Activation of Chk2, ATM, H2AX, and DNA-PK in response to TRAIL treatment. (A) Kinetics of the effects of TRAIL on DDR proteins in HCT116 cells. Cells were treated with 0.1 μg/ml TRAIL for the indicated times, and the indicated proteins were examined by Western blotting. The phosphospecific antibodies used are listed in Materials and Methods. Tubulin was used as a loading control. The asterisk corresponds to an unspecific cross-reactive protein for the phospho-T68-Chk2 antibody (P-Chk2 T68). Positive controls (+): P-p53-S20, HCT116 cells 1 h after 20 Gy; p21, HCT116 cells 20 h after 20 Gy; P-E2F1-S364, HT29 cells treated with etoposide (50 μM, 6 h); P-Chk1-S345, HT29 cells treated with camptothecin (1 μM, 0.5 h). (B) Quantification of apoptosis (by Hoechst staining) and DNA fragmentation (measured by filter elution assay). The pound sign for percent apoptosis at 20 h indicates an underestimation due to loss of signal due to dissolution of nuclei in advanced apoptotic cells. (C) Kinetics of the effects of TRAIL on Chk2, ATM, H2AX, and DNA-PK phosphorylations in HCT116 cells with Mre11 stable complementation (HCT116-Mre11). Cells were treated with 0.1 μg/ml TRAIL for the indicated times. Protein phosphorylations (P-Chk2 T68, P-ATM S1981, γ-H2AX, and P-DNA-PK T2609) were analyzed by Western blotting. Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated. The pound sign for percent apoptosis at 20 h indicates an underestimation due to dissolution of nuclei in advanced apoptotic cells. (D) Phosphorylation of histone H2AX (γ-H2AX) in response to TRAIL in HCT116 cells and in HCT116-Mre11 cells. γ-H2AX was analyzed by Western blotting. Tubulin was used as a loading control. (E) Activation of Chk2 in response to TRAIL in HCT116 cells and in HCT116-Mre11 cells. Chk2 phosphorylated on threonine 68 was analyzed by Western blotting. Tubulin was used as a loading control. (F) Concentration-dependent activation of Chk2, ATM, H2AX, and DNA-PK by TRAIL. HCT116-Mre11 cells were treated with the indicated TRAIL concentrations for 4 h. Protein phosphorylations (P-Chk2 T68, P-ATM S1981, γ-H2AX, and P-DNA-PK T2609) were analyzed by Western blotting. Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated. The asterisk corresponds to an unspecific cross-reactive protein for the phospho-T68-Chk2 antibody (P-Chk2 T68).

Analyses of γ-H2AX responses to TRAIL. (A) γ-H2AX confocal immunofluorescence staining in HCT116 cells treated with TRAIL. Images are representative of cells treated with 0.1 μg/ml TRAIL for 1 or 2 h. γ-H2AX was labeled in green, and nuclei were stained in red with propidium iodide (PI). (B) Single-cell analyses showing typical γ-H2AX patterns. From left to right: untreated cell, irradiated cell, and cells treated with TRAIL. The graph shows the relative distribution of the different γ-H2AX patterns. HCT116 cells were treated with 0.1 μg/ml TRAIL for 1 or 2 h. White columns correspond to peripheral nuclear staining (ring pattern, I), gray columns correspond to panstaining (flooded pattern, II), and black columns correspond to apoptotic bodies fully stained with γ-H2AX (III). The distribution of the three γ-H2AX patterns was significantly different for the 1- and 2-h TRAIL treatments (chi-square test, P < 0.05). (C) γ-H2AX confocal immunofluorescence staining in PrEC cells treated with TRAIL (0.1 μg/ml for 6 h). γ-H2AX was labeled in green, and nuclei were stained in red with propidium iodide. (D) Western blotting for γ-H2AX response to TRAIL in PrEC cells. Cells were treated with 0.1 μg/ml TRAIL for the indicated times. HCT116 cells treated with 0.1 μg/ml TRAIL for 2 h were used as a positive control. The percentage of γ-H2AX-positive cells (determined by immunofluorescence microscopy) is indicated for each time point.

