Regulation of Apoptosis and Oxidative Stress by LMP1 Oncoprotein of Epstein-Barr Virus in Patients with Low Grade B-Cell Leukemic Lymphomas

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5212-5212
Author(s):  
Panagiotis Theodorou Diamantopoulos ◽  
Katerina Polonyfi ◽  
Nikolaos Spanakis ◽  
Athanassios Galanopoulos ◽  
Georgia Diamantopoulou ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Cell Aging ◽  
Control Group ◽  
Low Grade ◽  
Qrt Pcr ◽  
Epstein Barr

Abstract Abstract 5212 Background: In all Epstein-Barr (EBV)-associated malignancies, the virus displays a latency program of infection and a restricted pattern of gene expression. Among the products of these genes, latent membrane protein 1 (LMP1) is a potent transforming protein with several different roles. LMP1 has been shown in cell lines to stimulate apoptosis. Survivin, a member of the inhibitor of apoptosis (IAP) family, is an important regulator of the mitochondrial apoptotic pathway, while oxidative stress (OS) is a cellular condition particularly relevant to cell aging. In the present study we enrolled patients with non-EBV-related low grade B-cell lymphoproliferative diseases. The aim was to detect (1) the viral load of EBV-positive patients, (2) the expression of LMP1 oncoprotein, (3) the possible apoptotic properties of LMP1 by correlating the levels of survivin with LMP1 expression, and (4) the levels of oxidative stress in LMP1-positive and negative patients. Patients and Methods: Forty eight Greek patients with EBV-unrelated low grade B-cell leukemic lymphomas, were enrolled in the study (chronic lymphocytic leukemia: 27, marginal zone lymphoma: 12, mantle cell lymphoma: 4, hairy cell leukemia: 2, follicular lymphoma: 2, lymphoplasmacytic lymphoma: 1). The majority of patients (61.2%) were treatment-naïve, while the rest had not received any treatment for at least 6 months. DNA from peripheral blood was tested by quantitative real time (qRT) PCR for the EBV-R gene. RNA from EBV-positive patients was examined by RT-PCR and qRT PCR for LMP-1, while using qRT PCR we measured survivin expression in all patients. Densitometric analysis (DA) was used for semi-quantification of the survivin gene expression. The results were expressed relative to the expression of ABL housekeeping gene. The control group included 30 EBV-negative healthy adults. Oxidative stress was measured in the serum of all patients using the PerOx (TOS/TOC) Kit, by Immunodiagnostik. Non parametric methods (Mann-Whitney test) were used for statistical analysis of the results. Results: Twenty five (25) men and 23 women, with a median age of 74 (51–87 years old) were studied. EBV positivity was detected in 19/48 (39.6%) patients, and LMP1 was expressed in 13/19 (68.4%) EBV-positive patients. Survivin levels were lower in LMP1-positive patients vs LMP1-negative patients (2-tailed p=0.009). The oxidative stress was lower (261.4 μmol/L) in LMP1-positive patients vs LMP1-negative patients (372.3 μmol/L), (2-tailed p=0.014). Discussion: The literature lacks information about the expression of LMP1 in the peripheral blood of patients with non-EBV-related low grade B-cell leukemic lymphomas. Previous studies in LMP1-positive lymphoma cell lines have shown the apoptotic functions of LMP1 during type II latency. In this study LMP1-positive patients express statistically significant lower levels of survivin vs LMP1-negative patients. This finding is in accordance to the hypothesis that LMP1 oncoprotein can induce apoptosis. LMP1-positive patients had lower levels of oxidative stress compared to LMP1-negative patients. According to our findings, in non-EBV-related lymphomas, LMP1 may increase apoptosis and decrease the levels of oxidative stress. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Modulating Gene Expression in Epstein‐Barr Virus (EBV)‐Positive B Cell Lines with CRISPRa and CRISPRi

2018 ◽  
Vol 121 (1) ◽  
Author(s):  
Liang Wei Wang ◽  
Stephen J. Trudeau ◽  
Chong Wang ◽  
Catherine Gerdt ◽  
Sizun Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Autoantibodies to neurofibrillary tangles and brain tissue in Alzheimer's disease. Establishment of Epstein-Barr virus-transformed antibody-producing cell lines.

