Figure 2.

EBP1 restoration ameliorates the hormone-refractory phenotype. A, Western blot analysis of C81 cells transfected with a vector control (V) or EBP1 (E) expression plasmid. B, left, growth in monolayer culture. Equal numbers (5 × 10⁴) of vector or EBP1 stably transfected cells were plated at day 0 in complete medium and viable cell numbers were determined at the indicated times. Points, mean of three wells; bars, SE. Representative of three experiments (right). Colony growth in soft agar. C81cells stably transfected with either pcDNA or EBP1 were plated in soft agar at the cell densities indicated and colony numbers were assessed at day 10. Point, mean of three dishes; bars, SE. Representative of three experiments. C, left, response of EBP1- and vector-transfected C81 cells to DHT. EBP1 and vector control C81-transfected cells and early-passage LNCaP cells were plated in complete medium at 5 × 10³ per well in 96-well plates for 1 d. The next day, cells were switched to steroid-reduced medium for 2 d as described in Materials and Methods. DHT was then added at the indicated concentrations. Cells were re-fed medium every 3 d. Total cell numbers were determined 7 d later by MTT assays. Points, mean of six wells; bars, SE. Representative of two experiments. Right, effect of bicalutamide on DHT-stimulated growth. Cells were plated as described in Materials and Methods. After 2 d in steroid-reduced medium, cells were fed with fresh steroid-reduced medium with or without 10 μmol/L bicalutamide (Casodex) in the presence or absence of 10 nmol/L DHT. Total cell numbers were assessed 7 d later by MTT assay. Cells were re-fed medium every 3 d. Points, mean of six wells; bars, SE. Similar results were found in two independent experiments. D, EBP1 expression sensitizes C81 cells to chemotherapeutic drugs. C81 vector control or EBP1 transfectants were plated in 96-well plates in complete medium and allowed to attach overnight. The cells were then placed in steroid-reduced medium for 2 d and then re-fed with steroid-reduced medium in the presence of the indicated concentrations of docetaxel (left) or mitoxantrone (right). Total cell numbers were determined by MTT assays 5 d later. Points, mean of six wells; bars, SE. Representative of two experiments.