scholarly journals Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL

Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 222-231 ◽  
Author(s):  
Jennifer S. Carew ◽  
Steffan T. Nawrocki ◽  
Yelena V. Krupnik ◽  
Kenneth Dunner ◽  
David J. McConkey ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cells ◽  
Protein Trafficking ◽  
Protein Transport ◽  
Brefeldin A ◽  
Secretory Protein ◽  
Lymphocytic Leukemia ◽  
Mtdna Mutations ◽  
Golgi Protein ◽  
Induced Apoptosis

Abstract Previous studies showed that chronic lymphocytic leukemia (CLL) cells exhibit certain mitochondrial abnormalities including mtDNA mutations, increased superoxide generation, and aberrant mitochondrial biogenesis, which are associated with impaired apoptosis and reduced sensitivity to fludarabine. Here we report that CLL cells and multiple myeloma cells are highly sensitive to brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport currently being developed as a novel anticancer agent in a prodrug formulation. Of importance, brefeldin A effectively induced apoptosis in fludarabine-refractory CLL cells. Disruption of protein trafficking by brefeldin A caused the sequestration of the prosurvival factors APRIL and VEGF in the ER, leading to abnormal ER swelling and a decrease in VEGF secretion. Such ER stress and blockage of secretory protein traffic eventually resulted in Golgi collapse, activation of caspases, and cell death. Notably, the cellular sensitivity to this compound appeared to be independent of p53 status. Taken together, these findings suggest that malignant B cells may be highly dependent on ER-Golgi protein transport and that targeting this process may be a promising therapeutic strategy for B-cell malignancies, especially for those that respond poorly to conventional treatments.

scholarly journals The ultrastructural localization of immunoglobulins in human b cells of immunoproliferative diseases

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1132-1140 ◽  
Author(s):  
MF Gourdin ◽  
JP Farcet ◽  
F Reyes
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cells ◽  
Plasma Cells ◽  
Simultaneous Detection ◽  
Ultrastructural Localization ◽  
Lymphocytic Leukemia ◽  
Cellular Distribution ◽  
Complex Plasma ◽  
Perinuclear Space ◽  
Human B Cells

Abstract The cellular distribution of immunoglobulins in human malignant and normal B cells was investigated by immunoelectron microscopy by direct incubation of fixed cells with electron microscopy by direct incubation of fixed cells with peroxidase-coupled antibody. These conjugates penetrated into the cell, resulting in the simultaneous detection of surface and cytoplasmic immunoglobulins. The latter were seen as specific intracisternal staining of the perinuclear space and endoplasmic reticulum and occasionally of the Golgi complex. Plasma cells were frequently characterized by a heterogeneity of reactivity of the endoplasmic reticulum. Minute amounts of cytoplasmic immunoglobulin were demonstrated in cells without developed secretory organelles, such as lymphoma cells and lymphocytes from chronic lymphocytic leukemia (CLL). The method allowed us to define several subsets of cells according to the expression of surface and cytoplasmic immunoglobulins and thus to determine the stage of maturation of cells involved in monoclonal proliferation.

Theophylline synergizes with chlorambucil in inducing apoptosis of B- chronic lymphocytic leukemia cells

Blood ◽  
1996 ◽  
Vol 88 (6) ◽  
pp. 2172-2182 ◽  
Author(s):  
F Mentz ◽  
MD Mossalayi ◽  
F Ouaaz ◽  
S Baudet ◽  
F Issaly ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Apoptotic Cell ◽  
Protein A ◽  
Phosphodiesterase Inhibitor ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Lymphocytic Leukemia ◽  
Interleukin 4 ◽  
Induced Apoptosis

We tested the effects of theophylline, a phosphodiesterase inhibitor inducing intracellular accumulation of cyclic adenosine monophosphate (cAMP), on malignant B cells from 15 patients with B-chronic lymphocytic leukemia (B-CLL). We observed a large increase in apoptotic cell numbers (mean, 90% v 20% in medium alone) in the presence of theophylline (100 micrograms/mL) or chlorambucil (10 mumol/L) after 72 hours of incubation. Maximal apoptosis (90%) was reached after 36 hours when the two drugs were used together at fourfold lower concentrations, indicating a synergistic effect; no effect was observed with normal B cells, suggesting that the combination might have therapeutic interest. Chlorambucil induced intracellular Ca+2 influx, pointing to the involvement of two signaling pathways that might explain its synergy with theophylline through their effects on oncogenes. The expression of bcl-2 protein, a proto-oncogene inhibiting apoptosis, decreased after incubation with the drugs, while c-myc, recently described as having a potent role in apoptosis, was overexpressed. For p53 we observed an overexpression in the presence of chlorambucil or both theophylline- chlorambucil and a decrease after theophylline incubation. Chlorambucil- and theophylline-induced apoptosis was partially inhibited by interleukin-4 (IL-4), which also abrogated the effects on oncogene expression. These results provide insight into the mechanisms underlying B-CLL apoptosis and suggest that the theophylline- chlorambucil combination may be of therapeutic value in this setting.

