FIG. 8.

Stimulation of hormone-independent cell proliferation by ACTR requires ACTR interaction with E2F1, but not ER. (A and B) T-47D cells were infected with adenovirus expressing wild-type and mutant forms of ACTR, as depicted. The expression levels of different ACTR proteins in the cells 2 days after infection were determined by Western blotting using α-HA antibody. Cell proliferation in the absence of hormone was monitored by MTT assay 4 days after infection as described in the legend for Fig. 2B, and the percentage of wild-type ACTR activity was determined by dividing the proliferation induced by the mutant ACTR with that of wild-type ACTR. (C and D) ER-negative cells (HCC-1806 and HCC-1937) infected with adeno-ACTR or adeno-GFP were monitored for cell proliferation by MTT assay. (E) T-47D and its derivative, A41 cells, hormone deprived for 2 days, were infected with an equal titer of the RNAi adenoviruses for E2F1 or GFP, as for Fig. 1A. Cells were then replenished with hormone-depleted medium either with (T-47D) or without (A41) 10⁻⁷ M E2. Cell proliferation was determined at 6 days after infection, with the values from Ri-GFP adenovirus-infected cells set as 100 arbitrarily. Cells were also collected at day 6 postinfection for Western analysis using α-E2F1 and α-β-actin. Note that no significant effect of GFP-RNAi vector on cell proliferation was observed when compared to mock infection. (F) Potential pathways for elevated levels of ACTR to stimulate breast cancer cell proliferation.