[Show abstract][Hide abstract] ABSTRACT: Activating mutations in the NOTCH1 gene are found in over 50 % of T-ALL cases. Since Notch signaling contributes to the leukemia cell survival and growth, targeting Notch signaling using γ-secretase inhibitors (GSI) has been proposed as a molecularly targeted therapy for the treatment of T-ALL. However, not all T-ALL with NOTCH1 activating mutations respond to GSI treatment. We examined whether GSI could enhance the cytotoxic effect of anti-leukemic agents in the GSI-resistant T-ALL cells although GSI does not have anti-tumor effect as a single agent. GSI significantly increased cell death induced by Vincristine (VCR) but not other anti-leukemic drugs (Methotrexate, Asparaginase, and Cytarabine). The GSI effect in enhancing VCR efficacy was not the result of inhibition of Notch signaling. GSI augmented VCR-induced mitotic arrest, followed by apoptosis. GSI accelerated VCR-triggered loss of mitochondrial membrane potential and caspase-mediated apoptosis. Our finding suggests that GSI has other functions besides inhibiting Notch signaling in T-ALL and incorporating GSI into the conventional regimen containing VCR may offer therapeutic advantage by potentiating VCR treatment in leukemia patients.
[Show abstract][Hide abstract] ABSTRACT: The germinal center (GC) is a dynamic microenvironment where antigen (Ag)- activated B cells rapidly expand and differentiate, generating plasma cells (PC) that produce high-affinity antibodies. Precise regulation of survival and proliferation of Ag-activated B cells within the GC is crucial for humoral immune responses. The follicular dendritic cells (FDC) are the specialized stromal cells in the GC that prevent apoptosis of GC-B cells. Recently, we reported that human GC-B cells consist of CD9+ and CD9- populations and that it is the CD9+ cells that are committed to the PC lineage. In this study, we investigated the functional role of CD9 on GC-B cells. Tonsillar tissue section staining revealed that in vivo CD9+ GC-B cells localized in the light zone FDC area. Consistent this, in vitro CD9+ GC-B cells survived better than CD9- GC-B cells in the presence of HK cells, an FDC line, in a cell-cell contact-dependent manner. The frozen tonsillar tissue section binding assay showed that CD9+ GC-B cells bound to the GC area of tonsillar tissues significantly more than the CD9- GC-B cells did and that the binding was significantly inhibited by neutralizing anti-integrin β1 antibody. Furthermore, CD9+ cells bound to soluble VCAM-1 more than CD9- cells did, resulting in activation and stabilization of the active epitope of integrin β1. All together, our data suggest that CD9 on GC-B cells contributes to survival by strengthening their binding to FDC through the VLA4/VCAM-1 axis.
FEBS Open Bio 04/2014; 4. DOI:10.1016/j.fob.2014.04.001 · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gamma secretase inhibitors (GSI), cell-permeable small-molecule inhibitors of gamma secretase activity, had been originally developed for the treatment of Alzheimer disease. In recent years, it has been exploited in cancer research to inhibit Notch signaling that is aberrantly activated in various cancers. We previously found that GSI could synergize with anti-microtubule agent, vincristine (VCR) in a Notch-independent manner. Here, we delineate the underlying cell cycle-related mechanism using HeLa cells, which have strong mitotic checkpoints. GSI enhanced VCR-induced cell death, although GSI alone did not affect cell viability at all. GSI augmented VCR-induced mitotic arrest in a dose-dependent manner, which was preceded by apoptotic cell death, as shown by an increase in Annexin V-positive and caspase-positive cell population. Furthermore, GSI amplified multi-polar spindle formation triggered by VCR. Altogether, we show the evidence that GSI enhances VCR-induced apoptosis in HeLa cells via multi-polar mitotic spindle formation, independent of Notch signaling. These data suggest that one or more GS substrates, yet to be identified, in a post-GS processed form, may play a role in maintaining functional centrosomes/mitotic spindles. More significantly, the synergistic effect of GSI in combination with VCR could be exploited in clinical setting to improve the efficacy of VCR.
[Show abstract][Hide abstract] ABSTRACT: A dynamic interaction occurs between the lymphoma cell and its microenvironment, with each profoundly influencing the behavior of the other. Here, using a clonogenic coculture growth system and a xenograft mouse model, we demonstrated that adhesion of mantle cell lymphoma (MCL) and other non-Hodgkin lymphoma cells to lymphoma stromal cells confers drug resistance, clonogenicity, and induction of histone deacetylase 6 (HDAC6). Furthermore, stroma triggered a c-Myc/miR-548m feed-forward loop, linking sustained c-Myc activation, miR-548m downregulation, and subsequent HDAC6 upregulation and stroma-mediated cell survival and lymphoma progression in lymphoma cell lines, primary MCL and other B cell lymphoma cell lines. Treatment with an HDAC6-selective inhibitor alone or in synergy with a c-Myc inhibitor enhanced cell death, abolished cell adhesion-mediated drug resistance, and suppressed clonogenicity and lymphoma growth ex vivo and in vivo. Together, these data suggest that the lymphoma-stroma interaction in the lymphoma microenvironment directly impacts the biology of lymphoma through genetic and epigenetic regulation, with HDAC6 and c-Myc as potential therapeutic targets.
The Journal of clinical investigation 10/2013; 123(11). DOI:10.1172/JCI64210 · 13.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The germinal center (GC) is the dymanic microenvironment where Ag-activated B cells rapidly expand and differentiate, generating plasma cells (PC) that produce high affinity antibodies. B cells within the GC have great heterogeneity, containing B cells at different stages of activation and differentiation. However, there are few surface markers that allow subsets of GC-B cells to be distinguished. In the present study, we show that GC-B cells in human tonsils contain two distinct populations regarding CD9 expression; CD9- and CD9+ cells. CD9+ GC-B cells are functionally more differentiated towards PC based upon the following evidence; (1) CD9+ cells express higher levels of PC transcription factor, Blimp-1 while lower levels of B cell transcription factors, Bcl-6 and Pax-5, compared to CD9- cells, (2) CD9+ cells differentiate into plasmablasts faster than CD9- cells in the presence of cytokines that generate PC, and (3) CD9 expression was induced in CD9- GC-B cells under PC generating condition and gradually increased in the course of PC differentiation. Taken together, our data suggest that CD9 is a novel marker for a human GC-B cell subset that is committed to PC lineage.
Biochemical and Biophysical Research Communications 01/2013; 431(1). DOI:10.1016/j.bbrc.2012.12.102 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Follicular dendritic cells (FDCs), an essential component of the lymph node microenvironment, regulate and support B-lymphocyte differentiation, survival, and lymphoma progression. Here, we demonstrate that adhesion of mantle cell lymphoma and other non-Hodgkin lymphoma cells to FDCs reduces cell apoptosis and is associated with decreased levels of the proapoptotic protein, Bim. Bim down-regulation is posttranscriptionally regulated via up-regulation of microRNA-181a (miR-181a). miR-181a overexpression decreases, whereas miR-181a inhibition increases Bim levels by directly targeting Bim. Furthermore, we found that cell adhesion-up-regulated miR-181a contributes to FDC-mediated cell survival through Bim down-regulation, implicating miR-181a as an upstream effector of the Bim-apoptosis signaling pathway. miR-181a inhibition and Bim upregulation significantly suppressed FDC-mediated protection against apoptosis in lymphoma cell lines and primary lymphoma cells. Thus, FDCs protect B-cell lymphoma cells against apoptosis, in part through activation of a miR-181a-dependent mechanism involving down-regulation of Bim expression. We demonstrate, for the first time, that cell-cell contact controls tumor cell survival and apoptosis via microRNA in mantle cell and other non-Hodgkin lymphomas. Regulation of microRNAs by B-cell-FDC interaction may support B-cell survival, representing a novel molecular mechanism for cell adhesion-mediated drug resistance and a potential therapeutic target in B-cell lymphomas.
[Show abstract][Hide abstract] ABSTRACT: Histological transformation, a pivotal event in the natural history of cancers including lymphomas, is typically associated with more aggressive clinical behavior. L3055, a B lymphoma cell line of germinal center (GC) origin, is dependent on follicular dendritic cells (FDCs) for survival and proliferation, similar to GC-B cells. However, L3055 cells become less FDC-dependent after prolonged culture, which is analogous to transformation in vivo. Comparison of two L3055 subclones (i.e., the FDC-dependent indolent clone 12 and the FDC-independent aggressive clone 33) by DNA microarray revealed that CD9 was the most differentially expressed gene (P = 0.05). L3055-12 expresses high levels of CD9 while L3055-33 does not. Reduced levels or loss of CD9 expression is also observed in other CD9-positive B lymphoma cell lines. The resultant CD9-negative cells grow faster than CD9-positive cells due to their greater resistance to apoptosis. Furthermore, CD9-negative cells are less dependent on FDCs for their survival and growth compared with CD9-positive cells. CD9 down-regulation in B lymphomas appears to be controlled epigenetically, mainly through histone modifications. These findings imply that CD9 is inversely correlated with B lymphoma progression, and CD9 inactivation may play an important role in B lymphoma transformation.
American Journal Of Pathology 07/2010; 177(1):377-86. DOI:10.2353/ajpath.2010.100048 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The GC is the anatomical site where antigen-activated B cells differentiate into PC, producing high-affinity antibodies in physiological and pathological states. PC differentiation is regulated by multiple factors within the GC microenvironment, including cytokines. IL-21, a recently identified type I cytokine produced by GC-Th cells, promotes differentiation of human B cells into ISC. In this study, we investigated in detail the functional role of IL-21 in the course of GC-B cell differentiation into terminally differentiated PC compared with that of IL-10, a well-known PC differentiation factor. IL-21 had a greater capacity to initiate PC differentiation from CD77(+) centroblasts than IL-10 by strongly inducing PC transcription factors through activation of STAT3; however, IL-10 was more potent than IL-21 in generating CD138(+) PC from CD20(-)CD38(++) plasmablasts in the terminal stage of GC-B cell differentiation. This differential effect of IL-21 and IL-10 was reflected in receptor expression on B cell subsets emerging in the course of differentiation. Our studies have revealed that IL-21 is a critical decision-maker for driving initial PC differentiation at the stage of CD77(+) centroblasts, yet IL-10 is more effective in producing IgG by generating terminally differentiated CD138(+) PC at the later stage of PC differentiation in the GC.
[Show abstract][Hide abstract] ABSTRACT: The Notch signaling pathway is one of the most conserved mechanisms to regulate cell fate in many tissues during development and postnatal life. In the immune system, Notch signaling regulates T and B cell development and modulates the differentiation of T and B cells. In this study, we investigated the functional roles of Notch signaling in human B cell differentiation within the germinal center (GC). Notch ligands, Delta-like 1 (Dll1) and Jagged 1 (Jg1), are expressed by follicular dendritic cells (FDC) but not by B cells in the GC, while GC-B cells express the Notch receptors, Notch1 and Notch2. The blockade of Notch signaling pathways using a gamma-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester), reduces the survival of GC-B cells in the presence of FDC/HK cells. Jg1 has a dominant effect on GC-B cell survival mediated by Notch signaling. Furthermore, Notch cooperates with another anti-apoptotic factor, BAFF/Blys produced by FDC to support GC-B cell growth. Taken together, our data shows the important role of Notch signaling provided by FDC in the survival of GC-B cells in vitro.
The Journal of Immunology 08/2009; 183(1):352-8. DOI:10.4049/jimmunol.0803183 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Follicular lymphoma, the neoplastic counterpart of germinal center B cells, typically recapitulates a follicular architecture. Several observations point to the crucial role of the cellular microenvironment in the development and/or progression of follicular lymphoma cells in vivo. The aim of our study was to characterize the spontaneous apoptosis of follicular lymphoma cells in vitro, and the modulation of this apoptosis by follicular dendritic cells.
We used a cell line derived from follicular dendritic cells to model the functional interactions of these cells and lymphoma cells in co-culture. Follicular lymphoma cells were isolated from tissue biopsies. Apoptosis was quantified by flow cytometry and apoptotic pathways were investigated by western blotting.
The spontaneous apoptosis of follicular lymphoma cells in vitro involves the activation of caspases-3 and -8 but not of caspase-9, occurs despite persistent high levels of BCL-2 and MCL-1, and is associated with down-regulation of c-FLIP(L). Spontaneous apoptosis of follicular lymphoma cells is partially prevented by co-culture with the follicular dendritic cells, which prevents activation of caspase-8, caspase-3 and induces an upregulation of c-FLIP(L). Using neutralizing antibodies, we demonstrated that interactions involving CD54 (ICAM-1), CD106 (VCAM-1) and CD40 are implicated in this biological process.
Follicular dendritic cells constitute a useful tool to study the functional interactions between follicular lymphoma cells and follicular dendritic cells in vitro. Understanding the molecular mechanisms involved in these protective interactions may lead to the identification of therapeutic agents that might suppress the survival and growth of follicular lymphoma cells.
[Show abstract][Hide abstract] ABSTRACT: Memory B cells provide rapid protection to previously encountered antigens; however, how these cells develop from germinal center B cells is not well understood. A previously described in vitro culture system using human tonsillar germinal center B cells was used to study the transcriptional changes that occur during differentiation of human memory B cells. Kinetic studies monitoring the expression levels of several known late B cell transcription factors revealed that BCL-6 is not expressed in memory B cells generated in vitro, and gene expression profiling studies confirmed that BCL-6 is not expressed in these memory B cells. Furthermore, ectopic expression of BCL-6 in human B cell cultures resulted in formation of fewer memory B cells. In addition, the expression profile of in vitro memory B cells showed a unique pattern that includes expression of genes encoding multiple costimulatory molecules and cytokine receptors, antiapoptotic proteins, T cell chemokines, and transcription factors. These studies establish new molecular criteria for defining the memory B cell stage in human B cells.
Journal of Experimental Medicine 04/2007; 204(4):819-30. DOI:10.1084/jem.20062104 · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: B cell-activating factor of the tumor necrosis factor family (BAFF/BLys) plays a critical role in B cell survival and immune responses through its three receptors: BAFF receptor (BAFF-R/BR3), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA). Using specific antibodies, we have investigated the expression of BAFF-R on human tonsillar B cells and their functional roles in naive and germinal center (GC) B cell differentiation. Our studies show that BAFF-R is the dominant receptor on naive B cells. However, three receptors are differentially modulated during in vitro GC-B cell differentiation. BAFF-R expression increased initially and then decreased with a corresponding induction of TACI and BCMA expression during differentiation to plasma cells (PCs). Consistently, blocking of BAFF-R alone with specific mAb inhibited GC-B cell proliferation and PC generation in the early period of their differentiation, whereas depletion of BAFF with TACI-Ig exhibited consistent inhibition throughout the differentiation. Finally, histological and molecular analyses of human tonsil tissue revealed that follicular dendritic cells produce BAFF. In conclusion, BAFF in the GC plays an important role through more than one receptor, and the three known receptors are differentially modulated as GC-B cells differentiate to PCs.
International Immunology 07/2005; 17(6):779-88. DOI:10.1093/intimm/dxh259 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The germinal centre is a dynamic microenvironment where antigen-activated B cells rapidly expand and differentiate, generating plasma cells and memory B cells. These cellular events are accompanied by dramatic changes in the antibody molecules that undergo somatic hypermutation and isotype switching. Follicular dendritic cells (FDCs) are the stromal cells located in the germinal centre. Although the capacity of FDCs to present antigen to B cells through antigen-antibody complexes has been recognized for many years, additional critical functions of FDCs have only recently been recognized. FDCs prevent apoptosis of germinal centre B cells and stimulate cellular interaction and proliferation. Here, we review the FDC signalling molecules that have recently been identified, some of which offer potential therapeutic targets for autoimmune diseases and B-cell lymphomas.
[Show abstract][Hide abstract] ABSTRACT: Factors that control the survival and proliferation of Ag-stimulated B cells within the germinal center (GC) are crucial for humoral immune responses with high affinity Abs against infectious agents. The follicular dendritic cell (FDC) is known as a key cellular component of the GC microenvironment for GC-B cell survival and proliferation. In this study, we report that IL-15 is produced by human FDC in vivo and by an FDC cell line, FDC/HK cells, in vitro. IL-15 is captured by IL-15Ralpha on the surface of FDC/HK cells. The surface IL-15 is functionally active and augments GC-B cell proliferation. Because GC-B cells have the signal-transducing components (IL-2/15Rbetagamma), but not a receptor for binding of soluble IL-15 (IL-15Ralpha), IL-15 signaling is possibly transduced by transpresentation from FDCs to GC-B cells via cell-cell contact. Together, these results suggest that IL-15 from FDC, in membrane-bound form, plays an important role in supporting GC-B cell proliferation, proposing a new target for immune modulation as well as treatment of B cell tumors of GC origin.
The Journal of Immunology 01/2005; 173(11):6676-83. DOI:10.4049/jimmunol.173.11.6676 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lymphoid follicle is a specialized microenvironment for the differentiation of antigen (Ag)-activated B cells; the major stromal cell components in lymphoid follicle are the follicular dendritic cells (FDCs). At the same time, most of the B-cell lymphomas originate from the germinal center, and the generation and blast transformation of B-cell lymphoma occurs in close association with FDCs in the early stage of tumorigenesis. To study the functional roles of FDCs in lymphomagenesis, we established an inducible tumor model. The human B-cell lymphoma cell line, L3055, formed solid tumors only when inoculated with an FDC line, HK. In addition, 2 FDC-signaling molecules (FDC-SMs), a novel protein 8D6 and 4G10/CD44, are required for tumor formation in vivo, because monoclonal antibodies (mAbs) specific to these 2 proteins inhibited lymphomagenesis completely when they were inoculated with L3055 and HK cells. However, these 2 FDC-SMs have distinct functional roles in tumor formation. FDC-SM-8D6 enhances L3055 cell proliferation, whereas FDC-SM-4G10/CD44 inhibits its apoptosis. Identification of the functional roles of these critical FDC-SMs may lead to the discovery of therapeutic drugs that suppress the survival and growth of lymphoma cells.
[Show abstract][Hide abstract] ABSTRACT: The Division of Cancer Biology of the National Cancer Institute hosted a workshop on hematological malignancies and the marrow microenvironment in Rockville, Maryland in the fall of 2002. There were 22 invited participants from the United States, Canada, and Europe, and the workshop was organized into disease-specific sessions. The sessions were designed to explore the basic science and therapeutic applications related to the stromal and nonstromal components of the marrow in malignant hematological diseases.
