The ZZ Domain of Sequestosome-1/p62 Plays An Important Role In Stromal Cell Support of Myeloma Cell Growth and Osteoclast Formation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 128-128
Author(s):  
Jumpei Teramachi ◽  
Jolene J. Windle ◽  
David Roodman ◽  
Noriyoshi Kurihara
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
P38 Mapk ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Bone Destruction ◽  
Mapk Signaling ◽  
Sequestosome 1 ◽  
Stromal Cell Line ◽  
Cell Support

Abstract Abstract 128 The marrow microenvironment provides a critical supportive role in myeloma (MM) and enhances both tumor growth and bone destruction through activation of multiple signaling pathways in stromal cells. We reported that sequestosome 1 (p62) plays a key role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with MM. These results suggest that p62 is a potential therapeutic target for blocking the supportive effects of the marrow microenvironment in MM. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling, as a means to develop inhibitory peptides/molecules as potential therapeutic agents for MM. To pursue this objective, we generated deletion constructs of p62 that lacked specific p62 domains: ΔSH2, ΔPB1, ΔZZ, Δp38, ΔTBS and ΔUBA domains. We then transfected these constructs into a p62-knockout (KO) stromal cell-line we established from p62-KO mice and examined their RANKL, IL-6, and VCAM-1 expression induced by TNF-a. GFP-labeled MM1.S myeloma cells or normal CFU-GM, a source of OCL precursors, were then co-cultured with the p62-KO cells transduced with the different p62 deletion constructs and compared to wild type (WT) stromal cells. IL-6 production and VCAM-1 expression induced by TNF-a was 50% lower in non-transduced p62-KO stromal cells compared to WT stromal cells. Further, in contrast to WT cells, RANKL was not induced by TNF-a in the p62-KO stromal cell-line, and OCL formation in co-cultures of p62-KO stromal cells with CFU-GM was very low. Transduction of p62-KO stromal cells with the ΔSH2, ΔPB1, Δp38 and ΔUBA constructs restored stromal cell support of MM growth, VCAM-1 and IL-6 production. However, p62-KO stromal cells transduced with the ΔZZ construct did not increase MM cell growth, or increase IL-6 and VCAM-1 expression, or fully restore the capacity of the p62-KO stromal cells to support OCL formation. These results demonstrate that the ZZ domain of p62 is required for stromal cell support of MM cell growth, increased IL-6 and VCAM-1 expression, and OCL formation. These results suggest that dominant negative constructs or small molecules that target the ZZ domain of p62 should block p62 function and inhibit support of MM cells and OCL formation by the marrow microenvironment. Disclosures: Roodman: Amgen, Celgene, Acceleron & Millennium: Consultancy.

p62 as a Therapeutic Target for Myeloma Cell Growth and Osteoclast Formation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2857-2857
Author(s):  
Fumito Ishizuka ◽  
Jolene Windle ◽  
David Roodman ◽  
Noriyoshi Kurihara
Keyword(s):  
Cell Growth ◽  
P38 Mapk ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Critical Role ◽  
Mapk Signaling ◽  
Marrow Stromal Cells ◽  
Fusion Peptides ◽  
Binding Domains ◽  
Cell Support

Abstract Abstract 2857 Poster Board II-833 We reported that sequestosome 1 (p62) plays a critical role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with multiple myeloma (MM), and that p62 is a potential therapeutic target for MM. In contrast to treating patients with inhibitors of each of the multiple signaling pathways activated in marrow stromal cells by MM cells (e.g. NF-kB or p38 MAPK), blocking the function of p62 should inhibit the activation of the multiple pathways mediated by p62 and have a broader effect on the bone marrow microenvironment. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling in marrow stromal cells when they interact with myeloma cells, and develop inhibitory peptides as potential therapeutic agents that interfere with p62's role in these signaling complexes. To pursue this objective, we transfected p62−/− stromal cells with p62 deletion constructs and assessed their effects on NF-kB and p38 MAPK signaling induced by MM cells or TNF-a. p62−/− stromal cells support of MM growth or OCL formation was significantly decreased compared to WT stromal cells. We made a series of 5' deletion constructs of p62 that lacked specific p62 domains: ΔPB1 (Δ1) lacks homodimerization domain and binding to PKCz, ΔPB1, ZZ (Δ2) lacks PB1 and RIP1 binding domains, and ΔPB1, ZZ, TBS, (Δ3) the PB1, RIP1, p38 and TRAF6 binding domains have been deleted. These constructs were tested for their capacity to restore p62 function in p62−/−stromal cells and support MM cell growth and OCL formation. GFP-labeled MM1.S myeloma cells were cocultured with p62−/− and WT marrow stromal cells transduced with the different p62 deletion constructs. Transduction of p62−/− stromal cells with the full-length p62 construct restored the capacity of p62−/− stromal cells to enhance the growth of MM cells to levels induced by WT stromal cells. Transduction of p62−/− stromal cells with the Δ1 construct also restored stromal cell support of MM growth. Therefore, the PB1 domain is not important for this function. Transduction of p62−/− stromal cells with the Δ2 construct, resulted in an inability of the stromal cells to support MM cell growth. Additional loss of the p38 and TRAF6 binding domains did not further impair p62−/− stromal cells support of MM cell growth. These results suggest that the RIP1 binding domain plays a critical role in supporting the growth of MM cells by marrow stromal cells. We then examined the capacity of p62−/− stromal cells transduced with various p62 deletion constructs to support OCL formation. Normal CFU-GM, a source of OCL precursors, were cocultured with p62−/− stromal cells transfected with the different p62 cDNA deletion constructs. The Δ1 construct completely rescued p62−/− support of OCL formation. However, Δ2 construct transduced p62−/− stromal cells only partly restored stromal cell support of OCL formation. Transduction of the Δ3 construct did not restore the capacity of the p62−/− stromal cells to support OCL formation. Similarly, transduction of the Δ2 and Δ3 construction decreased WT stromal cell support of MM cell growth. We then tested the feasibility of using transduction domain (PTD) fusion peptides as a potential means of delivering dominant negative p62 constructs into stromal cells in vitro and in vivo to block MM cell growth and VCAM-1 expression induced by marrow stromal cells. PTD binding domain fusion peptides containing NEMO binding protein that blocks NF-kB activity was used as a model system to determine the feasibility of transducing marrow stromal cells with p62 constructs. Addition of PTD-NEMO fusion peptides to stromal cells significantly inhibited WT stromal cell to enhance MM cell growth and VCAM-1 expression on stromal cells, which is the capacity of dependent, in part, on NF-kB signaling. These results demonstrate that the ZZ, p38 MAPK and TRAF-6 domains of p62 together are required for stromal cell support of MM cell growth and OCL formation and suggest that PTD constructs containing dominant negatives for p62 may be a feasible method for blocking p62 function in the MM marrow microenvironment. Disclosures: Roodman: Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy.

scholarly journals Increased signaling through p62 in the marrow microenvironment increases myeloma cell growth and osteoclast formation

Blood ◽  
2009 ◽  
Vol 113 (20) ◽  
pp. 4894-4902 ◽  
Author(s):  
Yuko Hiruma ◽  
Tadashi Honjo ◽  
Diane F. Jelinek ◽  
Jolene J. Windle ◽  
Jaekyoon Shin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Growth ◽  
Signaling Pathways ◽  
P38 Mapk ◽  
Stromal Cells ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Marrow Stromal Cells ◽  
Sequestosome 1 ◽  
Multiple Signaling

Adhesive interactions between multiple myeloma (MM) cells and marrow stromal cells activate multiple signaling pathways including nuclear factor κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and Jun N-terminal kinase (JNK) in stromal cells, which promote tumor growth and bone destruction. Sequestosome-1 (p62), an adapter protein that has no intrinsic enzymatic activity, serves as a platform to facilitate formation of signaling complexes for these pathways. Therefore, we determined if targeting only p62 would inhibit multiple signaling pathways activated in the MM microenvironment and thereby decrease MM cell growth and osteoclast formation. Signaling through NF-κB and p38 MAPK was increased in primary stromal cells from MM patients. Increased interleukin-6 (IL-6) production by MM stromal cells was p38 MAPK-dependent while increased vascular cell adhesion molecule-1 (VCAM-1) expression was NF-κB–dependent. Knocking-down p62 in patient-derived stromal cells significantly decreased protein kinase Cζ (PKCζ), VCAM-1, and IL-6 levels as well as decreased stromal cell support of MM cell growth. Similarly, marrow stromal cells from p62−/− mice produced much lower levels of IL-6, tumor necrosis factor-α (TNF-α), and receptor activator of NF-κB ligand (RANKL) and supported MM cell growth and osteoclast formation to a much lower extent than normal cells. Thus, p62 is an attractive therapeutic target for MM.

Blocking the ZZ Domain of Sequestosome 1/p62 Suppress the Enhancement of Myeloma Cell Growth and Osteoclast Formation by Marrow Stromal Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 888-888 ◽  
Author(s):  
Jumpei Teramachi ◽  
Kyaw Ze Yar Myint ◽  
Rentian Feng ◽  
Xiangqun Xie ◽  
Jolene J. Windle ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Mononuclear Cells ◽  
Apoptotic Cell ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Marrow Stromal Cells ◽  
Sequestosome 1

Abstract Abstract 888 The marrow microenvironment enhances both tumor growth and bone destruction in multiple myeloma (MM) through MM cell-induced activation of multiple signaling pathways in bone marrow stromal cells (BMSC) by TNFα. We reported that sequestosome-1 (p62) acts as a signaling hub for NF-kB, MAPK and PI3K activation in BMSC of MM patients and enhances MM growth and osteoclast (OCL) formation. p62 is composed of 5 domains that are involved in protein–protein interactions required for formation of these signaling complexes, but which domain of p62 mediates increase MM growth and OCL formation is unclear. Therefore, deletion constructs of p62 that lacked each of the 5 domains (PB1, ZZ, p38, TBS or UBA) were transfected into a p62−/− stromal cell line. We found that the ZZ domains mediated BMSC enhancement of MM cell growth, IL-6 production, VCAM-1 expression and OCL formation. Using virtual modeling of the ZZ domain, we identified 6 candidate p62-ZZ inhibitory molecules and tested them for their capacity to block enhanced MM cell growth, OCL formation, IL-6 production, and VCAM-1 expression on BMSC induced by TNFα. When MM1.S, RPMI8266, ANBL6 MM cell-lines or CD138+ primary MM cells were cultured in the absence of BMSC with p62-ZZ inhibitor #3 (10mM), this inhibitor directly induced cell death. The p62-ZZ inhibitor-induced cell death was characterized by an increase in reactive oxygen species (ROS) production by inhibiting NF-kB activation, and apoptotic cell death by triggering the activation of caspases 3, 7, and 9. This inhibitor had an IC50 of 4.6mM for MM1.S survival. In contrast, CFU-Blast formation by human CD34+ cells was not inhibited by p62-ZZ inhibitor #3 (10mM). p62-ZZ inhibitor #3 (10mM) treatment of human OCL precursors derived from CFU-GM, inhibited OCL formation by blocking precursor proliferation. To examine the specificity of the p62-ZZ inhibitor #3, we tested its effects on OCL formation by CD11b+ mononuclear cells from wild type (WT) and p62−/− mice cultured with TNFα for 7 days. p62-ZZ inhibitor #3 blocked WT OCL formation but did not block p62−/− OCL formation. The p62-ZZ inhibitor also blocked VCAM-1 expression and IL-6 production by normal and MM patient stromal cells induced by TNFα compared to vehicle. Importantly, the inhibitor (10mM) did not block stromal cell proliferation. Further, the inhibitor blocked TNFα induced PKCζ phosphorylation in stromal cells and MM1.S when the cells were pretreated with the p62-ZZ inhibitor for 3 hours. These results demonstrate that p62-ZZ inhibitor #3 specifically blocks both stromal cells independent and dependent MM cell growth and OCL formation but does not affect hematopoietic or stromal cell growth. These results support p62 as a potential novel therapeutic target for MM. Disclosures: Roodman: Amgen: Consultancy; Millennium: Consultancy.

scholarly journals Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 365-372 ◽  
Author(s):  
JP Wineman ◽  
S Nishikawa ◽  
CE Muller-Sieburg
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Stromal Cell Line ◽  
Cell Maintenance

We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).

scholarly journals Establishment of an immortalized stromal cell line derived from human Endometriotic lesion

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Zhi-Xiong Huang ◽  
Rong-Feng Wu ◽  
Xiao-Mei Mao ◽  
Shao-Min Huang ◽  
Tian-Tian Liu ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Life Span ◽  
Cell Line ◽  
Nude Mouse ◽  
Stromal Cells ◽  
Colony Formation ◽  
Stromal Cell ◽  
Primary Cells ◽  
Immortalized Cells ◽  
Stromal Cell Line

