MYC Responsible for down-Regulation of CD33 Expression in Human Myeloma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5116-5116
Author(s):  
Karim Shamsasenjan ◽  
Ken-ichiro Otsuyama ◽  
Mohd S. Iqbal ◽  
Maged S. Mahmoud ◽  
Michio M. Kawano
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Down Regulation ◽  
Myeloma Cells ◽  
Expression Levels ◽  
Stat3 Phosphorylation ◽  
Serum Crp ◽  
Human Myeloma Cells

Abstract Human myeloma cells from about 10% of cases with multiple myeloma expressed CD33 and have monocytoid morphology with convoluted nuclei, and all these patients had no increase in serum CRP values. In CD33(+) myeloma cells as well as myeloma cell lines, CD33 expression levels were correlated with the increased expression levels of CEBPA (C/EBPα) gene. This correlation was confirmed by the finding that transfection with the CEBPA gene induced CD33 expression in a CD33(−) myeloma cell line. As suggested by the lack of an increase in serum CRP values in CD33(+) myelomas, IL-6 down-regulated the expression of CD33 in CD33(+) myeloma cell lines along with the down-regulation of CEBPA gene expression. Cucurbitacin I (STAT3 inhibitor) but not U0126 (MAPK inhibitor) could abolish the effect of IL-6. Furthermore, IL-6 up-regulated the expression of MYC via STAT3 phosphorylation and MYC bound to the promoter region of CEBPA gene followed by the down-regulation of the CEBPA expression. It was confirmed that introduction of shRNA for MYC into a CD33(+) myeloma cell line blocked the IL6-induced down-regulation of CD33 and CEBPA expression. Therefore, these results indicate that IL-6 can reverse the expression level of CD33 by up-regulating MYC followed by the down-regulation of CEBPA expression.

Molecular Mechanism of CD56 Expression in Human Myeloma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5130-5130
Author(s):  
Mohd S. Iqbal ◽  
Ken-ichiro Otsuyama ◽  
Karim Shamsasenjan ◽  
Michio M. Kawano
Keyword(s):  
Transcription Factors ◽  
Cell Line ◽  
Cell Lines ◽  
Promoter Region ◽  
Cell Lineage ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Down Regulation ◽  
Myeloma Cells ◽  
Cd56 Expression

Abstract CD56 (NCAM1), one of the non-B cell lineage markers, is frequently detected on primary myeloma cells from more than 80% patients with overt myeloma. Microarray analysis of the CD56(+) myeloma cell lines showed markedly increased expressions of transcription factors involved in the neuronal cell lineage compared to the CD56(−) myeloma cell lines. There are three important binding sites of transcription factors such as HOX, SOX and PAX, present in the CD56 promoter region. Furthermore, ChIP analysis showed the specific bindings of HOXB9 and SOX1 in the CD56 promoter region of both the CD56(+) and CD56(−) myeloma cell line, while there was no significant difference observed in the PAX8 expression between the CD56(+) and CD56(−) myeloma cell lines. RT-PCR and western blot analysis confirmed that HOXB3 and HOXB9 as well as some of their potential cofactors were up-regulated in the CD56(+) myeloma cell lines with the concomitant down-regulation of the SOX1 and OCT3/4. First, we focused on the role of down-regulation of SOX1 in the CD56(−) myeloma cell line by the shRNA transfection. However, shRNA against SOX1 failed to induce CD56 expression in CD56(−) myeloma cell line. Therefore, these results indicate that the down-regulation of SOX1 alone may not be enough for the CD56 induction which might also require the overexpression of either HOXB9 or HOXB3, and the further study in the molecular mechanism of CD56 expression might contribute to the understanding of complex network of the interaction of transcription factors in human myeloma cells.

SDF-1 Is Responsible for the Constitutively High NF-kB Activity in Human Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4737-4737
Author(s):  
Abul Islam ◽  
Ken-ichiro Otsuyama ◽  
Jakia Amin ◽  
Saeid Abroun ◽  
Karim Shamsasenjan ◽  
...  
Keyword(s):  
Cell Lines ◽  
Target Genes ◽  
Plasma Cells ◽  
Plasma Concentrations ◽  
Myeloma Cell ◽  
Binding Activity ◽  
Myeloma Cells ◽  
Expression Levels ◽  
Human Myeloma Cells

Abstract 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<0.01). Furthermore, it was confirmed that addition of SDF-1 significantly increased the expression levels of CD54 in the in vitro culture of primary myeloma cells. Therefore, these results indicate that SDF-1 is responsible for high expression levels of CD54 and possibly the constitutively high NF-kB activity in primary myeloma cells.

