title: The adenovirus E4orf6 protein inhibits DNA double strand break repair and radiosensitizes human tumor cells in an E1B-55K-independent manner.
abstract: The adenoviral protein E4orf6 has been shown to inhibit both in vitro V(D)J recombination and adenoviral DNA concatenation, two processes that rely on cellular DNA double strand break repair (DSBR) proteins. Most of the known activities of E4orf6 during adenoviral infection require its interaction with another adenoviral protein, E1B-55K. Here we report that E4orf6, stably expressed in RKO human colorectal carcinoma cells or transiently expressed by adenoviral vector in U251 human glioblastoma cells, inhibits DSBR and induces significant radiosensitization in the absence of E1B-55K. Expression of a mutant form of E4orf6 (L245P) failed to radiosensitize RKO cells. E4orf6 reduced DSBR capacity in transfected and infected cells, as measured by sublethal DNA damage repair assay and phosphorylated H2AX (gamma-H2AX) levels, respectively. Consistent with the inhibitory effect of E4orf6 on DSBR, expression of wild-type but not mutant E4orf6 reduced recovery of a transfected, replicating reporter plasmid (pSP189) in 293 cells but did not increase the mutation frequency measured in the reporter plasmid. The kinase activity of DNA-PKcs (the DNA-dependent protein kinase catalytic subunit) toward heterologous substrates was not affected by expression of E4orf6; however, autophosphorylation of DNA-PKcs at Thr-2609 following ionizing radiation was prolonged in the presence of E4orf6 when compared with control-infected cells. Our results demonstrate for the first time that E4orf6 expression hinders the cellular DNA repair process in mammalian cells in the absence of E1B-55K or other adenoviral genes and suggest that viral-mediated delivery of E4orf6, combined with localized external beam radiation, could be a useful approach for the treatment of radioresistant solid tumors such as glioblastomas.
authors.affiliation: Department of Radiation Oncology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
authors.forename: Lori S
authors.initials: LS
authors.lastname: Hart
authors.forename: Steven M
authors.initials: SM
authors.lastname: Yannone
authors.forename: Christine
authors.initials: C
authors.lastname: Naczki
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Orlando
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Waters
authors.forename: Steven A
authors.initials: SA
authors.lastname: Akman
authors.forename: David J
authors.initials: DJ
authors.lastname: Chen
authors.forename: David
authors.initials: D
authors.lastname: Ornelles
authors.forename: Constantinos
authors.initials: C
authors.lastname: Koumenis
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000502:physiology*; D017501:Adenovirus E1B Proteins / Q000378:metabolism*; D017504:Adenovirus E4 Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000528:radiation effects*; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins; D016366:Open Reading Frames / Q000235:genetics*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011836:Radiation Tolerance; D011839:Radiation, Ionizing; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D017501:Adenovirus E1B Proteins; D017504:Adenovirus E4 Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D013912:Threonine; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases