MNNG exposure results in G₂ arrest; activation of ATM, Chk1, and Chk2; and inactivation of Cdc2. (A) NHFs were either mock treated (UT) or treated with 25 μM MNNG, harvested at the indicated time point, fixed, stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells with a 4N DNA content is indicated. (B) Logarithmically growing NHFs were either mock treated (top) or treated with MNNG (bottom). Forty-eight hours after MNNG exposure, demecolcine was added to the media and after an additional 24-h incubation the cells were harvested, stained, and analyzed for total DNA content (y-axis) and phosphohistone H3 (x-axis). The mitotic cell population is circled and percentage of the total cell population is indicated. (C) Extracts were formed from mock-treated (lane 1) and MNNG-treated (lane 2) NHFs 48 h after drug exposure and immunoblotted with phospho-Tyr15 specific anti-Cdc2 (top) and anti-total Cdc2 (bottom) to ensure equal protein abundance. (D) Extracts from untreated (lane 1) and MNNG-treated (lane 2) NHFs were subjected to immunoblotting with phospho-Ser317-specific anti-Chk1 (top) and anti-total Chk1 (bottom). (E) Extracts from untreated (lane 1) and MNNG-treated (lane 2) NHFs were subjected to immunoblotting with phospho-Thr68-specific anti-Chk2 (top) and anti-total Chk2 (bottom). (F) Extracts from untreated (lane 1) and MNNG-treated (lane 2) NHFs were subjected to immunoblotting with phospho-Ser1981–specific anti-ATM (top) and anti-total ATM (bottom).

MNNG-induced G₂ arrest occurs through ATM- and MMR-dependent and -independent mechanisms activated in a dose-dependent manner. (A) MLH1-deficient HCT116+ch2 (open bars) and matched MMR-proficient HCT116+ch3 (filled bars) cells were treated with the indicated dose of MNNG, harvested 48 h after drug, fixed, stained with propidium iodide, and analyzed for cell cycle arrest by flow cytometry. Graphed is the percentage of cells within the analyzed population containing a 4N DNA content (G₂/M fraction). (B) Isogenic ATM-deficient (EBS-7; open bars) and ATM-proficient (YZ-5; filled bars) cells were treated with indicated doses of MNNG and analyzed for cell cycle arrest as outlined in A. In each case, graphed is the average of five to eight independent experiments; error bar, SD.

ATM and MMR are required for MNNG-induced G₂ arrest. (A) EBS-7 (top) and YZ-5 (bottom) cells were treated with 5 μM MNNG, and cells were harvested 48 h later and along with cultures of untreated (UT) logarithmically growing cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. In addition, YZ-5 cells were treated with 5 mM caffeine (+C) before, during, and after MNNG exposure. The percentage of cells containing a 4N DNA content is noted. (B) MMR-deficient HCT116+ch2 (top) and isogenic MMR-proficient HCT116+ch3 (bottom) were treated with 5 μM MNNG with or without caffeine (+C) pretreatment, harvested 48 h after exposure, and analyzed by flow cytometry. (C) MMR-deficient HEC59 (top) and isogenic MMR-proficient HEC59+ch2 (bottom) were treated with 25 μM MNNG with or without caffeine (+C) pretreatment, harvested 48 h after exposure, and analyzed by flow cytometry. (D) EBS-7, YZ-5, HCT116+ch2, HCT116+ch3, HEC59, and HEC59+ch2 cells were either mock treated (open bars) or treated (filled bars) with MNNG and 48 h after drug, 0.4 μg/ml demecolcine was added to the medium. After a 24-h incubation, cells were fixed and stained for DNA and phosphohistone H3. Percentage of the total population of cells that progressed into mitosis during this time interval is graphed. The mean of three independent experiments is shown; error bars, 1 SD. Asterisk (^*) denotes p ≤ 0.001 (Student's t-test, two-tailed). (E) EBS-7 (lanes 1 and 2), YZ-5 (lanes 3 and 4), HCT116+ch2 (lanes 5 and 6), HCT116+ch3 (lanes 7 and 8), HEC59 (lanes 9 and 10), and HEC59+ch2 (lanes 11 and 12) cells were either mock treated (-) or treated with MNNG (+) and 48 h after drug exposure, cells were harvested, and lysates were formed and subjected to immunoblot analysis with antibodies against phospho-Tyr15 Cdc2 (top) or total Cdc2 (bottom).

Requirement for MMR and ATM in MNNG-induced activation of Chk1 and Chk2. (A) EBS-7 (lanes 1 and 2), YZ-5 (lanes 3 and 4), HCT116+ch2 (lanes 5 and 6), HCT116+ch3 (lanes 7 and 8), HEC59 (lanes 9 and 10), and HEC59+ch2 (lanes 11 and 12) were either mock treated (-) or treated with MNNG (+) (5 μM for EBS7/YZ-5, HCT116+ch2/HCT116+ch3; 25 μM for HEC59/HEC59+ch2). Forty-eight hours after drug, lysates were formed and subjected to immunoblot analysis with antibodies against phospho-Thr68 Chk2 (top) or total Chk2 (bottom). (B) Lysates outlined above were immunoblotted with anti-phospho Ser317 Chk1 (top) or anti-total Chk1 (bottom).

UCN-01 ablates MNNG-induced G₂ arrest. (A) HCT116+ch3 cells were pretreated with 500 nM UCN-01 for 45 min before 5 μM MNNG exposure, and UCN-01 was maintained in the culture medium during and after MNNG treatment. Mock treated (UT), MNNG-only treated, and MNNG/UCN-01–treated cells were harvested 48 h after MNNG exposure, fixed, stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells containing a 4N DNA content is indicated. (B) YZ-5 cells were treated and analyzed as outlined in A. Percentage of cell population containing a 4N DNA content is given.

