ALBERT

Description: 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.

Description: The International Prognostic Scoring System (IPSS) has been widely used to predict the prognosis of patients with myelodysplastic syndrome (MDS). However, IPSS does not always provide a sufficiently precise evaluation of patients to allow the appropriate choice of clinical interventions. Here, we analyzed the expression of Bmi-1, which is required to regulate the self-renewal in CD34+ cells from 51 patients with cases of MDS and acute myeloid leukemia preceded by MDS (MDS-AML). Higher positivity rate of Bmi-1 was preferentially seen in refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEB-T), and MDS-AML compared with refractory anemia (RA) and RA with ringed sideroblasts (RARS). IPSS score was positively correlated with the percentage of Bmi-1 expression. Patients with RA and RARS with a higher percentage of Bmi-1+ cells showed disease progression to RAEB. Here, we propose Bmi-1 as a novel molecular marker to predict the progression and prognosis of MDS.

Description: Bmi-1 is a member of the Polycomb group (PcG) of transcriptional repressor genes, which could have an essential role in embryogenesis and be expressed restrictedly in the stem cells and proliferating cells. Some studies have recently reported that Bmi-1 is required to regulate the adult self-renewing hematopoietic stem cells. Others have shown that overexpressed Bmi-1 gene could cause the neoplastic proliferation of cells. A series of evidence on Bmi-1 suggests that it might be closely associated with the progression of the hematopoietic malignancies. Myelodysplastic syndrome (MDS) is a clonal bone marrow disorder characterized by aberrant hematopoiesis susceptible to acute leukemia. This category is quite heterogeneous with a variable clinical course and prognosis. Therefore, it is difficult to decide when to initiate therapies because some patients survive for years without treatment whereas others come to die in rapid progress in overt leukemia. Thus, we investigated whether the expression of Bmi-1 protein in CD34+ cells could be correlated with disease progression of MDS. Primary cells from patients with RA, RAEB, and MDS-AML, were stained with anti-CD34 antibody-PE and then fixed in PFA followed by staining with anti-Bmi-1-antibody-FITC. These cells were subjected to flow cytometric analysis. Bmi-1 expression in CD34+ cells was preferentially seen in RAEB and MDS-AML compared with RA (13.86%±6.40% in RA(n=4), 63.05%±8.19% in RAEB(n=6), 73.99%±33.40% in MDS-AML(n=6)). Patients were dead with 〉70% at Bmi-1 expression level in CD34+. Moreover, two patients with RA at 〉10% of Bmi-1 positivity made disease progression to RAEB. One patient with RAEB at

Description: The mechanism of oncogenesis of plasma cells remains unclear. Since tumor cells are post-germinal center B cells, reciprocal chromosomal translocations between the IgH gene located on chromosome 14q32 and other chromosomal partners such as cyclin D1 located on chromosome 11q23, which are supposed to be candidate oncogene, arise in myeloma cells during the procedure of isotype switching. However, a recent study demonstrated that multiple myeloma (MM) with cyclin D1 overexpression belongs to the low-risk group. Previously, we reported that cyclin D1 overexpression downregulated cyclin D2 expression in myeloma cells and increased the sensitivity to treatment with anti-myeloma agents such as bortezomib (PS-341), dexamethasone, melphalan, and immunomodulatory thalidomide analogs in a comparison between RPMI8226 transfected cyclin D1 and mock. Here we have analyzed characteristics of RPMI8226 transfected cyclin D1. This cyclin D1 transfectant did not induce cell growth advantage, and cell cycle analysis by bromodeoxyuridine (BrdU) stimulation showed significant increase cell number in S-phase without an increase of that in G2/M-phase and decrease of cell number in G0/G1-phase. Therefore, Cyclin D1 overeexpression prolonged the S-phase. Western blot analysis demonstrated an increase in the hyperphosphorylated form of retinoblastoma protein (ppRb), and this ppRb was also found in KMS12BM, KMS21BM, in which myeloma cell line cyclin D1 overexpresion was detected due to t(11;14). Considering that ppRb releases free E2F, the increase in E2F could be expected to upregulate apoptosis-related genes. However, expressions of p53, Bcl-2, Bax, Bad, Bim, Mcl-1, p16, and CDK4 were not changed in cyclin D1 transfectant besides the decrease in p27 expression compared with those of the mock and parent cells. Furthermore, cyclin D1 overexpression that alone did not induce apoptosis because there were no such cells detected in sub-G0/G1 by BrdU stimulation without treatment by anti-myeloma agents. On the other hand, treatment with anti-myeloma agents induced both the intrinsic and extrinsic pathways earlier in the cyclin D1 transfectant. And this cyclin D1 transfectant cells easily lost viability in confluent samples. Interestingly, expression of the TRAIL receptors (DR4 & DR5) were significantly higher in cyclin D1 transfectant cells and treatment with recombinant TRAIL induced apoptosis earlier compared with those of mock and parent cells. There was no difference of TRAIL expression in these cells by western blot. These findings suggest that high sensitivity to anti-myeloma agents in myeloma cell with cyclin D1 overexpressin might be due to the prolonged S-phase duration and high expression of TRAIL receptor. We speculate that high ppRb induces gene instability via high E2F and leads to progressive disease in MM. This might be a reason why cyclin D1 overexpression caused by t(11;14) or hyperdiploidy is an early event in the progression of MM.

