title: 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.
abstract: 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.
authors.affiliation: National Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Dong-Mei
authors.initials: DM
authors.lastname: Wang
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhou
authors.forename: Hai-Jun
authors.initials: HJ
authors.lastname: Xie
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Ma
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.forename: Bing-Ren
authors.initials: BR
authors.lastname: Huang
mesh_terms: D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000235:genetics*; D005434:Flow Cytometry; D006801:Humans; D018613:Microscopy, Confocal; D009369:Neoplasms / Q000188:drug therapy; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency*; D014764:Viral Proteins / Q000235:genetics*
chemical_list: C121517:E4orf4 protein, adenovirus; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; D004815:Epidermal Growth Factor