PMC

Induction of apoptosis in Chk1- depleted HCT116 cells treated with hydroxyurea or CPT. (A) Western blot analysis of Chk1 in extracts of HCT116 cells treated with control or Chk1 siRNAs for 24 h. (B) Cell cycle distribution of siRNA-treated HCT116 cells exposed to hydroxyurea or 20 nM CPT for 48 h.

Effect of Chk1 silencing on cell cycle distribution of SW480 cells treated with thymidine. (A) Chk1 protein level 24 h after the transfection of indicated siRNAs. β-Actin levels are presented as a loading control. (B) Cell cycle analysis of SW480 cells transfected with control or Chk1 siRNAs and treated with 2 mM TdR for 24 or 40 h.

Effects of TdR or CPT on the induction of apoptosis. (A) HCT 116 cells were treated with the indicated concentrations of thymidine or CPT for 24 h and then analyzed for the binding of Annexin V/PI (percentages of Annexin V+/PI- cells are represented). (B-D) HCT 116 (B), SW480 (C), or HCT 116 + 3 cells (D) were treated with the indicated agents for 1 to 4 d, and the induction of apoptosis was analyzed by Annexin V/PI binding.

Thymidine treatment of Chk1-depleted cells induces apoptosis predominantly in cells in early S phase. HCT 116 cells were transfected with control or Chk1 siRNA and treated or not with 2 mM TdR for 48 h. Cells were then analyzed for caspase-3 activation and DNA content simultaneously. Cells presenting inactive and active forms of caspase-3 are gated and computed (R1 and R2 gates, respectively) in the cytogram panels on the left, and their corresponding cell cycle profiles are depicted in the panels on the right.

Role of Chk1 and p21 in prevention of cell death after replication fork stress. (A) DNA synthesis inhibitors activate Chk1 that protects replication forks and prevents S-phase apoptosis. (B) The lack of Chk1-induced protection favors the induction of apoptosis in the S phase. In this case, p21 activation triggers an alternative protective mechanism by preventing cells from entering S phase. (C) Depletion of both Chk1 and p21 eliminates the protection of G₁ phase cells and results in apoptosis of virtually the entire cell population.

Cells deficient in Chk2 do not undergo apoptosis after thymidine treatment. (A) Appearance of phosho-Chk2 (Thr68) in HCT 116 cells treated with 2 mM TdR for the indicated times. β-Actin levels in the extracts are included as loading controls. (B) Western blot analysis of Chk2 in extracts of HCT116 cells treated with control or Chk2 siRNAs for 24 h. (C) Effect of siRNA transfection on cell cycle distribution of HCT116 cells treated with 2 mM thymidine for 48 h. (D) DLD-1 cells were treated during the indicated periods of time with 2 mM thymidine and analyzed for cell cycle distribution. Cell cycle profiles and percentage of cells in each phase of a representative experiment are shown. (E) DLD-1 cells were treated for 24 or 48 h with 2 mM thymidine or 20 nM CPT and then analyzed for the binding of Annexin V/PI (percentages of Annexin V+/PI- cells are represented).

Chk1 depletion ablates TdR block by committing S-phase cells to apoptosis. (A) Appearance of phosho-Chk1 (ser345) and total Chk1 levels of HCT 116 cells treated with 2 mM thymidine for the indicated times. β-Actin levels in the extracts are included as loading controls. (B) Chk1 levels 24 h after the transfection of indicated siRNAs in HCT116 cells. β-Actin levels are presented as a loading control. (C and D) Effect of siRNA transfection on cell cycle distribution of HCT116 cells treated with 2 mM thymidine for 24 (C) or 48 (D) hours. (E) HCT 116 siRNA-transfected cells were treated with 2 mM thymidine for 48 h and with 0.2 μg/ml nocodazole (Noc) for the last 20 h of this period to trap cycling cells in mitosis. Cell were then harvested, fixed, and analyzed by flow cytometry for the level of phospho-Histone H3 (ser10) and DNA content simultaneously. Separated cell cycle profiles for each condition were also displayed in the panels on right.

Effect of Chk1 silencing on p53-/- and p21-/- cells. (A) Western blot analysis of p53, phospho-p53 (ser15), and p21 in wild-type, p53-/-, and p21-/- HCT116 cells treated with 2 mM thymidine for the indicated times. (B) Chk1 and β-actin protein levels 24 h after transfection of control or Chk1 siRNAs. (C) Cell cycle analysis of siRNA-transfected HCT116 p53-/- or p21-/- cells treated with 2 mM thymidine for 48 h. (D) Induction of apoptosis (as measured by Annexin V binding) in wild-type, p53-/-, and p21-/- HCT116 cells transfected with control or Chk1 siRNAs after 48 h treatment with 2 mM thymidine. Values represent the increase in the percentage of apoptotic cells in thymidine treated cells relative to cells not exposed to thymidine. (E) Survival of wild-type or p21-/- HCT116 cells treated with control or Chk1 siRNAs in increasing concentrations of thymidine in a colony-forming assay. (F) Western blot analysis of p21, phospho-p53 (ser15), chk1, and β-actin in wild-type (left) or p53-/- HCT 116 cells (right) transfected with control or Chk1 siRNAs and treated or not with 2 mM thymidine for 24 h. (G) Chk1-depleted cells treated with thymidine have an increased level of the p21 transcript. p21 mRNA levels were determined by RT-PCR analysis as described in Materials and Methods in HCT 116 cells transfected with control or Chk1 siRNAs and treated with thymidine as described above. p21 expression was quantified and expressed as fold induction relative to the control. β-Actin was used to normalize the amount of loaded RNA.