ALBERT

Description: PPAR (Peroxisome proliferator-activated receptor) ß is ubiquitously expressed in all cells and considered to be involved in the lipid metabolism and regulating the inflammatory response and cell proliferation. However, it remain to be clarified the role of PPARß in human myeloma cells. In order to identify the possible ligands for PPARß, we constructed the PPARß response element (PPREß)-lusiferase reporter gene and performed the reporter assay in Hela cell. We found that adrenal cortex hormones (DHEA and DHEA-S etc), dexamethasone (Dex) and baicalein augmented the expression of reporter gene. As already reported by us, baicalein, one of the major flavonoids in Scutellaria baicalensis (Chinese Skullcap), has the suppressive effects on cell proliferation and angiogenesis. Furthermore, baicalein as well as Dex upregulated the expression of PPARß target genes such as ILK and PPARg in myeloma cell lines. Moreover the combination of baicalein with Dex showed cooperative effect on the growth suppression in primary myeloma cells as well as myeloma cell lines. The expression of NF-kB target genes such as IL-6 was also markedly suppressed with the treatment with baiacalein and Dex in U266 cells. Since U266 cells constitutively express RelA(p65), RelB and p50 in the nucleus, we examined whether ligand (baicalein or Dex)-stimulated PPAR ß interacted with RelA in the nucleus of U266 cells or not. We detected physical interaction of PPARß with RelA in the nuclear fraction of U266 cells after stimulation with baicalein or Dex, and also confirmed that baicalein and Dex suppressed the DNA binding of RelA to kB site in the p65 ELISA. Therefore, these data suggest that baicalein combined with Dex can stimulate PPARß-mediated growth suppression in myeloma cells possibly through nuclear interaction between PPARß and RelA (NF-kB) more effectively.

Description: Since human myeloma cells specifically lose the expression of PAX-5 gene, the master gene of B cell lineage, we frequently detect the ectopic expressions of non-B cell lineage markers such as CD56 and CD33. As for the expression of CD33, we confirmed that CD33 expression was found in primary myeloma cells from about 12% of 120 cases with overt myeloma and also in 2 myeloma cell lines (ILKM8 and Liu01 (a subclone of U266)) in the protein and mRNA levels. In primary myeloma cells as well as CD33(–) myeloma cell lines, DMSO treatment could induce the expression of CD33 in the in vitro culture. In these CD33(+) myeloma cell lines and DMSO-induced myeloma cells, we found that expressions of C/EBPα and PU.1 were markedly increased by gene expression profiling. On the other hand, IL-6 down-regulated the expression of CD33 in CD33(+) myeloma cell lines accompanying with down-regulation of C/EBPα and PU.1 expressions. Also, we found that IL-6 up-regulated the expression of C-MYC and the increased C-MYC bound to the promoter region of C/EBPα gene followed by the down-regulation of C/EBPα expression. It was confirmed that introduction of sh-RNA for C-MYC to two CD33(+) myeloma cell lines blocked the IL-6-induced down-regulation of CD33 and C/EBPα expression. Therefore, these results indicate that IL-6 can reverse the ectopic expression of non-B cell lineage markers by up-regulating C-MYC followed by its down-regulation of C/EBPα expression and also suggest that constitutive activation of STAT3 by IL-6 may keep PAX-5(–) myeloma cells being uncommitted cells to any lineage; myeloma cells could be one of stem cell-like cancer cells.

Description: NF-κB has been described as a family of ubiquitously expressed transcription factors, composed of a dimer of distinct members of the Rel protein family (RelA/p65, cRel, RelB, p50, p52), and after stimulation, NF-κB dimer translocate into the nucleus to activate the transcription of various target genes. In the present study, we aimed to elucidate the mechanism of constitutive activation of NF-κB in human myeloma cells. For this purpose here, we used a human myeloma cell line, U266 cells, and performed gel shift assay and also examined the expression of different target genes of NF-κB by RT-PCR and determined the up-regulation of some of the anti-apoptopic genes (IAP-1, IAP-2, Survivin, Bcl-2, Bfl-1) and cell cycle regulators (Cox-2, Cyclin-D1, Cyclin-D2). This NF-κB-mediated gene expression provided an approach for investigating the protein expression of NF-κB family in U266. Our data revealed that the expression of p65, p50, RelB and cRel was strongly detected in the whole lysate of U266, while weak expression of p52 was found in U266. Furthermore, in the nuclear fractions of U266 cells, we strongly detected the expression of p65, RelB and p50, but not the cRel, for cRel was present in the cytoplasm. It has been recognized that dimerization is essential for NF-κB activity. Then, we performed immunoprecipitation-immunoblot analysis to demonstrate the dimer that might present in the U266, and we detected the possible dimerization such as p65-RelB in the nucleus. Therefore, these results suggest that U266 shows high constitutive activation of NF-kB, and possible hetero- and homodimer of NF-kB except for p52 may be responsible for the constitutive activation of NF-kB in U266 cells.

