Sato Kota, Yoshino Hironori, Sato Yoshiaki, Sasaki Fuki, Munakata Nanami, Tsuruga Eichi
Department of Radiation Science, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036-8564, Japan.
Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Biomed Rep. 2025 Aug 26;23(5):169. doi: 10.3892/br.2025.2047. eCollection 2025 Nov.
Cell senescence is a state of stable proliferation arrest characterized by morphological changes and high senescence-associated β-galactosidase (SA-β-gal) activity. Inducing senescence in cancer cells is beneficial for cancer therapy due to proliferation arrest, however, the mechanisms underlying this process remain insufficiently understood. Therefore, the present study investigated the mechanisms of radiation-induced cellular senescence in A549 human lung cancer cells, focusing on the DNA damage response and cell cycle regulation. Cellular senescence was estimated by activity of SA-β-gal, and cell cycle was analyzed by propidium iodide staining using a flow cytometer. Cell cycle synchronization was performed by the double thymine block method. First, the roles of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia mutated and Rad3-related (ATR), which are important factors for DNA damages response, in radiation-induced cellular senescence were investigated. ATM/ATR inhibitors suppressed radiation-induced G2/M phase arrest and decreased the percentage of senescent cells with high SA-β-gal activity, implying that G2/M arrest was associated with radiation-induced senescence. However, an analysis using inhibitors of checkpoint kinase 1 (Chk1) and Chk2, which function downstream of ATR and ATM, respectively, revealed that the Chk2, but not the Chk1, pathway was involved in radiation-induced senescence. To enhance radiation-induced senescence, radiation was combined with olaparib treatment, an inhibitor of DNA single-strand break repair. Olaparib increased the number of radiation-induced senescent cells. Additionally, cell cycle synchronization experiments revealed that irradiation of cells in S or G2/M phase resulted in higher senescent cell counts than irradiation in G1 phase. Taken together, the present results demonstrated that the ATM/Chk2 pathway and the DNA content are involved in the radiation-induced senescence of A549 cells.
细胞衰老状态是一种稳定的增殖停滞状态，其特征为形态学改变以及衰老相关β-半乳糖苷酶（SA-β-gal）活性升高。由于增殖停滞，诱导癌细胞衰老对癌症治疗有益，然而，这一过程的潜在机制仍未得到充分了解。因此，本研究探讨了A549人肺癌细胞中辐射诱导细胞衰老的机制，重点关注DNA损伤反应和细胞周期调控。通过SA-β-gal活性评估细胞衰老，使用流式细胞仪通过碘化丙啶染色分析细胞周期。采用双胸腺嘧啶阻断法进行细胞周期同步化。首先，研究了共济失调毛细血管扩张症突变基因（ATM）和共济失调毛细血管扩张症突变基因及Rad3相关基因（ATR）这两种DNA损伤反应的重要因子在辐射诱导细胞衰老中的作用。ATM/ATR抑制剂抑制了辐射诱导的G2/M期阻滞，并降低了具有高SA-β-gal活性的衰老细胞百分比，这意味着G2/M期阻滞与辐射诱导的衰老相关。然而，分别使用在ATR和ATM下游起作用的检查点激酶1（Chk1）和Chk2抑制剂进行的分析表明，Chk2而非Chk1途径参与了辐射诱导的衰老。为增强辐射诱导的衰老，将辐射与DNA单链断裂修复抑制剂奥拉帕利联合处理。奥拉帕利增加了辐射诱导的衰老细胞数量。此外，细胞周期同步化实验表明，S期或G2/M期细胞受照射比G1期细胞受照射产生的衰老细胞计数更高。综上所述，本研究结果表明，ATM/Chk2途径和DNA含量参与了A549细胞的辐射诱导衰老。
