Characterization of Bortezomib (Velcade ™) Resistance in a New Set of Human Cell Lines Obtained by Stepwise Selection.

Abstract 2741

Poster Board II-717

Bortezomib (Velcade ™, PS-341, BTZ) is a boronate dipeptide that reversibly inhibits the 26S proteasome, which is essential for the breakdown of ubiquitinated proteins and the regulation of normal cellular homeostasis. The activity of BTZ in treatment of newly diagnosed and refractory/relapsed multiple myeloma may be limited by the development of chemoresistance, the mechanisms of which are poorly understood. To investigate the molecular basis of Bortezomib resistance, BTZ-resistant (BTZ^r) cell lines were generated by stepwise selection procedures from HeLa, CCRF-CEM, and 4 multiple myeloma cells lines (8226S, U266, H929, and MM.1S), respectively. These BTZ-selected cell lines displayed varying degrees of elevated resistance (2 to 50 fold) to clinically relevant concentrations of BTZ. In addition, while most of the BTZ^r cells showed cross resistance to several other proteasome inhibitors (PIs), including MG-132 and Epoxomicin, they remained as sensitive to other chemotherapeutic drugs, such as anthracyclines, vinkalkaloids and etoposide, as their parental cells. The proteasome activity profiles are distinct among the cell lines. All parental cell lines displayed varying levels of chymotrypsin-like (CT-L) activity, which is the primary target of BTZ. Most BTZ^r lines showed markedly decreased CT-L activity, with a few exceptions. Moreover, the observed CT-L activity in all cell lines can be inhibited directly by BTZ and other PIs. In contrast, very low levels of caspase-like or post-glutamyl peptide hydrolyzing (PGPH) proteasome activity were detected in all cell lines. BTZ resistance in HeLa/BTZ cells was closely associated with increased resistance to PI-induced apoptosis, as shown by reduced number of Annexin V-stained cells and by delayed activation/cleavage of apoptosis proteins, such as Caspase-3 and Poly(ADP-ribose) Polymerase (PARP). Furthermore, the resistance to BTZ affected the mechanisms of cell stress responses. As for the parental HeLa cells, HeLa/BTZ^r cells retained the ability to form, in response to PI treatment, pro-survival foci in the cytoplasm known as stress granules (SGs). However, the drug concentrations required to induce SG formation in HeLa/BTZ^r cells are much higher (∼4 fold) than those for the parental HeLa, suggesting the development of stress-coping mechanisms in these BTZ^r cells. Gene expression profiling studies are in progress to identify transcriptomes individually or generally associated with BTZ resistance in these cell lines. Further characterization of these phenotypically similar, yet mechanistically distinct BTZ^r cell lines may elucidate diverse mechanisms of drug resistance to Bortezomib and other proteasome inhibitors.