doi: 10.3967/bes2023.119
Aurora A Kinase Plays a Key Role in Mitosis Skip during Senescence Induced by Ionizing Radiation
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Abstract:
Objective To investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation (IR). Methods Human melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora A inhibitor MLN8237 (MLN) and/or p21 depletion by small interfering RNA (siRNA). Cell cycle distribution was determined using flow cytometry and a fluorescent ubiquitin-based cell cycle indicator (FUCCI) system combined with histone H3 phosphorylation at Ser10 (pS10 H3) detection. Senescence was assessed using senescence-associated-β-galactosidase (SA-β-Gal), Ki67, and γH2AX staining. Protein expression levels were determined using western blotting. Results Tumor cells suffered severe DNA damage and underwent G2 arrest after IR treatment. The damaged cells did not successfully enter M phase nor were they stably blocked at G2 phase but underwent mitotic skipping and entered G1 phase as tetraploid cells, ultimately leading to senescence in G1. During this process, the p53/p21 pathway is hyperactivated. Accompanying p21 accumulation, Aurora A kinase levels declined sharply. MLN treatment confirmed that Aurora A kinase activity is essential for mitosis skipping and senescence induction. Conclusion Persistent p21 activation during IR-induced G2 phase blockade drives Aurora A kinase degradation, leading to senescence via mitotic skipping. -
Key words:
- Ionizing radiation /
- Senescence /
- G2 arrest /
- Tetraploid /
- Mitosis skipping
The authors declare no competing financial interests.
&These authors contributed equally to this work.
注释:1) AUTHOR CONTRIBUTIONS: 2) COMPETING INTERESTS: -
Figure 1. IR induces G2 arrest, mitosis skipping, and senescence in A375 cells. (A) Experimental design indicating main molecular targets and features of biological events on the experimental timeline; AO: acridine orange. (B) A375 cells were exposed to 5 Gy of X-rays, stained with PI, and analyzed by flow cytometry at the indicated time points. (C) The cell cycle distribution of cells in (B). (D) The protein level of mitosis marker pS10 H3 was examined by western blot analysis in A375 control (Ctrl) cells and cells exposed to 5 Gy of X-rays. Protein was harvested at the indicated time points after IR treatment and GAPDH was used as a loading control. (E) The percentage of binucleated cells among A375 cells treated with cytochalasin B only (Ctrl) or cytochalasin B together with 5 Gy of X-rays (5 Gy X-rays) at 24 h or 48 h post-irradiation. (F) A375 cells infected with FUCCI lentiviruses expressing Cdt1-RFP (red) and geminin-GFP (green) were irradiated with 5 Gy of X-rays. After 1 d of irradiation, representative micrographs at the indicated time points were captured (left) and the cell population was counted (right). Scale bar, 100 µm. (G) Representative images of γH2AX foci or Ki67-positive nuclei in A375 control (Ctrl) cells or irradiated cells (5 Gy of X-rays) at the indicated time points post-irradiation (left), and the percentage of γH2AX foci or Ki67-positive cells (right). Scale bar, 10 µm. (H) Representative images of control (Ctrl) cells or cells exposed to 5 Gy of X-rays and stained for SA-β-Gal at 5 d after treatment (left), and the calculated data (right). Scale bar, 100 µm. (I) Flow cytometric analysis of apoptotic cells in A375 control cells (Ctrl) and cells exposed to 5 Gy of X-rays at 48 h post-irradiation (upper panel). Representative images of single cell in different gating regions detected by flow cytometry (lower left panel). Quantification of apoptotic cells (lower right panel). ns, not significant; ***P < 0.001, compared to Ctrl.
Figure 2. IR activates p53-p21 pathway and reduces Aurora A protein expression. (A–B) Western blot analysis (left) and densitometric quantification (right) of p53, p21 (A), Aurora A, and pS10 H3 (B) expression (normalized to GAPDH) in lysates of A375 control (Ctrl) cells or cells treated with 5 Gy of X-rays. Protein was harvested at the indicated time points post-irradiation and GAPDH was used as loading control.
Figure 3. Knockdown of p21 rescues IR-induced Aurora A reduction and senescence induction. (A–B) Western blot analysis of A375 cells transfected with negative control siRNA (NC) or siRNA targeted p21 (sip21). Protein harvested at the indicated time points after X-rays irradiation were subjected to immunoblotting using anti-p53, p21 (A), Aurora A, and pS10 H3 (B) antibodies. (C) A375 cells were transfected with sip21 or directedly transfected with NC siRNA, then these cells were exposed to 5 Gy of X-rays. Representative images of γH2AX foci or pS10 H3 positive nuclei were obtained at the indicated time points post-irradiation (upper panel), and the percentage of γH2AX foci or pS10 H3 positive cells were calculated (lower panel). Scale bar, 10 µm. (D) A375 cells were infected with FUCCI lentiviruses expressing Cdt1-RFP (red) and geminin-GFP (green) and transfected with NC or sip21, then irradiated with 5 Gy of X-rays. Representative micrographs were captured at the indicated time points (upper panel) and the population was calculated (lower panel). Scale bar, 100 µm. (E) Flow cytometric analysis of apoptotic cells in A375 control cells (Ctrl) and cells transfected with sip21 or NC after exposure to 5 Gy of X-rays, at 48 h post-irradiation (left). Quantification of apoptotic cells (right). *P < 0.05, **P < 0.01, and ***P < 0.001, compared to Ctrl.
