scholarly journals Adhesion of human myeloma-derived cell lines to bone marrow stromal cells stimulates interleukin-6 secretion

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3712-3720 ◽  
Author(s):  
H Uchiyama ◽  
BA Barut ◽  
AF Mohrbacher ◽  
D Chauhan ◽  
KC Anderson
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Rgd Peptide ◽  
Beta 1 Integrin ◽  
Beta 2

Previous studies show that human myeloma-derived cell lines specifically adhere to fibronectin (FN) through very late antigen-4 (VLA-4; alpha 4 beta 1 integrin complex) and RGD-peptide mechanisms, which may contribute to the localization of tumor cells in bone marrow (BM). In these studies, we characterized the adhesion of myeloma- derived cell lines to both normal and myeloma BM stromal cells (BMSCs) and the effect of adhesion on DNA synthesis. Because interleukin-6 (IL- 6) plays an important role in the pathogenesis of multiple myeloma, we also examined the effects of tumor cell adhesion on IL-6 secretion by BMSCs. In 51chromium binding assays, the U266, ARH-77, and IM-9 cell lines showed 52% +/- 12%, 55% +/- 6%, and 47% +/- 7% specific adherence, respectively, to normal BMSCs and 74% +/- 4%, 60% +/- 3%, and 61% +/- 6% specific adherence, respectively, to myeloma BMSCs. In contrast, only 12% to 13% specific binding of HS-Sultan cells to BMSCs was noted. The binding of myeloma cells to BMSCs was partially blocked with anti-beta 1 monoclonal antibody (MoAb), anti-beta 2 integrin MoAb, and excess RGD peptide, suggesting multiple mechanisms for the adhesion of myeloma cell lines to BMSCs. Binding of cell lines to FN or myeloma BMSCs did not affect cell line proliferation; however, adhesion of myeloma cell lines to normal BMSCs decreased DNA synthesis, ie, stimulation indices are 0.1 +/- 0.04, 0.2 +/- 0.1, 0.2 +/- 0.07, and 0.1 +/- 0.06 for the adherent non-IL-6-dependent U266, ARH-77, HS- Sultan, and IM-9 cells, respectively (n = 5, P < .01). In contrast, adherence of IL-6-dependent B9 cells increased their proliferation (stimulation index, 3.2 +/- 0.7). Significant (twofold to eightfold) increases in IL-6 secretion were evident in cell line-adherent (> or = 12 hours) normal and myeloma BMSC cultures. Paraformaldehyde fixation of BMSCs before adhesion completely abrogated IL-6 secretion, suggesting that IL-6 secretion was triggered in BMSCs rather than in cell lines. Partial blocking of cell line adhesion to BMSCs, using anti- beta 1 integrin and anti-beta 2 integrin MoAbs and RGD peptide, also partially blocked the triggering of IL-6 secretion by BMSCs. When cell lines were placed in Transwell inserts and then cultured with either normal or myeloma BMSCs, permitting juxtaposition without cell to cell contact between myeloma cell lines and BMSCs, no increase in IL-6 secretion was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Adhesion of human myeloma-derived cell lines to bone marrow stromal cells stimulates interleukin-6 secretion

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3712-3720 ◽  
Author(s):  
H Uchiyama ◽  
BA Barut ◽  
AF Mohrbacher ◽  
D Chauhan ◽  
KC Anderson
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Rgd Peptide ◽  
Beta 1 Integrin ◽  
Beta 2