scholarly journals Poliovirus Protein 3A Inhibits Tumor Necrosis Factor (TNF)-Induced Apoptosis by Eliminating the TNF Receptor from the Cell Surface

2001 ◽  
Vol 75 (21) ◽  
pp. 10409-10420 ◽  
Author(s):  
Nickolay Neznanov ◽  
Anna Kondratova ◽  
Konstantin M. Chumakov ◽  
Brigitte Angres ◽  
Bakhyt Zhumabayeva ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Cell Surface ◽  
Protein Trafficking ◽  
Tumor Necrosis ◽  
Viral Infections ◽  
Brefeldin A ◽  
Tnf Receptor ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
Necrosis Factor

ABSTRACT Viral infections often trigger host defensive reactions by activating intrinsic (intracellular) and extrinsic (receptor-mediated) apoptotic pathways. Poliovirus is known to encode an antiapoptotic function(s) suppressing the intrinsic pathway. Here, the effect of poliovirus nonstructural proteins on cell sensitivity to tumor necrosis factor (TNF)-induced (i.e., receptor-mediated) apoptosis was studied. This sensitivity is dramatically enhanced by the viral proteinase 2A, due, most likely, to inhibition of cellular translation. On the other hand, cells expressing poliovirus noncapsid proteins 3A and 2B exhibit strong TNF resistance. Expression of 3A neutralizes the proapoptotic activity of 2A and results in a specific suppression of TNF signaling, including the lack of activation of NF-κB, due to elimination of the TNF receptor from the cell surface. In agreement with this, poliovirus infection results in a dramatic decrease in TNF receptor abundance on the surfaces of infected cells as early as 4 h postinfection. Poliovirus proteins that confer resistance to TNF interfere with endoplasmic reticulum-Golgi protein trafficking, and their effect on TNF signaling can be imitated by brefeldin A, suggesting that the mechanism of poliovirus-mediated resistance to TNF is a result of aberrant TNF receptor trafficking.

Novel targets for endoplasmic reticulum stress-induced apoptosis in B-CLL

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2713-2723 ◽  
Author(s):  
Emanuela Rosati ◽  
Rita Sabatini ◽  
Giuliana Rampino ◽  
Filomena De Falco ◽  
Mauro Di Ianni ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Apoptosis ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Minor Role ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Induced Apoptosis

Abstract A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8–mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.

scholarly journals TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER–Golgi intermediate compartments

2017 ◽  
Vol 114 (37) ◽  
pp. E7707-E7716 ◽  
Author(s):  
Michael G. Hanna ◽  
Samuel Block ◽  
E. B. Frankel ◽  
Feng Hou ◽  
Adam Johnson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Trafficking ◽  
Secretory Protein ◽  
Dependent Manner ◽  
Outer Coat ◽  
Concentration Dependent Manner ◽  
C Terminus ◽  
Intermediate Compartment ◽  
Fused Gene

The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER–Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.

scholarly journals Control of protein trafficking by reversible masking of transport signals

2016 ◽  
Vol 27 (8) ◽  
pp. 1310-1319 ◽  
Author(s):  
Omer Abraham ◽  
Karnit Gotliv ◽  
Anna Parnis ◽  
Gaelle Boncompain ◽  
Franck Perez ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Trafficking ◽  
Protein Transport ◽  
Protein Import ◽  
Binding Peptide ◽  
Protein Traffic ◽  
Alternative Approach ◽  
Targeting Signal ◽  
Regulated Trafficking ◽  
Streptavidin Binding

Systems that allow the control of protein traffic between subcellular compartments have been valuable in elucidating trafficking mechanisms. Most current approaches rely on ligand or light-controlled dimerization, which results in either retardation or enhancement of the transport of a reporter. We developed an alternative approach for trafficking regulation that we term “controlled unmasking of targeting elements” (CUTE). Regulated trafficking is achieved by reversible masking of the signal that directs the reporter to its target organelle, relying on the streptavidin–biotin system. The targeting signal is generated within or immediately after a 38–amino acid streptavidin-binding peptide (SBP) that is appended to the reporter. The binding of coexpressed streptavidin to SBP causes signal masking, whereas addition of biotin causes complex dissociation and triggers protein transport to the target organelle. We demonstrate the application of this approach to the control of nuclear and peroxisomal protein import and the generation of biotin-dependent trafficking through the endocytic and COPI systems. By simultaneous masking of COPI and endocytic signals, we were able to generate a synthetic pathway for efficient transport of a reporter from the plasma membrane to the endoplasmic reticulum.