Abstract Background Endometriosis is a benign gynecological disease with obviously feature of estrogen-dependence and inflammatory response. The applications of primary endometriotic stromal cells in research of endometriosis are restricted for short life span, dedifferentiation of hormone and cytokine responsiveness. The objective of this study was to establish and characterize immortalized human endometriotic stromal cells (ihESCs). Methods The endometriotic samples were from a patient with ovarian endometriosis and the primary endometriotic stromal cells were isolated from the endometriotic tissues. The primary cells were infected by lentivirus to establish telomerase reverse transcriptase (hTERT)-induced immortalized cells. Quantification of mRNA and proteins was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot. CCK-8 assay and EdU labeling assay were assigned to assess the growth of ihESCs. Karyotype assay was performed to detect the chromosomes of ihESCs. Colony formation assay and nude mouse tumorigenicity assay were used to evaluate colony-formation and tumorigenesis abilities. Results ihESCs continuously overexpressed hTERT via infection of lentivirus and significant extended the life span reaching 31 passages. The morphology, proliferation and karyotype of ihESCs remained unchanged. The expression of epithelial-mesenchymal transition (EMT) markers, estrogen-metabolizing proteins and estrogen/progesterone receptors (ERs and PRs) were unaltered. Furthermore, the treatment of estrogen increased the proliferation and EMT of ihESCs. Lipopolysaccharides (LPS) and IL-1β remarkably induced inflammatory response. The clonogenesis ability of ihESCs was consistent with primary cells, which were much lower than Ishikawa cells. In addition, nude mouse tumorigenicity assay demonstrated that ihESCs were unable to trigger tumor formation. Conclusion This study established and characterized an immortalized endometriotic stromal cell line that exhibited longer life span and kept the cellular morphology and physiological function as the primary cells. The immortalized cells remained normal feedback to estrogen and inflammatory response. Moreover, the immortalized cells were not available with tumorigenic ability. Therefore, ihESCs would be serviceable as in vitro cell tool to investigate the pathogenesis of endometriosis.

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 >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 > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

Stromal Cells Protect B-CLL Cells from Killing by Antigen-Specific Cytotoxic T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2814-2814
Author(s):  
Katja Zirlik ◽  
Meike Burger ◽  
Philipp Brantner ◽  
Gabriele Prinz ◽  
Maike Buchner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cd8 T Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cytotoxic T Cells ◽  
Protective Effects ◽  
Stromal Cell Line

Abstract B-cell malignancy-derived immunoglobulin (idiotype) and survivin, a member of the inhibitor of apoptosis gene family and a shared tumor-associated antigen, are expressed by B-CLL cells. Idiotype- and survivin-specific cytotoxic T cells (CTLs), capable of lysing primary autologous B-CLL cells, can be induced in patients with B-CLL. However, the leukemia cell microenvironment was shown to protect B-CLL cells from apoptosis. The protective effects of stromal cells can be reversed by CXCR4 antagonists in vitro and resensitize CLL cells to spontaneous and chemotherapy-induced apoptosis. The aim of the present study is to investigate whether stromal cell contact impairs CLL killing by CTLs raised against immunoglobulin- or survivin-derived peptides and whether the addition of CXCR4 inhibitors enhances T cell mediated cytotoxicity. To analyze the T cell response, we isolated CD8+ T cells and PBMCs from HLA-A2+ healthy donors. PBMCs were differentiated into dendritic cells (DCs) and CD40-activated B cells. CD8+ T cells were primarily stimulated with peptide-pulsed DCs and then restimulated weekly with peptide-pulsed CD40-activated B cells. Heteroclitic framework region (FR−), heteroclitic complementarity-determining region (CD−) derived peptides, and native and heteroclitic survivin-derived peptides were used for CTL induction. As expected, heteroclitic peptide modifications increased the binding affinity to HLA-A*0201 compared to the native peptide as predicted by the Parker Score (Median change of predicted half-time of dissociation to HLA class I molecules 1429 minutes) and measured by the T2 binding assay (Fluorescence Index (FI) native 0.2; FI heteroclitic 0.9). Cytotoxicity of T cells was assessed by chromium release assay and by flow cytometry against CFSE-labelled CLL cells alone and in co-culture with unlabelled stromal cells in the absence or presence of CXCR4 blocking agents. The induced CTLs efficiently lysed allogenic HLA-A2+ CLL cells (mean cytotoxicity at 30:1, 10:1, 3:1 effector-to-target (E:T) ratio: 15,5%+/−2,8; 7,5%+/−2,8; and 1,9%+/− 0,6), but not HLA-A2 negative CLL cells. Co-culture of CLL cells with the murine stromal cell line M2-10B4 resulted in protection of CLL cells from lysis by antigen-specific cytotoxic T cells in vitro, indeed suggesting a protective role of the microenvironment (mean cytotoxicity at 30:1, 10:1, 3:1 E:T ratio: 5,2%+/−4,1; 0,4%+/−1,6; 1,2%+/−2,0). In contrast to apoptosis induced by fludarabine, CXCR4 blocking agents did not reverse the protective effects of the stromal cell line on T cell mediated cytotoxicity (mean cytotoxicity 30:1, 10:1, 3:1 E:T ratio: 3,1%+/−2,4; 0,8%+/−2,5; 2,3%+/−1,6). These data indicate that the microenvironment may exert protective effects against immunotherapeutic strategies in CLL. However, the protective interaction is not entirely mediated by the CXCR4 - CXCL12 axis. Additional cell-cell interactions appear to play a role and need to be identified as therapeutic targets in order to effectively interrupt the protective effect of the microenvironment on T cell mediated cytotoxicity of B-CLL cells.

