Anti-Myeloma Activity of Two Novel N-Substituted and Tetraflourinated Thalidomide Analogues.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2406-2406
Author(s):  
Shaji Kumar ◽  
Noopur Raje ◽  
Teru Hideshima ◽  
Kenji Ishitsuka ◽  
Aldo Roccaro ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Myeloma Cell ◽  
Clinical Activity ◽  
Significant Activity ◽  
Protective Effects ◽  
Inhibition Of Proliferation ◽  
Myeloma Cells

Abstract The importance of bone marrow angiogenesis in multiple myeloma (MM) provided the initial rationale for using thalidomide in this disease, given its anti-angiogenic properties. Thalidomide (α-N-[phthalimido] glutarimide, C 13 H 10 N 2 O 4), a glutamic acid derivative, has proved useful in the treatment of both newly diagnosed and relapsed/refractory MM, however, its potential for teratogenicity lays a huge burden on its use as a therapeutic agent. Ongoing efforts have been directed towards development of analogues, which retain anti-MM activity without teratogenic effects. A product of cytochrome P450 2C19 isozyme biotransformation of thalidomide, 5′-OH-thalidomide, has been shown to be partly responsible for the anti-angiogenic effect of thalidomide. Based on the structure of this metabolite several N-substituted and tetraflourinated thalidomide analogues have been synthesized as potential anti-angiogenic agents and have shown pre-clinical activity in prostate cancer. In this study, we demonstrate potent anti-MM activity and delineate potential mechanisms of two such compounds, CPS11 (an N-substituted thalidomide) and CPS49 (a tetraflourinated thalidomide). Both CPS11 and CPS49 showed inhibition of proliferation of several MM cell lines as well as primary tumor cells from relapsed/refractory MM patients. These drugs had significant activity in myeloma cell lines resistant to conventional chemotherapy agents (dexamethasone, adriamycin, mitoxantrone and melphalan). Both the drugs were able to overcome the protective effects of growth factors like IL-6, IGF-1, and VEGF, and co-culture with bone marrow stromal cells (BMSCs), demonstrating their activity against myeloma cells in conditions simulating the natural microenvironment. In most of the cytotoxicity experiments, CPS49 was more potent compared to CPS11. An additive cytotoxic effect was noted when these agents were combined with dexamethasone and melphalan. Apoptosis was noted in MM1.S cells as evidenced by annexin and PI staining and a time- and dose-dependent increase in cleavage of poly ADP-ribosepolymerase (PARP) and caspase-8. Additionally, Z-VAD-FMK partially blocked these effects. Importantly, apoptosis triggered by these drugs was associated with down-regulation of anti-apoptotic proteins like Mcl1 and XIAP. In matrigel based angiogenesis assays both drugs demonstrated anti-angiogenic activity, CPS49 being more potent than CPS11. To further delineate their molecular mechanism of actions, we evaluated the effect of these drugs on various intracellular signaling pathways known to be important in myeloma. Both the drugs were able inhibit the PI3K/Akt and JAK/STAT pathways in MM1.S myeloma cells line. These encouraging in vitro data will provide the platform for testing these analogues initially in animal models and subsequently in the clinic.

Plasma Levels of SDF-1 and Expression of SDF-1 Receptor on Multiply Myeloma Cells of Human Multiply Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4874-4874
Author(s):  
Caixia Li ◽  
De Pei Wu ◽  
Junjie Cao ◽  
Xiaojin Wu ◽  
Xiao Ma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Levels ◽  
Myeloma Cell ◽  
Migration Ability ◽  
Myeloma Cells ◽  
And Migration ◽  
Cxcr4 Axis ◽  
Healthy Persons