scholarly journals The ultrastructural localization of immunoglobulins in human b cells of immunoproliferative diseases

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1132-1140
Author(s):  
MF Gourdin ◽  
JP Farcet ◽  
F Reyes
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cells ◽  
Plasma Cells ◽  
Simultaneous Detection ◽  
Ultrastructural Localization ◽  
Lymphocytic Leukemia ◽  
Cellular Distribution ◽  
Complex Plasma ◽  
Perinuclear Space ◽  
Human B Cells

The cellular distribution of immunoglobulins in human malignant and normal B cells was investigated by immunoelectron microscopy by direct incubation of fixed cells with electron microscopy by direct incubation of fixed cells with peroxidase-coupled antibody. These conjugates penetrated into the cell, resulting in the simultaneous detection of surface and cytoplasmic immunoglobulins. The latter were seen as specific intracisternal staining of the perinuclear space and endoplasmic reticulum and occasionally of the Golgi complex. Plasma cells were frequently characterized by a heterogeneity of reactivity of the endoplasmic reticulum. Minute amounts of cytoplasmic immunoglobulin were demonstrated in cells without developed secretory organelles, such as lymphoma cells and lymphocytes from chronic lymphocytic leukemia (CLL). The method allowed us to define several subsets of cells according to the expression of surface and cytoplasmic immunoglobulins and thus to determine the stage of maturation of cells involved in monoclonal proliferation.

Humanized Anti CD-40 Antibody SGN-40 Effectively Induces Cytotoxicity Against Chronic Lymphocytic Leukemia (CLL) Cells through Antibody Mediated Cytotoxicity and Demonstrates Modest Biologic Evidence of CD40 Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2966-2966 ◽  
Author(s):  
Aruna C. Gowda ◽  
Xiaobin B. Zhao ◽  
Carolyn Cheney ◽  
Najma Mehter ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Activation ◽  
Cd40 Ligand ◽  
Lymphocytic Leukemia ◽  
Extrinsic Pathway ◽  
Concurrent Administration ◽  
Cd40 Ligation ◽  
Cd40 Activation ◽  
Induced Apoptosis

Abstract The CD40 antigen is involved in cell survival and differentiation of B-cells and is uniformly expressed on chronic lymphocytic leukemia (CLL) cells. The CD40/CD40L interaction stimulates B-cells, dendritic cells and monocytes to proliferate, differentiate, up regulate co-stimulatory molecules and increase antigen presentation. While activation of CD40 can protect CLL cells against early fludarabine-induced apoptosis, these cells become sensitive to delayed death by extrinsic pathway apoptosis. (Blood, 105: 3193–8, 2005). SGN-40 is a humanized anti-CD40 antibody entering clinical trials and has been reported to have weak agonistic properties following CD40 ligation. To pursue rational clinical development of SGN-40, we studied the effects of this antibody in fresh, non-cryopreserved primary CLL cells. These studies included classic antibody mediated killing mechanisms and evidence of both CLL cell activation and protection against early fludarabine-mediated apoptosis. CLL cells treated with SGN-40 (10 mcg/ml) for 2 hours (hrs) in the presence of human serum promoted no complement mediated cytoxicity (CDC) in 8 pts tested. Direct SGN-40 induced apoptosis of human CLL cells with or without anti-Fc IgG cross-linking at 24, 48 and 72 hrs was not increased over that observed with the isotype control antibody trastuzumab in 8 pts studied. In contrast, SGN-40 induced antibody dependent cellular cytotoxicity (ADCC) against CLL cells an average of 12% (±11.39 SD, range 2–32%) killing at 4 hrs (effector to target cell ratio 25:1) in 6 pts tested. The SGN-40 induced ADCC against CLL cells were similar to that observed with alemtuzumab (average 19%, SD 6.9, range10–30%) or rituximab (average 18%, SD 12.48, range 8–42.5%). SGN-40 also mediated death in Raji and 697 lymphoblastic lymphoma cell lines via ADCC. Similar to reports by others with CD40 ligand, SGN-40 mediated activation was noted with modest up-regulation of CD80 and HLA-DR at 48hrs. When administered prior to fludarabine, SGN-40 also protected against death in 5 consecutive samples, although this was less than observed with CD40 ligand transfected HeLa cells, consistent with incomplete CD40 activation. Concurrent administration of SGN-40 and fludarabine did not protect from drug-mediated apoptosis. In conclusion, these findings suggest that SGN-40 has dual property of mediating cytotoxic effect by ADCC and partial CD40 activation. Development of SGN-40 as a therapeutic agent in CLL is justified and future studies by our group are focusing on enhancing SGN-40 mediated ADCC against CLL cells and potentially designing combination studies with SGN-40 to exploit this agent’s ability to engage the CD40/CD40L network.

Neuropilin-1 Receptor (NRP-1) Occupancy Induces Cell Death in Primary Chronic Lymphocytic Leukemia (CLL) B Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 586-586
Author(s):  
Grzegorz S. Nowakowski ◽  
Yean K. Lee ◽  
Neil Kay
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
B Cells ◽  
Gradient Centrifugation ◽  
Lymphocytic Leukemia ◽  
Vegf Receptor ◽  
Dependent Manner ◽  
Blocking Antibody ◽  
Induced Apoptosis ◽  
Blocking Antibodies

Abstract Background: We have previously shown that CLL B cells secrete vascular endothelial growth factor (VEGF) and express VEGF receptors: VEGFR-1 and VEGFR-2. Secreted VEGF protects CLL B cells from spontaneous and drug induced apoptosis via increased levels of Mcl-1 and XIAP; however, the exact mechanism of this process is unknown. In solid tumors, there is increasing evidence that signaling through VEGF receptor known as neuropillin-1 (NRP-1) is critical for VEGF induced resistance to apoptosis. Hypothesis: NRP-1 is expressed by CLL B cells and is critical for VEGF mediated protection from apoptosis. Methods: To demonstrate the presence of NRP-1 on CLL B cells, we conducted flow cytometry and immunoblotting. We then evaluated the ability of NRP-1 blocking antibodies to induce apoptosis of primary CLL B cells. To do this, circulating CLL B cells were isolated by density gradient centrifugation. Patient samples with greater than 80% of CD5+ CD19+ cells in a mononuclear cell population, as assessed by flow cytometry, were cultured in AIM-V media in 24-well plates at 1.5 x 106 cells/mL. To occupy NRP-1, we added NRP-1 blocking antibodies (Calbiochem, Darmstadt, Germany) at increasing concentrations (0.5 μg/mL -10 μg/mL) to cultured CLL B cells. Cell death was assessed using an annexin and propidium iodide flow assay after 24 h of in vitro culture. CLL cells cultured without antibodies and isotype nonspecific monoclonal antibodies were used as controls. Results: CLL B cells were found to express NRP-1 but not uniformly. Most patients had flow positive evidence for NRP-1 but a distinct percentage (35%) was very low (≤5%) or negative. However, immunoblot analysis revealed moderate to low levels of NRP-1 protein with evidence of tyrosine phosphorylation in all tested CLL patients (N=9). NRP-1 blocking antibodies, but not VEGF-R1 and -R2 blocking antibodies, induced apoptosis in a dose dependent manner in primary CLL cells (n=5, FIG.) at antibody concentrations starting at 1 μg/mL (p=0.003). The effect of the NRP-1 blockade varied between patients, with a median IC50, 1.5 μg/mL (range 0.5–2 μ/mL). Importantly, concentrations of 5 μg/mL and higher induced apoptosis in more than 90% of the CLL B cells. We also found that the sensitivity of CLL B cells to NRP-1 blocking antibody, in terms of apoptosis induction, was correlated with the number of NRP-1 receptors as assessed by flow cytometry. CLL B cell clones with no detectable NRP-1 had no induction of cell death when exposed to the NRP-1 blocking antibody. Finally, immunoprecipitation and immunoblot assays indicated that NRP-1 physically interacted with VEGF-R2 on CLL B cells. This suggests that NRP-1 could be enhancing VEGF binding affinity on VEGF-R2 to further increase the ability of VEGF to generate signals that lead to apoptosis modulation. Conclusion: We have found that NRP-1 blocking antibodies induce cell death in NRP-1 positive CLL B cells. Similar results using NRP-1 blocking peptides rather than blocking antibodies have been observed in breast cancer (Br J Cancer.2005 Jan 31;92(2):328–33). Our results suggest that NRP-1 represents an attractive therapeutic target in CLL and should be explored further. Figure Figure

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