1987 ◽  
Vol 165 (1) ◽  
pp. 245-250 ◽  
Author(s):  
F Gaskin ◽  
B S Kingsley ◽  
S M Fu
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
B Cell ◽  
Cell Lines ◽  
Neurofibrillary Tangles ◽  
Control Group ◽  
Antibody Activity ◽  
Barr Virus ◽  
Epstein Barr ◽  
And Control

Multiple EBV-transformed B cell lines were established from five patients with a clinical diagnosis of Alzheimer's disease (AD) and six age-matched controls. The supernatants were screened for antibody activity against SDS-treated isolated neurofibrillary tangles (NFT). Reactive supernatants were identified from both the AD and control group. The frequencies of anti-NFT antibody-secreting lines were 6.3 and 1.6% for the AD and the control groups, respectively. A proportion of these supernatants also stained NFT in situ and neurons and/or glia in sections of the frontal and the temporal cortexes of autopsied AD and normal brains, as well as cells from three cell lines (HeLa, fibroblast, and neuroblastoma). Several patterns of staining were revealed by these supernatants, indicating different reactive antigens. One supernatant stained NFT and astrocytes in sections from AD brains. It did not stain sections from two normal brains. This cell line is the result of the immortalization of a circulating B cell making antibody specific for an antigen in AD. The present approach may provide new insights in the pathogenesis of AD.

scholarly journals Synergistic Cytotoxicity of Ibrutinib and the BCL2 Antagonist, ABT-199(GDC-0199) in Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL): Molecular Analysis Reveals Mechanisms of Target Interactions

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 509-509 ◽  
Author(s):  
Craig A. Portell ◽  
Mark Axelrod ◽  
L. Kyle Brett ◽  
Vicki L Gordon ◽  
Brian Capaldo ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
B Cell ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Single Agent ◽  
Parp Cleavage ◽  
Synergistic Cytotoxicity ◽  
Transcriptional Changes ◽  
Induced Apoptosis

Abstract Bruton tyrosine kinase (BTK) is critical to both normal B-cell development and the pathogenesis of B-cell malignancies. Ibrutinib is a recently FDA-approved small molecule irreversible inhibitor of BTK. In Phase II studies of single-agent ibrutinib in MCL (Wang ML et al, NEJM 2013) and CLL (Byrd JC, et al, NEJM 2013) the overall response rate was 68% and 89% (CR, PR, and PR with lymphocytosis), respectively, with PR as the best response in the majority of patients. Thus, not all patients respond and complete responses are infrequent with single agent ibrutinib. We previously reported that the BCL2 inhibitor, ABT-199, and the proteasome inhibitor, carfilzomib, were highly synergistic with ibrutinib in MCL cell lines using a focused drug panel (Axelrod M et al, Leukemia 2014). We sought to confirm these findings in MCL and CLL patient samples and to determine the mechanisms of synergy. Peripheral blood buffy coat samples from patients with circulating tumor cells were exposed to ibrutinib, ABT-199, carfilzomib and the combinations of ibrutinib and ABT-199 and ibrutinib and carfilzomib at pharmacologically-achievable doses for 72 hours. Apoptosis was assessed using PARP cleavage by FACS analysis of CD3-, CD5+, CD19+ cells representing the neoplastic clones. The combination of Ibrutinib and ABT-199 substantially induced apoptosis compared to each single agent alone (combo: 23%, ibrutinib: 3.8%, ABT-199: 3.0%). Ibrutinib plus carfilzomib also substantially induced apoptosis compared to each single agent alone (combo: 5.5%, Ibrutinib 3.8%, carfilzomib 1.7%) though to a less degree than the ABT-199 combination. The normal B-cell population (CD3-, CD5-, CD19+) in these samples was too small for analysis, thus normal T-cells (CD3+, CD5+, CD19-) from the same patients were used to identify the effects on normal lymphocytes. Minimal apoptosis was seen in normal T-cells with the single agents or the combinations. In a cohort of CLL and normal donor samples, heterogeneity in response to the combination of ibrutinib and ABT-199 was seen. When evaluated by Bliss modeling, 5 of 9 CLL samples had a synergistic improvement in apoptosis with the combination with the other 4 having no change. No increased apoptosis was seen in two tested peripheral blood lymphocyte (CD3-, CD5-, CD19+) populations from healthy donors. Gene expression profiling with Illumina Bead Chip array was used to evaluate the mechanisms of synergy with ABT-199 plus ibrutinib after 6 hours of drug exposure. The MCL cell line JVM2 was exposed to pharmacologically-achievable doses of ibrutinib, ABT-199 and combinations of each dose. Ibrutinib alone induced transcriptional change whereas ABT-199 did little to change gene expression. The combination induced both potentiative transcriptional changes (changes present in isolation and enhanced by the combination) and emergent transcriptional changes (changes only seen with the combination, unchanged by each single agent). Protein-protein interaction networks generated using the drug targets (BTK and BCL2) and emergent genes as input to STRING revealed activation of apoptosis via p53 and BIM as mechanisms of synergy. In conclusion, Ibrutinib and ABT-199 induce synergistic apoptosis in MCL cell lines and leukemic patient samples. The combination also induced apoptosis in some, but not all, CLL patient samples. No apoptosis was seen with either drug or the combination in normal T-cells from patients, suggesting little off-target effect. Emergent changes were seen when combining ABT-199 with ibrutinib in MCL cell lines. These changes suggest activation of p53 and BIM as potential mechanisms of synergy. A clinical trial with ABT-199 and ibrutinib is planned. Disclosures Off Label Use: Pre-clinical data with ABT-199 for MCL and CLL, not FDA approved. Williams:Pharmacyclics, Janssen: Consultancy, Research Funding.