Abstract Multiple myeloma(MM) is a monoclonal expansion of malignant cells with a plasmablast-plasma cell morphology that is almost exclusively localized to the bone marrow, except at the final stages of disease, when they proliferate in the extramedullary area. The mechanisms of the selective homing of MM cells to the bone marrow compartment are poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 contribute to stem cell homing and play a role in trafficking of leukemic cells. In this study we have investigated expression and biological behavior of SDF-1/CXCR4 in MM-derived cell lines and primary MM cells. FACS and RT-PCR analysis was used to study the expression of CXCR4 and ICAM-1(CD54) on the surface of MM cells from 4 IL-6 dependant cell lines (XG1,XG2,XG6 and XG7) and 25 freshly isolated tumor samples from patients with diagnosed MM. Mononuclear cells were purified by positive selection of magnetical and FACS sorting. Chemotaxis assay through transwell bore polycaronate and ELISA assay were employed to monitor the SDF-1, IL-6, and sICAM-1 levels. We found that[circ1]Fresh MM cells and MM cell lines expressed various levels of functional CXCR4 ranging from 23.1% to 77.7%,which was correlated with the in vitro migration ability of MM cells[(23.2±1.08)%, P<0.01]; [circ2]SDF-1 levels in the bone marrow(BM) of MM patients were significantly higher than the those of healthy persons (3489.23±651.63)pg/ml, (2818.57±597.79)pg/ml, P<0.05; but plasma levels of SDF-1 in peripheral blood of MM patients were lower than those of healthy persons[(1973±133)pg/ml, (2334.857±574.92), P=0.062]; [circ3]Plasma levels of PCL(4097.14±680.71) were significantly higher than those of healthy persons, P<0.01. The results firstly demonstrated abnormal expression of SDF-1 and its receptor CXCR4 on Human MM cells, which is closely correlated with the migration of MM cells. Furthermore, we discovered that SDF-1 could up-regulate the expression of ICAM-1 on MM cells; the plasma level of soluble ICAM-1 was correlated with the expression of CXCR4 on MM cells. These findings suggested that SDF-1/CXCR4 axis play a key role on the trafficking of MM cells via mediating the effect of adhesion molecules. Moreover, we observed higher plasma levels of IL-6 in PB of 60% MM patients compared with those of healthy individuals. Finally, the levels of IL-6 were closely correlated with SDF-1 levels (γ=0.8, P<0.01), These data indicated that in the IL-6-dependent myeloma cell lines or fresh myeloma samples and myeloma cell growth triggered by SDF-1 maybe due to up-regulation of autocrine and paracrine IL-6 by myeloma cells and stromal cells in BM. The results suggested that the expression of CXCR4 have an essential role in the proliferation and migration of myeloma cells in patients with multiple myeloma.In conclusion, MM cells expressed various levels of functional CXCR4, which were correlated with the migration ability of MM cells in vitro; SDF-1/CXCR4 axis plays a key role in the trafficking of MM cells via mediating the effect of adhesion molecules; The plasma levels of IL-6 closely correlated with SDF-1 plasma levels, myeloma cell growth triggered by SDF-1 may be due to up-regulation of autocrine and paracrine IL-6 by myeloma cells and stromal cells in BM. All these suggested that the expression of CXCR4 play an essential role in the proliferation and migration of myeloma cells in patients with multiple myeloma.

Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1419-1419
Author(s):  
Soraya Wuilleme-Toumi ◽  
Nelly Robillard ◽  
Patricia Gomez-Bougie ◽  
Philippe Moreau ◽  
Steven Le Gouill ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Disease Severity ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Lymphocytic Leukemia ◽  
Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (&gt;20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5080-5080
Author(s):  
Shankaranarayana Paneesha ◽  
Raghu Adya ◽  
Hemali Khanji ◽  
Ed Leung ◽  
C. Vijayasekar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Human Myeloma Cell

Abstract Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection. Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE). Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.

Expression of CD21 antigen on myeloma cells and its involvement in their adhesion to bone marrow stromal cells

Blood ◽  
1995 ◽  
Vol 85 (12) ◽  
pp. 3704-3712 ◽  
Author(s):  
N Huang ◽  
MM Kawano ◽  
MS Mahmoud ◽  
K Mihara ◽  
T Tsujimoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Plasma Cells ◽  
Cell Types ◽  
Myeloma Cell ◽  
Adhesion Assay ◽  
Myeloma Cells ◽  
Stromal Cell Line