Abstract Previous studies show that human myeloma-derived cell lines specifically adhere to fibronectin (FN) through very late antigen-4 (VLA-4; alpha 4 beta 1 integrin complex) and RGD-peptide mechanisms, which may contribute to the localization of tumor cells in bone marrow (BM). In these studies, we characterized the adhesion of myeloma- derived cell lines to both normal and myeloma BM stromal cells (BMSCs) and the effect of adhesion on DNA synthesis. Because interleukin-6 (IL- 6) plays an important role in the pathogenesis of multiple myeloma, we also examined the effects of tumor cell adhesion on IL-6 secretion by BMSCs. In 51chromium binding assays, the U266, ARH-77, and IM-9 cell lines showed 52% +/- 12%, 55% +/- 6%, and 47% +/- 7% specific adherence, respectively, to normal BMSCs and 74% +/- 4%, 60% +/- 3%, and 61% +/- 6% specific adherence, respectively, to myeloma BMSCs. In contrast, only 12% to 13% specific binding of HS-Sultan cells to BMSCs was noted. The binding of myeloma cells to BMSCs was partially blocked with anti-beta 1 monoclonal antibody (MoAb), anti-beta 2 integrin MoAb, and excess RGD peptide, suggesting multiple mechanisms for the adhesion of myeloma cell lines to BMSCs. Binding of cell lines to FN or myeloma BMSCs did not affect cell line proliferation; however, adhesion of myeloma cell lines to normal BMSCs decreased DNA synthesis, ie, stimulation indices are 0.1 +/- 0.04, 0.2 +/- 0.1, 0.2 +/- 0.07, and 0.1 +/- 0.06 for the adherent non-IL-6-dependent U266, ARH-77, HS- Sultan, and IM-9 cells, respectively (n = 5, P < .01). In contrast, adherence of IL-6-dependent B9 cells increased their proliferation (stimulation index, 3.2 +/- 0.7). Significant (twofold to eightfold) increases in IL-6 secretion were evident in cell line-adherent (> or = 12 hours) normal and myeloma BMSC cultures. Paraformaldehyde fixation of BMSCs before adhesion completely abrogated IL-6 secretion, suggesting that IL-6 secretion was triggered in BMSCs rather than in cell lines. Partial blocking of cell line adhesion to BMSCs, using anti- beta 1 integrin and anti-beta 2 integrin MoAbs and RGD peptide, also partially blocked the triggering of IL-6 secretion by BMSCs. When cell lines were placed in Transwell inserts and then cultured with either normal or myeloma BMSCs, permitting juxtaposition without cell to cell contact between myeloma cell lines and BMSCs, no increase in IL-6 secretion was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological and Molecular Characterization by Integrative Genomics Approach of CMA-03/06, a Newly Established Interleukin-6 Independent Variant of the CMA-03 Human Myeloma Cell Line.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1814-1814
Author(s):  
Donata Verdelli ◽  
Lucia Nobili ◽  
Katia Todoerti ◽  
Laura Mosca ◽  
Sonia Fabris ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Molecular Characterization ◽  
Cell Lines ◽  
Stromal Cells ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Growth And Survival ◽  
Genome Wide