Description: Cyclin D is dysregulated in at least two-thirds of multiple myeloma (MM) tumors. In addition, recent reports showed that the dysregulation of cyclin D1 is frequent in the absence of a t(11;14) translocation in MM. However, as we also reported (Int J Oncol, 2004), there appears to be no obvious correlation between the expression of cyclin D1 and the proliferation index (PI) or Ki67 expression. Therefore, we thought that the down-regulation of cyclin D2 might offset the expression of cyclin D1 in myeloma cells with cyclin D1 overexpression in cDNA microarray, since primary myeloma cells or myeloma cell lines express cyclin D3 ubiquitously. Here we transfected cyclin D1 gene into a myeloma cell line (RPMI8226), originally not expressing cyclin D1, using a retrovirus-mediated gene transfer system. In this method we inserted a 1.1 kb fragment containing the open reading frame of cyclin D1 removing from a tet-cyclin D1 plasmid (kindly provided by Dr. Reed SI, MCB, 1994) into a retrovirus vector (pQCXIP). First, we analyzed the expression of cyclin D1 in the bulk culture of cyclin D1 transfectant. We detected the expression of cyclin D1 by western blot, and found that the limited numbers of transfectant expressed cyclin D1 protein by immunohistocytochemical staining. Subsequently, we separated the two types of cyclin D1 transfectant by limiting dilution. Both transfectants showed the expression of cyclin D1 mRNA in RT-PCR, however, one of the two did not show the expression of cyclin D1 protein in western blot and immunohistocytochemical staining. Interestingly, we clearly detected the down-regulation of cyclin D2 mRNA in the transfectant with cyclin D1 protein expression by RQ-PCR. Furthermore, we detected an increase of cells in S phase in the transfectant with cyclin D1 protein by flow cytometry. Unlike in the study of Lamb J et al. (Cell, 2003), we could not observe the induction of IL-6 by the transfection of cyclin D1 gene. Although the mechanism of the impairment of cyclin D1 translation is unclear, here we suggest that the lack of correlation between the expression of cyclin D1 and PI might be due to the impairment of cyclin D1 translation or the offset of the expression of cyclin D1 by the down-regulation of cyclin D2. We are now analyzing the effects of velcade and IMiDs on these transfectants, since we suspect that these differences would affect the response to chemotherapy for MM. Furthermore, we are going to analyze the difference of gene expression between these transfectants using cDNA microaray. Therefore, these transfectants could be useful materials to analyze the cyclin D1 dysregulation in myeloma cells.

Description: Since prognosis of patients with acute myeloid leukemia (AML) is highly variable even in a single subpopulation in FAB classification, it would be useful to find prognostic molecular markers for AML. Thus, we investigated Bmi-1 expression in AML cells by flow cytometry and analyzed whether it predicts prognosis in AML patients and further it is helpful to choose therapies in the modalities of treatment options, because it is known to be required for self-renewal mechanism of leukemic stem cells. Bmi-1 expression in bone marrow or peripheral blood cells was analyzed in 49 patients with AML (M0(n=5), M1(n=7), M2(n=6), M3(n=5), M4(n=8), M5(n=5), M6(n=1)), granulocytic sarcoma(n=1), MDS-AML (n=9), and secondary AML(n=2). Freshly isolated AML cells were stained with a PE-conjugated anti-CD34-antibody followed by fixation and then with anti-Bmi-1-antibody-FITC. All of patients with low Bmi-1 positivity ( 70%, n=19) except for AML(M3) progressed to death within two years, unless they were treated with highly intensive therapy such as high dose AraC or allogeneic stem cell transplantation (n=3). Patients with intermediate degree of Bmi-1 expression (35–60%, n=5) responded to standard intensity of chemotherapy (n=2) and are alive for more than two years. Interestingly, patients with MDS-AML (n=9) had high Bmi-1 expression (79%) and all of them have died within 20 months. Binary logistic regression model showed that significant correlation was found among survival status as dependent variable, Bmi-1, and treatment intensity as independent variable (p = 0.004). On the other hand, Univariate analysis did not reveal any relation of Bmi-1 expression to karyotype, age, WBC count, or FAB subtype. In conclusion, Bmi-1 expression could be an independent prognostic marker and useful tool to design therapy for the AML patients.