FIGURE 8.

Function of p21 upstream promoter in etoposide-treated HCT116 cells. A, effect of etoposide on the activity of the p21 upstream promoter and nuclear accumulation of TLP and p53. Cells were treated with 25 and 50 μm etoposide or vehicle (−) for 24 h. Panel a, RT-PCR for detection of p21 alt-a transcripts with a specific primer set. Panel b, Western blotting for detection of nuclear TLP and p21 protein. Nuclear extracts were prepared as described previously (46), and the amounts of three kinds of endogenous proteins were detected by Western blotting. TBP was analyzed as a constitutive nuclear protein. B, association of endogenous p53 (panel a) and TLP (panel b) with p21 upstream promoter. Wild-type HCT116 cells were treated with etoposide (E) or vehicle (V) and then analyzed by ChIP assay using specific antibodies for the p53-responsive element (RE) in the upstream promoter region (−2431 to −2196) and the far-upstream region (−7018 to −6833) as a negative control because this region had been shown not to bind to p53 (46). IgG and Ab, IgG- and specific antibody-precipitated materials, respectively; Inp, input. C, effect of overexpressed TLP on the association of p53 and TLP with the p21 upstream promoter. Etoposide-treated cells were transfected with FH-TLP (TLP) or vacant vector (vec) DNA, and association of p53 with the responsive element was analyzed by ChIP assay with a specific primer set as described above. IP, immunoprecipitation.