Summary Multiple myeloma (MM) is a neoplastic proliferation of plasma cells and remains an incurable disease because of the development of drug resistance. Histone deacytylase (HDAC) inhibitors are a new class of chemotherapeutic reagents that cause growth arrest and apoptosis of neoplastic cells. Depsipeptide, a new member of the HDAC inhibitors, was found to be safe in humans and has been shown to induce apoptosis in various cancers. In order to evaluate the effects of depsipeptide, a MM cell line, U266 [interleukin (IL)‐6 dependent], was analysed for viability and apoptosis. The combined effect of depsipeptide with melphalan and changes in BCL‐2 family proteins (BCL‐2, BCL‐XL, BAX and MCL‐1) were also investigated. In addition, the RPMI 8226 cell line (IL‐6 independent), and primary patient myeloma cells were also analysed for apoptosis after depsipeptide treatment. Depsipeptide induced apoptosis in both U266 and RPMI 8226 cell lines in a time‐ and dose‐dependent fashion, and in primary patient myeloma cells. We also demonstrated that depsipeptide had an additive effect with melphalan (10 μmol/l). BCL‐2, BCL‐XL and MCL‐1 showed decreased expression in depsipeptide‐treated samples. Based on recent clinical trials demonstrating minimal clinical toxicity, our study supports the future clinical utilization of depsipeptide in the management of MM.

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Heat shock protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated an antitumor effect in solid tumors. To test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analysed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55% also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins and akt in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL, MCL-1 and akt. Furthermore, although a low concentration of bortezomib resulted in no cell death, a combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.

Recent studies have suggested that protein kinase C (PKC) activation plays an important role in survival of chronic lymphocytic leukemia (CLL). In order to characterize the role of PKC in CLL, we investigated the expression pattern of PKC isoforms in CLL cells (7 cases) and evaluated the effect of PKC inhibition on the survival of CLL cells (20 cases). Expression of the classical PKC isoforms b and g, the novel isoform d and the atypical isoform z was seen in all analyzed patient samples by Western blot analysis. Expression of the PKC isoforms a, e, and i was variable. Following incubation with the PKC inhibitor, safingol, CLL cells underwent marked apoptosis in all cases. In order to characterize the molecular events associated with the apoptotic effect of PKC inhibition, gene expression patterns in CLL cells were evaluated by cDNA-microarray analysis. Following safingol treatment, several genes showed marked downregulation and PKCrelated proteins demonstrated decreased hybridization signals. Among these proteins, CREB and Daxx were further studied by using Western blotting, nuclear binding assay and confocal immunofluorescent microscopy. These studies showed significant inhibition of these proteins, consistent with the results of microarray gene analysis. Overall, these findings suggest that PKC activation is important for CLL cell survival and that inhibitors of PKC may have a role in the treatment of patients with CLL. Am.

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