Figure 4

Activation of transcription factors NF-κB and AP-1, and viral promoter LTR in A549 cells cocultured with MT-2 cells. (A) A549 cells were transfected with κB-LUC, AP-1-LUC or LTR-LUC. After 24 h, transfected A549 cells were cocultured with MMC-treated MT-2 or CCRF-CEM cells for 24 h before luciferase assay. Luciferase activity is presented as a fold induction relative to the basal level measured in A549 cells that were not cocultured with MT-2 or CCRF-CEM cells. Relative luciferase amounts were normalized to equivalent Renilla expression to control for transfection efficiency. Data are mean ± SD values of three independent experiments. (B) DNA-binding activities of NF-κB and AP-1 in A549 cells. A549 cells were cocultured with MMC-treated MT-2 or CCRF-CEM cells. At 3 and 5 days after cocultivation, A549 cells were harvested and then NF-κB and AP-1 DNA-binding activities were analyzed by EMSA. Specific bands are indicated by arrows. (C) Characterization of DNA-protein complexes present in nuclei of A549 cells cocultured with MT-2 cells. At 5 days after cocultivation, nuclear extracts were prepared from A549 cells. The competitors used were the NF-κB site of the IL-2 receptor α chain gene and the AP-1 site of the IL-8 gene. Supershift assay in the same nuclear extracts was also performed. Supershifted bands with Abs are indicated by arrowheads.