Description: Human primary myeloma cells show the marked heterogeneity of surface marker expression and morphology; possibly for myeloma cells lose the expression of PAX-5 and EBF, master genes of B cell lineage. As reported previously, we detected the expression of a myeloid marker, CD33, in the primary myeloma cells in about 10% out of 60 cases with overt myelomas as well as in 2 myeloma cell lines (Liu-01 and ILKM8 cells) by RT-PCR and flowcytometry using 4 different anti-CD33 antibodies. The expression of CD33 in these cells appeared to be well correlated to the monocytoid morphology with convoluted nuclei, and furthermore correlated to the increased expression of PU.1 and C/EBPα genes. Induction of CD33 expression was also found by the treatment with vitamin D3 in some primary myeloma cells as well as ILKM3 cell lines. This induction was not found in PAX-5(+) B cell lines (KUS and Raji cells). Interestingly, IL-6 down-regulated the expression of CD33 in Liu-01 and ILKM8 cell lines; if cultured with IL-6 (10 ng/ml) for 12 days, the expression of CD33 on Liu-01 cells completely disappeared. By IL-6 stimulation, the morphology of Liu-01 cells was also reversed to the morphology with round nuclei. Also, it is confirmed that IL-6 markedly suppressed the expression of PU.1 and C/EBPα genes in Liu-01 cells. Therefore, these data suggest that IL-6, a growth factor for myeloma cells, could reverse phenotypic conversion of non-B cell lineage such as CD33(+) myeloid/monocytoid cells to the B cell lineage.

Description: Human myeloma cells have the marked phenotypic heterogeneity of surface marker expressions, possibly because of loss of PAX-5 expression. Especially, ectopic expression of CD56, one of non-B cell lineage markers, is frequently detected on primary myeloma cells from more than 80% patients with overt myeloma. However, only 2 (NOP2 and AMO1) out of 10 myeloma cell lines were CD56(+). In primary myeloma cells as well as CD56(−) myeloma cell lines, the treatment with forskolin could induce the expression of CD56 in the in vitro culture. In most CD56(+) primary myeloma cells as well as myeloma cell lines, the expressions of neuronal cell markers such as neuron specific enolase (NSE), nestin, β-tubulin III or chromogranin A were found coincidentally. By gene expression profiling, CD56(+) myeloma cell lines showed the marked expressions of transcription factors involved in neuronal cell lineage. On the other hand, addition of IL-6 down-regulated the expression of CD56 in CD56(+) myeloma cell lines in the in vitro culture. In 13 out of 60 patients with overt myeloma, these myeloma cells showed CD56(−) and their values of plasma CRP were significantly increased and MPC-1(−)CD45(+) immature myeloma cells were also increased compared to those in CD56(+) myeloma cases. Therefore, these results indicate that the expression of CD56 is possibly due to phenotypic changes into neuronal cell lineage, and IL-6 can block these phenotypic changes, keeping PAX-5(−) myeloma cells being uncommitted cells to any lineage.

Description: [Purpose] It is considered that human myeloma cells have the constitutively high NF-kB activity involved in survival and proliferation. PPAR (Peroxisome proliferator-activated receptor) ß is ubiquitously expressed in all cells and considered to be involved in the lipid metabolism and regulating the inflammatory response and cell proliferation. We already have found that adrenal cortex hormones (DHEA and DHEA-S etc), dexamethasone (Dex) and baicalein augmented the expression of reporter gene. However, it remain to be clarified the role of PPARß in human myeloma cells. We focus on the mechanism of PPARß suppressed NF-kB activity. [Method] To know NF-kB activity of human myeloma cells, we performed EMSA with NF-kB consensus oligo. To investigate NF-kB and PPARß after stimulation of PPAR agonist, we did EMSA with NF-kB and PPAR consensus oligo. To confirm whether it is repression according the NF-kB activity suppression accompanying PPARß activation to the interaction of PPARß and NF-kB, we conducted immunoprecipitation - western blot analysis. To check whether the expression of NF-kB target genes (cIAP1, Bcl-xL, etc) were suppressed after stimulation of PPAR agonist, we performed RT-PCR analysis. [Result and discussion] It was suggested that human myeloma cell lines have constitutive NF-kB activity, and its activity mainly regulated p50. NF-kB activity and its target genes were repressed by stimulation of PPAR agonist. From the above, it was suggested that the activated PPARß interacted NF-kB and then its activity was suppressed.

Description: The chemokine, stromal cell-derived factor 1 (SDF-1; CXCL12) and its receptor, CXCR4 are considered to be essentially required for plasma cell homing to the bone marrow (BM). It is well known that plasma cells in the BM (long-lived plasma cells) survive for a long time and have the constitutively high NF-kB activity. Since human myeloma cells are considered to be derived from these committed long-lived plasma cells, we investigated the role of SDF-1 on the survival of primary myeloma cells from myeloma patients and the possible relationship with NF-kB activity. First, we confirmed that all primary myeloma cells expressed CXCR4 but not CCR9 or CCR10 receptors on their surface and the levels of CXCR4 expression apparently correlated with maturity of BM plasma cells; mature myeloma cells (MPC-1+) as well as polyclonal plasma cells expressed higher levels of CXCR4 than those on immature myeloma cells (MPC-1-). The production of SDF-1 was found strongly in BM stromal cells but not in primary myeloma cells as well as myeloma cell lines. On the other hand, high DNA binding activity of NF-kB was constitutively detected in primary myeloma cells as well as myeloma cell lines, and these NF-kB activities significantly correlated with the expression levels of CD54 on their surface, for CD54 gene is one of the strict NF-kB target genes. Based on the expression levels of CD54 protein, interestingly, primary myeloma cells showed weaker NF-kB activities than those in monoclonal plasma cells from MGUS and polyclonal plasma cells from polyclonal gammopathy. Plasma concentrations of SDF-1 were also significantly correlated to the expression levels of CD54 on primary myeloma cells significantly (P

