Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G₂ checkpoint arrest

Abstract

Besides the well-understood DNA damage response via establishment of G(2) checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re-entry. The aim of this study was to investigate the role of Chk1 in the recovery from G(2) checkpoint arrest in HCT116 (human colorectal cancer) wt, p53(-/-) and p21(-/-) cell lines following H(2) O(2) treatment. Firstly, DNA damage caused G(2) checkpoint activation via Chk1. Secondly, overriding G(2) checkpoint led to (i) mitotic slippage, cell cycle re-entry in G(1) and subsequent G(1) arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21(WAF1) causing mitotic catastrophe. We revealed subtle differences in the initial Chk1-involved G(2) arrest with respect to p53/p21(WAF1) : absence of either protein led to late G(2) arrest instead of the classic G(2) arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G(2) arrest correlated with downstream senescence, but late G(2) arrest led to mitotic catastrophe, although both cell cycle re-entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long-term DNA damage responses causing cell cycle re-entry. We propose that recovery from oxidative DNA damage-induced G(2) arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21(WAF1) . The general relevance of Chk1 as an important determinant of recovery from G(2) checkpoint arrest was verified in HT29 colorectal cancer cells.

Fig 1

H₂O₂ treatment induces establishment and override of Chk1-involved G₂ checkpoint arrest in HCT116 wt and p53^–/– cells. (A) After H₂O₂ treatment (30 mM, 3 min.), G₂ checkpoint arrest is established via Chk1 involvement reflected by the accumulation of active p-Chk1^Ser317, inactive p-cdc25C^Ser216 and p-cdc2^Thr14. Whole cell lysates were processed for Western blot analysis and probed with indicated antibodies. β-actin served as loading control. Fold expression changes are given below the blots. (B, C) G₂ checkpoint override causes Chk1-dependent long-term DNA damage responses in HCT116 wt (B, G₁ arrest) and p53^–/– cells (C, apoptosis). FACS analyses were performed at 24, 48 and 72 hrs after treatment. Twenty-four hours after transfection with Chk1 siRNA, cells were treated with 30 mM H₂O₂ for 3 min. and grown for 24, 48 or 72 hrs. A control siRNA was used as a negative control for targeted siRNA transfection. Differentially gated cell populations were counted; their percentage in the total cell populations was calculated and presented in the diagram. Dashed lines contribute to cell cycle analysis without H₂O₂ treatment to mark H₂O₂-induced G₂ arrest as well as reduced G₂ arrest following Chk1 siRNA transfection. Data are means of three independent experiments. (D) Whole cell lysates were subjected to caspase 3 Western blot analysis. β-actin served as loading control. Fold expression changes are given below the blots. (E) Analysis of cyclin B1 localization 24 hrs after treatment revealed its dominant cytoplasmic localization in wt cells (G₂ arrest) and its dominant nuclear localization in p53^–/– cells (late G₂ arrest). Cells were fixed, subsequently stained with anti-cyclin B1, and counterstained with DAPI.

Fig 2

H₂O₂ treatment alters expression of cell cycle regulatory proteins in HCT116 wt and p53^–/– cells. Cells were treated with 30 mM H₂O₂ for 3 min. and further grown for 1 hr up to 72 hrs. Whole cell lysates were analysed by Western blot and probed with indicated antibodies. β-actin was used to control protein loading. Fold expression changes are given below the blots.

Fig 3

Role of p53 in Chk1-dependent long-term DNA damage responses. (A) Comet assay analysis of nuclear DNA in HCT116 wt and p53^–/– cells revealed DNA strand breaks in cells exposed to 30 mM H₂O₂ for 3 min. and further grown for 1 hr and 24 hrs. (B) Formation of γ-H2AX foci in H₂O₂-treated wt and p53^–/– cells. Cells were fixed and subsequently stained with anti-γ-H2AX and counterstained with DAPI. (C) Accumulation of γ-H2AX in H₂O₂-treated wt and p53^–/– cells and PARP cleavage in p53^–/– cells. Whole cell lysates were subjected to Western blot analysis. β-actin was used to control protein loading. Fold expression changes are given below the blots. (D) Effect of H₂O₂ treatment on protein modification in HCT116 wt and p53^–/– cells. Cells were treated with 30 mM H₂O₂ for 3 min. Proteins having undergone oxidative modifications were detected after 1, 6 and 24 hrs after H₂O₂. Data are means ± S. D. of three independent experiments. *, P < 0.05 versus untreated cells.

Fig 4

Analysis of establishment and override of Chk1-dependent G₂ checkpoint arrest in HCT116 p21^–/– cells. (A) H₂O₂ treatment (30 mM, 3 min.) induces establishment of G₂ checkpoint arrest in p21^–/– cells via the Chk1 pathway. H₂O₂ also induces apoptosis as indicated by the expression level of cleaved caspase 3. Whole cell lysates were subjected to Western blot analysis. β-actin was used to control protein loading. Fold expression changes are given below the blots. (B) G₂ checkpoint override causes increased Chk1-dependent apoptosis as indicated by Pre-G₁ cell population. FACS analyses were performed at 24, 48 and 72 hrs after treatment. Twenty-four hours after transfection with Chk1 siRNA, cells were treated with 30 mM H₂O₂ for 3 min. and grown for 24, 48 or 72 hrs. A control siRNA was used as a negative control for targeted siRNA transfection. Differentially gated cell populations were counted; their percentage in the total cell populations was calculated and presented in the diagram. The dashed line contributes to cell cycle analysis without H₂O₂ treatment to mark H₂O₂-induced G₂ arrest as well as abrogation of G₂ arrest following Chk1 siRNA transfection. Data are means of three independent experiments. (C) H₂O₂ treatment alters expression of cell cycle regulatory proteins in p21^–/– cells. Whole cell lysates were subjected to Western blot analysis. β-actin was used to control protein loading. Fold expression changes are given below the blots. (D) Analysis of cyclin B1 localization 24 hrs after H₂O₂ revealed its dominant nuclear localization in p21^–/– cells (late G₂ arrest). Cells were fixed and subsequently stained with anti-cyclin B1 and counterstained with DAPI.

