Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany.
Department of Particle Therapy, University Hospital Essen, West German Proton Therapy Centre Essen (WPE), West German Cancer Center (WTZ), German Cancer Consortium (DKTK), 45147 Essen, Germany.
Cells. 2021 Dec 27;11(1):63. doi: 10.3390/cells11010063.
The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G-, S- or G-phase checkpoints, that ATR is only partly involved in the regulation of S- and G-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G-phase cells activate a G-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G checkpoint in cells irradiated either in the G or G phase. We report that ATM is the sole regulator of the G checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G-, G- and S-phase checkpoint regulation that may guide DSB processing decisions.
目前认为，PI3-激酶参与 DNA 损伤后的检查点反应，其中 ATM 是 G、S 或 G 期检查点的关键调节因子，ATR 仅部分参与 S 和 G 期检查点的调节，而 DNA-PKcs 不参与检查点调节。然而，最近对这些激酶在暴露于电离辐射（IR）的细胞中对检查点反应的贡献的进一步分析揭示了 ATM、ATR 和 DNA-PKcs 之间的惊人整合和相互作用，这些整合和相互作用不仅适应于细胞被照射的细胞周期阶段，而且还适应于 DNA 双链断裂（DSB）的负荷，大概是为了优化其处理。具体来说，我们发现 G 期细胞中的低剂量 IR 激活了由遗传上相互作用的 ATM 和 ATR 调节的 G 检查点。因此，抑制任一种激酶几乎完全抑制其激活。在高剂量 IR 下，遗传上相互作用的 ATM/ATR 耦合松弛，产生协同调节。因此，单一激酶抑制部分抑制，只有联合抑制完全抑制 G 检查点激活。有趣的是，DNA-PKcs 与 ATM/ATR 整合在 G 检查点控制中，但以剂量独立的方式发挥作用。引人注目地，S 期照射独立于剂量激活仅依赖于 ATR 的 G 检查点。在这里，ATM 与 DNA-PKcs 结合以调节检查点恢复。在本工作中，我们扩展了这些研究，并研究了这些 PI3-激酶在 G 期或 G 期照射的细胞中 G 检查点激活中的组织和功能。我们报道说，ATM 是暴露于低剂量 IR 后 G 检查点的唯一调节剂。在高剂量 IR 下，ATM 仍然占主导地位，但 ATR 的贡献也变得可检测，并与 DSB 处有限的 ATM/ATR 依赖性末端切除相关。在这些条件下，只有联合抑制 ATM+ATR 才能完全阻断检查点和切除。在这些实验中，DNA-PKcs 和 CHK2 对 G 检查点的调节贡献不明显，并且可能被用于检查点分析的终点和暴露于 DNA-PKcs 抑制剂的细胞正常通过细胞周期的进展中的扰动所掩盖。结果拓宽了我们对整个细胞周期的组织以及 ATM/ATR/DNA-PKcs 模块随着 IR 剂量的增加调节检查点反应的适应的理解。它们强调了 G、G 和 S 期检查点调节之间的显著相似性和明显差异，这可能指导 DSB 处理决策。
