Bmps(Bone Morphogenetic Proteins) Inhibit Growth in Multiple Myeloma Cells by p53 Activation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3354-3354
Author(s):  
Anders Sundan ◽  
Torstein B. Ro ◽  
Janne Bonhorst ◽  
Anders Waage ◽  
Magne Borset
Keyword(s):  
Multiple Myeloma ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Target Genes ◽  
Myeloma Cell ◽  
Resistant Cell ◽  
P53 Mutation ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Myeloma Cells

Abstract Only a few naturally occurring cytokines are able to inhibit myeloma cell growth, and among them BMP-4 is a potent inhibitor of growth as well as an inductor of apoptosis in myeloma cells. To study the mechanism behind BMP-4-induced growth inhibition, we performed gene expression profiling by microarrays in two human myeloma cell lines after BMP-4 stimulation for 4 hours. We found that BMP-4 upregulated several known p53 target genes like p21/Cip1, Bax, cyclin G, Gadd45 and dual specificity phosphatases, suggesting a role for p53 in BMP-mediated growth inhibition of myeloma cells. p53 is activated by several post-translational modifications, including phosphorylation at several serine/threonine residues. We found that BMP-4 treatment lead to rapid phosphorylation of p53 at Serine 15. Furthermore, we were able to co-precipitate phosphorylated Smad-1/5/8 and p53 from nuclear extracts of cells treated with BMP by applying a p53 binding element from the Gadd45 promoter. Because some myeloma cell lines are resistant for BMP-4-induced growth inhibition, we examined whether this BMP-4 resistance was associated with p53 mutations in the BMP-4-resistant cell lines. We sequenced the p53 cDNA from eight myeloma cell lines, and resistant cells all had mutated or deleted p53. Introduction of normal p53 by adenoviral constructs in the p53 mutated cell lines restored BMP-4 responsiveness to some degree. Furthermore, in a myeloma cell line with a temperature-sensitive p53 mutation, the cells were sensitive towards BMP-induced growth inhibition at the permissive temperature but resistant when p53 was inactive. In conclusion, we show that normal p53 activity is involved in BMP-4-induced growth inhibition of multiple myeloma cells.

Perifosine Induces DR4/DR5 Expression Leading to Apoptosis That Can Be Enhanced with Exogenous TRAIL, and Blocked with Strategies Directed at DR4/DR5 Inhibition.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3446-3446 ◽  
Author(s):  
Ebenezer David ◽  
Rajni Sinha ◽  
Jonathan L. Kaufman ◽  
Sagar Lonial
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Rt Pcr ◽  
Myeloma Cells

Abstract Background: Perifosine is an oral AKT inhibitor which exerts a marked cytotoxic effect on human tumor cell lines. It is currently being tested in several phase II trials for the treatment of major cancers including multiple myeloma. While the proposed mechanism of action relates to downregulation of AKT expression, overepxression of constitutively active AKT does not abrogate perifosine induced cell death suggesting alternative mechanisms. Hypothesis: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2 ligand) effectively kills multiple myeloma cells in vitro after binding to their membrane specific receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). It is our hypothesis that DR4/DR5 upregulation occurs in response to perifosine treatment, and thus may be additive with exogenous TRAIL. Materials and Methods: TRAIL-sensitive myeloma cell lines (MM.1S, RPMI8226, MM.1R) and TRAIL- resistant myeloma cell lines (U266) were used in this study. Apoptosis was assessed by annexin-V binding assay using flow-cytometry and cell death was assessed by MTT assay. Recombinant human TRAIL, chimeras of DR4 and DR5 were obtained from R&D systems. Results: Perifosine alone(5μM and 10μM) induced apoptosis of MM.1S in 40% and 50% of the treated cells as measured by flow cytometry, that increased to 81% and 91% when 50ng/ml of TRAIL was added to 5μM and 10 μM of perifosine. TRAIL alone induced only nominal apoptosis. Use of the TRAIL resistant U266 cell line showed only minimal apoptosis in response to perifosine, TRAIL, or the combination of both agents. Perifosine also induced DR4 and DR5 expression in less than 2hrs upon the Perifosine exposure in MM.1S as shown by RT-PCR. The combination of perifosine and TRAIL was not sequence specific. Furthermore, we observed that the enhanced apoptosis induced by perifosine and TRAIL in combination was almost or partially blocked by the administration of the DR4 and DR5 blocking antibodies only in the case of MM.1S, MM.1R, RPMI8226 TRAIL sensitive cells lines. Apoptosis was completely blocked in the case of U266 TRAIL resistant cell line when the chimera antibodies were used with perifosine alone or in combination with TRAIL. Conclusion and future directions: Perifosine, an agent proposed to function via inhibition of p-AKT and PDK-1, may have other effects on cell cycle regulation and it pro-apoptotic effects may be partially related to the TRAIL pathway. Our data suggests that an additional mechanism of action relates to the effect perifosine has on DR4 and DR5 expression thus directly effecting apoptosis via the TRAIL mediated effects. The limited response the trail resistant cell line U266 cells suggest that the TRAIL resistant myeloma cells have less DR4 or DR5 surface receptors as compared to the TRAIL sensitive cell lines, MM.1S, MM.1R, and RPMI8226 further validating this alternative mechanism. Further experiments such as inhibition of DR4, DR5, and FADD by small interfering RNAs, RT-PCR, the response in primary myeloma cells and also using more TRAIL resistant cell lines to support our preliminary observations are currently in progress.