RNAi-induced depletion of Chk1 or Chk2 results in attenuation of the G₂ arrest activated by MNNG. (A) HCT116+ch3 cells were either mock transfected (no siRNA included in transfection, lane 1) or transfected with luciferase-specific siRNA (lane 2) or Chk1-specific siRNA (lane 3), and after transfection, cell extracts were immunoblotted with anti-total Chk1 (top), anti-total Chk2 (middle), or anti-tubulin (bottom) to confirm equivalent loading. (B) HCT116+ch3 cells were either mock-transfected or transfected with Chk1 or luciferase-specific siRNA, treated with 5 μM MNNG (+) or were mock treated (-), harvested 48 h later, and analyzed by flow cytometry. The percentage of cells containing a 4N DNA content is indicated. (C) HCT116+ch3 cells were either mock transfected (lane 1) or transfected with luciferase-specific siRNA (lane 2) or Chk2-specific siRNA (lane 3). After transfection, cell extracts were immunoblotted with anti-total Chk2 (top), anti-total Chk1 (middle), or anti-tubulin (bottom). (D) HCT116+ch3 cells were either mock transfected or transfected with Chk1 or luciferase-specific siRNA, exposed to 5 μM MNNG (+) or mock treated (-), harvested 48 h later, and analyzed by flow cytometry. (E) HCT116+ch3 cells were either mock transfected (lane 1), transfected with the control shRNA plasmid pKD-NegCon-v1 (lane 2), the Chk1 shRNA plasmid pKD-CHK1-v1 (lane 3), the Chk2 shRNA plasmid pKD-CHK2-v3 (lane 4), or cotransfected with both CHK1 shRNA and CHK2 shRNA plasmids (lane 5) for 72 h. After this, cells were harvested and extracts were immunoblotted with anti-total Chk1 (top), anti-total Chk2 (middle), or anti-tubulin (bottom). (F) HCT116+ch3 cells were either mock transfected or transfected with indicated shRNA plasmids for 72 h. After transfection, cells were either treated with 5 μM MNNG (+) or were mock treated (-), and 48 h later were analyzed by flow cytometry. The percentage of cells containing a 4N DNA content is indicated. (G) HCT116+ch3 cells were either mock transfected (lanes 1 and 2), transfected with the control shRNA plasmid pKD-NegCon-v1 (lanes 3 and 4), the Chk1 shRNA plasmid pKD-CHK1-v1 (lanes 5 and 6), the Chk2 shRNA plasmid pKD-CHK2-v3 (lanes 7 and 8), or cotransfected with both CHK1 shRNA and CHK2 shRNA plasmids (lanes 9 and 10) for 72 h. Subsequently, cells were cells were either treated were mock treated (lanes 1, 3, 5, 7, and 9) or treated with 5 μM MNNG (lanes 2, 4, 6, 8, and 10), and after a 48-h incubation, cells were harvested and extracts immunoblotted with antibodies against phospho-Tyr15 Cdc2 (top) or total Cdc2 (bottom).

Nuclear exclusion of Cdc25C occurs in response to MNNG through an ATM- and MMR-dependent mechanism. (A) NHFs (lanes 1–3) were either mock treated (lane 1), 5 μM MNNG treated (lane 2), or irradiated with 5 Gy of IR (lane 3). Cells were harvested 48 h after MNNG exposure or 1 h after irradiation, and total cell lysates were formed and subjected to immunoblot analysis with anti-Cdc25A (top) or tubulin (bottom). (B) ATM-deficient (lanes 1 and 2) and ATM-proficient (lanes 3 and 4) as well as MMR-deficient (lanes 5 and 6) and MMR-proficient (lanes 7 and 8) cells were either mock treated (lanes 1, 3, 5, and 7) or 5 μM MNNG treated (lanes 2, 4, 6, and 8), total cell lysates were prepared 48 h after drug, and Cdc25A abundance was assessed by immunoblotting. (C) NHFs were either mock treated (lane 1) or treated with 5 μM MNNG (lanes 2 and 3). Cells were fractionated into nuclear (lanes 1 and 2) or cytoplasmic (lane 3) components and immunoblotted with anti-Cdc25C (top), SMC1 (middle; nuclear fraction control), or tubulin (bottom; cytoplasmic fraction control). (D) EBS-7 (lanes 1 and 2), YZ-5 (lanes 3 and 4), HCT116+ch2 (lanes 5 and 6), and HCT116+ch3 (lanes 7 and 8) cells were either mock treated (-) or treated with 5 μM MNNG (+). Nuclei were isolated 48 h after MNNG and immunoblotted with anti-Cdc25C (top) or anti-SMC1 (bottom) to ensure equal loading.

Chk1 and Chk2 coprecipitate with the MMR protein MSH2. MSH2-deficient HEC59 (lanes 1 and 2) and MSH2-proficient HEC59+ch2 (lanes 3 and 4) cells were either mock treated (lanes 1 and 3) or treated with 25 μM MNNG (lanes 2 and 4) for 1 h. After a 48-h incubation cells were harvested, and extracts were formed and MSH2 was immunoprecipitated. The resultant immunocomplexes were subjected to immunoblot analysis with anti-MSH2 (top), anti-total Chk1 (middle), or anti-total Chk2 (bottom).