scholarly journals Proteoglycan synthesis in two murine bone marrow stromal cell lines

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1777-1783 ◽  
Author(s):  
SL Kirby ◽  
SA Bentley
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Dermatan Sulfate ◽  
Cell Layer ◽  
Gradient Centrifugation ◽  
Exchange Chromatography ◽  
Proteoglycan Synthesis ◽  
Murine Bone Marrow

There is evidence indicating that stromal proteoglycans are an important functional component of the hematopoietic microenvironment. Proteoglycan synthesis was therefore investigated in the MS3–2A and D2XRII hematopoietic stromal cell lines. These lines differ in their capacity to support hematopoiesis in vitro, D2XRII supporting in vitro hematopoiesis, whereas MS3–2A does not. Cells were labeled with 35S- sulfate as precursor, and 4 mol/L guanidine HCl extracts of cells and media were analyzed by ion-exchange chromatography, cesium chloride density gradient centrifugation, and molecular sieve chromatography. Proteoglycans were further examined by enzymatic and chemical digestions. MS3–2A cells produced at least three proteoglycan species. Two chondroitin/dermatan sulfate (CS/DS) proteoglycans, Kav = 0.40 and Kav = 0.68 on Sepharose CL-2B, were present primarily in the medium. The respective glycosaminoglycan molecular weight (mol wt) values were 38 kd and 40 kd. A heparan sulfate (HS) proteoglycan of Kav = 0.58 and glycosaminoglycan mol wt 36 kd was present primarily in the cell layer extract. D2XRII cells synthesized two HS proteoglycans. The larger (Kav = 0.45; glycosaminoglycan mol wt, 30 kd) was of low density on gradient centrifugation and more prominent in the cell layer extracts, whereas the smaller (Kav = 0.68; glycosaminoglycan mol wt, 38 kd) was dense and present mainly in the culture medium. A single CS/DS proteoglycan species of Kav 0.78 and average glycosaminoglycan of mol wt 18 kd was present in roughly equal amounts in the medium and in the cell layer. MS3–2A and D2XRII thus appear phenotypically distinct with respect to proteoglycan synthesis. These differences are discussed in relation to the microenvironmental function of bone marrow stromal elements.

scholarly journals Proteoglycan synthesis in two murine bone marrow stromal cell lines

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1777-1783 ◽  
Author(s):  
SL Kirby ◽  
SA Bentley
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Dermatan Sulfate ◽  
Cell Layer ◽  
Gradient Centrifugation ◽  
Exchange Chromatography ◽  
Proteoglycan Synthesis ◽  
Murine Bone Marrow

Abstract There is evidence indicating that stromal proteoglycans are an important functional component of the hematopoietic microenvironment. Proteoglycan synthesis was therefore investigated in the MS3–2A and D2XRII hematopoietic stromal cell lines. These lines differ in their capacity to support hematopoiesis in vitro, D2XRII supporting in vitro hematopoiesis, whereas MS3–2A does not. Cells were labeled with 35S- sulfate as precursor, and 4 mol/L guanidine HCl extracts of cells and media were analyzed by ion-exchange chromatography, cesium chloride density gradient centrifugation, and molecular sieve chromatography. Proteoglycans were further examined by enzymatic and chemical digestions. MS3–2A cells produced at least three proteoglycan species. Two chondroitin/dermatan sulfate (CS/DS) proteoglycans, Kav = 0.40 and Kav = 0.68 on Sepharose CL-2B, were present primarily in the medium. The respective glycosaminoglycan molecular weight (mol wt) values were 38 kd and 40 kd. A heparan sulfate (HS) proteoglycan of Kav = 0.58 and glycosaminoglycan mol wt 36 kd was present primarily in the cell layer extract. D2XRII cells synthesized two HS proteoglycans. The larger (Kav = 0.45; glycosaminoglycan mol wt, 30 kd) was of low density on gradient centrifugation and more prominent in the cell layer extracts, whereas the smaller (Kav = 0.68; glycosaminoglycan mol wt, 38 kd) was dense and present mainly in the culture medium. A single CS/DS proteoglycan species of Kav 0.78 and average glycosaminoglycan of mol wt 18 kd was present in roughly equal amounts in the medium and in the cell layer. MS3–2A and D2XRII thus appear phenotypically distinct with respect to proteoglycan synthesis. These differences are discussed in relation to the microenvironmental function of bone marrow stromal elements.

Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells

1986 ◽  
Vol 6 (9) ◽  
pp. 3221-3231
Author(s):  
R C Schwartz ◽  
L W Stanton ◽  
S C Riley ◽  
K B Marcu ◽  
O N Witte
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Retroviral Vectors ◽  
Soft Agar ◽  
Lymphoid Cells ◽  
Neoplastic Progression ◽  
Direct Role ◽  
Murine Bone Marrow ◽  
Myc Oncogene

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.

scholarly journals Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 447-455 ◽  
Author(s):  
D Zipori ◽  
J Toledo ◽  
K von der Mark
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cell ◽  
Leukemic Cells ◽  
Matrix Composition ◽  
Mouse Bone Marrow ◽  
Type I

Abstract Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

Neutrophil Elastase Is Important for Several Activities of PML-RARα in Early Myeloid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 486-486
Author(s):  
Andrew A. Lane ◽  
Timothy J. Ley
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Neutrophil Elastase ◽  
Fusion Gene ◽  
Myeloid Cells ◽  
Full Length ◽  
Intrinsic Defect ◽  
Murine Bone Marrow

Abstract Acute promyelocytic leukemia (APL) is associated with the accumulation of promyelocytes that usually carry the t(15;17) translocation, generating the PML-RARα fusion gene. APL develops in mice only if PML-RARα expression is targeted to early myeloid cells; when PML-RARα is expressed in all hematopoietic cells, the only malignancy that develops is APL. These observations have suggested that there may be a component(s) of the early myeloid environment that is important for the actions of PML-RARα. We recently showed that murine bone marrow extracts and a human early myeloid cell line, U937, contain a serine protease activity that cleaves PML-RARα. The dominant cleaving activity was attributable to an early myeloid-associated protease, neutrophil elastase (NE). The penetrance of APL in mice expressing PML-RARα in early myeloid cells was significantly reduced in NE-deficient animals (Lane and Ley, Cell 115:305–318, 2003). To determine whether measurable functions of PML-RARα require NE, we performed a series of experiments. Using a GFP-PML-RARα fusion construct, we determined that expression of PML-RARα caused disruption of PML oncogenic domains (PODs) in several human hematopoietic cell lines, regardless of whether they expressed NE. However, when PML-RARα was expressed at high levels in cell lines that contained NE activity, substantial toxicity was observed. In contrast, PML-RARα did not cause toxicity in cell lines without NE activity. Similarly, when we expressed an NE-resistant PML-RARα cDNA (containing valine to arginine mutations at the P1 amino acid of the two preferred NE cleavage sites), toxicity was abrogated in clonogenic assays. The toxic effects of expressing full-length PML-RARα could not be recapitulated by expressing either dominant cleavage fragment alone, or in combination, which suggests that NE-induced cleavage may not directly create the “active” fragment of PML-RARα. Primary human and mouse primary APL cells contain abundant NE activity and PML-RARα-cleaving activity. However, we determined that two commonly used APL cell line models (NB4 and U937-PR9 cells) contain abundant full length PML-RARα protein, but neither contains NE activity nor PML-RARα-cleaving activity. As expected, high levels of expression of PML-RARα in PR9 cells did not cause toxicity. Using an in vitro G-CSF-dependent myeloid differentiation assay, we found that purified hematopoietic progenitors from mCGPML-RARα knock-in mice demonstrated markedly increased proliferation of early (blasts and promyelocytes) myeloid cells compared to wild type progenitors. This increase was not observed in progenitors from mCGPML-RARαNE−/− animals. The difference was not due to an intrinsic defect in myeloid development due to NE deficiency, since NE−/− progenitors developed normally in response to G-CSF in vitro. To extend these results, we performed competitive repopulation bone marrow transplants using NE+/+ and NE−/− mice as bone marrow donors; at 3 weeks after hematopoietic reconstitution, there was no difference in the contributions of either genotype to multi-lineage hematopoiesis. Together, these data strongly suggest that NE is important for several of the measurable activities of PML-RARα in early myeloid cells; we are therefore exploring pharmacologic NE inhibition as an approach to reduce the growth of APL cells. Our data also suggest that the physiologic activities of PML-RARα should be studied in early myeloid cells that contain NE activity.

