Global Proteomic Profiling of Endemic Versus Sporadic Epstein-Barr Virus (EBV) Positive Burkitt Lymphoma (BL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4779-4779
Author(s):  
Nader El-Mallawany ◽  
Janet Ayello ◽  
Nancy Day ◽  
Carmella van de Ven ◽  
Kevin P Conlon ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
High Energy ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Apoptosis Pathway ◽  
Cellular Processes ◽  
Ebv Infection

Abstract Abstract 4779 Background EBV infection of normal B-cells is commonly associated with the pathogenesis of BL (Brady et al, Clin Path, 2007). Endemic BL (eBL) is characteristically positive (100%) for EBV, contrasting with sporadic BL (sBL), where approximately 30% of cases are positive for EBV. eBL vs. sBL have significantly different breakpoint regions within c-myc (Shiramizu/Magrath et al, Blood, 1991). Overexpression of c-myc is the sine quae non of BL. C-myc interactions with other genes/proteins is multilayered and complex (Basso/Della-Favera, Nat Gen, 2005). Apoptotic pathway disruption is propelled by EBV and is critically important in c-myc deregulation and subsequent lymphomagenesis that occurs in EBV+ eBL vs. sBL (Ruf et al, J Vir, 2000). Global analysis of proteins expressed in EBV+ eBL vs. sBL may provide insights into biologic, pathogenetic, and molecular differences between the two subtypes of lymphoma, and potentially identify targets for the development of therapeutic agents. Objectives To compare the proteomic expression profile and signal transduction pathways of EBV+ eBL vs. sBL. Methods Whole cell lysates obtained from the EBV+ eBL cell line Raji and the EBV+ sBL cell line NC37 were digested and labeled with iTRAQ” labeling reagents, following manufacturer's protocol. The peptides were resolved by 2D-LC technique (off-line Strong cation exchange followed by on-line reverse-phase liquid chromatography). Data-dependant High energy C-trap Dissociation MS/MS spectra were acquired using an Orbitrap XL Tandem Mass Spectrometer (ThermoFisher). The MS/MS data was searched using X!Tandem/TPP software suite against human IPI database (v3.50) appended with decoy (reverse) sequences. iTRAQ” ratios of proteins (ProteinProphet probability of >0.9) were normalized and differentially expressed proteins were determined through Mixture Modeling. Protein interactions were further analyzed using the GoMiner and Ingenuity pathway analysis tools. Results Over 400 proteins were identified as being differentially expressed by a ≥ 1.25 fold change between the two cell lines. We identified differentially expressed proteins in both cell lines that are involved in a wide array of cellular processes as exhibited in Figure 1. Cellular processes uniquely involved by proteins over-expressed in eBL included immune response, hematopoiesis, cell proliferation, heat shock, and B-cell activation, while those uniquely identified in sBL included cell division, response to virus, and NF-kB cascade proteins. Specific cell-regulatory pathways implicated by the differential protein profile expressions (with associated proteins in parentheses) included the p53 apoptosis pathway (PCNA, MSH6, C1QBP, MAP4, and BAX), the caspase network of apoptosis (HCLS1, ACIN1, and AIFM1), the tumor suppressor protein RB network (MCM7, PA2G4, and API5), general apoptotic pathways (HSP90 and PDCD4), B-cell differentiation and proliferation pathways (TPD52 and IKBKG), and the ubiquitin-proteasome pathway (UBE2J1, UBE2C, and UBE2S). Seven of these proteins are c-myc target genes. Ingenuity protein network analysis revealed nine proteins identified in the experiment with interactions connected through the p53, caspase, and tumor necrosis factor apoptosis pathways. Conclusion Proteomic profile analysis of EBV+ eBL and sBL revealed over and under-expression of multiple proteins that may be implicated in the multi-factorial nature of disease pathogenesis. This is the first MS-based direct proteomic comparison of eBL and sBL. Our results suggest that there are potentially different mechanisms driving cell proliferation and resistance to apoptosis in eBL versus sBL and that EBV infection may be involved in the processes that drive lymphomagenesis. Ultimately, identification of proteins unique to the distinct disease subtypes will serve to establish tumor markers that may enable development of new diagnostic, prognostic, and therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

The Suppressor of Cytokine Signaling-3 Gene Is Overexpressed in Human De Novo Follicular Lymphomas and Promotes IL-3-Independent Proliferation of the BaF3 Pro-B Cell Line.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1107-1107
Author(s):  
Jacqueline C. Barrientos ◽  
Sofya Rodov ◽  
Arthur W. Zieske ◽  
K. Gary J. Vanasse
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
De Novo ◽  
Myeloid Cells ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Socs3 Protein