scholarly journals Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2612-2617 ◽  
Author(s):  
Y Gunji ◽  
T Sudo ◽  
J Suda ◽  
Y Yamaguchi ◽  
H Nakauchi ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Liver Cells ◽  
B Cell Differentiation ◽  
Stromal Cell Line ◽  
Fetal Liver Cells

We compared the development of B-cell progenitors with that of myeloid progenitors in fetal liver cells at various gestational ages. Day 12 to 14 fetal liver cells did not form pre-B-cell colonies. Pre-B-cell colonies were developed from day 15 fetal liver cells. The incidence of colonies increased with increases in gestational age and reached a maximum on days 18 to 19. In contrast, the incidence of myeloid colonies formed in the presence of interleukin-3 (IL-3) and erythropoietin did not change significantly during days 13 to 21 of gestation. After coculturing day 13 fetal liver cells with IL-7- producing stromal cell line ST-2, they could respond to IL-7 and proliferate. Analysis of the phenotypes showed that day 13 fetal liver cells were B220-, IgM-, while culturing day 13 fetal liver cells with ST-2 and untreated day 18 fetal liver cells contained the population of B220+ cells. Even in the presence of IL-7-defective stromal cell line FLS-3, IL-7-responsive cells could be induced from day 13 fetal liver cells. IL-7 acted on B220+ cells and induced pre-B-cell colonies that contained IgM+ cells in the methylcellulose culture. IL-7 mRNA was expressed in days 13 and 18 fetal liver cells but not in pre-B cells or adult liver cells. From these findings, it is suggested that stromal cells or stromal-derived factors but not IL-7 were required for the differentiation from B220- cells to B220+ cells. In the second stage, B220+, IgM- cells proliferated and some of them differentiated to IgM+ cells in the presence of IL-7 alone. The two-step model can apply to in vivo early B lymphopoiesis.

scholarly journals Integrin β2 (CD18)-Mediated Cell Proliferation of HEL Cells on a Hematopoietic-Supportive Bone Marrow Stromal Cell Line, HESS-5 Cells

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1263-1271 ◽  
Author(s):  
Takashi Tsuji ◽  
Iwao Waga ◽  
Katsunari Tezuka ◽  
Masafumi Kamada ◽  
Kimio Yatsunami ◽  
...  
Keyword(s):  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Cell Contact ◽  
Β2 Integrin ◽  
Hel Cells ◽  
Stromal Cell Line ◽  
Direct Cell ◽  
The Common

Abstract Cellular interactions between hematopoietic cells and stromal cells play important roles in the proliferation and differentiation of hematopoietic cells. The proliferation of a human erythroleukemia cell line, HEL cells, which can differentiate into macrophage- and megakaryocyte-like cells, and erythroid precursors was dramatically induced on coculture with a hematopoietic-supportive stromal cell line, HESS-5 cells, which can support long-term hematopoiesis in vitro without fetal bovine serum. HEL cells proliferated when they were cocultured with but not without direct cell contact. Because the coculture supernatants with direct cell contact and cytokines such as interleukins and growth factors did not exhibit growth-stimulating activity toward HEL cells, it was suggested that some molecule that has growth-stimulating activity exists on the surface of the cells. Extracellular matrix components such as fibronectin, laminin, vitronectin, and collagen did not affect the proliferation of HEL cells. An anti-CD18 monoclonal antibody, which recognizes the common β chain of the β2 integrin subfamily, induced dramatic proliferation of HEL cells. Moreover, the proliferation of HEL cells was inhibited by an antisense oligonucleotide of CD18 mRNA. As judged from these observations, the proliferation of HEL cells was mediated by CD18 molecules expressed on HEL cells. On the contrary, the common counter-receptor of the β2 integrin subfamily, intercellular adhesion molecule-1, which is expressed on CHO-K1 cells, did not stimulate the growth of HEL cells. It is known that other counter molecules of the β2 integrin subfamily, such as complement C3bi and fibrinogen, are not produced by stromal cells. These findings suggest that the proliferation of HEL cells may be induced through an interaction between a novel molecule of the β2 integrin subfamily on HEL cells and the counter-receptor on HESS-5 cells. The β2 integrin subfamily may regulate the growth of hematopoietic cells in hematopoiesis in vivo and/or cause the abnormal growth of leukemia cells.

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