scholarly journals Low Dietary n6/n3 Ratio Attenuates Changes in the NRF 2 Gene Expression, Lipid Peroxidation, and Inflammatory Markers Induced by Fructose Overconsumption in the Rat Abdominal Adipose Tissue

Antioxidants ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2005
Author(s):  
Petra Roškarić ◽  
Marcela Šperanda ◽  
Tomislav Mašek ◽  
Donatella Verbanac ◽  
Kristina Starčević
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Adipose Tissue ◽  
Fatty Acid ◽  
Female Rats ◽  
Control Group ◽  
Plain Water ◽  
Low Grade ◽  
Male And Female Rats ◽  
High Fructose

The objective of this study was to examine the benefits of different n6/n3 polyunsaturated fatty acid ratios on the lipid metabolism, insulin resistance, and oxidative stress in the adipose tissue of rats fed a high-fructose diet. Male and female rats were divided into four groups: a control group (CON) (n6/n3 ratio ~7), a high-fructose group (HF) (n6/n3 ratio ~7), an N6-HF group (n6/n3 ratio ~50), and the DHA-HF group (n6/n3 ratio ~1, with the addition of docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid). The CON group received plain water and the HF group received 15% fructose in their drinking water. Fructose induced an increase in the content of serum triglycerides, serum cholesterol, and HOMA-IR index. Among the fatty acids, elevated proportions of C18:1n9 and C16:1n7, as well as an increase in total monounsaturated fatty acid (MUFA), were found in the adipose tissue of the HF group. Fructose treatment also changed oxidative parameters, including a marked increase in the serum malondialdehyde (MDA) content. Meanwhile, DHA supplementation caused a significant decrease in the serum MDA concentration in comparison with the HF group. In addition, DHA/EPA supplementation attenuated oxidative stress by increasing NRF 2 gene expression. Fructose treatment also significantly decreased the adiponectin level, while DHA supplementation ameliorated it. The changes observed in this trial, including the decrease in the content of DHA and EPA, the decreased EPA/ARA ratio, and the increase in the expression of inflammatory genes, are characteristics of the low-grade inflammation caused by fructose treatment. These changes in the rat adipose tissue could be prevented by dietary intervention consisting of DHA supplementation and a low n6/n3 ratio.

Induction of Epstein-Barr virus (EBV) lytic cycle in vitro causes oxidative stress in lymphoblastoid B cell lines

2008 ◽  
Vol 324 (1-2) ◽  
pp. 55-63 ◽  
Author(s):  
Bochra Gargouri ◽  
Jos Van Pelt ◽  
Abd El Fatteh El Feki ◽  
Hammadi Attia ◽  
Saloua Lassoued
Keyword(s):  
Oxidative Stress ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Defective Epstein-Barr Virus Genomes and Atypical Viral Gene Expression in B-Cell Lines Derived from Multiple Myeloma Patients

2021 ◽  
Author(s):  
Fang Lu ◽  
Kayla A. Martin ◽  
Samantha S. Soldan ◽  
Andrew V. Kossenkov ◽  
Priyankara Wickramasinghe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Viral Genomes ◽  
Barr Virus ◽  
Epstein Barr