Abstract The recent generation of mice lacking functional SOCS3 in hepatocytes, macrophages, and neutrophils reveals SOCS3 to be an essential regulator of IL-6 signaling via mediation of gp130-related cellular complexes, as well as a negative regulator of G-CSF signaling in myeloid cells. Although SOCS3 would appear to be a critical physiologic regulator of inflammatory responses, its possible role in hematologic malignancies and the underlying mechanisms which regulate its expression in B cells remain to be clearly defined. We previously showed that CD19+ B cells isolated from Eμ-Bcl-2 transgenic mice express high levels of SOCS3 in addition to overexpression of Bcl-2. Moreover, hematopoietic cell lines transduced to stably overexpress Bcl-2 exhibited marked induction of SOCS3 compared to controls, suggesting Bcl-2-associated pathways may play a role in the induction of SOCS3. In the current study, we describe SOCS3 overexpression limited to neoplastic follicular lymphoma (FL) cells in Bcl-2-associated human de novo FL and show that overexpression of SOCS3 is capable of stimulating cytokine-independent cellular proliferation of the BaF3 pro-B cell line. We measured SOCS3 protein levels by immunohistochemistry in paraffin-embedded biopsies from twelve patients diagnosed with de novo, untreated histologic grade I or II FL which harbored t(14;18) and Bcl-2 overexpression. In 9/12 de novo FL cases examined, immunostaining with two distinct antibodies to SOCS3 revealed marked overexpression of SOCS3 protein that, within the follicular center cell region, was limited to neoplastic FL cells and co-localized with Bcl-2 primarily in the nucleus of positive cells. In contrast, SOCS3 protein was not detected by immunostaining in germinal center follicular B cells from benign hyperplastic tonsil tissue. To further evaluate the role of SOCS3 in B cell biology, the IL-3-dependent BaF3 pro-B cell line was stably transduced with either a retroviral expression construct containing a 675bp human SOCS3 cDNA (BaF3SOCS3) or with vector only control (BaF3Δ). Whereas no SOCS3 protein was detected in control cells, high level expression of SOCS3 in transduced BaF3SOCS3 cells was confirmed by Western analysis using SOCS3 anti-sera. Furthermore, Bcl-2 protein was not detected in either BaF3SOCS3 or control cell lines. 2 x 105 BaF3SOCS3, BaF3Δ, and non-transduced BaF3 cell lines were initially grown in the presence 10% fetal bovine serum (FBS) and 5% WEHI 3B cell-conditioned medium as a source of IL-3. IL-3 was then removed by washing with DMEM/10% FBS. Cell viability was then measured by recording absorbance at 490nm using incorporation of the MTS tetrazolium compound. Interestingly, BaF3SOCS3 cells overexpressing SOCS3 did not undergo apoptosis but were able to proliferate in the absence of IL-3, with percent viable cells approaching 400% at > 96 hours, which represented the final time-point measured. In contrast, BaF3Δ and non-transduced BaF3 cells underwent apoptotic cell death between 8 and 36 hours in response to IL-3 withdrawal. Thus, SOCS3 overexpression confers IL-3-independent cell proliferation to the BaF3 cell line. These data indicate that unlike its negative regulatory effect on G-CSF signaling in myeloid cells, overexpression of SOCS3 in B cells may promote B cell proliferation rather than growth suppression and may play an important role in the pathogenesis of de novo FL in humans.

Quantitative Proteomic Identification of Novel Diagnostic Biomarkers and Therapeutic Targets for Classical Hodgkin Lymphoma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1462-1462
Author(s):  
Mihaela D. Chiselite ◽  
Damian Fermin ◽  
Venkatesha Basrur ◽  
Kevin P. Conlon ◽  
Charles Seiler ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Line ◽  
Cell Lines ◽  
Total Cell ◽  
Negative Regulator ◽  
Differentially Expressed ◽  
Tandem Mass ◽  
Differentially Expressed Proteins ◽  
Classical Hodgkin Lymphoma ◽  
False Discovery

