Figure 1

E2 inhibits and RA enhances DNA damage-mediated apoptosis in human breast cancer cell lines. (a) Estrogen receptor (ER)-positive human breast cancer cell lines (MCF7 and T47D) and ER-negative lines (MDA-MB-231 and MDA-MB-468) were treated with 17β-estradiol (E2) or all-trans retinoic acid (RA) alone or in combination (E2/RA) prior to etoposide induced DNA damage. Vehicle-treated cells were used as the negative control (con). Apoptotic cells were identified by TdT-mediated dUTP nick end labeling (TUNEL) assay. Error bars indicate SEM. (b) The human breast cancer cell lines identified above were treated with E2 or RA alone or in combination (E2/RA)before treatment with 3 Gy of ionizing radiation to induce double-strand DNA breaks. Vehicle-treated cells were used as the negative control (con). Apoptotic cells were identified by TUNEL assay. Error bars indicate SEM. (c) The pro-survival effects of E2 were not mediated by MAPK, PKC, phosphoinositide 3-kinase, phospholipase Cγ, or Akt/PKB signaling in human breast cancer cell lines. ER-positive breast cancer cell lines MCF7 and T47D were pretreated with PD98059 (PD), SP600125, (SP), SB203580 (SB), SH5, (Akt/PKB inhibitor), Go6976 (Go), LY294002 (LY), or U73122 (U) as indicated in Materials and Methods. Vehicle-treated cells were used as the negative control (con) for the TUNEL assay. (d) E2 inhibits and RA increases the extent of DNA damage in etoposide-treated human breast cancer cell lines. The indicated ER-positive and ER-negative cell lines were treated with E2 or RA alone or in combination (E2/RA), as described above, before exposure to etoposide. Vehicle-treated cells were used as the negative control (con). Relative DNA damage was quantified as described in Materials and Methods. (e) E2 enhances and RA inhibits DNA double-strand break repair in human breast cancer cell lines. The indicated cell lines were treated with E2 or RA alone or in combination (E2/RA) as described above. Vehicle-treated cells were used as the negative control (con). The plasmid end-joining assay was used to quantify DNA repair activity. (f) E2 or RA treatment does not affect double-strand break repair protein expression in human breast cancer cell lines. The indicated lines were treated with E2 or RA as described above. Vehicle-treated cells were used as the negative control (con). Protein extracts from treated cells were subjected to western blotting with the anti-human antibodies indicated at the left. (g) Complex formation of BRCA1 with CBP and ERα in E2-treated but not RA-treated T47D cells (upper panel). Vehicle-treated cells were used as the negative control. The CBP coactivator protein was immunoprecipitated from treated cultures with anti-CBP antibody (IP CBP). Preimmune IgG was used as the negative control for immunoprecipitation. Immunoprecipitates were subjected to western blotting to detect interaction with ERα, retinoic acid receptor α (RARα), and BRCA1 by using the antibodies indicated at the left. Blots were stripped and incubated with anti-CBP antibody to detect relative amounts of immunoprecipitated protein in each lane. These experiments were performed three times with similar results; representative blots are shown.