Materials and

methods Cell culture, animal and reagents Chemicals employed were obtained from the following sources: MNNG and PMA from Sigma Chemical Co. (St. Louis, MO, USA). These chemicals were dissolved in dimethyl sulfoxide (DMSO, from Sigma Protein Tyrosine Kinase inhibitor Chemical Co.) before addition to the cultures. The final concentration of DMSO was 0.1%. Antibodies against acetylated histone H3 and GAPDH were from SantaCruz (California, USA). The rat Oligo-GE-Array (9.2 version) was supplied Exiqon (Denmark). Male Balb/c nude mice, 6–8 weeks of age, were obtained from The Animal Facility of Third Military Medical University (Chongqing, China). Animals were housed under controlled temperature, humidity and day-night cycle with food and water. All animal experiments were conducted according to the Cancer Statement for the Use of Animals in Cancer Research,

and approved by the institutional committee for animal research of Third Military Medical University, Chongqing, China. Cell culture and cell transformation IEC-6 cells (ATCC, USA) ERK inhibitor were cultured in DMEM (Logan, USA) containing 10% fetal calf serum (Hyclone), penicillin (100 U/mL), and streptomycin (100 μg/mL). For cell transformation, exponentially growing cells were seeded at a density of 105 cells per 60-mm dish in 5 ml of culture medium. Twenty-four hours after seeding, the cells were treated with enough 1 μg/ml MNNG for 8 h and then grown in normal medium for 3 days. Then the cell culture was grown in a medium containing PMA at concentrations of 100 ng/ml for 3–4 days of promotion stage. The MNNG/PMA treatment was repeated 11 times and

the finally treated IEC-6 cells were tested for transformation properties. Normal IEC-6 cells were used as negative control. Achorage dependence The efficiency of colony formation in semisolid medium was measured by the procedure described by MacPherson [21]. Cells suspended in 3.0 ml of 0.3% agar with complete medium and were plated in 60-mm dishes over a layer of 0.7% agar containing complete medium. A final concentration was 1 × 104 cells per dish and allowed to harden. Plates were incubated at 37°C in a 5% CO2 humidifed atmosphere for 21 days and scored for clones. Colony formation efficiency in semisolid agar was expressed as the percentage of total cells that formed colonies containing at least 50 cells. Tumor development in nude mice Normal or transformed IEC-6 cells were trypsinized and collected by centrifugation. Male Balb/c nude mice were inoculated subcutaneously with 5 × 105 IEC-6 cells in the dorsal aspect of the neck (4 mice in each group). Human colon cancer SW480 cells were used as positive control, and the same amount of cells were inoculated in nude mice as well. All the mice were further raised for 4–8 weeks, and the tumor weight was scored after the mice were kindly sacrificed.