Figure 5

E2-dependent effects on DNA damage and cell survival are mediated by the expression of ERα. (a) ER-negative MDA-MB-468 cells were stably transfected with estrogen receptor α (ERα) expression vector (ER) or neomycin resistance plasmid (neo). ER-positive T47D neomycin-resistant control clones (neo) were used for comparison of ERα expression. Blots were stripped and incubated with anti-β-actin antibody to determine relative amounts of protein in each lane. (b) Complex formation of BRCA1 with CBP and ERα in MDA-MB-468 ERα stable clones treated with E2 but not with RA. 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. The experiments were performed three times with similar results; representative blots are shown. (c) E2 inhibits DNA damage in MDA-MB-468 clones stably expressing ERα. Neomycin-resistant control clones (neo) were used as controls. ER-positive T47D neomycin-resistant control clones (T47D neo) were used for comparison. Cultures were treated with E2 or RA alone or in combination (E2/RA) as described in Materials and Methods. Vehicle-treated clones were used as negative controls (con). Relative DNA damage was quantified with the single-cell gel electrophoresis assay. Error bars indicate SEM. (d) Ectopic ER expression in ER-negative MDA-MB-468 cells is sufficient to mediate the effects of E2 on DNA repair activity. Neomycin-resistant control clones (neo) were used as controls. ER-positive T47D neomycin resistant control clones (T47D neo) were used for comparison. Cultures were treated with E2 or RA alone or in combination (E2/RA) as described in Materials and Methods. Vehicle-treated clones were used as negative controls (con). Relative DNA repair activity was quantified by the plasmid end-joining assay. Error bars indicate SEM. (e) E2 increases cell survival in MDA-MB-468 clones stably expressing ERα. Neomycin-resistant control clones (neo) were used as controls. ER-positive T47D neomycin-resistant control clones (T47D neo) were used for comparison. Cultures were treated with E2 or RA alone or in combination (E2/RA) as described in Materials and Methods. Vehicle-treated clones were used as negative controls (con). Apoptotic cells were identified by TdT-mediated dUTP nick end labelling assay. Error bars indicate SEM.