Cyclin D1 Overexpression Increases Chemosensitivity Via the Induction of Bim in Myeloma Cells.

Recent study analyzing the gene expression in myeloma cells using cDNA microarray showed that myeloma cells are divided into 7 groups. Two showed a high expression of cyclin D1 together with a low expression of cyclin D2, and they belong to the low-risk group when analyzed based on event-free and overall survival. Since cyclin D1 promotes the cell cycle progression, we sought to explain why cyclin D1 overexpression appears to be a favorable prognostic variable for multiple myeloma (MM) patients treated with high-dose chemotherapy and single or double autologous transplantation. It is not clear whether the down-regulation of cyclin D2 in these myeloma cells might offset cyclin D1 overexpression in cell biology. We have established a myeloma cell line (RPMI8226) with cyclin D1 overexpression by transfection of the cyclin D1 gene using a retrovirus vector to analyze biological changes in myeloma cells with cyclin D1 overexpression. In comparison with myeloma cells transfected with the vector only (mock), the analysis of gene expression by cDNA microarray showed the down-regulation of cyclin D2 and MCL-1 and the up-regulation of CED6 in cyclin D1-transfected myeloma cells. However, there were no significant changes in Bcl-related genes between them. And as we expected, cyclin D1-transfected myeloma cells showed high proliferative activity, increased number of cells in S-phase, and increased pRb protein. Next we analyzed their sensitivity for bortezomib (Millennium Pharmaceuticals, Inc., USA), immunomodulatory thalidomide analogs (IMiD1, D2, D3) (Celgene Corporation, USA) and dexamethasone. Bortezomib and dexamethasone induced apoptosis at an earlier time point (12hr) in cyclin D1 transfectant compared to mock transfectant, concomitant with decreased expression of MCL-1, but with increased expression of Bim. Furthermore, we confirmed in cell culture condition with a low concentration of FCS that these results were not due to just promotion of the cell cycle caused by cyclin D1 overexpression. Given that myeloma cells with cyclin D1 overexpression easily undergo apoptosis upon bortezomib or dexamethasone treatment, this may explain why patients with those myeloma cells are in the low-risk group. With this finding, we then analyzed the expression of cyclin D1 by RQ-PCR and immunocytostaining before and after 2 or 3 cycles of VAD (vincristine, doxorubicin, dexamethasone). There was no significant difference between the response to VAD and the reduction rate of cyclin D1 positive myeloma cells or the decrease of cyclin D1 expression in RQ-PCR. Interestingly, cyclin D2 expression increases relatively in progressive disease (PD) after chemotherapy containing high-dose melphalan followed by autologous stem cell transplantation compared with those of cyclin D1 and D3 by RQ-PCR. Therefore, cyclin D1-induced chemosensitivity may be due to the induction of Bim, which consequently inhibits the function of MCL-1. New strategies to down-regulate of MCL-1 might be useful in the treatment of MM.