Abstract Abstract 1814 Poster Board I-840 Background The growth and survival of multiple myeloma (MM) cells in the bone marrow microenvironment is regulated by functional complex interactions between the tumor cells and the surrounding bone marrow stromal cells mediated by adhesion molecules and the production of several cytokines of which interleukin-6 (IL-6) has been identified as the most important. Major advances in the investigation of MM biology were made possible by the availability of human myeloma cell lines (HMCLs). The IL-6-dependent CMA-03 cell line was established in our laboratory from a peritoneal effusion of a refractory relapsed MM patient. By gradually decreasing the IL-6 added to the culture, an IL-6-independent variant, CMA-03/06, could be obtained. Aims. To perform a biological and molecular characterization of this novel cell line, and to provide insights into the signaling pathways and target genes involved in the growth and survival of CMA-03/06. Methods. The growth, immunophenotypic, cytogenetic and fluorescence in situ hybridization (FISH) characterization of CMA-03/06 cell line was performed by means of standard procedures. IL-6 production into the culture media was determined using a high sensitivity IL-6 specific ELISA. Genome-wide profiling data were generated by means of Affymetrix GeneChip® Human Mapping 250K Nsp arrays; copy number (CN) alterations were calculated using the DNAcopy Bioconductor package, based on circular binary segmentation method. Global gene expression profiling (GEP) was performed by means of the GeneChip® Human Gene 1.0 ST Arrays (Affymetrix); the supervised analyses were done using the SAM software version 3.0. Results Unlike CMA-03, the addition of IL-6 to the culture medium of CMA-03/06 cells or co-culture with multipotent mesenchymal stromal cells did not induce an increase in CMA-03/06 proliferation. IL-6 was not detected in the supernatants from either CMA-03 or CMA-03/06 cell lines within 48 h, suggesting that the IL-6 independence of CMA03/06 cells is not a result of the development of an autocrine IL-6 loop. Nevertheless, IL-6 induced the activation of STAT3 and STAT1 in both cell lines, even if a slight constitutive STAT3 phosphorylation was found in CMA-03/06. The immunophenotypic analysis showed a significant difference in the expression of three antigens in the 2 cell lines: CD45 was considerably reduced in CMA-03/06 cells, whereas they were found positive for both chains of IL-6 receptor, CD126 and CD130, almost undetectable in CMA-03. Conventional cytogenetic and FISH analyses did not reveal differences between the 2 HMCLs. The genome-wide analysis allowed the identification of about 100 altered chromosomal regions common to both HMCLs, mostly DNA gains. Comparison of CMA-03/06 and CMA-03 cells evidenced a different CN in only 15 small chromosomal regions, 8 of which did not contain any transcript, whereas few genes were located on the other ones. GEP analysis of CMA-03/06 compared with CMA-03 identified 21 upregulated and 47 downregulated genes, many of which particularly relevant for MM biology, mainly involved in cellular signaling, cell cycle, cell adhesion, cell development, regulation of transcription, immunologic, inflammatory or defense activity, apoptosis. None of the genes differentially expressed in CMA-03/06 compared with CMA-03 except 1 were positioned on the chromosomal regions showing a different CN. Finally, CMA-03/06 cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA-03 cells. Conclusions Our data show the IL-6 independence of CMA-03/06 cell line in the absence of an autocrine IL-6 loop; the cells, however, maintain the IL-6 signaling pathway responsiveness. A consistent number of genes particularly relevant for MM biology were found deregulated in CMA-03/06 cell line compared with CMA-03. Furthermore, CMA-03/06 cell line shows an increased resistance to apoptosis. The novel CMA03/06 cell line may thus represent a suitable model for studies investigating molecular mechanisms involved in clonal evolution towards IL-6 and/or stroma-independent growth and survival of myeloma cells. Disclosures No relevant conflicts of interest to declare.

Functional P2X7 Receptors in Myeloma Cell Lines and Myeloma Patient Bone Marrow Samples Can Be Blocked by Specific P2X7 Receptor Antagonists: Implications for Myeloma Therapy and Chemotherapy Induced Inflammation.

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1845-1845
Author(s):  
Kathleen A. Donovan ◽  
Laurie L. Moon-Tasson ◽  
Alexander E. Hromockyj ◽  
Debra M Meyer ◽  
Andrew D Stone ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
P2x7 Receptor ◽  
Myeloma Cell ◽  
Rt Pcr ◽  
P2x7 Receptors ◽  
Dose Dependent