The mature myeloma cells express very late antigen 5 (VLA-5) and MPC-1 antigens on their surface and adhere to bone marrow (BM) stromal cells more tightly than the VLA-5-MPC-1-immature myeloma cells in vitro. The VLA-5 and MPC-1 antigens possibly function as two of the molecules responsible for interaction of mature myeloma cells with BM stromal cells. However, the immature myeloma cells do interact with BM stromal cells, and it is unclear which adhesion molecules mediate their interaction. In this study, we found that both immature and mature myeloma cells expressed CD21, an adhesion molecule known to bind to CD23. CD21 was also detected on normal plasma cells. To evaluate the role of CD21 expression on myeloma cells, two myeloma cell lines, NOP-2 (VLA-5-MPC-1-) and KMS-5 (VLA-5+MPC-1+), were used as representatives of immature and mature myeloma cell types, respectively, and an adhesion assay was performed between the myeloma cell lines and BM stromal cells. Antibody-blocking results showed that adhesion of the mature type KMS-5 to KM102, a human BM-derived stromal cell line, or to short-term cultured BM primary stromal cells was inhibited by monoclonal antibodies (MoAbs) against CD21, VLA-5, and MPC-1, and inhibition of adhesion of the immature type NOP-2 to KM102 by the anti-CD21 MoAb was observed as well. Furthermore, CD23 was detected on KM102. Treatment of KM102 with an anti-CD23 MoAb also inhibited adhesion of either KMS-5 or NOP-2 to KM102. Therefore, we propose that CD21 expressed on myeloma cells likely functions as a molecule responsible for the interaction of immature myeloma cells as well as mature myeloma cells with BM stromal cells, and CD23 may be the ligand on the stromal cells for the CD21-mediated adhesion.

1′-Acetoxychavicol Acetate (ACA) Is a Novel NF-κB Inhibitor with Significant Activity Against Multiple Myeloma in Vitro and in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 765-765 ◽  
Author(s):  
Keisuke Ito ◽  
Tomonori Nakazato ◽  
Yoshitaka Miyakawa ◽  
Ming Ji Xian ◽  
Taketo Yamada ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Serine Phosphorylation ◽  
Significant Activity ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract 1′-acetoxychavicol acetate (ACA) is a component of traditional Asian condiment, obtained from rhizomes of the commonly used ethno-medicinal plant Languas galanga (Zingiberacetate). Recent extensive studies revealed that ACA has potent chemopreventive effects against various tumors. More recently, we have reported that ACA induces apoptosis of myeloid leukemic cells via mitochondrial- and Fas-mediated dual pathway. The transcription factor NF-κB confers significant survival potential in myeloma cells; therefore, it has emerged as a therapeutic target for the treatment of multiple myeloma. Multiple myeloma is an incurable hematological disorders, which has been fatal outcome despite of high dose chemotherapy with stem cell transplantation; therefore, a novel biologically based therapeutic approach is desired. In this study, we investigated the effects of ACA on myeloma cells in vitro and in vivo, and further examined the molecular mechanisms of ACA-induced apoptosis in myeloma cells. ACA dramatically inhibited cellular growth of various human myeloma cell lines (RPMI8226, U266, IM9, and HS-Sultan) as well as freshly isolated myeloma cells from patients, but not normal bone marrow cells, in a dose (0-20 μM)- and time (0-24 h)-dependent manner. Cultivation with 10 μM ACA rapidly increased the population of cells in the G0/G1 phase with a reduction of cells in the S phase, and a strong induction of apoptosis was shown by the appearance of a hypodiploid DNA peak with sub-G1 DNA content 3 h after treatment. Treatment with ACA induced both caspase-3, -9, and caspase-8 activities, suggesting that ACA-induced apoptosis in myeloma cells mediates both mitochondrial- and Fas-dependent pathways. Furthermore, we investigated the effects of ACA on NF-κB activity in myeloma cells, and were able to demonstrate that ACA significantly inhibited serine phosphorylation and degradation of IκBα in a time-dependent manner. ACA rapidly decreased the nuclear expression of NF-κB, but increased the accumulation of cytosol NF-κB in RPMI8226 cells, indicating that ACA inhibits translocation of NF-κB from the cytosol to the nucleus. In addition, we also confirmed the inhibitory effects of ACA on NF-κB activation by ELISA in myeloma cell lines and fresh samples. ACA had a synergistic proapoptotic effect with another NF-κB inhibitor, MG-132 and TLCK. In contrast, NF-κB activator, PMA, dramatically abrogated ACA-induced apoptosis in myeloma cells. These in vitro studies prompted us to examine whether the effects of ACA are equally valid in vivo. To evaluate the effects of ACA in vivo, RPMI8226-transplanted NOD/SCID mice were treated with ACA. Tumor weight decreased in the mice that were injected ACA (mean weight: 0.04±0.06 g in the ACA-treated group vs. 0.63±0.29 g in the control group; p<0.01). During the treatment, ACA-treated mice appeared healthy, and pathological analysis at autopsy revealed no ACA-induced tissue changes in any of the organ, indicating that ACA might be developed as a new potent anti-cancer agent for the management of multiple myeloma. In conclusion, ACA has an inhibitory activity of NF-κB, and induces apoptosis of myeloma cells in vitro and in vivo. Therefore, ACA provides the new biologically based therapy for the treatment of multiple myeloma patients as a novel NF-κB inhibitor.