Sphingolipids as a novel target for the treatment of multiple myeloma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e19534-e19534
Author(s):  
Yubin Kang ◽  
Jagadish Kummetha Venketa
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Myeloma Cell ◽  
Sphingolipid Metabolism ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Novel Target

e19534 Background: Multiple myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ~10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding the disease’s molecular pathways and for identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in tumor cell proliferation and in tumor sensitivity to anticancer drugs. We hypothesize that altered sphingolipid metabolism plays an important role in the pathogenesis of MM, thus providing a novel target in the treatment of MM. Methods: We first assayed sphingolipid metabolism including sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+myeloma cells, and in publically available dataset. We then tested the efficacy of the selective SK2 inhibitor (ABC294640) and the SK2 shRNA in killing myeloma cells in vitro. Results: 1) Compared to immortalized B cells, the levels of pro-apoptotic ceramides were decreased whereas the proliferative sphingosine 1-phosphate (S1P) was increased in myeloma cell lines. 2) The expression of several key sphingolipid-metabolizing genes including sphingosine kinase (SK) 1 and 2 was altered in freshly isolated human primary bone marrow myeloma cells and in publically available microarray dataset. 3) The selective SK2 inhibitor (ABC294640) induces apoptotic cell death and inhibits myeloma cell growth with an IC50of ~20 μM in 9 myeloma cell lines. 4) Interestingly, OPM-1 myeloma cell line was extremely sensitive to ABC294640 with an IC50of <5 µM whereas U266 myeloma cell line was resistant to ABC294640. SK2 shRNA induced apoptotic cell death in OPM-1, but not in U266 cells. We are currently investigating the molecular mechanisms underlying the resistance of U266 myeloma cells to ABC294640. Conclusions: Our data demonstrated that sphingolipid metabolism provides an attractive target in the treatment of refractory/relapased multiple myeloma.

HLA-Cw6 Alloreactive CTL Recognizing HLA-CW6+ Myeloma Cells but Not Non-Myeloma HLA-Cw6+ Cells Are Peptide Dependent.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2474-2474
Author(s):  
Alexandrine Geffroy ◽  
Agnes Moreau-Aubry ◽  
Regis Bataille ◽  
Catherine Pellat-Deceunynck
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cytotoxic T Lymphocytes ◽  
Myeloma Cell ◽  
Unrelated Donor ◽  
Myeloma Cell Line ◽  
Specific Lysis ◽  
Myeloma Cells ◽  
Target Ratio ◽  
Effector Target

Abstract We investigated the generation of myeloma-specific cytotoxic T lymphocytes (CTL) from unrelated normal donors non-HLA matched with the myeloma cell line SBN. The aim was to obtain alloreactive CTL specific of peptides to identify genes preferentially expressed by to myeloma cells. After coculture of SBN with PBL of unrelated donor, the T-cell line obtained was cloned and each CTL was assessed against SBN and SB-EBV (B-EBV cell line obtained by infecting B cells of SB patient with EBV) simultaneously. Only the CTL recognizing SBN but not SB-EBV were kept for further study. Among more than 200 clones screened, we isolated two different CTL CD8+ recognizing SBN only (60% of specific lysis at effector: target ratio 5). Their cytotoxicity was blocked by antibodies against HLA-I molecules and more precisely by mAb against HLA-B/Cw molecules. Both CTL recognized also other MM cell lines but only those matched with SBN for HLA-Cw0602 (XG6, BCN) suggesting an HLA-Cw6 recognition. Furthermore, antiserum directed against HLA-Cw6 abrogated the recognition of SBN, XG6 and BCN. Both CTL were not cytotoxic against BC-EBV nor against 3 other B-EBV cell lines derived from HLA-Cw0602 donors. This unreactivity was not restricted to B-EBV cell lines since PBL from 2 normal HLA-Cw0602 donors were also not recognized (cytotoxicity, TNF secretion). We directly measured cell surface HLA-Cw6 expression of both B-EBV and myeloma cells with a scFv directed against HLA-Cw6 and we showed that HLA-Cw6 was more expressed by B-EBV cells as compared to myeloma cells (2fold). So, the lack of recognition of B-EBV cells was not related to HLA-Cw6 expression level. We also looked for KIR expression by the CTL since KIR molecules could have blocked B-EBV recognition that expressed more HLA-Cw6 molecules as compared to myeloma cells. No KIR molecules (p58.1, p58.2, CD94) were found to be expressed by the CTL. To investigate whether the recognition of myeloma cells was peptide dependent, we performed two experiments i.e., acid elution and cold target competition. Acid elution (glycine 0.3M, pH=2.5) abrogated myeloma recognition and cold target competition showed that B-EBV cells were not recognized by the CTL. These data suggest that the CTL recognize a peptide or a set of peptides restricted to myeloma cells or that the CTL recognition is activated by tumor cells only. Anyway, such alloreactive CTL could be helpful for GVL reaction in allotransplanted patients with myeloma.

