Institute of Medical Radiation Biology, University Duisburg-Essen Medical School, Essen, Germany.
Int J Radiat Oncol Biol Phys. 2012 Oct 1;84(2):e237-43. doi: 10.1016/j.ijrobp.2012.03.060. Epub 2012 May 30.
It has long been known that the proliferation status of a cell is a determinant of radiation response, and the available evidence implicates repair of DNA double-strand breaks (DSBs) in the underlying mechanism. Recent results have shown that a novel, highly error-prone pathway of nonhomologous end joining (NHEJ) operating as backup (B-NHEJ) processes DSBs in irradiated cells when the canonical, DNA-PK (DNA-dependent protein kinase)-dependent pathway of NHEJ (D-NHEJ) is compromised. Notably, B-NHEJ shows marked reduction in efficiency when D-NHEJ-deficient cells cease to grow and enter a plateau phase. This phenomenon is widespread and observed in cells of different species with defects in core components of D-NHEJ, with the notable exception of DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Using new, standardized serum-deprivation protocols, we re-examine the growth requirements of B-NHEJ and test the role of epidermal growth factor receptor (EGFR) signaling in its regulation.
DSB repair was measured by pulsed-field gel electrophoresis in cells maintained under different conditions of growth.
Serum deprivation in D-NHEJ-deficient cells causes a rapid reduction in B-NHEJ similar to that measured in normally growing cells that enter the plateau phase of growth. Upon serum deprivation, reduction in B-NHEJ activity is evident at 4 h and reaches a plateau reflecting maximum inhibition at 12-16 h. The inhibition is reversible, and B-NHEJ quickly recovers to the levels of actively growing cells upon supply of serum to serum-deprived cells. Chemical inhibition of EGFR in proliferating cells inhibits only marginally B-NHEJ and addition of EGFR in serum-deprived cells increases only a marginally B-NHEJ.
The results document a rapid and fully reversible adaptation of B-NHEJ to growth activity and point to factors beyond EGFR in its regulation. They show notable differences in the regulation of error-prone DSB repair pathways between proliferating and non proliferating cells that may present new treatment design opportunities in radiation therapy.
长期以来，人们一直认为细胞的增殖状态是辐射反应的决定因素，现有证据表明，DNA 双链断裂（DSB）的修复是其潜在机制。最近的研究结果表明，在经典的、依赖 DNA 依赖性蛋白激酶（DNA-PK）的非同源末端连接（NHEJ）途径（D-NHEJ）受损时，一种新的、高度易错的非同源末端连接（NHEJ）途径（B-NHEJ）作为备用途径（B-NHEJ）在照射细胞中处理 DSB。值得注意的是，当缺乏 D-NHEJ 的细胞停止生长并进入平台期时，B-NHEJ 的效率明显降低。这种现象很普遍，在不同物种的细胞中都有观察到，这些细胞存在 D-NHEJ 的核心成分缺陷，DNA-PKcs（DNA 依赖性蛋白激酶，催化亚基）是一个显著的例外。利用新的标准化血清剥夺方案，我们重新检查了 B-NHEJ 的生长要求，并测试了表皮生长因子受体（EGFR）信号在其调节中的作用。
通过脉冲场凝胶电泳在不同生长条件下维持的细胞中测量 DSB 修复。
在缺乏 D-NHEJ 的细胞中血清剥夺会导致 B-NHEJ 迅速减少，类似于正常生长的细胞进入生长平台期时测量到的情况。血清剥夺后，4 小时即可观察到 B-NHEJ 活性降低，12-16 小时达到最大抑制的平台期。这种抑制是可逆的，当向血清剥夺的细胞供应血清时，B-NHEJ 会迅速恢复到活跃生长细胞的水平。在增殖细胞中化学抑制 EGFR 仅略微抑制 B-NHEJ，而在血清剥夺的细胞中添加 EGFR 仅略微增加 B-NHEJ。
这些结果记录了 B-NHEJ 对生长活性的快速和完全可逆的适应，并指出了其调节中除 EGFR 以外的因素。它们显示了在增殖细胞和非增殖细胞中易错 DSB 修复途径的调节存在显著差异，这可能为放射治疗提供新的治疗设计机会。
