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.
Wang
Dong-Mei
DM
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.
Zhou
Yu
Y
Xie
Hai-Jun
HJ
Ma
Xiao-Li
XL
Wang
Xin
X
Chen
Hong
H
Huang
Bing-Ren
BR
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*
C121517:E4orf4 protein, adenovirus; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; D004815:Epidermal Growth Factor