Bax and caspases are required for the activation of Chk2, ATM, H2AX, and DNA-PK in response to TRAIL. (A) Bax requirement for TRAIL-induced DDR response. Bax^+/− or Bax^−/− HCT116 cells were treated with TRAIL as indicated. Protein phosphorylations (P-Chk2 T68, P-ATM S1981, and γ-H2AX) were analyzed by Western blotting. Tubulin was used as a loading control. (B) Representative immunofluorescence experiment showing γ-H2AX staining after TRAIL treatment (0.1 μg/ml, 1 h) in Bax^+/− or Bax^−/− HCT116 cells. γ-H2AX was labeled in green, and nuclei were stained in red with propidium iodide. (C) Caspase requirement for TRAIL-induced DDR response. HCT116 cells were treated with Z-VAD-fmk for 1 h prior to the addition of TRAIL. Protein phosphorylations (P-Chk2 T68, P-ATM S1981, γ-H2AX, and P-DNA-PK T2609) were analyzed by Western blotting. Tubulin was used as a loading control. The asterisk corresponds to an unspecific cross-reactive protein for the P-Chk2-T68 antibody. (D) Representative immunofluorescence experiment showing γ-H2AX and P-Chk2 T68 staining after TRAIL treatment in HCT116-Mre11 cells pretreated with Z-VAD-fmk. The cells were treated with Z-VAD-fmk at 100 μM for 1 h prior to exposure to TRAIL at 0.1 μg/ml for 2 h. γ-H2AX and P-Chk2 were labeled in green, and nuclei were stained in red with propidium iodide.

Cross talk between ATM and DNA-PK in response to TRAIL. (A) Effect of the DNA-PKi on ATM phosphorylation on S1981 and effect of the ATMi on DNA-PK phosphorylation on T2609. HCT116 cells were treated with the ATMi and/or the DNA-PKi for 1 h prior to the addition of TRAIL. Protein phosphorylations (P-ATM S1981, P-DNA-PK T2609, and P-DNA-PK S2056) were analyzed by Western blotting. Tubulin was used as a loading control. (B) Decreased ATM phosphorylation on S1981 in response to TRAIL in cells transfected with siRNA against DNA-PK. (Upper panel) HCT116 cells were treated with TRAIL (0.1 μg/ml, 3 h) 48 h after transfection with siRNA against DNA-PK or a negative control siRNA. P-ATM S1981 was analyzed by Western blotting. Tubulin was used as a loading control. (Lower panel) Western blotting showing the efficiency of DNA-PK downregulation by siRNA. Total DNA-PK was analyzed by Western blotting. Tubulin was used as a loading control. (C) Immunofluorescence experiment showing the colocalization of P-Chk2 T68 with γ-H2AX, P-ATM S1981, and P-DNA-PK T2609 in response to TRAIL. HeLa cells were treated with TRAIL at 0.1 μg/ml for 2 h. γ-H2AX, P-ATM S1981, or P-DNA-PK T2609 was labeled in red, and P-Chk2 T68 was labeled in green. The dashed circles define the nuclei. (D) Tables showing the relationships between the numbers of cells positive for P-Chk2 T68, γ-H2AX, P-ATM S1981, and P-DNA-PK T2609. Double-positive, single-positive, and double-negative cells were scored as shown in panel C. Expected numbers from contingency tables are in parentheses (purple). Results of chi-square tests are shown at the right (in all three cases, P < 0.001).

