E2F-1 up-regulates c-Myc and p14(ARF) and induces apoptosis in colon cancer cells.

Although overexpression of E2F-1 can induce apoptosis in a variety of tumor cell lines, the mechanisms by which E2F-1 induces apoptosis remain ambiguous. In this study, we examine the ability of E2F-1 to induce apoptosis in colon cancer and the molecular mechanisms underlying E2F-1-mediated apoptosis. HT-29 and SW-620 colon adenocarcinoma cells (both mutant p53) were treated by mock infection or adenoviral vectors Ad5CMV (empty vector), Ad5CMVLacZ (beta-galactosidase), and Ad5CMVE2F-1 (E2F-1) at multiplicity of infection of 100. Western blot analysis confirmed marked overexpression of E2F-1 in both cell lines. By 5 days after infection, E2F-1 overexpression resulted in >25-fold reduction in cell growth and >90% loss of cell viability in both cell lines. Cell cycle analysis of Ad-E2F-1-infected cells revealed an increase in G(2)/M and sub-G(1) populations. By in situ terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling analysis, evidence of apoptosis was observed including internucleosomal DNA fragmentation and the formation of apoptotic bodies. In addition, caspase-3 and poly(ADP-ribose) polymerase apoptotic fragments were detected by 48 h after treatment with Ad-E2F-1. Of mechanistic importance, overexpression of E2F-1 caused a G(2)/M arrest followed by increased levels of c-Myc and p14(ARF) proteins. Additionally, expression of the antiapoptotic Bcl-2 family member Mcl-1 was down-regulated in E2F-1-overexpressing cells. In conclusion, E2F-1 overexpression initiates apoptosis and suppresses growth in HT-29 and SW620 colon adenocarcinoma cells. Overexpression of E2F-1 triggers apoptosis and is associated with up-regulation of c-Myc and p14(ARF) proteins and down-regulation of Mcl-1. Therefore, E2F-1 is a potentially active gene therapy agent for the treatment of colon cancer.