FIG.2.

Overexpression of ACTR stimulates hormone-independent cell proliferation and confers antiestrogen resistance. (A) Ectopic expression of ACTR in T-47D cells by the adenovirus vector (a) or by stable transfection (b). ACTR protein levels in T-47D cells infected with adeno-ACTR (labeled with an A) or adeno-GFP (labeled with a G) or mock infected (Mock) at different days after infection (a), or levels in ACTR stable transfectants of T-47D cells (A6, A8, and A41) and an empty vector transfectant (labeled with a V) (b) were determined by Western analysis of whole-cell lysates from cells grown in hormone-depleted medium. (B) T-47D cells were hormone deprived for 60 h before infection by adenovirus vectors for ACTR or GFP with expression at an estimated multiplicity of infection of 100 and treated with 10⁻⁸ M E2 (+E2) or the solvent (-E2). Different days after infection, cell proliferation was measured by the colorimetric MTT assay in six replicates. (C) T-47D cells were hormone deprived for 24 h and treated with 10⁻⁸ M ICI for 36 h prior to adenovirus vector infection. Cells were then maintained in medium supplemented with 10⁻⁸ M ICI. Cell proliferation was monitored as described for panel B. (D) MDA-MB 361 cells were treated and analyzed as for panels B (except no E2) and C. (E) T-47D cells grown in regular medium (RPMI plus 10% FBS) were treated with 10⁻⁷ M ICI for 36 h prior to adenoviral infection. Cell proliferation was measured as described in the legend for panel B. (F and G) The same number of T-47D stable transfectant cells was plated. Different days after plating, cell proliferation was measured by cell enumeration in triplicates in the absence of hormone (F) or in the presence of 10⁻⁸ M ICI (G).