scholarly journals Altered expression of Pax-5 gene in human myeloma cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4311-4315 ◽  
Author(s):  
MS Mahmoud ◽  
N Huang ◽  
M Nobuyoshi ◽  
IA Lisukov ◽  
H Tanaka ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Altered Expression ◽  
Myeloma Cells ◽  
Normal Plasma ◽  
Human Myeloma Cells

Recent phenotypic analysis of plasma cells showed that normal plasma cells do express the B-cell lineage-specific molecule CD19, but their malignant counterpart (myeloma cells) are CD19-. To clarify the meaning of loss of CD19 antigen on myeloma cells, we first compared the expression of CD19 and Pax-5 genes among B cells, normal plasma cells, myeloma cell lines, and primary myeloma cells, because the Pax-5 gene was reported to encode the transcriptional factor, B-cell-specific activating protein (BSAP), necessary for CD19 gene expression. Neither CD19 nor Pax-5 mRNA could be detected in those primary myeloma cells and cell lines, whereas normal plasma cells did express both CD19 and Pax-5 mRNA. Furthermore, we could confirm that BSAP-binding activity was not detected in the nuclear extract from CD19- myeloma cell line (KMS-5) but was detected in CD19+ B-cell line (Raji) by gel-shift assay. We further examined the expression of E2A and Id genes, because E2A and Id are considered to be positive and negative regulators in the expression of Pax-5 gene, respectively. However, no significant differences in the expression of these E2A and Id-2 genes could be observed between myeloma cells and normal plasma cells. Therefore, these data suggest that the altered expression of Pax-5, but not E2A or Id, is responsible for the loss of CD19 expression in human myeloma cells, although the underlying mechanism of the altered Pax-5 gene expression remains to be clarified.

scholarly journals Heterogeneous expression of a novel MPC-1 antigen on myeloma cells: possible involvement of MPC-1 antigen in the adhesion of mature myeloma cells to bone marrow stromal cells

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3721-3729 ◽  
Author(s):  
N Huang ◽  
MM Kawano ◽  
H Harada ◽  
Y Harada ◽  
A Sakai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cell Line ◽  
Bone Marrow Stromal Cells ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Human Myeloma Cell Line ◽  
Heterogeneous Expression

Abstract Recent immunophenotypic analysis has shown that the heterogeneous expression of the adhesion molecule VLA-5 classifies myeloma cells into VLA-5+ mature and VLA-5- immature subpopulations. To further clarify the two myeloma subpopulations, we generated a monoclonal antibody, MPC- 1, by immunizing mice with an adherent human myeloma cell line, KMS-5. The MPC-1 antibody recognized a 48-Kd surface antigen on KMS-5 but not on U-266, a nonadherent human myeloma cell line. Specificity characterization showed that MPC-1 antigen was expressed on mature myeloma cells, normal plasma cells, and mature B cells, whereas pre-B cells and germinal center B cells lacked its expression. Monocytes and a human bone marrow stromal cell line, KM102, also expressed this antigen. Two subclones of MPC-1+ VLA-5+ (KMS-5Ad) and MPC-1-VLA-5+ (KMS- 5NAd) were separated from the KMS-5 cell line. The KMS-5NAd adhered to KM102 more tightly than did the KMS-5NAd, and the U-266 (MPC-1-VLA-5-) displayed almost no adherence to the KM102. The adhesion of the KMS-5Ad was partially inhibited by the MPC-1 antibody. These results, taken together, suggest that the MPC-1 antigen serves as a differentiation marker for B-lineage cells, including plasma cells, and may function as an adhesion molecule involved in the interaction of mature myeloma cells with bone marrow stromal cells.