Abstract Abstract 1845 Inflammation is a driving factor in the pathogenesis of many cancers including multiple myeloma (MM). The active role that cytokines play, IL-1β in particular, during the early stages of MM has been extensively characterized. Myeloma is a plasma cell tumor whose early stages are characterized by cytokine dependent growth, predominantly driven by IL-6. We have demonstrated that IL-1β is a key inducer of IL-6 expressed by bone marrow stromal cells in a paracrine fashion during the progression of multiple myeloma from smoldering myeloma (SMM) to active disease (Mayo Clinic Proc 84:114 (Feb. 2009)). Elevated ATP levels have been documented in vivo in tumor micro-environments and is a potential contributing factor to IL-1β induced inflammation. The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by cells of the hematopoetic lineage that has been shown to play a critical role in IL-1b processing and secretion. We investigated the role this receptor may have in the release of IL-1β in the bone marrow microenvironment using bone marrow samples from both chemotherapy treated and untreated patients. Myeloma cell lines were evaluated by RT-PCR for the expression of P2X7R mRNA. Expression was detected and confirmed by sequencing in ANBL-6, MM1.S, U266, RPMI-8226 and KAS-6/1 and was comparable to U937, a previously characterized P2X7R positive cell line. A newly derived plasmacytoma cell line, PCYT3, showed very low levels of message expression. The ability of ATP, the P2X7R agonist, to trigger the processing and release of IL-1β by activation of the P2X7 channel was studied using KAS-Pro, a myeloma cell line stably transfected with a pro- IL-1β gene. IL-1β, measured by ELISA, was released in a dose dependent fashion in response to increasing amounts of ATP ranging from.6mM to 5mM. Four P2X7R specific antagonists were tested for their effect on ATP stimulated IL- 1β release from KAS-Pro; all 4 compounds demonstrated inhibition in a dose dependent manner with varying degrees of potency correlating with P2X7R inhibition. In addition fresh patient samples (8 MGUS/7SMM/9 newly diagnosed MM as well as 12 Treated MM) were evaluated for receptor expression and function. All stages of disease expressed the receptor by RT-PCR. Analysis of samples from all stages of MM, as measured directly by IL-1β ELISA as well as indirectly by measurement of IL-1β induced IL-6 production from normal bone marrow stromal cells, showed functional receptor activation in response to ATP stimulation. IL-6 production detected from MGUS patient samples had a mean result of 2,253 pg/ml, the mean for SMM was 5,495 pg/ml, MM were 6,875 pg/ml while treated MM samples showed a mean result of 15,219 pg/m. Greater than 90% inhibition of IL-1β release by the P2X7R antagonists was seen for all patients who demonstrated a positive ATP induced IL-1β release (see figure). We observed a greater than 2 fold increase in IL-1β release between the treated MM samples and the newly diagnosed MM samples, suggesting a chemotherapy induced inflammation process may be ongoing in these patients. These results confirm the activity of functional P2X7 receptors on myeloma cell lines and in fresh patient samples and suggest a role for the use of P2X7 receptor antagonists in the therapy of myeloma, particularly in patients undergoing chemotherapy. Disclosures: Hromockyj: Pfizer Corporation: Employment. Meyer:Pfizer Corporation: Employment.

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.

scholarly journals Role of interleukin-6 in the proliferation of human multiple myeloma cell lines OCI-My 1 to 7 established from patients with advanced stage of the disease

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 1996-2004 ◽  
Author(s):  
JK Hitzler ◽  
H Martinez-Valdez ◽  
DB Bergsagel ◽  
MD Minden ◽  
HA Messner
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Receptor Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Populations ◽  
Thymidine Uptake ◽  
Auxiliary Cell

Abstract Interleukin-6 (IL-6) has been shown to stimulate the proliferation of multiple myeloma cells purified to a high degree from human bone marrow. IL-6 production in multiple myeloma has been attributed to cells belonging to the myeloma clone, thus supporting a mechanism of autostimulation. In addition, it has been shown that IL-6 may be produced by auxiliary cell populations of the bone marrow that are not part of the myeloma clone. A definitive separation of both putative sources for IL-6 may be difficult to achieve in fresh patient IL-6 growth requirement and production by pure myeloma cell populations using seven human myeloma cell lines (OCI-My 1 to 7) that were established from patients with advanced disease. The proliferative response of each line to recombinant IL-6 was measured in a clonogenic assay providing human plasma and methylcellulose as a viscous support and by 3H-thymidine uptake in liquid suspension culture. We observed marked heterogeneity, ranging from IL-6-dependent colony formation by OCI-My 4, to IL-6-independent growth. All lines expressed mRNA for the IL-6 receptor. Expression of IL-6 mRNA was analyzed after amplification by polymerase chain reaction and was present in five of seven lines. IL- 6 protein was detected by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of two lines (OCI-My 3 and 2). Its functional activity was confirmed in a bioassay using the IL-6-dependent murine hybridoma line B 13.29. This activity was neutralized by anti-IL-6 antibody. Two lines did not express mRNA for IL-6. The remaining three lines expressed mRNA for IL-6, but did not secrete IL-6 protein. Immunoprecipitation experiments with lysates of one of these three lines did not detect the presence of IL-6 protein. These results suggest that autocrine stimulation by IL-6 may occur in some cell lines derived from patients with multiple myeloma. However, it does not represent a universal mechanism in myeloma cell growth.