scholarly journals NCOR2 Mutations Are Associated with IMiD Resistance in Human Myeloma Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4
Author(s):  
Tomoaki Mori ◽  
Cristina Panaroni ◽  
Chukwuamaka Onyewadume ◽  
Noopur S. Raje
Keyword(s):  
Cell Lines ◽  
Protein Interaction ◽  
Enrichment Analysis ◽  
Myeloma Cell ◽  
Resistant Cell ◽  
Ppi Network ◽  
Myeloma Cells ◽  
Knock Out ◽  
Protein Protein Interaction ◽  
Bet Inhibitor

The immunomodulatory drug thalidomide, and its analogs, lenalidomide, and pomalidomide (IMiDs) have significantly changed the treatment paradigm of multiple myeloma (MM). Despite this progress, IMiD resistance develops in the majority of patients resulting in the development of refractory disease. Cereblon (CRBN), a direct target, has been implicated in IMiD resistance. However, alternate mechanisms of IMiD resistance independent of CRBN remain largely unknown. To understand and study the mechanisms responsible for the development of IMiD resistance, we created lenalidomide-resistant (Len-R) and pomalidomide-resistant (Pom-R) human myeloma MM.1s cell lines, by continuous culture in the presence of lenalidomide or pomalidomide for 3 months. Whole genome sequencing of these 2 resistant cell lines compared with parental MM.1s revealed 172 genes with exonic mutations in both Len-R and Pom-R myeloma cells. Furthermore, a protein-protein interaction (PPI) network was constructed based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The PPI network demonstrated 8 genes that scored a high degree of protein-protein interaction. Among these genes, we identified NCOR2, a corepressor that negatively regulates gene expression, as a downregulated gene in resistant cell lines. To study this further, we created NCOR2 knock out MM.1s cell lines using CRISPR/cas9 gene modification. Our data demonstrates that depletion of NCOR2 confers IMiD resistance independent to CRBN. Interestingly, Len-R, Pom-R and NCOR2 knock out MM.1s showed increased MYC protein expression, which is essential for myeloma cell survival and proliferation. A BET inhibitor, known to disrupt the binding of BRD4 to chromatin, inhibited the proliferation of Len-R and Pom-R and NCOR2 knock out MM.1s by completely suppressing MYC expression. These results indicate that NCOR2 down regulation in IMiD resistant cells induces MYC upregulation which may in part result in IMiD resistance. Our findings reveal a novel molecular mechanism associated with IMiD resistance, independent of CRBN and suggest that NCOR2-MYC pathway may be a new target for IMiD refractory patients. Disclosures Raje: Celgene: Consultancy.

scholarly journals An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti–IL-6 antibody-induced apoptosis

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1829-1837 ◽  
Author(s):  
Karène Mahtouk ◽  
Michel Jourdan ◽  
John De Vos ◽  
Catherine Hertogh ◽  
Geneviève Fiol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Egf Receptor ◽  
Myeloma Cell ◽  
Factor Activity ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

CD40 activation mediates p53-dependent cell cycle regulation in human multiple myeloma cell lines

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 1039-1046 ◽  
Author(s):  
G. Teoh ◽  
Y.-T. Tai ◽  
M. Urashima ◽  
S. Shirahama ◽  
M. Matsuzaki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Luciferase Reporter ◽  
Temperature Sensitive ◽  
Multiple Myeloma Cell ◽  
Cd40 Activation ◽  
Human Multiple Myeloma ◽  
Rpmi 8226

