Collis S J, Ketner G W, Hicks J L, Nelson W G, Demarzo A M, Deweese T L
Radiation Biology Program, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
Int J Radiat Biol. 2003 Jan;79(1):53-60.
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.
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.
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.
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.
腺病毒E4orf6 34 kDa蛋白（E4-34k）因与细胞DNA依赖性蛋白激酶的催化亚基（DNA-PK(cs)）相互作用而破坏V(D)J重组，DNA-PK(cs)是DNA双链断裂（DSB）修复的主要参与者。先前的研究表明，由于DNA-PK(cs)活性减弱而导致DSB修复和V(D)J重组受损的细胞表现出辐射敏感表型。目前尚不清楚E4-34k蛋白是否也能改变细胞对电离辐射的反应。为了开发一种改变细胞辐射反应的新型基因治疗策略，我们试图确定腺病毒E4-34k蛋白的表达是否会导致对临床相关剂量电离辐射的敏感性增加。
为了尽量减少选择程序导致的潜在偏差，我们在瞬时转染表达E4-34k和/或E1B-55k的质粒后，对DU 145前列腺癌细胞、RKO结肠癌细胞和293肾细胞进行了克隆形成存活试验。使用蛋白质免疫印迹和免疫组织化学分析来证明转染细胞内E4-34k的表达。进行荧光激活细胞分选（FACS）以富集用表达E4-34k和增强型绿色荧光蛋白的质粒转染的细胞。
结果表明，E4-34k的表达不影响DU 145前列腺癌细胞系或RKO结肠癌细胞系瞬时转染群体的细胞放射敏感性。同样，组成性表达E1B-55k蛋白的人胚肾293细胞的放射敏感性也未受影响。共转染表达E4-34k和E1-55K质粒的DU 145细胞的放射敏感性没有变化，这表明腺病毒E1B-55k蛋白不会增强E4-34k可能对DNA-PK(cs)活性产生的任何影响。
E4-34k表达缺乏放射增敏作用相当令人费解，因为已知E4-34k与DNA-PK(cs)的相互作用会导致V(D)J重组的破坏，这是一个依赖于DSB重新连接的过程。这些数据表明，在未来的研究中，需要优先靶向DNA-PK(cs)的DSB活性来影响细胞放射敏感性。