Enforced CD19 Expression Leads to Growth Inhibition and Reduced Tumorigenicity

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3551-3558 ◽  
Author(s):  
Maged S. Mahmoud ◽  
Ryuichi Fujii ◽  
Hideaki Ishikawa ◽  
Michio M. Kawano
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Myeloma Cell ◽  
Inhibitory Effect ◽  
Myeloma Cell Line ◽  
Growth Inhibitory Effect ◽  
Myeloma Cells ◽  
Growth Inhibitory ◽  
Human Myeloma Cell Line

In multiple myeloma (MM), the cell surface protein, CD19, is specifically lost while it continues to be expressed on normal plasma cells. To examine the biological significance of loss of CD19 in human myeloma, we have generated CD19 transfectants of a tumorigenic human myeloma cell line (KMS-5). The CD19 transfectants showed slower growth rate in vitro than that of control transfectants. They also showed a lower capability for colony formation as evaluated by anchorage-independent growth in soft agar assay. The CD19 transfectants also had reduced tumorigenicity in vivo when subcutaneously implanted into severe combined immunodeficiency (SCID)-human interleukin-6 (hIL-6) transgenic mice. The growth-inhibitory effect was CD19-specific and probably due to CD19 signaling because this effect was not observed in cells transfected with a truncated form of CD19 that lacks the cytoplasmic signaling domain. The in vitro growth-inhibitory effect was confirmed in a nontumorigenic human myeloma cell line (U-266). However, introduction of the CD19 gene into a human erythroleukemia cell line (K-562) also induced growth inhibition, suggesting that this effect is CD19-specific, but not restricted to myeloma cells. These data suggest that the specific and generalized loss of CD19 in human myeloma cells could be an important factor contributing to the proliferation of the malignant plasma cell clones in this disease.

Expression of TRAIL Receptors on the Multiple Myeloma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4868-4868
Author(s):  
Juan Li ◽  
Junhe Li ◽  
Shaokai Luo ◽  
Yin Zhao
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Mononuclear Cells ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Killing Effect ◽  
Myeloma Cells ◽  
Decoy Receptors ◽  
Trail Receptors ◽  
Chemotherapy Agents

Abstract Objective To study the different expression of death receptors and decoy receptors on mononuclear cells from patients with multiple myeloma and myeloma cell line KM3 and compare the different expression of TRAIL receptors after chemotherapy or exposure to doxorubicin, to explore the mechanisms by which TRAIL selectively kills tumor cells. Methods Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry was used to investigate the expression of four receptors on mononuclear cells in 23 multiple myeloma patients and myeloma cell line KM3 and 15 controls, we furthermore compared the changes of expression mode after chemotherapy and incubation of KM3 cell with sub-clinical concentration of Doxorubicin. Results There finds only DR4 and DR5 on KM3 cell line without the expression of DcR1 and DcR2. Expression of DR4 and DR5 on mononuclear cells of MM patients is higher than that of controls (P&lt;0.05), but DcR1 and DcR2 expression was lower than that of controls (P&lt;0.05), after chemotherapy and exposure to Doxorubicin, the expression of DR5 on MM cells was up-regulated (P&lt;0.05) Conclusions The expression of four receptors on myeloma cells and normal controls was significantly different, which might account for the selective killing effect of TRAIL on MM cells. DR5 was up-regulated on KM3 when incubating with Doxorubicin and after chemotherapy which suggests chemotherapy agents might enhance the apopotosis of MM cells through up-regulating of DR5 receptor.