Further characterization of the functional impact of Chk2 with HCT116 derivatives. (A) Effect of Chk2 on sub-G₁. HCT116 cells or Chk2^−/− HCT116 cells were treated with TRAIL at 0.1 μg/ml for 3, 6, or 20 h. The y axis represents the percentage of cells with sub-G₁ DNA. ***, P < 0.001, unpaired t test; n = 3. (B) The effect of Chk2 on caspase 3 and 8 activities is independent of p53. p53^−/− and p53^+/+ HCT116 cells transfected with siRNA against Chk2 or a negative control siRNA were treated with TRAIL at 0.1 μg/ml for 2 h before measurement of caspase 3 or 8 activity. The y axis represents the reduction of caspase activity by Chk2 siRNA normalized to the control siRNA (percent). p53^+/+ HCT116 cells are in gray, and p53^−/− HCT116 cells are in white.

Effect of TRAIL on DDR proteins is observed at all phases of the cell cycle and is not restricted to HCT116 cells. (A) Relationships between γ-H2AX cellular content and cell cycle. Untreated (upper left panel) and TRAIL-treated (0.1 μg/ml, 2 h) HCT116 cells (upper right panel) were analyzed by FACScan flow cytometry. The x axis indicates the DNA content (as determined by propidium iodide staining), and the y axis indicates γ-H2AX content (cells positive for γ-H2AX are in red, and the negative cells are in gray). Results are quantitated in the lower panel. (B) Effects of TRAIL on Chk2, ATM, H2AX, and DNA-PK phosphorylations in HeLa cells. Protein phosphorylations were analyzed by Western blotting. The asterisk corresponds to an unspecific cross-reactive protein for the phospho-T68-Chk2 antibody (P-Chk2 T68). Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated at the bottom. (C) Activation of Chk2 in response to TRAIL in Jurkat cells. Cells were treated with 0.1 μg/ml TRAIL for the indicated times, and the phosphorylation of Chk2 on T68 was analyzed by Western blotting. Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated.

Peripheral distribution of nuclear γ-H2AX in response to TRAIL (0.1 μg/ml for 1 h). (A) γ-H2AX and heterochromatin confocal immunofluorescence staining in HCT116 cells treated with TRAIL. Heterochromatin was labeled with histone H3 trimethyl K9 antibody (green), and γ-H2AX was labeled in red. (B) Relative distribution of γ-H2AX and heterochromatin in a single cell positive for γ-H2AX. Top: confocal microscopy image. Bottom: intensity tracing. (C) γ-H2AX and lamin B1 confocal immunofluorescence staining in HCT116 cells treated with TRAIL. (D) Representative 3D image of a γ-H2AX-positive cell double stained with γ-H2AX (red) and lamin B1 (green). Note that the green signal intensity has been reduced to visualize the γ-H2AX signal. (E) Relative distribution of γ-H2AX and lamin B1 in a single cell positive for γ-H2AX. Top, confocal microscopy image; bottom, intensity tracing. (F) Differential staining patterns for 53BP1 (green) and γ-H2AX (red) in response to TRAIL and IR (3 Gy) in HCT116 cells. AU, arbitrary units.

