cancer • Apoptosis-inducing factor • Mitochondrion

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