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2328-2328
Author(s):  
Katja C. Weisel ◽  
Ying Gao ◽  
Jae-Hung Shieh ◽  
Lothar Kanz ◽  
Malcolm A.S. Moore
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stromal Cell Line ◽  
Mes Cells

Abstract The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated &gt;100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of &gt; 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

scholarly journals Characterization of adhesion molecules on human myeloma cell lines

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2306-2314 ◽  
Author(s):  
H Uchiyama ◽  
BA Barut ◽  
D Chauhan ◽  
SA Cannistra ◽  
KC Anderson
Keyword(s):  
Adhesion Molecules ◽  
Cell Lines ◽  
Plasma Cells ◽  
Specific Binding ◽  
Myeloma Cell ◽  
Rgd Peptide ◽  
Beta 1 Integrin ◽  
Marrow Stroma ◽  
And Function ◽  
Alpha V Beta 3

In multiple myeloma, malignant plasma cells are localized in marrow and rarely circulate in peripheral blood. To investigate the role of adhesion proteins in this process, we determined the expression and function of adhesion molecules on cell lines derived from patients with myeloma. The U266, ARH-77, IM-9, and HS-Sultan cell lines strongly expressed beta 1 and alpha 4 integrins (89% to 98% positive), confirming that VLA-4 is the principal integrin on these cell lines. The U266 and IM-9 cell lines also expressed alpha 3 integrin on 15% to 20% cells. In contrast, all lines lacked cell surface alpha 2, alpha 5, and alpha 6 integrin expression (< 5% positive). These cell lines adhered to fibronectin (20% to 40% specific binding), without significant binding to either collagen or laminin. Adhesion of these cell lines to fibronectin was partially blocked with either anti-beta 1 integrin monoclonal antibody (MoAb) (75% inhibition), anti-alpha 4 integrin MoAb (75% inhibition), or RGD peptide (50% inhibition), but was unaffected by anti-alpha v beta 3 or anti-alpha IIb beta 3 MoAbs. Moreover, the combination of anti-beta 1 plus RGD peptide or anti-alpha 4 plus RGD peptide inhibited binding to fibronectin by 80% and 95%, respectively. Finally, pretreatment and coculture of the IM-9 cell line with interleukin-6 (IL-6) resulted in a 52% decrease in specific binding to fibronectin (30% +/- 6% to 15% +/- 6%; P = .001), associated with a decrease in the number of cells expressing VLA-4 and a decrease in intensity of VLA-4 expression. These data suggest that myeloma cells adhere to fibronectin through VLA-4 as well as through RGD-dependent mechanisms, and that this binding can be downregulated by IL-6. Future studies of binding of both myeloma cell lines and freshly isolated tumor cells to extracellular matrix proteins and to marrow stroma may enhance our understanding of localization and trafficking of cells within the bone marrow microenvironment.

scholarly journals Over-expression of c-src or v-src in bone marrow stromal cells stimulates hematopoiesis in long-term bone marrow culture

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3079-3089
Author(s):  
J Mladenovic ◽  
SM Anderson
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conditioned Media ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf

The S17 murine stromal cell line was infected with retroviral vectors encoding the v-src and c-src oncogenes and cells expressing high levels of either pp60v-src or pp60c-src were isolated. Long-term bone marrow cultures (LTBMCs) established with these different stromal cell lines showed that progenitor cells proliferated to a greater extent in cultures with stromal cells that over-expressed either c-src or v-src. An increase in the number of granulocytes, monocytes, and colony- forming units granulocyte-macrophage (CFU-GM) in the nonadherent cell population of LTBMCs prepared with S17/v-src or S17/c-src stromal cells was observed. Conditioned media from the S17/v-src and S17/src stromal cell lines stimulated the formation of CFU-GM in the absence of additional hematopoietic cell growth factors. Conditioned media from S17/v-src and S17/c-src stimulated proliferation of the granulocyte- macrophage colony-stimulating factor (GM-CSF)-responsive cell line FDCP-1 and this stimulation was inhibited by neutralizing antisera to murine GM-CSF. An increase in the concentration of GM-CSF was confirmed by enzyme-linked immunosorbent assay. No secretion of interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha was detected by any of the stromal cell lines. There was no increase in the secretion of either CSF-1 or IL-6 by either S17/v-src or S17/c-src. The addition of 1 micrograms/mL monoclonal anti-GM-CSF antibody to LTBMCs caused a decrease in the number of nonadherent cells in cultures established with each of the different stromal cell lines. Northern blot analysis showed no difference in the level of GM-CSF RNA among the different stromal cell lines. These studies suggest that the increased proliferation of hematopoietic progenitor cells in LTBMCs with S17/v-src or S17/c-src cells may result from a posttranscriptional event that elevates production of GM-CSF by the S17/c-src and S17/v-src stromal cells.

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.

scholarly journals Characterization of adhesion molecules on human myeloma cell lines

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2306-2314 ◽  
Author(s):  
H Uchiyama ◽  
BA Barut ◽  
D Chauhan ◽  
SA Cannistra ◽  
KC Anderson
Keyword(s):  
Adhesion Molecules ◽  
Cell Lines ◽  
Plasma Cells ◽  
Specific Binding ◽  
Myeloma Cell ◽  
Rgd Peptide ◽  
Beta 1 Integrin ◽  
Marrow Stroma ◽  
And Function ◽  
Alpha V Beta 3

Abstract In multiple myeloma, malignant plasma cells are localized in marrow and rarely circulate in peripheral blood. To investigate the role of adhesion proteins in this process, we determined the expression and function of adhesion molecules on cell lines derived from patients with myeloma. The U266, ARH-77, IM-9, and HS-Sultan cell lines strongly expressed beta 1 and alpha 4 integrins (89% to 98% positive), confirming that VLA-4 is the principal integrin on these cell lines. The U266 and IM-9 cell lines also expressed alpha 3 integrin on 15% to 20% cells. In contrast, all lines lacked cell surface alpha 2, alpha 5, and alpha 6 integrin expression (< 5% positive). These cell lines adhered to fibronectin (20% to 40% specific binding), without significant binding to either collagen or laminin. Adhesion of these cell lines to fibronectin was partially blocked with either anti-beta 1 integrin monoclonal antibody (MoAb) (75% inhibition), anti-alpha 4 integrin MoAb (75% inhibition), or RGD peptide (50% inhibition), but was unaffected by anti-alpha v beta 3 or anti-alpha IIb beta 3 MoAbs. Moreover, the combination of anti-beta 1 plus RGD peptide or anti-alpha 4 plus RGD peptide inhibited binding to fibronectin by 80% and 95%, respectively. Finally, pretreatment and coculture of the IM-9 cell line with interleukin-6 (IL-6) resulted in a 52% decrease in specific binding to fibronectin (30% +/- 6% to 15% +/- 6%; P = .001), associated with a decrease in the number of cells expressing VLA-4 and a decrease in intensity of VLA-4 expression. These data suggest that myeloma cells adhere to fibronectin through VLA-4 as well as through RGD-dependent mechanisms, and that this binding can be downregulated by IL-6. Future studies of binding of both myeloma cell lines and freshly isolated tumor cells to extracellular matrix proteins and to marrow stroma may enhance our understanding of localization and trafficking of cells within the bone marrow microenvironment.

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