Identification of Vacuolar H+- ATPase as a Target for Myeloma Therapeutics.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 257-257
Author(s):  
Yuan Xiao Zhu ◽  
Chung Xin ◽  
Sheng Ben Liang Lian ◽  
Wee-Joo Chng ◽  
Suzanne Trudel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Viability ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Normal Donor ◽  
Bafilomycin A1 ◽  
Myeloma Cells ◽  
Treatment Conditions

Abstract From a high throughput RNAi screen of the human druggable genome targeting the KMS11 cell line, we identified the suppression of the vacuolar H+- ATPase (V-ATPase) family as cytotoxic to myeloma cells. In the screen, two oligos against each gene for the V-ATPase subunits ATP6V1A and ATP6V1B1 resulted in suppression of cell growth (50% and 60% inhibition of cell viability respectively). We further confirmed this result using both lentiviral shRNA knockdown and two small molecule inhibitors specific for V-ATPase. Silencing of ATP6V1A by lentiviral shRNA knock-down in KMS11 and in OPM1 myeloma cell lines caused 75–80% reduction of cell viability at 5 days post infection (measured by MTT assay). Consistent with this result, the V-ATPase specific inhibitors, bafilomycin A1 and REATA 203, both inhibited the growth of a genetically heterogeneous and standardized panel of 14 human myeloma cell lines in vitro with an IC50 ranging from 2.2 – 8.9 nM (mean 5.25 nM) for bafilomycin A1 and 46–1594 nM (mean 542.5 nM)) for REATA 203. We further demonstrated that patient samples (n=10) were sensitive to 20nM bafilomycin A1 which induced a mean of 58% of MM cells to undergo apoptosis (range 10% to 93%) after 24 hours of treatment. Similar to bafilomycin A1, treatment of primary patient-derived MM cells with 500 nM REATA 203 for 72 hours resulted in a mean 69% apoptosis (range 24% to 97%). In contrast, non-myeloma cells (the CD138- fractions of the bone marrow samples) were less sensitive - mean 9% apoptosis (range from 0% to 34%) under the same treatment conditions. Of high interest, however, unlike most drugs we have studied in pre-clinical myeloma models, the cytotoxicity induced by bafilomycin A1 in MM cell lines is abrogated by co-culture with patient bone marrow stromal cells but is not affected by IL-6 or IGF-1 treatment. Dexamethasone- or melphalan-resistant MM cell lines were also highly sensitive to both bafilomycin A1 and REATA 203. In a xenographic JJN3 mouse model, bafilomycin A1 suppresses and delays growth of tumor in a dose-dependent fashion. Gene expression analysis of normal-donor bone marrow plasma cells (n=19), primary tumor samples from MM patients (n=107) and normal somatic tissues demonstrates ubiquitous expression of most subunits of V-ATPase, however, some subunits are preferentially expressed in myeloma cells compared with normal plasma cells, including ATP6V1F (84% vs. 11%), ATP6V1E1 (29% vs. 5%), ATP6V1G2 (17% vs. 0%) and ATP6V0E 2 (36% vs. 16%). In conclusion, our data indicate that vacuolar H+-ATPase inhibitors are of interest as potential therapeutics for MM.

Bone Morphogenic Protein 6: A Prognostic Factor Expressed by Normal Plasma Cells and Multiple Myeloma Cells Inhibiting Their Proliferation and Angiogenesis Induction

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2701-2701
Author(s):  
Anja Seckinger ◽  
Tobias Meißner ◽  
Jérôme Moreaux ◽  
Hartmut Goldschmidt ◽  
Axel Benner ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Overall Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
High Dose Chemotherapy ◽  
High Dose ◽  
Myeloma Cells

