Apoptotic Signaling in Response to a Single Type of DNA Lesion, O6-Methylguanine  Mark J Hickman, Leona D Samson  Molecular Cell  Volume 14, Issue 1, Pages 105-116 (April 2004) DOI: 10.1016/S1097-2765(04)00162-5

Figure 1 O6MeG/MutSα-Dependent Apoptosis (A) Western blot showing MGMT expression in TK6 cells or in TK6 cells expressing MGMT from the MMP retroviral vector (TK6 + MGMT). (B) Methyltransferase activity in TK6 (■), TK6 [MutSα−] (○), or TK6 + MGMT (▴) cell extracts, measured as described previously (Sassanfar et al., 1991). (C) O6MeG/MutSα-dependent apoptosis in TK6 cells. TK6 (■), TK6[MutSα−] (○), or TK6 + MGMT (▴) cells were treated with 0, 0.01, 0.02, 0.05, or 0.1 μg/ml MNNG. A representative of several experiments is shown. (D) Cells were left untreated (□) or treated with α-Fas (■). Shown are the averages and standard deviations for three experiments. Molecular Cell 2004 14, 105-116DOI: (10.1016/S1097-2765(04)00162-5)

Figure 2 O6MeG/MutSα-Dependent Caspase Processing (A) MNNG-induced caspase cleavage. TK6, TK6[MutSα−], or TK6 stably expressing MGMT (+MGMT) were treated with MNNG and caspase or substrate cleavage monitored by Western blot. (B) α-Fas-induced caspase cleavage. Cells were left untreated (−) or treated with α-Fas (+). (C) Caspase-2 cleavage induced by MNNG or α-Fas. TK6 cells expressing GFP or MGMT were left untreated (−) or treated with MNNG or α-Fas (+). Molecular Cell 2004 14, 105-116DOI: (10.1016/S1097-2765(04)00162-5)

Figure 3 O6MeG-Induced Caspase Activity (A) Activity of caspase-2 (■), caspase-3 (□), caspase-8 (•), and caspase-9 (○) in TK6 cells after MNNG treatment. (B) Caspase activity after MNNG treatment of TK6 + GFP (■) or TK6 + MGMT (□) cells. (C) Caspase activity after α-Fas treatment of TK6 + GFP (■) or TK6 + MGMT (□) cells. For all panels, representative experiments are shown. Molecular Cell 2004 14, 105-116DOI: (10.1016/S1097-2765(04)00162-5)

Figure 4 Role of Death Receptor Signaling in O6MeG/MutSα-Dependent Apoptosis (A) Western blot of TK6 clones stably expressing dnFADD from the pMMP-iresGFP vector or with the pMMP-eGFP vector alone (+MMP). The same clones were used in (B) and (C). (B) α-Fas-induced apoptosis of TK6 + MMP (■) or TK6 + dnFADD (○) clones. Shown are the means and standard deviations for at least two experiments. (C) O6MeG-induced apoptosis of TK6 + MMP (■) or TK6 + dnFADD (○) clones. Apoptosis was also monitored by microscopy with similar results (data not shown). Shown are the means and standard deviations for at least two experiments. (D) Western blot of TK6 clones stably expressing mouse c-FLIPL or with the MIG vector alone (+MIG). The same clones were used in (E) and (F). (E) Cells were left untreated (□) or treated with α-Fas (■). Shown are the means and standard deviations for at least three experiments. (F) O6MeG-induced apoptosis of TK6 + MIG (■) or TK6 + c-FLIP (○) clones. Shown are the means and standard deviations for five experiments. Apoptosis was also monitored by microscopy with similar results (data not shown). (G) After sFasL treatment in the presence of FasL or control antibodies, apoptosis was quantitated by microscopy. A representative graph is shown. (H) Cells were treated with MNNG, and apoptosis was quantitated by flow cytometry. Shown are the means and standard deviations for three experiments. Apoptosis was also monitored by microscopy with similar results (data not shown). For (G) and (H), antibodies were added to the medium at 1 μg/ml (for sFasL) or 30 μg/ml (for MNNG) just before treatment. A range of antibody concentrations was used with no effect on O6MeG-induced apoptosis (data not shown). In addition, an antibody to the Fas receptor did not block O6MeG-induced apoptosis, while it did inhibit apoptosis induced by sFasL (data not shown). The antibodies are stable over the 48 hr treatment period since sFasL-induced apoptosis was still fully blocked when the antibodies were added 48 hr before sFasL treatment (data not shown). Molecular Cell 2004 14, 105-116DOI: (10.1016/S1097-2765(04)00162-5)

Figure 5 Role of Mitochondrial Signaling in O6MeG/MutSα-Dependent Apoptosis (A) Western blot of TK6 clones stably expressing Bcl-2 or vector alone (+GFP). (B) Western blot of TK6 clones stably expressing Bcl-xL or vector alone (+GFP). (C) O6MeG-induced apoptosis in TK6 + GFP (▴), TK6 + Bcl-2 clone 5 (■), TK6 + Bcl-2 clone 6 (□), TK6 + Bcl-xL clone 1 (•), and TK6 + Bcl-xL clone 3 (○). Shown are the means and standard deviations for at least three experiments. (D) The same clones used in (C) were monitored by microscopy after MNNG treatment. Shown are the means and standard deviations for two experiments. (E) Cells were left untreated (□) or treated with α-Fas (■). Shown are the means and standard deviations for three experiments. (F) TK6 cells expressing GFP, Bcl-2, or Bcl-xL were left untreated (■) or treated with MNNG (□) and then monitored for mitochondrial depolarization. (G) TK6 cells expressing GFP (black bars), Bcl-2 (gray bars), or Bcl-xL (white bars) were left untreated or treated with MNNG, and the fold increase in caspase activity was determined. (H) Western blot showing caspase-2 and caspase-8 processing. Cells were left untreated (−) or treated with MNNG (+). Representative experiments are shown. TK6 + Bcl-2 (clone 5) and TK6 + Bcl-xL (clone 1) were used in (F)–(H). Molecular Cell 2004 14, 105-116DOI: (10.1016/S1097-2765(04)00162-5)

Figure 6 Role of Caspases in O6MeG/MutSα-Dependent Apoptosis (A) MNNG-induced apoptosis, in the presence of 10 μM (dark gray bars), 20 μM (light gray bars), or 50 μM (white bars) of inhibitor or equal volume of DMSO (black bars). (B) Same as (A), but apoptosis was monitored by flow cytometry. (C) Apoptosis after α-Fas treatment. For all panels, the averages and standard deviations for two or more experiments are shown. *p < 0.05 (one-sided t test), when comparing DMSO control to sample with caspase inhibitor. Molecular Cell 2004 14, 105-116DOI: (10.1016/S1097-2765(04)00162-5)

Figure 7 Apoptotic and Nonapoptotic Cell Death Triggered by O6MeG (A) Percent survival of TK6 cells expressing GFP (■), MGMT (□), Bcl-2 (•), or Bcl-xL (○). Shown are the means and standard deviations of three experiments. (B) Time course of apoptosis after MNNG treatment. Shown is a representative experiment. (C) Time course of cell survival after MNNG treatment. Shown is a representative experiment. TK6 + Bcl-2 (clone 5) and TK6 + Bcl-xL (clone 1) from Figure 5 were used in (A)–(C). (D) Model for O6MeG/MutSα-dependent cell death. Molecular Cell 2004 14, 105-116DOI: (10.1016/S1097-2765(04)00162-5)