Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany.
Radiat Res. 2021 May 1;195(5):412-426. doi: 10.1667/RADE-20-00195.1.
Alternative end-joining (alt-EJ) is a DNA end resection-dependent, error-prone pathway utilized by vertebrate cells to repair DNA double-strand breaks (DSBs), but its engagement is linked to chromosomal translocations and genomic instability. Here, we report that when proliferating cells are exposed to ionizing radiation, treatment with nucleoside analogs (NAs) causes strong radiosensitization by increasing engagement of alt-EJ, while at the same time suppressing homologous recombination (HR) in S- and G2phase cells. This NA-mediated pathway shift may reflect a passive compensatory engagement of alt-EJ following HR suppression that is specific for S- and G2-phase cells, and/or the direct activation of alt-EJ throughout the cell cycle. To distinguish between these possibilities, we utilize here a cell culture model that exploits genetic and cell cycle-dependent inactivation of DSB repair pathways, to exclusively study alt-EJ and its modulation by NAs in murine and human cell lines. To this end, we allow LIG4-/--deficient cells to accumulate in G1/G0 phase by transfer to serum-deprived media and obtain cells deficient in c-NHEJ owing to the genetic LIG4 knockout, deficient in HR owing to the absence of S- or G2-phase cells, and compromised in their ability to carry out alt-EJ owing to their accumulation in G0. We find that in these cells irradiation and treatment with the NA, β-arabinofuranosyladenine (araA), and to a lesser degree with other NAs, promptly activates suppressed alt-EJ that now functions at levels approximating those of c-NHEJ in wild-type cells. Results at high dose (20 Gy) generated using pulsed-field gel electrophoresis (PFGE) are corroborated by results at low dose (1 Gy) generated by scoring 53BP1 foci. Strikingly, araA treatment activates a normally undetectable DNA-end-resection at DSBs, which requires ATR activity, but proceeds unimpeded after CtIP knockdown. Treatment with araA increases the formation of chromosomal aberrations and enhances radiation-induced cell killing. The results support direct stimulation of resection by NAs and alt-EJ as a mechanism of their documented radiosensitizing potential. We propose that this stimulation also occurs in repair-proficient cells and that it occurs throughout the cell cycle. It may therefore be harnessed to develop protocols combining NAs with radiation to treat human cancer.
非同源末端连接（alt-EJ）是一种依赖于 DNA 末端切除的易错途径，脊椎动物细胞利用该途径修复 DNA 双链断裂（DSBs），但它的参与与染色体易位和基因组不稳定性有关。在这里，我们报告说，当增殖细胞暴露于电离辐射下时，用核苷类似物（NAs）处理会通过增加 alt-EJ 的参与来强烈增强放射敏感性，同时抑制 S 和 G2 期细胞中的同源重组（HR）。这种 NA 介导的途径转变可能反映了 HR 抑制后被动补偿性参与 alt-EJ，这是 S 和 G2 期细胞特有的，和/或整个细胞周期中直接激活 alt-EJ。为了区分这些可能性，我们在这里利用细胞培养模型，该模型利用遗传和细胞周期依赖性 DSB 修复途径失活，专门研究 alt-EJ 及其在小鼠和人类细胞系中 NAs 的调节。为此，我们通过转移到血清剥夺培养基中使 LIG4-/-缺陷细胞积累在 G1/G0 期，并通过遗传 LIG4 敲除获得缺乏 c-NHEJ 的细胞，由于缺乏 S 期或 G2 期细胞而缺乏 HR，并且由于其在 G0 期的积累而使其进行 alt-EJ 的能力受损。我们发现，在这些细胞中，照射和用 NA（β-阿拉伯呋喃糖腺嘌呤，araA）处理，以及用其他 NA 处理程度较小，会立即激活被抑制的 alt-EJ，现在的功能水平近似于野生型细胞中的 c-NHEJ。使用脉冲场凝胶电泳（PFGE）在高剂量（20 Gy）下产生的结果通过评分 53BP1 焦点在低剂量（1 Gy）下得到证实。引人注目的是，araA 处理会激活通常无法检测到的 DSBs 的 DNA 末端切除，这需要 ATR 活性，但在 CtIP 敲低后仍能无阻地进行。用 araA 处理会增加染色体畸变的形成并增强辐射诱导的细胞杀伤。结果支持 NA 直接刺激切除和 alt-EJ 作为其有记录的放射增敏潜力的机制。我们提出，这种刺激也发生在修复功能正常的细胞中，并且发生在整个细胞周期中。因此，可以利用它来开发将 NAs 与辐射相结合治疗人类癌症的方案。