Abstract BACKGROUND: Pathogenesis of multiple myeloma is partly attributed to an aberrant expression of proliferation-, pro-angiogenic and bone-metabolism modifying factors by malignant plasma-cells. AIM. Given the long and variable time-span from first diagnosis of early-stage plasma-cell dyscrasias to overt myeloma and the low proliferation rate of malignant plasma-cells, we hypothesize these to concomitantly express a novel class of anti-proliferative factors of potential prognostic relevance. Here, bone morphogenic proteins (BMPs) represent possible candidates, as they inhibit proliferation, stimulate bone formation, and have an impact on the survival of cancer patients. PATIENTS AND METHODS. We assessed expression of BMPs and its receptors by Affymetrix DNA-microarrays (n=434) including CD138-purified primary myeloma-cell-samples, normal bone-marrow plasma-cell-samples, polyclonal plasmoblasts-samples, human myeloma-cell-lines (HMCL), and whole bone-marrow. Presence and differential gene expression was determined by PANP-algorithm and empirical Bayes statistics. Event-free (EFS) and overall survival (OAS) were investigated for the 168 patients undergoing high-dose chemotherapy (HM-group) using Cox’s proportional hazard model. Findings were validated using the same strategy on an independent group of 345 patients from the Arkansas-group. For validation, quantitative real-time PCR and flow cytometry were performed. In vitro induction of angiogenesis was assessed using the AngioKit-assay. Effect of BMP6 on proliferation of HMCL was assessed by 3H-thymidine uptake. RESULTS. BMP6 is the only BMP expressed by normal- (13/14 samples) and malignant plasma-cells (228/233 samples). It is significantly lower expressed in proliferating non-malignant plasmablastic cells and human myeloma cell-lines. In vitro, BMP6 significantly inhibits proliferation of myeloma-cell-lines with an IC50 ranged from 0.08–2.15μg/ml, survival of primary myeloma-cells, and in vitro tubule formation down to the level of the negative control. High BMP6-expression in malignant plasma cells delineates significantly superior overall-survival for patients undergoing high-dose chemotherapy in both independent series of patients (n=168, P=.02 and n=345, P=.03, respectively, see below). CONCLUSION. With BMP6 we report for the first time the autocrine expression of a prognostically relevant anti-angiogenic and anti-proliferative factor and its receptors by normal and malignant plasma-cells. Figure Figure

A Method for Measurement of Drug Sensitivity of Myeloma Cells Co-Cultured with Bone Marrow Stromal Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1373-1373
Author(s):  
Kristine Misund ◽  
Katarzyna Anna Baranowska ◽  
Toril Holien ◽  
Christoph Rampa ◽  
Dionne Klein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Stromal Cell ◽  
Drug Sensitivity ◽  
Myeloma Cell ◽  
Marrow Stromal Cells ◽  
Myeloma Cells

Abstract Abstract 1373 The aim of this work was to establish a robust and simple method for the measurement of drug sensitivity in myeloma cells under conditions mimicking aspects of the bone marrow microenvironment. In particular we wanted to measure drug sensitivity in myeloma cells cultivated in the presence of stromal cells. The tumor microenvironment can profoundly affect tumor cell survival as well as alter antitumor drug activity, and it is generally believed that growth and survival of myeloma cells is critically dependent on the bone marrow microenvironment. Bone marrow stromal cells (BMSC) have been shown to protect myeloma cells from common cytostatic or cytotoxic drugs in vitro. Common in vitro assays used for high-throughput drug screening cannot easily discriminate between stromal and tumor cell responses in co-cultures. Although a few recent studies have overcome this problem (Ramasamy K. et al., 157(5):564–79,2012, McMillin D. et al., 16(4):483–9, 2010), the application of stable transfection for labeling of cells limits the practical application of these co-culture studies to cell lines, excluding primary myeloma cells that inherently may be hard to transduce even by retroviral vectors. Here, we analyzed survival of myeloma cells co-cultured with BMSC using an automated fluorescence microscope, ScanR. ScanR is a microscope based screening station. By staining the cell nuclei with DRAQ5, we were able to discriminate between BMSC and myeloma cells, based on their staining intensity and nuclear shape. Using the apoptotic marker YO-PRO-1, the effects of drug treatment on the viability of the myeloma cells in the presence of stromal cells could be measured. The main advantages of this method are the non-necessity of cell manipulation before co-culture and the low number of myeloma cells (5000 primary cells) that are needed per measurement, which makes the method ideal for experiments with primary myeloma cells. In fact, the analysis was easier and more robust when using slowly growing cells, i.e. by using primary myeloma cells compared to more rapidly proliferating myeloma cell lines. This method should be well-suited for high throughput analysis, as the cells are stained in situ with no washing, centrifugation, or fixation steps before analysis. The method was compared to a conventional method for detecting cell viability; flow cytometry where annexin V labeling was used to detect apoptotic cells. As shown in figure 1, the dose-response curves obtained for ANBL-6 cells treated with different doses of melphalan were similar and showed the same trends for both methods. However, the effects of melphalan treatment were more evident analyzed by the ScanR system than by flow cytometry (EC50 YO-PRO-1 = 11μM versus EC50Annexin V= 15μM). The stromal cell population applied in this study was able to support IL-6 dependent myeloma cell lines without addition of IL-6. This as IL-6 dependent INA-6 cells cultivated in the presence of BMSC survived in the absence of added IL-6. This study shows the importance of stromal cell support for primary myeloma cell survival in vitro, as half of the cell samples had a marked increase in their viability when cultured in the presence of BMSC. Stromal cell-induced protection against common myeloma drugs was also observed with this method. For instance, experiments with primary myeloma cells from patient MM7, showed that in the presence of BMSC, the EC50 for the common myeloma drug cyclophosphamide was increased from 5 μM to approximately 10 μM (figure 2). Figure 1 Figure 1. Figure 2 Figure 2. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Plasma cells induce apoptosis of pre-B cells by interacting with bone marrow stromal cells

