Bortezomib (Velcade™) is a reversible proteasome inhibitor that is approved for 
the treatment of multiple myeloma (MM). Despite its demonstrated clinical 
success, some patients are deprived of treatment due to primary refractoriness 
or development of resistance during therapy. To investigate the role of the 
duration of proteasome inhibition in the anti-tumor response of bortezomib, we 
established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous 
exposure of bortezomib. These cells were ~30-fold resistant to bortezomib. Two 
novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the 
binding site in a helix critical for drug binding, and Arg24Cys, found in the 
propeptide region were found in all resistant clones. The latter mutation is a 
natural variant found to be elevated in frequency in patients with MM. 
Proteasome activity and levels of both the constitutive and immunoproteasome 
were increased in resistant cells, which correlated to an increase in subunit 
gene expression. These changes correlated with a more rapid recovery of 
proteasome activity following brief exposure to bortezomib. Increased recovery 
rate was not due to increased proteasome turnover as similar findings were seen 
in cells co-treated with cycloheximide. When we exposed resistant cells to the 
irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery 
of proteasome activity as compared to bortezomib in both parental and resistant 
cells. Importantly, carfilzomib maintained its cytotoxic potential in the 
bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be 
overcome with irreversible inhibitors, suggesting prolonged proteasome 
inhibition induces a more potent anti-tumor response.