Adenovirus-Mediated Expression of Truncated E2F-1 Suppresses Tumor Growth In Vitro and In Vivo

Adenovirus (Ad) vectors that express the transcription factor E2F-1 (Ad-E2F-1) efficiently induce apoptosis in cancer cells in vitro and in vivo.1–5 However, it is believed that E2F-1 is an oncogene, presumably by virtue of its ability to stimulate cell cycle progression from the G1 phase to the S-phase.6,7 Thus, the use of wild type (wt) E2F-1 in gene therapy has been controversial because of its oncogenic potential. To circumvent this barrier, we deleted the transactivation domain of wt E2F-1 and generated a truncated form of the E2F-1 gene (E2Ftr) (amino acids 1 through 375). Transactivation-deficient E2F-1 is unable to induce cell cycle progression compared with full-length E2F-1 but still is a potent inducer of apoptosis.8,9 To generate adenoviral vectors for efficiently transferring the therapeutic E2Ftr gene into tumors, we previously tried to use a cytomegalovirus (CMV) promoter to control E2Ftr. However, unlike an adenoviral vector that expresses the wt E2F-1 gene, the E2Ftr adenoviral vector could not be rescued from the plasmids with the adenoviral backbone in Ad host HEK-293 human embryonic kidney cells despite numerous attempts. It is likely that high levels of E2Ftr expression driven by the CMV promoter may be toxic to HEK-293 cells and may inhibit adenoviral replication. Next, we applied the tetracycline (Tet)-off regulated expression system10 to control the expression of E2Ftr in adenoviral vectors. We developed new vectors in which the Tet-controlled transactivator (tTA) and the E2Ftr gene were integrated into the same Ad construct. Under regulation of the Tet-responsive promoter (phcmv), E2Ftr is activated by the tTA only in the absence of Tet.11 This system overcame the toxic effects of transgenic gene on HEK-293 cells, allowing us to construct E2Ftr-expressing adenoviruses. In this report, we demonstrate that the inducible expression system in our new vectors resulted in high-level E2Ftr expression in cancer cells. Compared with Ad-E2F-1–expressing wt E2F-1, vectors that expressed E2Ftr induced greater cancer cell cytotoxicity and lower cytotoxicity in normal cells. In addition, E2Ftr significantly suppressed tumor growth in a nude mouse melanoma tumor xenograft model. Our studies indicate that this novel Ad that expresses E2Ftr has potent antitumor effects with significant potential for clinical application.