Epstein-Barr virus (EBV) is a human γ-herpesvirus that is causally associated with various lymphomas and carcinomas. Although EBV is not typically associated with multiple myeloma (MM), it can be found in some B-cell lines derived from multiple myeloma patients. Here, we analyzed two EBV+ MM-patient derived cell lines IM9 and ARH77 and found defective viral genomes and atypical viral gene expression patterns. We performed RNA-seq transcriptomics to characterize the viral and cellular properties of the two EBV+ cell lines compared to canonical MM cell line 8226. Principal component analyses indicated that IM9 and ARH77 clustered together and distinct from 8226. ImmGen analysis designate these cells as stem-cell and bone marrow derived. IM9 and ARH77 displayed atypical viral gene expression, including a leaky lytic cycle gene expression with absence of lytic DNA amplification. Genome sequencing revealed that EBV genomes in ARH77 contain large deletions, while IM9 has copy number losses in multiple EBV loci. Both IM9 and ARH77 show EBV genome heterogeneity suggestive of cell harboring multiple and variant viral genomes. We identified atypical high-level expression for lytic genes BLRF1 and BLRF2. We demonstrate that shRNA depletion of BLRF2 alters viral and host gene expression, including a reduction in lytic gene activation and DNA amplification. These findings demonstrate that aberrant viral genomes and lytic gene expression persist in rare B-cells derived from MM tumors, and suggest that EBV may contribute to etiology of MM. Importance Epstein-Barr Virus (EBV) is an oncogenic herpesvirus but its mechanisms of oncogenesis are not fully understood. A role for EBV in multiple myeloma has not yet been established. We analyzed EBV positive B-cell lines derived from multiple myeloma patients and found these cells harbor defective viral genomes with aberrant viral gene expression patterns and cell gene signatures for bone marrow derived lymphoid stem cells. These findings suggest that aberrant EBV latent infection may contribute to the etiology of multiple myeloma.

Specific Micro-RNA Expression Profile in Hodgkin Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 381-381
Author(s):  
Lu Ping Tan ◽  
Geert Harms ◽  
Tjasso Blokzijl ◽  
Rikst Nynke Schakel ◽  
Johan Gibcus ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Differentially Expressed ◽  
Cell Phenotype ◽  
Qrt Pcr

Abstract Introduction Classical Hodgkin lymphoma (cHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) differs not only in the form of histology and reactive background but also in the phenotypes of the tumor cells. Although tumor cells from both HL subtypes are originated from the germinal center (GC) B cell, gene expression studies show that lymphocytic and histiocytic (L&H) cells from NLPHL resembles normal B cells while Hodgkin/Reed-Sternberg cells (H/RS) from cHL demonstrate a loss of B cell phenotype and have significant overlap with primary mediastinal B cell lymphoma (PMBL). Recently, a new class of small RNAs, namely the micro-RNAs (miRNAs), has been identified. It is now known that at the post-transcriptional level, miRNAs negatively regulate gene expression in a sequence specific manner. Unique miRNAs expression patterns have been reported in various tissue types and also during a wide range of physiological states, such as cell proliferation, development, differentiation, apoptosis and hypoxia. As miRNAs play important roles in many cellular processes, it is proposed that there is a link between aberrant miRNA expression and loss of B cell phenotype in cHL. Methods In this study, miRNA profiles from cell lines of various B cell lymphoma subtypes were examined by qRT-PCR. Also, several B cell subsets were sorted from tonsil by FACS and the miRNA profiles studied by qRT-PCR. Some of the miRNAs are analyzed by in situ hybridization (ISH) in both HL tissue and tonsil samples. Results The miRNA profiling data indicated that cHL cell lines cluster together with PMBL while DEV, an NLPHL cell line, clusters together with CB. Upon validation of differentially expressed miRNAs on a cell line panel of 33 cell lines by monoplex qRT-PCR, 5/8 miRNAs identified as differentially expressed between cHL and GC B cells, were confirmed. Four out of six miRNAs differentially expressed between cHL and PMBL, were also confirmed as being differentially expressed in a larger cell panel. A high degree of overlap was observed between the most abundantly expressed miRNAs in the four HL cell lines. Expression of these miRNAs in HRS cells was verified by ISH in HL tissue samples. miRNA profiles of naive, GC and memory B cells display unique patterns. The overall miRNA expression levels were much lower than observed in the cell lines. Results of miRNA ISH in tonsil tissue demonstrated a specific staining pattern for each miRNA. These data indicate that miRNAs are particularly important for subsets of lymphocytes. Conclusion Several miRNAs that are expressed specifically in Hodgkin lymphoma have been identified. However, the effect of the aberrant expressions of these miRNAs in HL is yet to be elucidated, as the targets of these miRNAs remain unknown.