Fig 5

Chk1 directs downstream senescence and mitotic catastrophe in HCT116 wt and p53^–/– or p21^–/– cells after H₂O₂ treatment. (A) HCT116 wt cells became senescent after they were treated with 30 mM H₂O₂ for 3 min. and grown for 72 hrs. Cells were fixed and subsequently stained for β-galactosidase activity. Cells grew larger, assumed a flattened shape and expressed senescence-associated β-galactosidase (blue areas). In contrast, p53^–/– and p21^–/– cells went into mitotic catastrophe 72 hrs after treatment. Cells were fixed and stained with DAPI. Multinucleation is marked. (B)–(D) Twenty-four hours after transfection with Chk1 siRNA, cells were treated with H₂O₂ and further analysed 1 and 72 hrs after H₂O₂ treatment. (B) Chk1 navigates HCT116 wt cells to senescence. Down-regulation of p-Chk1^Ser317 in wt cells causes time-delayed down-regulation of senescence-associated G₁ arrest markers p-p53^Ser15, p21^WAF1 and cyclin D1 after 72 hrs. (C) Chk1 navigates HCT116 p53^–/– cells to mitotic catastrophe. Down-regulation of p-Chk1^Ser317 in p53^–/– cells causes down-regulation of mitotic markers p-H3^Ser10, cdc2 and cyclin B1 after 72 hrs. (D) Chk1 navigates HCT116 p21^–/– cells to mitotic catastrophe. Down-regulation of p-Chk1^Ser317 in p21^–/– cells causes up-regulation of mitotic markers p-H3^Ser10, cdc2 and cyclin B1 after 72 hrs. The transfection medium alone (TR) and a control siRNA were used as negative controls for targeted siRNA transfection. Whole cell lysates were subjected to Western blot analysis. Fold expression changes are given below the blots. β-actin was immunoblotted to control protein loading. (E) Chk1 knockdown affects cell morphology of H₂O₂-treated HCT116 cells. Twenty-four hours after transfection with Chk1 siRNA, cells were treated with 30 mM H₂O₂ for 3 min. and grown for 72 hrs. Cells were fixed and subsequently stained for β-galactosidase activity (blue areas: wt cells) or with DAPI (multinucleation is marked: p53^–/–, p21^–/– cells). A control siRNA was used as a negative control for targeted siRNA transfection.

Fig 6

Chk1 regulates long-term DNA damage response in HT29 cells. (A) H₂O₂ treatment causes activation of the Chk1 pathway, apoptosis induction and altered expression of cell cycle regulatory proteins in HT29 cells. Whole cell lysates were processed for Western blot analysis and probed with indicated antibodies. β-actin served as loading control. Fold expression changes are given below the blots. (B) G₂ checkpoint override causes Chk1-dependent apoptosis induction in HT29 cells. FACS analyses were performed at 24, 48, 72 hrs, and 6 days after treatment. Twenty-four hours after transfection with Chk1 siRNA, cells were treated with 30 mM H₂O₂ for 3 min. and grown until 6 days. A control siRNA was used as a negative control for targeted siRNA transfection. Differentially gated cell populations were counted; their percentage in the total cell populations was calculated and presented in the diagram. Dashed lines contribute to cell cycle analysis without H₂O₂ treatment to mark H₂O₂-induced G₂ arrest as well as reduced G₂ arrest following Chk1 siRNA transfection. Data are means of three independent experiments. (C) Chk1 knockdown affects cell morphology of H₂O₂-treated HT29 cells. Twenty-four hours after transfection with Chk1 siRNA, cells were treated with 30 mM H₂O₂ for 3 min. and grown for 72 hrs. Cells were fixed and subsequently stained with DAPI. Multinucleation is marked. A control siRNA was used as a negative control for targeted siRNA transfection. (D) Chk1 navigates HT29 cells to mitotic catastrophe. Down-regulation of p-Chk1^Ser317 in HT29 cells causes up-regulation of mitotic markers p-H3^Ser10, cdc2 and cyclin B1 after 72 hrs. The transfection medium alone (TR) and a control siRNA were used as negative controls for targeted siRNA transfection. Whole cell lysates were subjected to Western blot analysis. Fold expression changes are given below the blots. β-actin was immunoblotted to control protein loading.

Fig 7

Proposed model of how Chk1 regulates oxidative DNA damage in HCT116 colorectal cancer cells. (A) H₂O₂-induced DNA damage activates Chk1 in wt cells, which prevents progression into mitosis via G₂ arrest. Cells with non-repairable DNA damage go into apoptosis, whereas cells with repairable DNA damage slip through mitosis and arrest in G₁ associated with senescence. (B) H₂O₂-induced DNA damage activates Chk1 in p53^–/–/ p21^–/– cells, preventing progression into mitosis via late G₂ arrest. Because of a lack or low levels of p53/p21^WAF1, respectively, cells undergo premature mitosis with their DNA largely unrepaired, which drives these cells into mitotic catastrophe. Importantly, Chk1 is required for recovery from G₂ checkpoint arrest, leading to long-term senescence (A) and mitotic catastrophe (B).