Tamoxifen downregulated transcription of CIP2A. (A) After cells were treated with 100 μg/ml translation inhibitor cycloheximide (CHX) in the presence (left) or absence (right) of tamoxifen (7.5 μM) for the indicated periods, the stability of CIP2A protein in whole-cell lysates was assessed by Western blot analysis. In tamoxifen-sensitive cells, the addition of tamoxifen did not affect CIP2A degradation. (B) Tamoxifen affects CIP2A transcription. Cells were treated with tamoxifen at the indicated doses for 36 hours, after which total RNA was isolated and CIP2A mRNA was assayed by quantitative RT-PCR. Columns, mean values (n = 3); bars, SD. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (C) Identification of CIP2A proximal promoter regions that were affected by tamoxifen treatment. MDA-MB-468 cells were transfected for 24 hours with Firefly luciferase reporter vectors carrying CIP2A promoters of different lengths and Renilla vectors and then treated for 24 hours with 5 μM tamoxifen or DMSO. Cell lysates were then assayed for dual-luciferase activity as described in the Methods section. Columns, mean values (n = 3); bars, SD; *P < 0.05. (D) Tamoxifen disturbed binding of Elk1 to the CIP2A promoter region. Chromatin immunoprecipitation assays of the CIP2A promoter were performed as described in the Methods section. Soluble chromatin was immunoprecipitated with Elk1, Ets1 or immunoglobulin G (negative control (NC)) antibodies. Immunoprecipitates were subjected to PCR with primer pairs specific to CIP2A promoter (-16 to -139 bp). The gel shown is representative of three independent experiments. Anti-RNA polymerase II antibody and GAPDH primers were used as a positive control (PC). (E) Tamoxifen affected Elk1 expression. Nuclear and cytoplasmic extracts were prepared from MDA-MB-468 cells treated with tamoxifen (7.5 μM) or DMSO for 24 hours. Cell lysates were examined by Western blotting for Elk1. Lamin B and tubulin were used as loading controls.