Lower Levels of Survivin in Patients with Leukemic Low Grade B-Cell Lymphomas Expressing LMP1 Oncoprotein of Epstein-Barr Virus

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1560-1560
Author(s):  
Panagiotis Theodorou Diamantopoulos ◽  
Katerina Polonyphi ◽  
Maria Sofotasioiu ◽  
Athanassios Galanopoulos ◽  
Nikolaos Spanakis ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Low Grade ◽  
B Cell Lymphomas ◽  
Barr Virus ◽  
Qrt Pcr ◽  
Epstein Barr ◽  
Rna Levels

Abstract Abstract 1560 Background Epstein-Barr virus (EBV) is a ubiquitous pathogen that chronically infects B lymphocytes and is implicated in the pathogenesis of lymphoproliferative diseases. Latent membrane protein 1 (LMP1), the major oncoprotein of the virus, has been shown to inhibit apoptosis and trigger survivin expression in malignant cell lines. Although EBV has not been implicated in the pathogenesis of low grade B-cell lymphomas, LMP1-mRNA has been detected in a significant proportion of patients with chronic lymphocytic leukemia (CLL). LMP1 is known for its antiapoptotic properties, but recent data show that LMP1 can simultaneously induce and inhibit apoptosis in B-cells. These opposite functions of LMP1 have not been studied in patients with low grade B-cell lymphomas. Objectives Our objectives were to detect LMP1-mRNA in patients with leukemic low grade B-cell lymphomas ant to investigate the postulated apoptotic properties of the protein, by correlating its expression to survivin levels. Patients and Methods Peripheral whole blood from 64 patients with leukemic low grade B-cell lymphomas was tested by qRT-PCR for the presence of BXLF-1 gene of EBV. The patients' characteristics are shown in table 1. All positive samples were tested by conventional PCR for LMP1-mRNA. Subsequently, survivin m-RNA levels were measured by qRT-PCR in all samples and compared between LMP1 positive and negative patients (Mann-Whitney U Test). Results The BXLF-1 gene was detected in 27/64 (42.1%) patients. LMP1-mRNA was detected in 23/64 (35.9%) patients and in 23/27 (85.2%) EBV-positive patients. Among CLL patients, LMP1-mRNA was detected in 19/44 (43.2%). Finally, surviving-mRNA levels were found to be 8.37 times higher in EBV-negative vs EBV-positive patients, (p=0.002) and 7.19 times higher in LMP1-negative vs LMP1-positive patients (p=0.009). The results are reported in detail in Table 1. Discussion Data from this year's studies suggest that LMP1 may exert both antiapoptotic and apoptotic functions. While the carboxy-terminal domain of LMP1 drives the proliferation and survival of EBV-infected B cells in vitro and in vivo, LMP1 may activate, through its amino-terminal six-transmembrane domains (6TM), the transmembrane receptor proteins PERK, ATF6 and IRE-1, leading to unfolded protein response (UPR) induction. UPR is a cellular stress response that promotes apoptosis. In different environments, LMP1 signaling may show differences regarding its apoptotic effects on B lymphocytes. In our study, we detected LMP1-mRNA in 43.2% of CLL patients, a proportion significantly higher than previously reported (14%). Moreover, for the first time, LMP1-mRNA was detected in patients with other than CLL low grade B-cell lymphomas (Table 1). In patients with leukemic low grade B-cell lymphomas, in the pathogenesis of which EBV is not causally implicated, LMP1 may have apoptotic instead of anti-apoptotic properties, as evidenced by the lower survivin m-RNA levels in LMP1-positive patients. This finding deserves further investigation, in order to reveal the clinical significance of the different functions of LMP1 in non-EBV related lymphomas. Disclosures: No relevant conflicts of interest to declare.

Common and Idiosyncratic Patterns of Cytokine Gene Expression by Epstein-Barr Virus Transformed Human B Cell Lines

1997 ◽  
Vol 10 (4) ◽  
pp. 183-195 ◽  
Author(s):  
ROSEMARY ROCHFORD ◽  
MARTIN J. CANNON ◽  
REBECCA E. SABBE ◽  
KALYANI ADUSUMILLI ◽  
GASTON PICCHIO ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cytokine Gene Expression ◽  
Cytokine Gene ◽  
Barr Virus ◽  
Epstein Barr ◽  
Human B Cell
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