Cytotoxicity of a recombinant fusion protein of adenovirus early region 4 open reading frame 4 (E4orf4) and human epidermal growth factor on p53-deficient tumor cells.

Adenovirus early region 4 open reading frame 4 (E4orf4) protein is a novel cell death factor that selectively induces p53-independent apoptosis in cancer cells, but not in normal human cells. This study presents an approach for inhibiting p53-deficient tumor cell growth by using protein-based E4orf4 that had been genetically fused to epidermal growth factor (EGF) to ensure selective targeting of EGF receptor-overexpressing tumor cells. EGF-E4orf4 enables binding onto the cell surface and is then internalized into Saos-2 cells. The success of the process had been demonstrated by immunofluorescence assay and confocal laser microscopy. After prolonged exposure, E4orf4 remained mostly in the nuclei. EGF-E4orf4 treatment of Saos-2 cells showed dose-dependent cytotoxicity. Nearly 50% of the Saos-2 cells were killed at a concentration of 250 nmol/l. In contrast, EGF-E4orf4 showed no significant inhibitory effect iresn primary cells of human umbilical vein endothelial cells. To confirm the ability of EGF-E4orf4 to induce apoptosis, DNA fragmentation was detected using BrdUTP end-labeling. Flow cytometric analysis revealed a significant increase of apoptotic cells in Saos-2 cells treated with EGF-E4orf4, but not in the case of cells cultured in plain medium (t=0.028, P<0.05). In conclusion, these preliminary results indicate that EGF-E4orf4 could show promise as a new reagent that is more efficient and less toxic in anti-cancer therapy.