Re-Educating Myeloma Associated Macrophages with Lenalidomide

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2034-2034 ◽  
Author(s):  
Heiko Bruns ◽  
Hanna Gehlen ◽  
Jens Nolting ◽  
Shirin Pasemann ◽  
Peter Brossart ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Plasma Cells ◽  
In Vivo Studies ◽  
Effector Cells ◽  
Effector Functions ◽  
Anti Inflammatory

Abstract Introduction: The bone marrow niche plays a critical role in determining the fate of malignant plasma cells in multiple myeloma (MM). Macrophages are an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Conversely, macrophages can directly kill tumor cells and participate in antitumor immune responses as effector cells. Moreover, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies. Lenalidomide, an immunomodulatory drug (IMiD®) is used for the treatment of MM, also in the combination with therapeutic antibodies. Lenalidomide is thought to target the stromal support, but its precise influence on the phenotype or the effector functions of macrophages is still unclear. Methods: To investigate the effect of lenalidomide on the interaction between macrophages and malignant plasma cells in vitro, we coincubated lenalidomide pretreated macrophages with several MM cell lines, and analysed the viability, proliferation and phenotype. For in vivo studies we utilized 5TMM mice, a suitable animal model for MM. Animals were treated with lenalidomide (50 mg/kg 5days/week) for 3 weeks, and the effector functions and phenotype of isolated bone marrow macrophages were analyzed. In addition, macrophages in the bone marrow of MM patients treated with lenalidomide were characterized by immunohistochemistry and flow cytometry. Results: We showed, that infiltrating macrophages in the bone marrow of MM patients display an anti-inflammatory M2-like phenotype characterized by the expression of surface marker CD163, CD206, PD-L1 and cytokine/chemokine secretion (e.g. IL10, CXCL10, APRIL, BAFF and RANKL). Incubation of macrophages with lenalidomide in vitro, substantially changed their transcriptional program (e.g. downregulation of IRF4 and upregulation of IRF5) and their phenotype (e.g. downregulation of the surfaces marker CD163, CD206, and upregulation of CD16, CD64, CD40 and CD86). Furthermore, we show that lenalidomide treatment decreases the expression of RANKL, BAFF and APRIL, while tumoricidal effector molecules (e.g. TRAIL, cathelicidine, Granzyme B) were increased. When lenalidomide treated macrophages were cocultured with MM cells significant cytotoxicity was detected, for all MM cell lines tested. In contrast, untreated macrophages promote tumor growth and viability of MM cells. Conclusion: Lenalidomide in vitro influences macrophages by reverting an anti-inflammatory M2 like profile to a more immunogenic phenotype. In addition it impacts on the support function by decreasing the secretion of important growth factors for B-cells. Similar results were observed in first in vivo studies. Taken together our results imply that lenalidomide interrupts an important stromal cell function thereby influencing survival of MM cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression of alpha-smooth muscle actin in murine bone marrow stromal cells

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 304-309 ◽  
Author(s):  
A Peled ◽  
D Zipori ◽  
O Abramsky ◽  
H Ovadia ◽  
E Shezen
Keyword(s):  
Bone Marrow ◽  
Smooth Muscle ◽  
Cell Lines ◽  
Smooth Muscle Actin ◽  
Muscle Actin ◽  
Alpha Smooth Muscle Actin ◽  
Murine Bone Marrow ◽  
Cellular Origin ◽  
Hematopoietic Activity

Human fibrotic bone marrow (BM) stroma has been shown to contain alpha- smooth muscle actin (alpha-SMA)-positive cells. These closely resemble myofibroblasts that were described in other fibrotic tissues. We studied the expression of alpha-SMA in a series of murine BM-derived stromal cell lines to investigate the cellular origin and functional significance of myofibroblast-like cells in hematopoietic tissues. Although these cell lines differed in their biologic properties, most of them expressed alpha-SMA under certain conditions. Cells expressing alpha-SMA constituted a minor population in post-confluent, growth- arrested cultures. However, the incidence of cells expressing alpha-SMA increased significantly when cultures were transferred to nonconfluent conditions. A similar increase in alpha-SMA-positive cells occurred after a strip of cells was scraped away from the confluent cell layer; the cells of the affected area acquired alpha-SMA-positive contractile phenotype. The relationship between alpha-SMA expression and hematopoietic activity was studied using a cloned cell line of BM origin (14F1.1). The ability of these endothelial-adipocyte cells to support hematopoiesis in vitro was maximal under confluent conditions, whereas their expression of alpha-SMA under such conditions was residual. Moreover, in long-term BM cultures supported by confluent 14F1.1 cells, stromal areas associated with proliferating hematopoietic precursors, known as “cobblestone areas,” were devoid of alpha-SMA- positive cells. These observations suggest that the expression of alpha- SMA is reversible and inversely related to hematopoietic activity.

P5. In vitro properties of two immortalised stromal cell lines derived from rabbit bone marrow

Bone ◽  
1994 ◽  
Vol 15 (2) ◽  
pp. 231
Author(s):  
E Brown ◽  
C Simpson ◽  
ME Nuttall ◽  
B Ashton ◽  
D Johnstone
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Rabbit Bone
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