Expression of the DNA-PK binding protein E4-34K fails to confer radiation sensitivity to mammalian cells.
OBJECTIVE
The adenovirus E4orf6 34 kDa protein (E4-34k) is known to disrupt V(D)J recombination as a result of its interaction with the catalytic subunit of cellular DNA-dependent protein kinase (DNA-PK(cs)), a major participant in the repair of DNA double-strand breaks (DSB). Previous studies have shown that cells with disrupted DSB repair and V(D)J recombination due to attenuation of DNA-PK(cs) activity exhibit a radiation-sensitive phenotype. It is not known at present whether the E4-34k protein can also modify cellular response to ionizing radiation. In an attempt to develop a novel gene therapy strategy to modify cellular radiation response, we sought to determine if expression of the adenovirus E4-34k protein resulted in sensitization to clinically relevant doses of ionizing radiation.


METHODS
In order to minimize potential bias resulting from selection procedures, we performed clonogenic survival assays on DU 145 prostate cancer cells, RKO colorectal cancer cells and 293 kidney cells following transient transfection of E4-34k- and/or E1B-55k-expressing plasmids. Western blots and immunohistochemical analyses were used to demonstrate E4-34k expression within transfected cells. FACS sorting was carried out to enrich cells transfected with a plasmid that expresses both E4-34k and enhanced green fluorescent protein.


RESULTS
It is shown that E4-34k expression does not affect cellular radiosensitivity of transiently transfected populations of either DU 145 prostate or RKO colon cancer cell lines. Similarly, the radiosensitivity of human embryonic kidney 293 cells, which constitutively express the E1B-55k protein, was also unaffected. The radiosensitivity of DU 145 cells co-transfected with E4-34k- and E1-55K-expressing plasmids was unchanged, suggesting that the adenovirus E1B-55k protein does not augment any effects E4-34k might have on DNA-PK(cs) activity.


CONCLUSIONS
The lack of radiosensitization by E4-34k expression is quite intriguing as it is known that E4-34k interaction with DNA-PK(cs) causes disruption of V(D)J recombination, a process dependent on DSB rejoining. These data suggest that for future studies, preferential targeting of DNA-PK(cs) DSB activity will be required to influence cellular radiosensitivity.
Collis
S J
SJ
Radiation Biology Program, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
Ketner
G W
GW
Hicks
J L
JL
Nelson
W G
WG
Demarzo
A M
AM
Deweese
T L
TL
D017501:Adenovirus E1B Proteins / Q000502:physiology; D017504:Adenovirus E4 Proteins / Q000502:physiology*; D002470:Cell Survival / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005803:Genes, Immunoglobulin; D006801:Humans; D008297:Male; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011836:Radiation Tolerance; D011995:Recombination, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017501:Adenovirus E1B Proteins; D017504:Adenovirus E4 Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases