Differentiated Acute Promyelocytic Leukemic Cells-Derived Microparticles Inhibit Cell Migration through Annexin A1

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2750-2750
Author(s):  
Hui-Chi Hsu ◽  
Wen-Hui TSai ◽  
Hon-Yu Chien ◽  
Shau-Chieh Hsu
Keyword(s):  
Specific Antibody ◽  
Conflicts Of Interest ◽  
Control Cell ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Granulocytic Differentiation ◽  
Nb4 Cells ◽  
Mediators Of Inflammation ◽  
Anti Inflammatory

Abstract Abstract 2750 Neutrophil-derived microparticles (MP) have become important mediators of inflammation, coagulation and vascular homeostasis. MP carry surface antigens, proteins and adhesion molecules from their originating cell and can mediate intercellular cross-talk. Human neutrophil contain high levels of the anti-inflammatory protein annexin 1 (AnxA1), which contributes to mechanisms activated in the host to keep under control cell activation and trafficking in the resolution of inflammation. We investigated the role of MP released by all-trans retinoic acid (ATRA)-treated acute promyelocytic leukemic (APL; NB4) cells during the process of granulocytic differentiation. Materials & Methods: We determined the expression of AnxA1 and its receptor (FPRL1) on the NB4 cells and their MP by flowcytometry, real-time PCR and western blotting. The anti-inflammatory effect of AnxA1was determined by the transmigration assay. Results: AnxA1 was constitutively expressed on the surface of ATRA-untreated NB4 cells. ATRA treatment of NB4 cells can significantly enhance their surface expression of Anx-A1 in a time dependent manner, but did not change their mRNA expression or release of free AnxA1 into the conditioning medium. We further determined the amount of MP in the CM by flowcytometry. Significantly higher number of MP was released by the ATRA-NB4 cells, as compared with those by ATRA-untreated NB4 cells (p<0.05). Further study demonstrated that AnxA1was expressed on the surface of MP, and ATRA can also enhance the release of Anx-A1 (+) MP from ATRA-NB4 cells. Transmigration studies indicated that exogenous Anx-A1 protein can inhibit the transmigration of ATRA-NB4 cells in a time- and dose-dependent manner (p<0.05 & p<0.05; respectively). AnxA1 receptor (FPRL-1) was expressed on the surface of both ATRA-treated and ATRA-untreated NB4 cells. Blocking the FPRL1 on the surface of ATRA-NB4 cells with its specific antibody can further enhance their transmigration activity, indicating that the important anti-migratory role of AnxA1-FPRL1 axis on the ATRA-treated NB4 cells. We further demonstrated that ATRA-treated NB4 cells-derived MP can also significantly inhibit the transmigration of ATRA-NB4 cells (p<0.05). Similarly, anti-migratory effect of MP can be attenuated when their surface Anx-A1 was blocked with its specific antibody, implying that AnxA1 mediates the rapid anti-migratory effects of MP. We further determined the mode of action that Anx-A1(+) MP acted on the recipient cells. MPs were pre-labeled with FITC-conjugated Anx-A1 monoclonal antibody before incubating with recipient ATRA-treated NB4 cells. Flowcytometry demonstrated that FITC-labeled Anx-A1 was detected on 87% of recipient ATRA-treated NB4 cells, indicating that Anx-A1 (+) MP can fuse into the surface of the recipient ATRA-treated NB4 cells. Conclusion: differentiated APL cells-derived microparticles contain functionally active AnxA1 that confers them anti-migratory properties which contributes to the control mechanism of cell recruitment. Disclosures: No relevant conflicts of interest to declare.

CD14 Mediates Phagocytic Activity during the Granulocytic Differentiation Process in Acute Promyelocytic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4955-4955 ◽  
Author(s):  
Hui-Chi Hsu ◽  
Wen-Hui Tsai ◽  
Yu-Chieh Lin
Keyword(s):  
Phagocytic Activity ◽  
Promyelocytic Leukemia ◽  
Time Dependent ◽  
Apoptotic Cells ◽  
Dependent Manner ◽  
Atra Treatment ◽  
Granulocytic Differentiation ◽  
Nb4 Cells ◽  
Pre Treatment

Abstract All-trans retinoic acid (ATRA) can induce acute promyelocytic leukemia (APL) cells differentiation into mature granulocytes. CD14 and Toll-like receptor 4 (TLR-4) play an important role in the phagocytic activity of macrophage, however, their role during granulopoiesis is still unclear. In this study, we determined the role of CD14/TLR-4 in the development of phagocytic activity in NB4 APL cells after induction into the process of granulocytic differentiation by ATRA. Flow cytometry analysis demonstrate that, during ATRA treatment for 6 days, the phagocytic activity of NB4 cells in engulfing either fluorescein-latex beads or idarubicin-induced apoptotic cells increased in a time-dependent manner, and the level of CD14 expression on NB4 cells was also significantly increased in a time dependent manner, though its level was only minimally expressed in ATRA-untreated NB4 cells. However, TLR-4 was constitutionally expressed in ATRA-untreated cells and its level did not changed significantly during the first 5 days of ATRA treatment. Further study demonstrates that the phagocytic activity of ATRA-NB4 cells was significantly inhibited by pre-treating cells with antibodies specific to either CD14 or TLR-4 before phagocytosis assay. In exploring the role of CD14/TLR4 associated signal transduction mediators, NF-κB and IRF-3, we further demonstrate that the phagocytic activity of ATRA-NB4 cells in engulfing beads was significantly inhibited when cells were pretreated with either a NF-κB inhibitor (BAY 11-7082) or an IRF-3 inhibitor (SP600125). However, this activity in engulfing apoptotic cells was only significantly inhibited by pretreatment with BAY11-7082, but not by pre-treatment with SP600125. Finally, our results indicate that the level of CD14(+) microparticles (MPs) released by ATRA-NB4 cells was significantly enhanced when those cells were induced into the process of apoptosis by pre-treatment with idarubicin. Moreover, by incubation with MPs derived from apoptotic ATRA-NB4 cells, the phagocytic activity of living ATRA-NB4 cells in engulfing apoptotic cells was significantly enhanced, and this phagocytic activity was also significantly inhibited by pre-treating MPs with antibody specific to CD14 before phagocytic assay. We conclude that CD14 contributes to the phagocytic activity of APL cells during the process of granulocytic differentiation. Disclosures No relevant conflicts of interest to declare.

HSP27 Protects Malignant Hematopoietic Cells Against VP-16-Induced Apoptosis through Modulation of P38 and C-JUN.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1276-1276
Author(s):  
Hein Schepers ◽  
Marjan Geugien ◽  
Marco van der Toorn ◽  
Anton L. Bryantsev ◽  
Harm H. Kampinga ◽  
...  
Keyword(s):  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Apoptosis Pathway ◽  
Hsp27 Expression ◽  
Cd34 Cells ◽  
Induced Apoptosis ◽  
And Function ◽  
Jnk Activation

Abstract In the present study, expression and function of Heat Shock Protein 27 (HSP27) was analyzed in acute myeloid leukemia (AML), since HSP27 expression is linked to unfavourable prognosis. HSP27 protein was predominantly expressed in monocytic blasts (M4-M5, 91%, N = 11) and absent in myeloid leukemic blasts (M1-M2, N = 5). A similar lineage restricted expression was observed in normal hematopoietic cells: high expression in normal CD34+ cells and monocytes, and absent in granulocytes. To study the functional role of HSP27, RNA interference (RNAi) studies were performed in the leukemic TF-1 cell line. These experiments demonstrated a twofold increase in VP-16-induced apoptosis after HSP27 siRNA. In contrast, CD95 Fas-induced apoptosis remained the same, as a result of CD95 Fas-mediated upregulation of HSP27. Additional investigations demonstrated that the increased VP-16-induced apoptosis after HSP27 RNAi, was associated with an enhanced phosphorylation of p38 and c-Jun. This VP-16-induced phosphorylation was subsequently followed by the release of cytochrome c into the cytoplasm, which increased twofold after siRNA treatment. These results indicate that HSP27 plays an important role in the protection against VP-16-induced apoptosis through the modulation of p38 and JNK activation, probably through interference with DAXX-mediated ASK1 activation. This was further underscored by co-immunoprecipitation studies, demonstrating complex formation of DAXX and HSP27 in an ASK1-dependent manner. However, in the investigated AML samples, VP-16-mediated apoptosis correlated moderately with HSP27 expression, although HSP27 was highly expressed and phosphorylated and activated in primitive monocytic AML blasts. This is likely due to the co-expression of p21Waf1/Cip1, which is in the majority of the monocytic AML M4-M5 blasts constitutively localised in the cytoplasm and interferes with apoptosis via the DAXX-ASK1-dependent pathway. Preliminary data indicate that overexpression of a cytoplasmic form of p21 is able to reduce the VP-16-induced apoptosis after RNAi for HSP27 as compared to controls, suggesting a predominant anti-apoptotic role of p21 over HSP27. In summary, we demonstrate a role for HSP27 in the survival of leukemic cells by modulation of the DAXX/p38/JNK apoptosis pathway. This survival advantage can further be promoted by the co-expression of cytoplasmic localised p21Waf1/Cip1 protein, indicating that strategies in AML treatment should be focused on targeting multiple signal transduction pathways.

Predominant Complement Mediated Lysis of B-CLL Cells by Therapeutic MAbs Rituximab and Campath-1H in Whole Blood Assays.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3445-3445 ◽  
Author(s):  
Josee Golay ◽  
Luca Bologna ◽  
Elisa Gotti ◽  
Alessandro Rambaldi ◽  
Renato Bassan ◽  
...  
Keyword(s):  
Target Cell ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Human Igg ◽  
Blood Samples ◽  
Expression Levels ◽  
Cd20 Expression

Abstract Abstract 3445 Poster Board III-333 The mechanism of action of unconjugated MAbs such as Rituximab and Campath-1H in vivo is still a matter of debate. Most in vitro assays with antibodies rely upon purified effector cells or proteins taken outside their natural context, and on target cell lines rather than patients cells. In order to analyse the activity of therapeutic MAbs on circulating leukemic cells in more physiological conditions and in a system the least manipulated as possible, we have set up a whole blood assays using Rituximab and Campath-1H. Peripheral blood samples were drawn from B-CLL patients or normal donors in sodium citrate and antibodies were directly added at different concentrations. We first demonstrated that neither apoptosis, induced by cross-linked anti-CD20 antibody, nor complement mediated cytotoxicity (CDC) induced by Campath-1H or Rituximab were significantly inhibited by citrate used at the standard concentration (0.1 M). We then performed a number of experiments using whole blood samples in citrate, into which increasing concentrations of Rituximab or Campath-1H were added. Lysis was analysed by FACS analysis after different incubation times at 37°C. We observed that Campath-1H very rapidly and efficiently lysed normal B cells or B-CLL targets in vitro in whole blood: maximal lysis was reached within 4 hours and was observed already with 1 and 10 μg/ml antibody (61 %), even though it was still more effective at 25 or 50 μg/ml (up to 90 % lysis). 25 μg/ml is known to be reached in the circulation after 30mg infusions of the antibody 3 times a week. Lysis by Campath-1H was fully complement dependent since it was inhibited by 90% in presence of excess blocking anti-C5 antibody Eculizumab (200 μg/ml). Eculizumab alone in contrast had no effect on cell viability. We then analysed the efficacy of increasing concentrations of Rituximab in the same assay conditions. We observed in general a much reduced lysis with Rituximab compared to Campath-1H, even using antibody up to 200 μg/ml, a concentration that is reached in the circulation after standard 375 mg/m2 administration of the antibody once a week. Lysis showed also slower kinetics, with limited lysis at 4 hours (mean 6.4%) and maximal lysis with Rituximab reached only after 24 hours incubation (mean 18.8%). Also in this case, target cell death was inhibited by at least 90% in presence of Eculizumab, suggesting a major role of complement. Lysis by Rituximab correlated directly with CD20 expression levels (R=0.8) in 13 B-CLL samples analysed, as expected for a mechanism complement dependent. Indeed a mean 29.3% and 73.2% killing could be observed in the two CD20 bright B-CLL, at 4 and 24 hours respectively, whereas a mean of 3.1% and 10.9% lysis was observed in the 11 low-intermediate CD20 samples analysed at the same time points. These data in whole blood confirm our previously published results on the role of CD20 expression levels in CDC of isolated B-CLL cells (Golay et al., Blood 98, 3383-3389, 2001). In contrast to CDC and apoptosis, ADCC was strongly inhibited by citrate as well as several anti-coagulants tested and therefore could not be analysed in this type of assay. Nonetheless in B-CLL samples, NK cells were below detection limit (<0.1%) in most cases analysed, suggesting that ADCC in the circulation is not a major mechanism of lysis in this disease subtype. Finally we determined the effect of citrate on phagocytosis mediated by Rituximab and in vitro differentiated human macrophages. Phagocytosis could be observed in presence of 0.1M citrate (31%, compared to 44% in absence of citrate). Phagocytosis of B-CLL in whole blood was therefore analysed by layering samples directly onto the macrophages. We observed that phagocytosis of B-CLL targets in whole blood was very low (less than 1% over background) compared to a mean of 47% for purified B-CLL targets phagocytosed in normal culture medium. Phagocytosis in whole blood was low presumably due to the presence of high concentration of human IgG in whole blood since as low as 50 μg/ml human IgG is known to inhibit phagocytosis by 90%. We conclude that the major activity of Campath-1H and Rituximab in the circulation is through complement. Apoptosis, ADCC and phagocytosis appear to play a marginal role in this context but may become more important in tissues. The method presented could be used to rapidly screen novel antibodies for their efficacy through either as apoptosis or CDC directly on unmanipulated patients material. Disclosures No relevant conflicts of interest to declare.

Anti-Leukemia and Multiple Myeloma Selective Activity of CXCR4 Antagonist 4F-Benzoyl-TN14003 Involves Apoptotic Death Pathway.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3857-3857
Author(s):  
Katia Beider ◽  
Michal Begin ◽  
Michal Abraham ◽  
Hanna Wald ◽  
Ido Weiss ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Hematological Malignancies ◽  
Conflicts Of Interest ◽  
K562 Cells ◽  
Jurkat Cells ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Hl60 Cells

Abstract Abstract 3857 Poster Board III-793 The chemokine receptor CXCR4 and its ligand CXCL12 are involved in the development and progression of a diverse number of hematological malignancies, including leukemia, lymphoma and multiple myeloma (MM). Binding CXCL12 to CXCR4 activates a variety of intracellular signal transduction pathways and effector molecules that regulate cell chemotaxis, adhesion, survival, apoptosis and proliferation. It was previously shown that CXCR4 signaling can directly induce caspase-independent cell apoptosis through the interaction with the HIV gp120 envelope protein. In the present study we investigated the effect of CXCR4 specific antagonists 4F-benzoyl-TN14003 (T140) and AMD3100 on the survival and proliferation of different human hematological cancer cells. Here, we demonstrate that T140, but not AMD3100, exhibits preferential cytotoxicity towards malignant cells of hematopoietic origin, as compared to primary normal cells or solid prostate and breast tumor cells. The in vitro treatment with T140, but not with AMD3100, significantly decreased the number of viable chronic myeloid leukemia K562 cells, acute T cell leukemia Jurkat cells, acute promyelocytic leukemia NB4 and HL60 cells, and four different MM cell lines (U266, NCI-H929, RPMI8226 and ARH77), demonstrating the highest sensitivity to T140 (p<0.01). Notably, T140 inhibited the growth of freshly isolated leukemia and MM cells obtained from consenting patients. T140 inhibits the growth of MM and leukemic cells by inducing their apoptotic cell death. The apoptotic changes in the cells were associated with morphological changes, phosphatidylserine externalization, sub-G1 arrest, DNA double-stranded breaks, decrease in mitochondrial membrane potential, release of cytochrome c, and caspase 3 activation. The important role of CXCR4 in T140-mediated cell death was confirmed by demonstrating that CXCR4 over-expression in NB4 and K562 cells increased their sensitivity to T140. Furthermore, pretreatment of NB4 and HL60 cells with AMD3100 abolishes the effect of T140 on these cells, indicating the involvement of CXCR4 in T140-induced apoptosis. Importantly, the combination with novel anti-myeloma agent bortezomib significantly augments anti-myeloma activity of T140. The anti leukemic and MM effect of T140 was confirmed in xenograft in vivo tumor models. Subcutaneous (s.c.) or intra-peritoneal (i.p.) injections of T140 (100 or 300 mcg/mouse) significantly reduced, in a dose-dependent manner, the tumor size in immuno-deficient mice that were previously inoculated s.c. with human acute leukemia cells NB4 or MM cells RPMI8226 (p<0.01). Tumors from animals treated with T140 had smaller sizes and weights, larger necrotic areas and high apoptotic scores. Taken together, these data support the unique anti-cancer effect of T140 in hematological malignancies and indicate the potential therapeutic role of T140 in MM and leukemia patients. Disclosures: No relevant conflicts of interest to declare.

Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2474-2474
Author(s):  
Jesus Duque-Afonso ◽  
Aitomi Essig ◽  
Leticia M Solari ◽  
Tobias Berg ◽  
Heike L. Pahl ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Functional Role ◽  
Target Gene ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation

Abstract Abstract 2474 Background: The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells. Methods: To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively. Results: The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2. Conclusions: In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype. Disclosures: No relevant conflicts of interest to declare.

Role of Hypoxia and Hypoxia-Inducible-Factor 1α (HIF1A) In the BM Microenvironment-Mediated Protection of Leukemic Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2586-2586
Author(s):  
Rodrigo Jacamo ◽  
Juliana Benito ◽  
Olga Frolova ◽  
Ye Chen ◽  
Hongbo Lu ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Conflicts Of Interest ◽  
Lactic Acid Production ◽  
Hypoxia Inducible Factor ◽  
Leukemic Cells ◽  
Hypoxia Inducible Factor 1Α ◽  
Hypoxic Conditions ◽  
Control Mscs ◽  
Culture Supernatants

Abstract Abstract 2586 Resistance to chemotherapy can be mediated by genetic, epigenetic and microenvironmental causes. Only recently the connection between leukemia growth and survival and the hypoxic state of the BM microenvironment has been appreciated, by work conducted by us and others (Fiegl M et.al. Blood 2009; 113: 1504–1512; Harrison JS et. al., Blood 2002; 99). In extension of this concept we investigated the role of Hypoxia-Inducible-Factor 1α (HIF1A), the master regulator of hypoxia induced responses, in the microenvironment and its relevance for leukemia progression. Here we focused on the role of hypoxia and HIF transcription factors in cells contributing to the BM microenvironment, the mesenchymal stromal cells (MSC). Co-culture of lymphoid (NALM6) and myeloid (OCI-AML3) leukemic cell lines with BM-derived MSC under hypoxic conditions (1% O2) stimulated the secretion of a number of pro-survival cytokines and chemokines (including IL-6, VEGF, Beta-NGF and SDF-1α) that were quantified in co-culture supernatants by Luminex flow cytometry (Table 1). These findings suggest that hypoxia, and possibly its main mediator, the transcription factor HIF1A, may be responsible for the increased production of these factors. Since the chemokine stromal cell-derived factor-1α (SDF-1α) is involved in the attraction of leukemic cells towards cells of the BM microenvironment, we next investigated the role of HIF1A expression in MSC and its effect on SDF-1 secretion and migration of leukemic cells under hypoxic conditions. To this end, we generated primary human BM MSC stably transduced with lentiviral-encoded shRNA against HIF1A. SDF-1α transcription levels measured by qRT-PCR were diminished (∼30%, p<0.01) in HIF1A-silenced MSCs compared to control MSCs expressing non-silencing shRNA. This correlated with significantly reduced transwell migration of OCI-AML3 cells towards HIF1A-silenced MSCs compared with control (non-silencing) MSCs (∼35%, p<0.05) under hypoxic conditions. We next examined the contribution of hypoxia and HIF1A in the protective role of the BM microenvironment against standard chemotherapy with AraC and Doxorubicin. To this end, we performed in vitro experiments co culturing OCI-AML3 cells with either HIF1A-silenced MSCs or control MSCs under hypoxic conditions. After 48h of drug treatment a significant decrease in chemotherapy-induced apoptosis in leukemic cells co-cultured with control MSCs compared to leukemic cells cultured alone was observed. In turn, chemoresistance was reduced in OCI-AML3 co-cultured with HIF1A-silenced MSC, suggesting that hypoxia mediates chemoresistance largely through its effects on cells of the BM microenvironment. It has been shown that leukemic cells seem to exhibit increased dependency on glycolysis for ATP generation, which is frequently associated with resistance to therapeutic agents. Therefore, we measured the production of lactic acid (LA) in leukemic cells co-cultured with MSC in hypoxia compared to normoxia. In agreement with previous observations, we found that REH and primary ALL cells produced more LA when they were co-cultured with MSC under hypoxia compared to normoxia (∼1.8 fold, p<0.05). When REH cells were co-cultured with HIF1A-silenced MSCs in hypoxic conditions the lactic acid production was slightly but significantly reduced (∼20%, p<0.05) compared with the values observed in REH-control MSCs co-culture supernatants. Altogether, these findings strongly point to hypoxia and HIF1A as pivotal components in the protection from chemotherapy mediated by the BM microenvironment. We propose that targeting HIF1A and hypoxia in the protective cells of the bone marrow niches may represent a new approach to increase chemosensitivity of leukemic cells and hopefully improve the existing therapeutic strategies. Table 1: Fold increase observed in leukemic cells-MSC co-culture supernatants in hypoxia compared to normoxia. OCI-AML3+MSC NALM6+MSC IL-6 ∼3.1 ∼1.2 VEGF ∼3 ∼2 B-NGF ∼8 ∼10 SDF-1 ∼1.5 ∼1.5 Disclosure: No relevant conflicts of interest to declare.

Oral Supplementation with Omega3 Fatty Acids Inhibits NFkB Activation In Chronic Lymphocytic Leukemia (CLL) Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4607-4607
Author(s):  
Oscar F. F Ballester ◽  
Johannes Fahrmann ◽  
Theodore Witte ◽  
Gabriela Ballester ◽  
W. Elaine Hardman
Keyword(s):  
Conflicts Of Interest ◽  
Early Stage ◽  
Gradient Centrifugation ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Red Cell ◽  
Omega 3 ◽  
Dose Dependent

Abstract Abstract 4607 Introduction: Nuclear factor kappa B (NFkB) is a critical transcription factor involved in the growth and survival of CLL cells. NFkB is recognized as an important target for the development of novel therapies for the treatment of various malignancies. In vitro and in experimental animal models, OMEGA-3 fatty acid (O3FA) supplementation has been shown to inhibit NFkB activity. Patients and Methods: Patients with early stage CLL (Rai stages 0-II) who required no therapy, where accrued to this phase I-II trial. O3FA supplements were given for a total of 12 months at doses ranging from 2250 mg (EPA plus DHA), escalated to 4500 mg and 6750 mg per day as tolerated. NFkB activity was measured in peripheral blood samples after separation of mononuclear cell by gradient centrifugation and expressed as luminescence units/μ g of protein. Baseline and multiple serial samples were obtained during the study period. In-vitro cytotoxicity assays to doxorubicin were conducted using standard LD50 methods. Compliance was monitored by analysis of red cell and lymphocyte membrane lipid composition by gas chromatography. Results: Fifteen patients have been accrued to the trial, 8 of them have currently completed the planned 12 months of the study period. No significant clinical changes in disease activity were noted. O3FA was well tolerated. Supplementation resulted in a dose-dependent increase of O3FA composition of red cell and lymphocyte membranes in a dose dependent manner. At baseline, CLL patients had NFkB above the range observed in normal controls (2.05 × 104 to 2.32 × 105 NFkB lum units/μ g). The median value in CLL patients at baseline was 11.60 × 106 NFkB lum units/μ g (range 0.9 × 105 to 23.12 × 106). Among 5 patients with the highest baseline levels of NFkB, a decrease in NFkB activity ranging from 0.02 to 0.19 of the baseline value, was noted at the 2 higher doses of O3FA supplementation. Similar results were seen in patients with relatively lower levels of baseline NFkB activity (0.9 × 105 to 2.96 × 106 lum units/μ g). In vitro, significant doxorubicin cytotoxicity (>50%) was noted in samples obtained during supplementation, at μ gM concentrations which produced no detectable cell kill in baseline samples. Conclusions: O3FA supplementation resulted in significant inhibition of NFkB activity in leukemic cells from patients with CLL. In-vitro, after O3FA supplementation CLL cells became more sensitive to doxorubicin. Preliminary analysis of whole genome micro arrays revealed significant down-regulation of multiple genes associated with O3FA supplementation. Disclosures: No relevant conflicts of interest to declare.

Apolipoprotein(a)-Dependent Inhibition of Pericellular Plasminogen Activation Is Mediated by Specific Cellular Receptors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2236-2236
Author(s):  
Rocco Romagnuolo ◽  
Michael B Boffa ◽  
Marlys L Koschinsky
Keyword(s):  
Cell Surface ◽  
Plasminogen Activator ◽  
Conflicts Of Interest ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Plasminogen Activation ◽  
Apolipoprotein A ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract Abstract 2236 Lipoprotein(a) [Lp(a)] has been identified as an independent risk factor for cardiovascular diseases such as coronary heart disease. Lp(a) levels vary over 1000-fold within the human population and Lp(a) possesses both proatherogenic and prothrombotic properties due to the LDL-like moiety and apolipoprotein(a) [apo(a)] components, respectively. Apo(a) is highly homologous to plasminogen and thus can potentially interfere with plasminogen activation. Plasmin generated in the context of fibrin mediates the breakdown of blood clots, which are the causative factors in heart attacks and strokes. Plasmin generated on the surface of vascular cells plays a role in cell migration and proliferation, two of the fibroproliferative inflammatory events that underlie atherosclerosis. Previous studies have suggested that apo(a) may inhibit pericellular plasminogen activation on the basis of observations that apo(a) decreases plasminogen binding to cells. We have undertaken analysis of the mechanism by which apo(a) may interfere with pericellular plasminogen activation to allow for a more definitive description of the role of Lp(a) within the vasculature. Plasminogen activation was found to be markedly inhibited by the recombinant apo(a) variant 17K, in a dose dependent manner, on human umbilical vein endothelial cells (HUVECs), human monocytic leukemia cells (THP-1), THP-1 macrophages, and smooth muscle cells. The strong lysine binding site in kringle IV type 10, as well as kringle V appear to be required for this effect since apo(a) variants lacking these elements (17KΔAsp and 17KΔV, respectively) failed to inhibit activation. However, the role of lysine-dependent binding of apo(a) itself to the cells is not clear. Carboxypeptidase treatment of cells did not decrease apo(a) binding, and apo(a) does not compete directly for plasminogen binding to the cells. Rather, apo(a) and plasminogen may bind to the cells as a complex. We next attempted to identify the cell-surface receptor(s) that mediate plasminogen activation on the cell surface as well as its inhibition by apo(a). Urokinase-type plasminogen activator receptor (uPAR) has been previously shown to bind to urokinase-type plasminogen activator (uPA), vitronectin, and β3 integrins. uPAR is involved in the remodeling of the extracellular matrix (ECM) through regulation of plasminogen activation. We found evidence that uPAR is a potential receptor for both plasminogen and apo(a). Knockdown of uPAR in HUVECs results in decreased binding of plasminogen, 17K and, to a lesser extent, 17KΔAsp and 17KΔV. Similar experiments in SMCs revealed no changes in binding. A decrease in tPA-mediated plasminogen activation following uPAR knockdown occurred in HUVECs, and addition of 17K did not result in any further decrease. Overexpression of uPAR in THP-1 macrophages leads to greater than a two fold increase in 17K and plasminogen binding. Plasminogen activation increases over two-fold as a result of overexpression of uPAR, while 17K blunts the effect of uPAR overexpression. These results indicate that uPAR plays a crucial role in both plasminogen and apo(a) binding to the cell surface of specific cells and inhibition by apo(a) of plasminogen activation. Macrophage-1-antigen (Mac-1) receptor consists of CD11b (αM) and CD18 (β2) integrin and has been previously shown to recognize uPA and control migration and adhesion. Furthermore, αVβ3 has been previously shown to bind to vitronectin and the uPA-uPAR complex which promotes cell adhesion through binding of both vitronectin and αVβ3 integrins. We found that blocking the αM, β2, or αVβ3 receptors with monoclonal antibodies in THP-1 cells leads to a decrease in plasminogen activation, as well as a blunting of the inhibitory effects of apo(a) on plasminogen activation. These results indicate a role for Mac-1 and αVβ3 in apo(a) binding and inhibition of plasminogen activation. In conclusion, we have demonstrated, for the first time, the role of specific receptors in binding of apo(a) to vascular cell surfaces and in mediating the inhibitory effect of apo(a) on pericellular plasminogen activation. Disclosures: No relevant conflicts of interest to declare.

Increased CD39 Expression on CD4+ T-Lymphocytes Has Clinical and Prognostic Significance in Chronic Lymphocytic Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3895-3895
Author(s):  
Yair Herishanu ◽  
Inbal Hazan-Hallevi ◽  
Sigi Kay ◽  
Varda Deutsch ◽  
Aaron Polliack ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Anti Inflammatory

Abstract Abstract 3895 Chronic lymphocytic leukemia (CLL) cells depend on their microenvironment for proliferation and survival. Ectonucleotidase CD39 has anti-inflammatory properties as it hydrolyzes pro-inflammatory extra-cellular ATP, generates anti-inflammatory adenosine and also protects regulatory T cells from ATP-induced cell death. In this study we investigated the clinical significance of CD39 expression on CD4+T-cells in 45 patients with CLL as well as its compartmental regulation and explored the possible mechanisms for its induction. Compared to healthy individuals, CD4+CD39+ lymphocytes were increased in the peripheral blood of patients with CLL (4.6%±2.28 vs. 17.3%±12.49, respectively, p=0.004), and correlated with advanced stage of disease (9.72%±5.76, 18.15%±12.03 and 25.90%±16.34, of CD4+ lymphocytes, in patients with Rai stages 0, 1+2 and 3+4, respectively, p=0.019). CD4+CD39+ cells were also higher in patients with CLL who needed therapeutic intervention (untreated; 12.99%±10.63 vs treated; 22.21%±12.88, p=0.01) and in those who were ZAP70+ or had b2-microglobulin levels>3g/L. There were more CD4+CD39+ lymphocytes in the bone marrow compartment (22.25%±16.16) than in the peripheral blood (16.60%±15.84, p=0.009). In-vitro studies showed that CD39 can be induced on CD4+cells by exposure to ATP or indirectly, following B-cell receptor (BCR) engagement (CD4+CD39+ lymphocytes increased by 1.56 fold, in the BCR engaged samples compared to their paired controls; 20.27%±11.3 vs. 13%±9.42, respectively, p=0.0006). Conclusions: Increased CD39 expression on CD4+ T-lymphocytes in CLL associates with an aggressive disease. This may reflect the ability of the leukemic cells to suppress the surrounding immune environment, and contribute to a poorer prognosis. CD39+ may also serve as a future target for the development of novel therapies with immune modulating anti–tumor agents in CLL. Disclosures: No relevant conflicts of interest to declare.

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