Figure 4. Aurora A kinase inhibition leads to G2 blockade and senescence in A375 cells. (A) Cell cycle distribution of A375 control (Ctrl) cells and cells treated with Aurora A inhibitor MLN (0.5 µmol/L) for 12 h, 24 h, 48 h, or 72 h. (B) Data analysis of cells in (A). (C) Cell lysates of A375 cells treated with MLN for the indicated times were subjected to immunoblotting using anti-p53, p21, Aurora A, and pS10 H3 antibodies. (D) Representative immunostaining images of anti-γH2AX and Ki67-positive nuclei in control (Ctrl) cells or cells treated with MLN, analyzed at the indicated time points (left), and the percentage of γH2AX foci or Ki67-positive cells (right). Scale bar, 10 µm. (E) The percentage of binucleated cells in A375 cells treated with cytochalasin B (Ctrl) or cytochalasin B combined with MLN. (F) A375 cells were infected with FUCCI lentiviruses expressing Cdt1-RFP (red) and geminin-GFP (green). Cells were treated with MLN for the indicated time periods and representative images from the indicated time points were acquired after MLN treatment (left). The graph (right) shows the population analysis data. Scale bar, 100 µm. (G) Representative images of cellular morphology of A375 cells treated with MLN for the indicated time periods and released from MLN by fresh medium washing for 1 d (5 d + 1 d), or SA-β-Gal staining performed 5 d after treatment with MLN (5d/SA-β-Gal). Scale bar, 100 µm. (H) Flow cytometric analysis of apoptotic cells in A375 control cells (Ctrl) and cells exposed to 5 Gy of X-rays or 0.5 μmol/L of MLN (left). Representative images of single cells from different gating regions detected by flow cytometry (middle). Quantification of apoptotic cells (right). (I–J) Representative images of control (Ctrl) cells, 5 Gy-irradiated, or MLN-treated cells stained for SA-β-Gal at 5 d after treatment, and the calculated data (J). Scale bar, 100 µm. *P < 0.05, **P < 0.01, and ***P < 0.001, compared to Ctrl.
Figure 5. Effect of p21 knockdown on MLN-induced senescence in A375 cells. (A) Cell lysates of A375 cells treated with MLN for the indicated time periods were subjected to immunoblotting using anti-p21 antibodies. GAPDH was used as loading control. (B) Cell lysates of A375 cells transfected with NC or sip21 at 48 h after MLN treatment were subjected to immunoblotting using anti-p21 antibodies. GAPDH was used as loading control. (C) A375 cells were transfected with NC or sip21 and infected with FUCCI lentiviruses expressing Cdt1-RFP (red) and geminin-GFP (green). Cells were treated with MLN for the indicated time periods. The resulting cells were photographed 3 d after treatment. Representative images at the indicated time points are shown (left), and the cell population was analyzed (right). Scale bar, 100 µm. (D) A375 cells were transfected with NC or sip21. Representative immunostaining micrographs of anti-pS10 H3-positive nuclei and γH2AX foci in untreated A375 control (Ctrl) cells or cells treated with MLN for the indicated time periods are shown (left), and the data were measured (right). Scale bar, 10 µm. (E) A375 cells were transfected with NC or sip21. Representative images of NC, 5 Gy-irradiated, and MLN-treated cells stained for SA-β-Gal at 5 d after treatment (left), with the analyzed data shown in (F). Scale bar, 100 µm. (G) Flow cytometric analysis of apoptotic cells in A375 cells transfected with NC or sip21 and treated with 0.5 μmol/L of MLN (left). Quantification of apoptotic cells (right). *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 6. MLN-induced senescence in p21-depleted A375 cells is independent of DNA damage. (A) A375 cells were transfected with sip21 prior to treatment with 5 Gy of X-rays and MLN or only transfected with negative control siRNA (NC). Representative immunostaining images of anti-pS10 H3-positive nuclei and γH2AX foci in untreated cells (NC) or cells treated with MLN combined with X-rays, captured at the indicated time points, are shown (upper panel), and the data were analyzed (lower panel). Scale bar, 10 µm. (B–C) A375 cells were transfected with NC or sip21 prior to treatment with 5 Gy of X-rays and MLN. Cell lysates harvested at the indicated time points were subjected to immunoblotting using the indicated antibodies. (D) A375 cells were transfected with NC or sip21, and then treated with 5 Gy of X-rays, MLN, or 5 Gy of X-rays combined with MLN. Representative images of cells stained for SA-β-Gal at 5 d after treatment, and the calculated data are shown in (E). Scale bar, 100 µm. (F) Schematic of the proposed model describing the fate determination of G2-arrested cells induced by IR. *P < 0.05, **P < 0.01, and ***P < 0.001, compared to Ctrl.
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22403+Supplementary Materials.pdf