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3375-3383 ◽  
Author(s):  
T Tsujimoto ◽  
IA Lisukov ◽  
N Huang ◽  
MS Mahmoud ◽  
MM Kawano
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Cell Lines ◽  
Stromal Cells ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
B Cell Survival

By using two-color phenotypic analysis with fluorescein isothiocyanate- anti-CD38 and phycoerythrin-anti-CD19 antibodies, we found that pre-B cells (CD38+CD19+) signifcantly decreased depending on the number of plasma cells (CD38++CD19+) in the bone marrow (BM) in the cases with BM plasmacytosis, such as myelomas and even polyclonal gammopathy. To clarify how plasma cells suppress survival of pre-B cells, we examined the effect of plasma cells on the survival of pre-B cells with or without BM-derived stromal cells in vitro. Pre-B cells alone rapidly entered apoptosis, but interleukin-7 (IL-7), a BM stromal cell line (KM- 102), or culture supernatants of KM-102 cells could support pre-B cell survival. On the other hand, inhibitory factors such as transforming growth factor-beta1 (TGF-beta1) and macrophage inflammatory protein- 1beta (MIP-1beta) could suppress survival of pre-B cells even in the presence of IL-7. Plasma cells alone could not suppress survival of pre- B cells in the presence of IL-7, but coculture of plasma cells with KM- 102 cells or primary BM stromal cells induced apoptosis of pre-B cells. Supernatants of coculture with KM-102 and myeloma cell lines (KMS-5) also could suppress survival of pre-B cells. Furthermore, we examined the expression of IL-7, TGF-beta1, and MIP-1beta mRNA in KM-102 cells and primary stromal cells cocultured with myeloma cell lines (KMS-5). In these cells, IL-7 mRNA was downregulated, but the expression of TGF- beta1 and MIP-1beta mRNA was augmented. Therefore, these results suggest that BM-derived stromal cells attached to plasma (myeloma) cells were modulated to secrete lesser levels of supporting factor (IL- 7) and higher levels of inhibitory factors (TGF-beta1 and MIP-1beta) for pre-B cell survival, which could explain why the increased number of plasma (myeloma) cells induced suppression of pre-B cells in the BM. This phenomenon may represent a feedback loop between pre-B cells and plasma cells via BM stromal cells in the BM.

The Expression of CD56 Is Frequently Accompanied with the Expression of Neuronal Cell Markers and Its Down-Regulation Is Induced by IL-6 in Human Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3534-3534
Author(s):  
Mohd S. Iqbal ◽  
Ken-ichiro Otsuyama ◽  
Karim Shamsasenjan ◽  
Saeid Abroun ◽  
Jakia Amin ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Cell Lines ◽  
Cell Lineage ◽  
Neuronal Cell ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Cell Markers ◽  
Phenotypic Changes ◽  
Human Myeloma Cells

Abstract 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.

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