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Impact of the ATM/Chk2 pathway and cell cycle phase on radiation‑induced senescence in A549 human lung cancer cells

  • Authors:
    • Kota Sato
    • Hironori Yoshino
    • Yoshiaki Sato
    • Fuki Sasaki
    • Nanami Munakata
    • Eichi Tsuruga
  • View Affiliations / Copyright

    Affiliations: Department of Radiation Science, Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori 036‑8564, Japan
    Copyright: © Sato et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 169
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    Published online on: August 26, 2025
       https://doi.org/10.3892/br.2025.2047
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Abstract

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.
View Figures

Figure 1

Effect of caffeine on SA-β-gal
activity and cell cycle arrest in irradiated A549 cells. A549 cells
treated with caffeine were irradiated with X-rays. After 48 h,
SA-β-gal activity was analyzed. (A) Representative histogram of
SA-β-gal. (B) Proportion of cells with high SA-β-gal activity. (C)
Representative cell cycle histogram. **P<0.01.
SA-β-gal, senescence-associated β-galactosidase; CTRL, control.

Figure 2

Effect of Chk1 or Chk2 inhibition on
senescence and cell cycle arrest in irradiated A549 cells. (A) A549
cells treated with Chk1 or Chk2 inhibitor were irradiated with
X-rays. After 8 h, cell cycle was analyzed. After 48 h, the cells
were harvested to analyze SA-β-gal activity. (B) Representative
SA-β-gal histogram. (C) Proportion of cells with high SA-β-gal
activity. (D) Cells were harvested for western blotting. (E)
Representative immunoblot of Chk2 of A549 cells transfected with
siRNA targeting Chk2. (F) Chk2-knockdown A549 cells were irradiated
with X-rays. After 48 h, SA-β-gal activity was analyzed. (G) A549
cells treated with a Chk2 inhibitor and (H) Chk2-knockdown cells
were irradiated with X-rays. After 48 h, SA-β-gal activity and FS
were analyzed. *P<0.05, **P<0.01.
SA-β-gal, senescence-associated β-galactosidase; Chk, checkpoint
kinase; CTRL, control; si, small interfering; p-, phosphorylated;
IR, ionizing radiation.

Figure 3

Effect of olaparib on SA-β-gal
activity in A549 cells. (A) A549 cells treated with olaparib were
irradiated with X-rays. After 0.5 and 3 h, γH2AX and H2AX
expression were analyzed. After 4 days, SA-β-gal activity, FS and
protein expression were analyzed. (B) Representative SA-β-gal
histogram. (C) Proportion of cells with high SA-β-gal activity. (D)
Representative immunoblots. Arrow represents non-specific band. (E)
After 8 and 24 h, cell cycle was analyzed. (F) A549 cells treated
with olaparib were irradiated with X-rays. After 4 days, SA-β-gal
activity and FS were analyzed. *P<0.05,
**P<0.01. SA-β-gal, senescence-associated
β-galactosidase; H2AX, H2A histone family member X; Chk, checkpoint
kinase; p-, phosphorylated; IR, ionizing radiation; FS, forward
scatter.

Figure 4

Effect of nutlin-3α on SA-β-gal
activity in irradiated A549 cells. (A) Representative immunoblot of
p53 in A549 cells transfected with siRNA targeting p53. (B)
p53-knockdown A549 cells were irradiated with X-rays. After 48 h,
SA-β-gal activity was analyzed. (C) Pifithrin-α treated cells were
irradiated with X-rays. After 48 h, SA-β-gal activity was analyzed.
(D) A549 cells treated with nutlin-3α were irradiated with X-rays.
After 1 day, the cells were harvested for western blotting. (E)
After 4 days, SA-β-gal activity was analyzed. (F) Proportion of
cells with high SA-β-gal activity. (G) A549 cells treated with
nutlin-3α were irradiated with X-rays. After 8 h, the cell cycle
distribution was analyzed. (H) A549 cells treated with nutlin-3α or
olaparib, in the absence or presence of a Chk1 inhibitor, were
irradiated with X-rays. After 24 h, the cell cycle distribution was
analyzed. P<0.05, **P<0.01. SA-β-gal,
senescence-associated β-galactosidase; Chk, checkpoint kinase; si,
small interfering; CTRL, control; p-, phosphorylated; IR, ionizing
radiation.

Figure 5

Effect of the cell cycle phase at the
time of irradiation on SA-β-gal activity in A549 cells. (A)
Following synchronization of the cell cycle by the double thymidine
block method, cell cycle analysis was performed at 0, 3, 4, 6 and 8
h. (B) Cells were irradiated with X-rays at 0 (G1 phase-rich), 3 (S
phase-rich) and 6 h (G2/M phase-rich) following cell cycle
synchronization. After 48 h, SA-β-gal activity was analyzed.
**P<0.01 vs. 0 Gy, ##P<0.01. SA-β-gal,
senescence-associated β-galactosidase.
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Copy and paste a formatted citation
Spandidos Publications style
Sato K, Yoshino H, Sato Y, Sasaki F, Munakata N and Tsuruga E: Impact of the ATM/Chk2 pathway and cell cycle phase on radiation‑induced senescence in A549 human lung cancer cells. Biomed Rep 23: 169, 2025.
APA
Sato, K., Yoshino, H., Sato, Y., Sasaki, F., Munakata, N., & Tsuruga, E. (2025). Impact of the ATM/Chk2 pathway and cell cycle phase on radiation‑induced senescence in A549 human lung cancer cells. Biomedical Reports, 23, 169. https://doi.org/10.3892/br.2025.2047
MLA
Sato, K., Yoshino, H., Sato, Y., Sasaki, F., Munakata, N., Tsuruga, E."Impact of the ATM/Chk2 pathway and cell cycle phase on radiation‑induced senescence in A549 human lung cancer cells". Biomedical Reports 23.5 (2025): 169.
Chicago
Sato, K., Yoshino, H., Sato, Y., Sasaki, F., Munakata, N., Tsuruga, E."Impact of the ATM/Chk2 pathway and cell cycle phase on radiation‑induced senescence in A549 human lung cancer cells". Biomedical Reports 23, no. 5 (2025): 169. https://doi.org/10.3892/br.2025.2047
Copy and paste a formatted citation
x
Spandidos Publications style
Sato K, Yoshino H, Sato Y, Sasaki F, Munakata N and Tsuruga E: Impact of the ATM/Chk2 pathway and cell cycle phase on radiation‑induced senescence in A549 human lung cancer cells. Biomed Rep 23: 169, 2025.
APA
Sato, K., Yoshino, H., Sato, Y., Sasaki, F., Munakata, N., & Tsuruga, E. (2025). Impact of the ATM/Chk2 pathway and cell cycle phase on radiation‑induced senescence in A549 human lung cancer cells. Biomedical Reports, 23, 169. https://doi.org/10.3892/br.2025.2047
MLA
Sato, K., Yoshino, H., Sato, Y., Sasaki, F., Munakata, N., Tsuruga, E."Impact of the ATM/Chk2 pathway and cell cycle phase on radiation‑induced senescence in A549 human lung cancer cells". Biomedical Reports 23.5 (2025): 169.
Chicago
Sato, K., Yoshino, H., Sato, Y., Sasaki, F., Munakata, N., Tsuruga, E."Impact of the ATM/Chk2 pathway and cell cycle phase on radiation‑induced senescence in A549 human lung cancer cells". Biomedical Reports 23, no. 5 (2025): 169. https://doi.org/10.3892/br.2025.2047
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