ATM promotes apoptosis and suppresses tumorigenesis in response to Myc

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   Fig. 1.

   ATM-dependent accumulation and phosphorylation of p53 in response to Myc. (A) Western blot analysis was performed on epidermal protein lysates from nontransgenic (lanes 1-3) and K5 Myc-transgenic (lanes 4 and 5) mice that were wild-type (lanes 1, 2, and 4) or nullizygous (lanes 3 and 5) for Atm. The wild-type mouse in lane 1 was treated with 10 Gy of IR 30 min before it was killed. Antisera specific to total p53 (Top), phospho-serine-15 p53 (Middle), or β-tubulin (Bottom) were used as indicated. (B) NHFs (lanes 1-4) or primary fibroblasts from an AT patient (AT, lanes 5-8) were mock-treated (lanes 1 and 5), exposed to 10 Gy of IR (lanes 2 and 6), or infected with AdGFP (lanes 3 and 7) or AdMyc (lanes 4 and 8) at a multiplicity of infection of 100. Cells were harvested for protein extract 1 h after IR or 24 h after infection, and Western blot analysis was performed by using antisera or antibody specific for phospho-serine-1981 ATM, total ATM, phospho-serine-15 p53, activated caspase-3, or β-actin as indicated.

   
   
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   Fig. 2.

   K5 Myc-transgenic tissue stains for markers of DNA double-strand breaks. Immunofluorescent staining was performed on skin sections from untreated wild-type mice, wild-type mice exposed to 3 Gy of IR 20 min before killing, K5 Myc mice, and K5 Myc, Atm ^-/- mice by using antibodies specific for ATM phosphorylated at serine-1981 (p-ATM), γH2AX, or SMC1 phosphorylated at serine-957 (p-SMC1).

   
   
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   Fig. 3.

   Inactivation of Atm reduces apoptosis in K5 Myc-transgenic mice. (A) Skin sections taken from mice with the indicated genotypes were immunohistochemically stained with an antibody specific for the activated form of caspase-3. The average number of positive epidermal cells per 10 mm of skin was determined microscopically from at least four independent mice in each group. The number of caspase-3-positive cells in K5 Myc-transgenic mice null for Atm is statistically different from the number in K5 Myc-transgenic mice wild-type for Atm by unpaired t test (P = 0.0175). (B) Three wild-type and three Atm-null mice were treated with 200 mJ/cm² UVB, and skin sections were taken 24 h later. Skin sections were immunohistochemically stained for activated caspase-3, and the average number of positive cells per 10 mm of epidermis was determined microscopically for each group.

   
   
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   Fig. 4.

   Inactivation of Atm accelerates epithelial tumorigenesis in K5 Myc-transgenic mice. K5 Myc-transgenic mice wild-type (+/+), hemizygous (+/-), or nullizygous (-/-) for Atm were monitored for spontaneous tumor development for 1 year. Only tumors from squamous epithelial tissues are included. Tumor incidence in K5 Myc-transgenic mice that are Atm ^-/- is statistically different from transgenic mice that are Atm ^+/+ or Atm ^+/- by univariate ANOVA (P < 0.05).

   
   
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   Fig. 5.

   Thymic lymphoma cell lines from Atm knockout mice overexpress Myc. Western blot analysis for Myc and β-actin was performed by using extracts from primary thymocytes isolated from 4-week-old wild-type (lanes 1 and 2) or Atm-null (lanes 3 and 4) mice or from three independent thymic lymphoma cell lines derived from Atm ^-/- mice (lanes 5-7).