It has been reported that the activation of multiple myeloma (MM) cells by CD40 induces proliferation, growth arrest, and apoptosis. To determine whether the biologic sequelae of CD40 activation in MM cells depends on p53 function, we identified temperature-sensitive p53 mutations in the RPMI 8226 (tsp53E285K) and the HS Sultan (tsp53Y163H) MM cell lines. These cells were then used as a model system of inducible wtp53-like function because wild-type-like p53 is induced at permissive (30°C) but not at restrictive (37°C) temperatures. Using p21-luciferase reporter assays, we confirmed that CD40 induces p53 transactivation in RPMI 8226 and HS Sultan cells cultured under permissive, but not restrictive, conditions. Furthermore, CD40 activation of these MM cells under permissive, but not restrictive, temperatures increased the expression of p53 and p21 mRNA and protein. Importantly, CD40 activation induced the proliferation of RPMI 8226 and HS Sultan cells at restrictive temperatures and growth arrest and increased subG1 phase cells at permissive temperatures. These data confirmed that CD40 activation might have distinct biologic sequelae in MM cells, depending on their p53 status.

Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 610-618 ◽  
Author(s):  
Inge Tinhofer ◽  
Ingrid Marschitz ◽  
Traudl Henn ◽  
Alexander Egle ◽  
Richard Greil
Keyword(s):  
Multiple Myeloma ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Constitutive Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Cytotoxic Treatment ◽  
Interleukin 15 ◽  
Induced Apoptosis

Interleukin-15 (IL-15) induces proliferation and promotes cell survival of human T and B lymphocytes, natural killer cells, and neutrophils. Here we report the constitutive expression of a functional IL-15 receptor (IL-15R) in 6 of 6 myeloma cell lines and in CD38high/CD45low plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, we detected IL-15 transcripts in all 6 myeloma cell lines, and IL-15 protein in 4/6 cell lines and also in the primary plasma cells of 8/14 multiple myeloma patients. Our observations confirm the existence of an autocrine IL-15 loop and point to the potential paracrine stimulation of myeloma cells by IL-15 released from the cellular microenvironment. Blocking autocrine IL-15 in cell lines increased the rate of spontaneous apoptosis, and the degree of this effect was comparable to the pro-apoptotic effect of depleting autocrine IL-6 by antibody targeting. IL-15 was also capable of substituting for autocrine IL-6 in order to promote cell survival and vice versa. In short-term cultures of primary myeloma cells, the addition of IL-15 reduced the percentage of tumor cells spontaneously undergoing apoptosis. Furthermore, IL-15 lowered the responsiveness to Fas-induced apoptosis and to cytotoxic treatment with vincristine and doxorubicin but not with dexamethasone. These data add IL-15 to the list of important factors promoting survival of multiple myeloma cells and demonstrate that it can be produced and be functionally active in an autocrine manner.

Induction of Multiple Myeloma-Specific Cytotoxic T Lymphocytes Using HLA-A2.1-Specific CD19 and CD20 Peptides.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2477-2477
Author(s):  
Jooeun Bae ◽  
Jeff A. Martinson ◽  
Hans G. Klingemann ◽  
Steven Treon ◽  
Kenneth C. Anderson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Lymphocytes ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Cytotoxic T Lymphocytes ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Specific Ctls ◽  
Ifn Γ ◽  
Hla A2.1

Abstract We have identified novel CD19 and CD20 antigen-derived HLA-A2.1-specific immunogenic peptides, CD19150–158 (KLMSPKLYV) and CD20188–196 (SLFLGILSV), for generating cytotoxic T lymphocytes (CTLs) against malignant B-cell diseases. Initial testing showed that the CTLs displayed antigen-specific and HLA-A2.1-restriced cytotoxic activity against both Burkitt’s lymphoma and chronic lymphoid leukemia cell lines. The observed cytotoxic activity of the CTLs was shown to be specific to the CD19150–158 or the CD20188–196 peptides. Additionally, the CTLs displayed a distinct phenotype (majority CD69+/CD45RO+) along with a significant (p<0.05) increase in cell proliferation and IFN-γ release following re-stimulation with HLA-A2.1+/CD19+/CD20+ tumor cell lines. Based on emerging information that clonogenic myeloma cells express CD19 and/or CD20, we evaluated the activity of the CD19 and CD20 peptide specific-CTLs against several multiple myeloma cell lines. Five of 10 myeloma cell lines evaluated were HLA-A2.1-positive and expressed both CD19 and CD20 antigens. CD19 peptide specific-CTLs generated from normal donors were able to specifically lyse CD19+/HLA-A2.1+ MM cell lines (30% lysis; 10:1 E:T ratio) but did not lyse CD19−/HLA-A2.1+ or CD19+/HLA-A2.1− cell lines. Similarly, the CD20-specific CTLs generated from normal donors lysed CD20+/HLA-A2.1+ MM cell lines (25% lysis; 10:1 E:T ratio), in a manner restricted to HLA-A2.1 and specific to antigens. We next showed IFN-γ production by the CTLs after exposure to CD19+/HLA-A2.1+ or CD20+/HLA-A2.1+ MM cells. Moreover, we have demonstrated the ability to expand CD20-CTLs under serum-free culture conditions while maintaining their cytotoxic activity (28–49%). In ongoing studies, we are evaluating the ability of CD19- and CD20-specific CTLs to eliminate clonogenic myeloma cells in vitro and in vivo in a SCID mouse model of myeloma. These preclinical studies strongly suggest that immunogenic CD19 and CD20 peptide-based vaccines represent a promising immunotherapeutic approach in myeloma.

