Abstract

The response to initial glucocorticoid (gc) therapy in childhood acute lymphoblastic leukemia (ALL) reliably predicts the response to multiagent chemotherapy. In a recent study, we identified the valosin-containing protein (VCP) as a part of the ubiquitin proteasome degradation pathway (UPDP) as one of the proteins overexpressed in prednisone poor responder (PPR) patients. Therefore, we investigated whether treatment of ALL cell lines with the proteasome inhibitor bortezomib acted synergistically with glucocorticoid treatment. Human B-cell precursor leukemic cell lines MHH cALL 2 (PPR) and MHH cALL 3 (PGR) were treated with prednisone(6.3μM) as baseline and also with different concentrations of the proteasome inhibitor bortezomib for 96hours (h). To study drug effects, cells were sampled every 24h for immunofluorescence (IF) staining, protein and RNA extraction, viability (Trypan blue, WST-1) and apoptosis assays. Western blot analyses using an anti-p97 antibody were performed on whole cell lysates (wcl) and fractions and separated by differential detergent fractionation. VCP RNA expression was analyzed by real-time PCR. Single bortezomib treatment with 3nM or higher concentrations led to a significant decline in vitality of both cell lines. Within 24h, the PPR cell line lost about half and the PGR about one-fourth of their vitality. In combination with prednisone, 1.5nM bortezomib reduced the vitality by about 50% within 96h for both cell lines. Combining both drugs decreased the vitality rate by about 10% in the PPR cell line, whereas the PGR cells showed no decrease compared to single gc treatment. In FACS analyses, stages of different quantities of apoptosis were detected in PPR and PGR cells. PPR cells treated with both drugs showed a strong increase of necrotic cells at 24h. PGR cells started with an accession of apoptotic cells and initially had no necrotic cells, but started to rise from 48h on. We hence propose that the PPR cells react more quickly to the combined therapy. Under single gc treatment, VCP RNA expression increased in the PPR cells to a maximum of about 1.8- and in PGR cells to 1.5-fold. In PGR cells treated only with 1.5nM or 3nM bortezomib, VCP RNA rose to 1.4- and 2-fold respectively. Drug combination led to a 1.4-fold increase of VCP RNA in PPR compared to untreated cells, whereas RNA was reduced compared to single gc-treated cells. Protein levels of VCP in PPR cells remained high during drug treatment. VCP increased to a maximum of 1.6-fold in the cytosol of PGR cells, using bortezomib only. In the combination experiments, the amount doubled within 48h and thence decreased to initial levels. Single gc treatment caused a VCP increase to 1.5-fold within 24h. In the wcl, we found the VCP levels for the PGR cells converted to the cytosolic patterns. The results of IF staining supported the different VCP concentrations and exposed formation of aggresome-like complexes in the PPR cell line. The results of this study suggest that the multiagent chemotherapy resistance is indicated by differentially expressed VCP and related to the deregulation of the UPDP. Using inhibitors appears to chemisensitize the PPR for gc treatment. Therefore, drug targeting the proteasome, as in other hematological cancer therapies, might improve the overall therapy outcome.

Author notes

Disclosure:Research Funding: This study was supported by the German José Carreras Leukaemia Foundation (DJCLS R05/16v). Honoraria Information: German José Carreras Leukaemia Foundation.