Fig. 1. Tissue differentiation is associated with an increase in (6)4 integrin-dependent Rac activity and enhanced resistance to apoptosis. (A) Dose-response curves showing that nonmalignant HMT-3522 S1 and MCF10A MECs acquire resistance to apoptosis induced by chemotherapeutics including taxol and immune receptor activators such as Trail following their rBM-induced differentiation into 3D polarized acini. The percentage apoptosis was calculated by scoring the number of activated caspase-3-positive cells 48 hours after treatment divided by the total cell number. MECs were either plated on top of a 1:100 diluted rBM for 48-96 hours (2D) or differentiated by embedment within rBM for 10-12 days (3D) followed by exposure to increasing doses of apoptotic stimuli. (B) Representative immunoblot of immunoprecipitated Pak-associated Rac (GTP-Rac), total Rac (Rac) and E-cadherin in MECs plated either on top (2D) as monolayers or within (3D) rBM to assemble acini. The data indicate that total Rac decreases noticeably following rBM-induced differentiation, whereas GTP-loaded Rac increases dramatically. (C) Average relative specific activity of Rac in 3D mammary acini calculated by densitometric analysis of immunoblots of GTP-Rac divided by total cellular Rac following E-cadherin normalization, as shown in B. (D) Representative immunoblot of GTP-Rac, total Rac (Rac) and E-cadherin in vector control and tail-less 4 integrin (4cyto) 3D mammary acini grown within rBM (10-14 days). The data illustrate that Rac activity diminishes significantly in mammary acini that express the signaling-defective tail-less 4 integrin. (E) Average relative specific activity of Rac in control mammary acini versus mammary tissues expressing the tail-less 4 integrin calculated as above in C and shown in D. Results are the mean±s.e.m. of three to five separate experiments. ^*P0.05; ^**P0.01; ^***P0.001.