Multiple Myeloma: Interleukin-15 Antagonizes Bone Morphogenetic Protein-Induced Apoptosis and Growth Inhibition.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3444-3444
Author(s):  
Magne Rekvig ◽  
Anne-Tove Brenne ◽  
Torstein Baade Ro ◽  
Anders Waage ◽  
Magne Borset ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Growth Inhibition ◽  
Cell Line ◽  
Caspase 3 ◽  
Myeloma Cell ◽  
Stimulus Figure ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract Multiple myeloma has two distinct features: Expansion of malignant plasma cells within the bone marrow accompanied by skeletal destruction. Bone morphogenetic proteins (BMPs) have been shown to induce apoptosis and inhibit growth in myeloma cells. BMPs are members of the TGF-β superfamily of proteins capable of inducing bone formation, and regulate proliferation, differentiation and apoptosis. We have investigated myeloma cell apoptosis and proliferation with BMP-4 and −6 in concert with the myeloma cell growth factors interleukin (IL)-2, IL-6, IL-10, IL-15, IL-21, tumor necrosis factor (TNF)-α and insulin-like growth factor (IGF)-1. Eight samples of highly purified myeloma cells from patients and a human myeloma cell line, IH-1 (Brenne AT et al. Blood. 2002 May 15;99(10):3756–62.), were used in this study. Cytokine concentrations used in the referred experiments were for BMP-4 20ng/ml, BMP-6 250ng/ml, IL-15 20ng/ml and IL-6 0,1ng/ml, respectively. Growth inhibition was measured in a proliferation assay by methyl-[3H]-thymidine incorporation and apoptosis by annexin V- FITC-binding/PI-uptake on flow cytometry. IL-15 antagonized growth inhibition (Figure A) and prevented apoptosis induced by BMP-4 (Figure B) and BMP-6 in the myeloma cell line IH-1. IL-15 also antagonized the growth inhibition induced by BMP-4 and/or BMP-6 in three out of eight patient samples. Neither IL-6, nor any of the other investigated cytokines were able to rescue the myeloma cells from growth inhibition and apoptosis induced by BMP-4 and -6. Among the investigated cytokines, we found that IL-15 has a unique capability to antagonize BMP- induced apoptosis and growth inhibition in myeloma cells. We examined cleavage of the proapoptotic protein caspase-3 and found that BMP-4 activated caspase-3 in the IH-1 cell line. This activation of caspase-3 was blocked by IL-15 but not by IL-6. We have demonstrated a possible mechanism for myeloma cells to escape apoptosis and growth-inhibition within the bone marrow. Intramedullar levels of IL-15 and BMPs may play a role in the pathogenesis of multiple myeloma. Figure A. Proliferation in response to BMP-4 stimulus Figure A. Proliferation in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus Figure B. Apoptosis in response to BMP-4 stimulus

PU.1 Is Downregulated in a Subset of Multiple Myeloma by the Methylation of Its -17 kb Upstream Regulatory Region and the Promoter.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3417-3417
Author(s):  
Yutaka Okuno ◽  
Hiro Tatetsu ◽  
Shikiko Ueno ◽  
Hiroyuki Hata ◽  
Yasuhiro Yamada ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Promoter Region ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Upstream Region ◽  
Cell Growth Arrest ◽  
Myeloma Cells ◽  
Expression Levels

Abstract It has been reported that disruption of transcription factors critical for hematopoiesis, such as C/EBPa and AML1, is involved in leukemogenesis. PU.1 is a transcription factor important for both myeloid and lymphoid development. We reported that mice in which the levels of PU.1 were 20% of that of wild-type developed acute myeloid leukemia, T cell lymphoma, and a CLL-like disease. These findings strongly suggest that PU.1 has tumor suppressive activity in multiple hematopoietic lineages. Last year, we reported that PU.1 is downregulated in a majority of multiple myeloma cell lines and and freshly isolated CD138 positive myeloma cells from certain number of myeloma patients, and that tet-off inducible exogenous expression of PU.1 in PU.1 negative myeloma cell lines induced cell growth arrest and apoptosis. Based on their PU.1 expression levels, we divided the myeloma patients into two groups, namely PU.1 high and PU.1 low-to-negative, (cutoff index of 25th percentile of the PU.1 expression level distribution among all patients). The PU.1 low-to-negative patients had a significantly poorer prognosis than the PU.1 high patients. To elucidate the mechanisms of downregulation of PU.1, we performed sequence and epigenetic analysis of the promoter region and the -17 kb upstream region that is conserved among mammalians and important for proper expression of PU.1. There are no mutations in these regions of all five myeloma cell lines. In contrast, the -17 kb upstream region was highly methylated in 3 of 4 PU.1 negative myeloma cell lines, while the promoter region was also methylated to various levels in all five myeloma cell lines including one PU.1 positive cell line. These data suggested that the downregulation of PU.1 in myeloma cell lines might be dependent on the methylation of both regulatory regions of PU.1 gene, especially the -17 kb upstream region. We also evaluated the mechanisms of cell growth arrest and apoptosis of myeloma cell lines induced by PU.1. Among apoptosis-related genes, we identified that TRAIL was upregulated after PU.1 induction. To evaluate the effect of upregulation of TRAIL, we stably introduced siRNA for TRAIL into myeloma cell lines expressing PU.1, and we found that apoptosis of these cells was partially suppressed by siRNA for TRAIL, suggesting that apoptosis of myeloma cells induced by PU.1 might be at least partially due to TRAIL upregulation. We are currently performing DNA microarray analysis to compare the expression levels of genes between before and after PU.1 induction, in order to further elucidate the mechanisms of cell growth arrest and apoptosis.

Sign in / Sign up

Export Citation Format

Share Document