Cancer Research 12/2003; 63(21):7539-41. · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) B cells have defects in apoptosis pathways and therefore accumulate in vivo. However, when removed from the patient and cultured in vitro, these malignant cells rapidly undergo apoptosis. Recent studies suggest that leukemia cell survival is influenced by interactions with nonleukemia cells in the microenvironment of lymph nodes, marrow, and other tissues. To model such cell-cell interactions in vitro, we cultured freshly isolated CLL B cells with a follicular dendritic cell line, HK. CLL B cells cocultured with HK cells were protected from apoptosis, either spontaneous or induced by treatment with anticancer drugs. Protection against spontaneous apoptosis could also be induced by coculturing the CLL B cells with normal dendritic cells (DCs) or with a CD40-ligand (CD154)-expressing fibroblast cell line. Examination of the expression of several apoptosis-regulatory proteins revealed that coculture with HK cells or DCs induced up-regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in CLL B cells, whereas CD40 ligation increased expression of Bcl-X(L). Cell-cell contact was required for HK-induced protection, and introducing neutralizing antibodies against various adhesion molecules showed that CD44 was involved in HK-mediated survival, whereas CD40, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were not. Anti-CD44 antibodies also blocked Mcl-1 induction by HK cells. Mcl-1 antisense oligonucleotides reduced leukemia cell expression of Mcl-1, and significantly suppressed HK-induced protection against apoptosis, whereas control oligonucleotides had no effect. Thus, HK cells protect CLL B cells against apoptosis, at least in part through a CD44-dependent mechanism involving up-regulation of Mcl-1, and this mechanism is distinct from that achieved by CD40 ligation. Consequently, the particular antiapoptotic proteins important for CLL survival may vary depending on the microenvironment.
[Show abstract][Hide abstract] ABSTRACT: Innate immunity has recently gained renewed interest in its ability to regulate adaptive immunity. Among the innate immune signals, CpG DNA has revealed its potential as a vaccine adjuvant. However, the cellular mechanism for the effect of CpG DNA on the humoral immune response is not well understood. Here, we investigated the effects of CpG DNA on human B cell differentiation using highly purified B cell subsets: naive, germinal center (GC), and memory B cells. In the in vitro culture system that mimics the primary or secondary immune response in vivo, CpG DNA markedly augmented the proliferation and generation of plasma cells from naive and memory B cells. CpG DNA dramatically increased plasma cell generation from GC B cells. However, CpG DNA did not have effect on memory B cell generation from GC B cells. These results suggest that CpG DNA potentiates the B cell adaptive immune response by enhancing terminal differentiation, but does not affect the generation of memory B cells.
The Journal of Immunology 10/2002; 169(5):2368-73. DOI:10.4049/jimmunol.169.5.2368 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study the functional role of follicular dendritic cells (FDC) in germinal center (GC)-B cell differentiation, we have established an in vitro experimental model that mimics GC-reactions in vivo. Using this culture system, we have characterized the distinct function of FDC from activated T cells. The primary function of FDC is to prevent apoptosis and support proliferation of GC-B cells, whereas activated T cells induce the differentiation of GC-B cells by providing CD40L and cytokines. Furthermore, we have identified two FDC-signaling molecules that mediate proliferation of GC-B cells in vitro and in vivo.
Seminars in Immunology 09/2002; 14(4):259-66. DOI:10.1016/S1044-5323(02)00058-1 · 5.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies suggest that tumor necrosis factor (TNF) family members such as TNFalpha and lymphotoxin alphabeta (LTalpha1beta2) are important in the development of follicular dendritic cells (FDCs) and maintenance of FDC function. In this study we used FDC-like cells (FDC-LC) cultured from normal human tonsil and investigated the effects of TNF and LTalpha1beta2 on expression of adhesion molecules and the production of cytokines and chemokines. TNF and LTalpha1beta2 both increased the expression of VCAM-1 and ICAM-1 on FDC-LC. In addition, IL-4 with LTalpha1beta2 synergistically increased the expression of VCAM-1, but not ICAM-1. Cytokine IL-6 and IL-15 mRNAs were induced following stimulation with TNF and LTalpha1beta2. These two cytokines were present in FDC-LC supernatants by ELISA and increased following TNF and LTalpha1beta2 stimulation. We also examined FDC-LC for chemokines, which affect B cells, including IL-8, SDF-1, MIP3beta/ELC, and BCA-1/BLC. SDF-1 mRNA and protein were expressed by FDC-LC, and following stimulation with TNF and LTalpha1beta2, decreases in both were observed. Therefore, TNF and LTalpha1beta2, which are produced by activated B cells, increased the expression of adhesion molecules and cytokines from FDC-LC, potentially providing key signals to support germinal center B cell survival and differentiation.