FIG. 6.

The deletion construct without the 5′-flanking region from −1481 to −170 bp reverses the suppressive effect of GFLX on IL-8 promoter activity. PC-3 cells were plated at 4 × 10⁴ cells/well in 24-well plates. After 24 h of incubation, the cells were transiently transfected with 179.1 ng of a luciferase reporter gene construct containing sequentially deleted 5′-flanking regions of the IL-8 gene and 20.8 ng of pRL-TK. The constructs of the deletion mutants (D-415, D-240, D-188, D-170, D-129, and D-62) are shown to the left. Twenty-four hours after transfection, GFLX was added at 16 μg/ml and the cells were further incubated for 24 h. The medium was then exchanged for serum-free RPMI 1640 medium. GFLX at the same concentration and TNF-α at 10 ng/ml were added, and the cells were incubated for 2 h. After the incubation, the IL-8 gene promoter activity was finally determined by the dual luciferase reporter assay described in Materials and Methods. White bars, control without stimulation; black bars, incubation in the presence of TNF-α; gray bars, incubation in the presence of TNF-α and GFLX. The data shown are the means ± SDs of three experiments. *, P < 0.05 compared with the results for the luciferase activity of TNF-α-stimulated cells.