H2AX is phosphorylated by DNA-PK and Chk2 is phosphorylated by both DNA-PK and ATM in response to TRAIL. (A) Effect of the DNA-PKi NU7441 on the phosphorylations of Chk2 and H2AX after TRAIL treatment. HCT116 cells were treated with the DNA-PKi (1 h) prior to the addition of TRAIL. Protein phosphorylations (P-Chk2 T68 and γ-H2AX) were analyzed by Western blotting. Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated. (B) Effect of the ATMi KU-55933 on the phosphorylations of Chk2 and H2AX after TRAIL treatment. HCT116 cells were treated with the ATMi (1 h) prior to the addition of TRAIL. Protein phosphorylations (P-Chk2 T68 and γ-H2AX) were analyzed by Western blotting. Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated. (C) Inhibition of γ-H2AX formation by the DNA-PKi (10 μM, 1 h) in TRAIL-treated HCT116 cells (0.1 μg/ml, 3 h). Cells were analyzed by FACScan flow cytometry. The x axis indicates γ-H2AX content (γ-H2AX-positive cells are circled in gray), and the y axis indicates the number of cells. The percentage of γ-H2AX-positive cells was significantly reduced by pretreatment with the DNA-PKi (n = 3; unpaired t test, P < 0.001). (D) Defective induction of γ-H2AX after TRAIL treatment in Fus9 (without DNA-PK) compared to Fus1 (with DNA-PK) M059J cells. Cells were treated as indicated. Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated. (E) Decreased H2AX phosphorylation in response to TRAIL in cells transfected with siRNA against DNA-PK. (Upper panel) HCT116 cells were treated with TRAIL (0.1 μg/ml, 3 h) 48 h after transfection with siRNA against DNA-PK or a negative control siRNA. γ-H2AX was analyzed by Western blotting. Tubulin was used as a loading control. (Lower panel) Western blotting showing the efficiency of DNA-PK downregulation by siRNA. Total DNA-PK was analyzed by Western blotting. Tubulin was used as a loading control. (F) Effects of both ATMi and DNA-PKi on the phosphorylations of Chk2 and H2AX after TRAIL treatment. HCT116 cells were treated with the ATMi or/and the DNA-PKi (10 μM, 1 h) prior to the addition of TRAIL. P-Chk2 T68, total Chk2, γ-H2AX, and total H2AX were analyzed by Western blotting. Tubulin was used as a loading control. The numerical values, obtained by densitometry analysis (ImageQuant software), represent the ratio of P-Chk2 T68 or γ-H2AX to tubulin.

Functional impact of Chk2 on TRAIL-induced apoptosis. (A) Western blotting of a representative experiment showing the efficiency of Chk2 downregulation by siRNA. HCT116 cells were transfected with siRNA against Chk2 or negative control siRNA. Total Chk2 was analyzed by Western blotting. Tubulin was used as a loading control. (B) Effect of Chk2 on cell detachment. HCT116 cells transfected with siRNA against Chk2 or a negative control siRNA were treated with TRAIL (0.1 μg/ml) for the indicated times. (Left panels) Representative microscopic pictures under visible light. Values under the panels correspond to the fractions of attached cells (± standard deviation). (Right panel) The y axis represents the percentage of attached cells. The cells transfected with negative control siRNA are in black, and those transfected with siRNA against Chk2 are in gray. ***, P < 0.001, unpaired t test; n = 3. (C) Effect of Chk2 on cell survival. HCT116 cells transfected with siRNA against Chk2 or negative control siRNA were treated with TRAIL for 1 h (0.1 μg/ml). Ten thousands cells were then seeded, and clonogenic assays were performed. Two independent experiments are plotted; the black bars correspond to negative control siRNA, and the gray bars correspond to siRNA against Chk2. The y axis indicates the number of colonies. ***, P < 0.001, chi-square test. (D, upper panels) Representative 3D single-cell analyses showing the different γ-H2AX patterns (Fig. 3B). (Lower panel) HCT116 cells transfected with siRNA against Chk2 or a negative control siRNA were treated with 0.1 μg/ml TRAIL for 1 or 2 h. White columns correspond to peripheral nuclear staining (ring pattern, I), gray columns correspond to panstaining (flooded pattern, II), and black columns correspond to apoptotic bodies fully stained with γ-H2AX (III). The distribution of the three γ-H2AX patterns was significantly different for the Chk2 siRNA- and control siRNA-treated cells (chi-square tests, P < 0.05). (E) Effect of Chk2 on caspase activities. HCT116 cells transfected with siRNA against Chk2 or a negative control siRNA were treated with TRAIL at 0.1 μg/ml for 1 to 6 h, and caspase 2, 3, 8, and 9 activities were measured (AU, arbitrary units). The cells transfected with negative control siRNA are in black, and those transfected with siRNA against Chk2 are in gray.

Schematic representation of the caspase- and Bax-dependent activation of DNA-PK, ATM, H2AX, and Chk2 in response to TRAIL. Open arrows correspond to activation.