Description: [Purpose] NF-kB has a key function in the transformation, proliferation and invasion of cancer cells as well as in resistance to radiotherapy and chemotherapy. Although constitutive NF-kB activation has been reported in many human tumors, the underlying factors and mechanisms responsible for constitutive NF-kB activation in myeloma cells has not been known. The purpose of this study was to determine the mechanism of constitutive NF-kB activity in myeloma cell lines and quantification of NF-kB activity in primary myeloma cells by expression of CD54 (a NF-kB target gene). [Methods] We checked the constitutive expression of NF-kB family proteins by western blot analysis and possible dimer formation of different NF-kB family members by Immunoprecipitation-western blot reaction. Constitutive NF-kB DNA-binding activity and dimers that are responsible for NF-kB activity were analyzed by electrophoretic mobility shift assay (EMSA). Moreover, expression of different NF-kB target genes was done by RT-PCR. [Results and discussion] Constitutive NF-kB activity was determined in myeloma cell lines and our results suggested that p50/RelB, p50/p50, p50/p52 dimers might be responsible for this. We also analyzed several NF-kB target gene expressions and found that the intensity of CD54 expression was positively correlated with total NF-kB DNA binding activity. Although there is no method to quantified NF-kB activity, it can be determine in terms of its target genes e.g. CD54. Therefore, this study provide the frame work for understanding the molecular mechanism of constitutive activation of NF-kB and would help to quantify NF-kB activation in primary myeloma cells.

Description: In order to evaluate NF-kB activity in the primary myeloma cells, we examined the expressionlevels of CD54 by a flow cytometer. First, we confirmed that the expression levels of CD54 showed good correlation with the levels of DNA binding activity for NF-kB in human myeloma cell lines. Primary myeloma cells unexpectedly revealed lower expression levels of CD54 compared to normal BM plasma cells, while plasma cells from MGUS had high expression levels of CD54 as same as normal BM plasma cells. Furthermore, the expression levels of CD54 on these plasma cells showed the significantly positive correlation with the plasma levels of CXCL12 (SDF-1α) in their BM aspirates, and the expression levels of CXCR4, the receptor for CXCL12, decreased on primary myeloma cells compared to normal BM plasma cells. It was also confirmed that addition of CXCL12 in the in vitro culture significantly induced up-regulation of CD54 expressions in primary myeloma cells. Furthermore, myeloma cells with lower expressions of CD54 were unstable in the in vitro culture, resulting in the marked reduction of viable cell number; this was also the case where cell lines with lower or no expression of CD54 were sensitive to H2O2 treatment. Therefore, these data suggest that primary myeloma cells have lower expression levels of CD54, possibly lower NF-kB activities and different stability of in vitro survival from normal BM plasma cells, depending on expression levels of CD54.

Description: To elucidate the mechanism of constitutive NF-κB activity in human myeloma cell lines, constitutive expression of all the NF-κB family, RelA (p65), c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52), genes in different human myeloma cell lines, such as IL-6-independent (AMO1, NOP2), IL-6-dependent (ILKM2, ILKM8) and IL-6-responsive cell line (U266), were first checked by RT-PCR and western blot analysis. In the electrophoretic mobility shift assay (EMSA) for κB main consensus, total DNA binding activity levels for NF-κB were higher in IL-6-dependent, IL-6-responsive and IL-6-independent human myeloma cell lines respectively. Supershift analysis with anti-NF-κB family antibodies and immunoprecipitation followed by western blotting, confirmed that p50/p50, p50/RelB and p50/p52 as the principal player responsible for constitutive activation of NF-κB in IL-6-independent cell lines. However, beside these major dimers, p50/p65 dimer was also involved in the constitutive NF-κB activation in IL-6-dependent and IL-6-responsive cell lines. Moreover, on the basis of microarray data in human myeloma cell lines, we confirmed that the expression levels of NF-κB target genes, such as NFKB2, RELB, NFKBIA, MALT1 and CD54, showed correlation with the level of DNA binding activity for NF-κB. Therefore, these data suggest that the difference of total DNA-binding activities for NF-κB partly depended on the different p50/p65 activity among the IL-6-independent, IL-6-dependent and IL-6-responsive human myeloma cell lines, and further studies in the different NF-κB-dimers activity levels might contribute to the clarification of the regulation of NF-κB target genes expression in human myeloma cell lines.