Singhal Jyotsana, Singhal Sharad S, Yadav Sushma, Suzuki Sumihiro, Warnke Molly M, Yacoub Adly, Dent Paul, Bae Sejong, Sharma Rajendra, Awasthi Yogesh C, Armstrong Daniel W, Awasthi Sanjay
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Int J Radiat Oncol Biol Phys. 2008 Oct 1;72(2):553-61. doi: 10.1016/j.ijrobp.2008.06.1497.
To determine the role of RLIP76 in providing protection from radiation and chemotherapy. In the present report, we used RLIP76 to refer to both the mouse (Ralbp1) and the human (RLIP76) 76-kDa splice variant proteins (RLIP76) for convenience and to avoid confusion. In other reports, Ralbp1 refers to the mouse enzyme (encoded by the Ralbp1 gene), which is structurally and functionally homologous to RLIP76, the human protein encoded by the human RALBP1 gene.
Median lethal dose studies were performed in RLIP76(-/-) and RLIP76(+/+) C57B mice after treatment with a single dose of RLIP76 liposomes 14 h after whole body radiation. The radiosensitivity of the cultured mouse embryonic fibroblasts and the effects of buthionine sulfoximine (BSO), amifostine, c-jun N-terminal kinase (JNK), protein kinase B (Akt), and MAPK/ERK kinase (MEK) were determined by colony-forming assays. Glutathione-linked enzyme activities were measured by spectrophotometric assays, glutathione by dithiobis-2-nitrobenzoic acid (DTNB), lipid hydroperoxides by iodometric titration, and aldehydes and metabolites by thiobarbitauric acid reactive substances and liquid chromatography-mass spectrometry (LCMS).
RLIP76(-/-) mice were significantly more sensitive to radiation than were the wild-type, and RLIP76 liposomes prolonged survival in a dose-dependent manner in both genotypes. The levels of 4-hydroxynonenal and glutathione-conjugate of 4-hydroxynonenal were significantly increased in RLIP76(-/-) tissues compared with RLIP76(+/+). RLIP76(-/-) mouse embryonic fibroblasts were markedly more radiosensitive than RLIP76(+/+) mouse embryonic fibroblasts, despite increased glutathione levels in the former. RLIP76 augmentation had a remarkably greater protective effect compared with amifostine. The magnitude of effects of RLIP76 loss on radiation sensitivity was greater than those caused by perturbations of JNK, MEK, or Akt, and the effects of RLIP76 loss could not be completely compensated for by modulating the levels of these signaling proteins.
The results of our study have shown that RLIP76 plays a central role in radiation resistance.
确定RLIP76在提供辐射和化疗防护中的作用。在本报告中，为方便起见并避免混淆，我们使用RLIP76同时指代小鼠（Ralbp1）和人类（RLIP76）76 kDa的剪接变体蛋白。在其他报告中，Ralbp1指小鼠酶（由Ralbp1基因编码），其在结构和功能上与人类RALBP1基因编码的人类蛋白RLIP76同源。
在用全身辐射14小时后，对RLIP76（-/-）和RLIP76（+/+）C57B小鼠给予单剂量RLIP76脂质体后进行半数致死剂量研究。通过集落形成试验确定培养的小鼠胚胎成纤维细胞的放射敏感性以及丁硫氨酸亚砜胺（BSO）、氨磷汀、c-jun氨基末端激酶（JNK）、蛋白激酶B（Akt）和丝裂原活化蛋白激酶/细胞外信号调节激酶（MEK）的作用。通过分光光度法测定谷胱甘肽相关酶活性，用二硫代双-2-硝基苯甲酸（DTNB）测定谷胱甘肽，用碘量滴定法测定脂质过氧化物，用硫代巴比妥酸反应物质和液相色谱-质谱联用（LCMS）测定醛类和代谢物。
RLIP76（-/-）小鼠对辐射的敏感性明显高于野生型，并且RLIP76脂质体在两种基因型中均以剂量依赖性方式延长了生存期。与RLIP76（+/+）相比，RLIP76（-/-）组织中4-羟基壬烯醛及其谷胱甘肽缀合物的水平显著升高。尽管RLIP76（-/-）小鼠胚胎成纤维细胞中的谷胱甘肽水平升高，但其放射敏感性明显高于RLIP76（+/+）小鼠胚胎成纤维细胞。与氨磷汀相比，RLIP76增强具有显著更大的保护作用。RLIP76缺失对放射敏感性的影响程度大于JNK、MEK或Akt扰动所引起的影响，并且通过调节这些信号蛋白的水平不能完全补偿RLIP76缺失的影响。
我们的研究结果表明，RLIP76在辐射抗性中起核心作用。
