Increasing proteasome expression specifically enhances resistance toward PIs. U266 cells were treated for 5 days with low, nontoxic doses of MG-132 (2 days at 1 nM followed by 3 days at 10 nM). Cells were then washed and cultured in fresh media for 4 more days, and then assayed for overall proteasome activity and apoptotic sensitivity to bortezomib. (A) Significant increases of proteasome-specific activities in MG-132–conditioned MMCs. Conditioning treatment resulted in doubled chymotryptic and caspase-like activities, and an approximately 50% increase in trypsin-like activity. *P < .05. (B) Conditioned cells show enhanced PI resistance. MG-132–conditioned and vehicle-treated cells were exposed to the indicated doses of bortezomib (Btz) for 48 hours, and apoptosis was assessed by FACS as the proportion of annexin V⁺ propidium iodide⁻ cells. The line graph averages (± SD) 2 representative experiments. P < .05. (C) Conditioned cells are not protected from UV-induced apoptosis. MG-132–conditioned and vehicle-treated cells were exposed to UV rays for the indicated times, and apoptosis assessed after 24 hours as in panel B. One representative experiment is shown. (D) MG-132 conditioning treatment fails to induce a detectable heat shock response. MG-132–conditioned and vehicle-treated cells were sampled at the indicated days from the beginning of the treatment, and mRNA levels for inducible HSP70 was quantitated by real-time RT-PCR. A total of 1 hour of exposure to 43°C (heat) significantly increased HSP70i mRNA in U266 and HeLa cells. One representative experiment is shown.