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.

Clarithromicin Induces Autophagy in Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3509-3509 ◽  
Author(s):  
Miki Nakamura ◽  
Takahiro Kamimoto ◽  
Tamotsu Yoshimori ◽  
Hiroaki Mitsuya ◽  
Hiroyuki Hata
Keyword(s):  
Electron Microscopy ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Fluorescent Microscopy ◽  
Myeloma Cell ◽  
Pi3 Kinase ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Akt Phosphorylation ◽  
Dose Dependent

Abstract Introduction Some macrolide antibiotics exert effects other than anti-bacterial activity on the growth and viability of certain cancer cells. The presence of cytoplasmic vacuoles is one the salient features of autophagy, a cellular event believed to recycle cellular ingredients under nutrient-starved conditions. Such vacuoles (autophagosomes) fuse with lysozomes, generating autolysozomes toward later stages of autophagy, digesting organelles and degenerated proteins. Our own and others’ findings that a macrolide antibiotic clarithromicin (CAM) occasionally shows anti-myeloma effects when combined with thalidomide and/or dexamethasone prompted us to examine CAM for its effects on myeloma cells in vitro. Methods Four myeloma cell lines (12PE, KHM-11, KMM-1 and U266) and primary myeloma cells purified by CD138-conjugated immune-magnetic beads (Miltenvi Biotec, Auburn, CA) were utilized. Clarithromicin was obtained from Taisho-Toyama pharmaceuticals (Tokyo, JAPAN). Morphology was analyzed either by May-Giemza staining or electron microscopy. Autolysozome was stained with Lysotracker (Invitrogen, Carlsbad, CA) and analyzed using fluorescent microscopy. Antibody to LC3 was obtained from Dr. T. Yoshimori (Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University). Results and discussion CAM induced vacuoles in the cytoplasm of both myeloma cell lines and primary myeloma cells at concentrations ranging from 10 to 50 mg/ml at a dose-dependent manner after ~18 hours treatment. Electron microscopy revealed that those vacuoles morphologically resemble autolysozomes. To further confirm the identity of autolysozomes, cells were stained with Lysotracker, which specifically stains acid lysozome. After the treatment with CAM, the accumulation of vacuoles in the cytoplasm, stained with Lysotacker, was observed. Since initiation of autophagy depends on PI3-kinase, we investigated whether CAM induced AKT phosphorylation. AKT phosphorylation was readily observed, and moreover, the emergence of vacuoles stainable with Lysotracker was inhibited when the cells were pretreated with PI3-kinase inhibitors, 3MA or LY294002, strongly suggesting that vacuolation is indeed mediated with PI3-kinase. To further confirm that autopahgy is induced by CAM, the process of LC3-I to LC3-II, a hallmark of autophagy, was examined. We found that the induction of LC3-II by CAM occurred at a dose-dependent manner. Taken together, these findings strongly suggest that CAM induces autolysozome accumulation through activating PI3-kinase. Finally, we examined whether CAM induced apoptosis when combined with thalidomide. Three myeloma cells lines, which abundantly expressed Bcl-2, showed no growth inhibition, while KHM-11, which was defective in Bcl-2, showed marked apoptosis and growth inhibition with the combination of CAM and thalidomide, suggesting that CAM might potentially augment anti-myeloma activity of thalidomide although the mechanisms are to be determined. Taken these observations together, the manipulation of certain autophagy processes with reagents such as macrolides (i.e., CAM) might represent a new therapeutic approach in the treatment of myeloma. We hypothesize that CAM dually functions in the event of autophagy, i.e., it initiates autophagy while it suppresses autophagy at later stages. Further study under the hypothesis is currently underway.

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.

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