Abstract There is significant morphologic, immunophenotypic and molecular overlap between some subtypes of diffuse large B-cell lymphoma and classical Hodgkin lymphoma (cHL). In addition, recent studies reveal similar gene expression profiles in PMBCL and cHL suggesting a common cell of origin. We utilized a differential isotopic strategy to determine the global proteomic differences between cell lines derived from cHL (L428), DLBCL (SUDHL-9) and PMBCL (Karpas 1106P). Protein was collected from cell lysates and subjected to labeling by isobaric tags (iTRAQ) for relative quantification and analyzed by reverse-phase liquid chromatography and electrospray ionization tandem mass spectrometry. 200 mg of total cell lysates from each cell line was used and liquid chromatography and tandem mass spectrometry (LC-MS/MS) analyses were performed in duplicate. The proteins were identified using X!Tandem. After normalization, quantitative data were subjected to false discovery rate (FDR) calculation to identify differentially expressed proteins through Mixture Modeling. Significantly differentially expressed proteins were scored at a false discovery rate (FDR) cut-off of ≤ 0.13. This approach yielded 66 proteins with differential expression patterns that were discriminative for the 3 different cell lines. Several proteins that have been previously reported to be differentially expressed between cHL and PMBCL were identified including Fascin, Galectin-1, Galectin-3, STAT1 and SWAP70. In addition, several previously unreported proteins involved in numerous cellular functions were differentially expressed (cell adhesion, signaling, immunity). We have validated a subset of these by Western blot (WB) analysis and immunohistochemistry. For example, the π class of glutathione S-transferases (GSTP), was over-expressed six-fold in K1106P cells vs. L428 cells, and virtually absent in the SUDHL-9 cell line, as confirmed by WB. This enzyme class has been shown to be overexpressed in many human cancers and to be involved in therapy refractoriness. More recently, GSTP has been implicated as a negative regulator of cellular death/apoptosis through the MAP kinase pathway. These factors, and its unusual absence in the SUDHL-9 cell line made GSTP a rational candidate for further functional analysis. Briefly, SUDHL-9 cells and the cHL cell lines L428 and KMH2 were exposed to increasing concentrations (20–80 μM) of the GSTP inhibitor and thiol modifier molecule ethacrynic acid (EA), and subjected to viability assays (WST-1) and to WB for apoptotic markers. Both cHL cell lines were equally resistant to EA concentrations up to 60μM by WST-1 assays, while SUDHL-9 cells were exquisitely sensitive, undergoing total cell necrosis at the lowest (20 μM) concentration. WB analysis for markers of apoptosis showed a higher level of apoptosis in SUDHL-9 cells compared to the HL-derived KMH2 and L428 cells, as evidenced by poly (ADP-ribose) polymerase (PARP) cleavage into its 85 kDa apoptosis-related fragment. L428 cells, expressing 40% the GSTP levels of KMH2 cells by WB, were more susceptible to apoptosis at 60μM EA than KMH2 cells, as evidenced by decrease in S-phase Kinase-associated Protein 2 (p45SKP2), with concomitant increase in p27Kip1, a negative regulator of G1-S progression. These results demonstrate an inverse relationship between GSTP levels and susceptibility to EA-induced cell death in lymphoma. This study demonstrates the utility of large-scale mass spectrometry-based proteomics for the discovery of proteins that may serve as potential diagnostic marker panels for the distinction of PMBCL, DLBCL and cHL, as well as candidate therapeutic targets.

scholarly journals Epstein-Barr virus transformation induces B lymphocytes to produce human interleukin 10.

1993 ◽  
Vol 177 (2) ◽  
pp. 295-304 ◽  
Author(s):  
N Burdin ◽  
C Péronne ◽  
J Banchereau ◽  
F Rousset
Keyword(s):  
Cell Proliferation ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Interleukin 10 ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr ◽  
Human B Cell

Interleukin 10 (IL-10) is a pleiotropic factor that enhances proliferation of activated human B lymphocytes and induces them to secrete high amounts of immunoglobulins. Here we show that several human B cell lines were able to constitutively secrete human (h)IL-10. Whereas none of the pre-B nor the plasmocytic cell lines tested produced hIL-10, 25 of the 36 tested mature B cell lines (lymphoblastoid and Burkitt lymphoma cell lines) secreted hIL-10. Moreover, 24 of these 25 hIL-10-producing B cell lines contained the Epstein-Barr virus (EBV) genome, suggesting a relationship between hIL-10 production by human B cell lines and EBV expression. Accordingly, whereas polyclonal activation via triggering of surface immunoglobulins or CD40 antigen induced highly purified normal human B lymphocytes to produce only low (0.3-0.4 ng/ml) but significant amounts of hIL-10, EBV infection induced them to secrete high amounts of hIL-10 (4-9 ng/ml). Furthermore, addition of exogenous hIL-10, simultaneously to EBV infection, potentiated cell proliferation, whereas a blocking anti-IL-10 antiserum inhibited it. Thus, hIL-10 produced by infected human B lymphocytes appears to be involved in the mechanisms of EBV-induced B cell proliferation.

scholarly journals Transformation-associated alterations in interactions between pre-B cells and fibronectin

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2311-2320 ◽  
Author(s):  
FM Lemoine ◽  
S Dedhar ◽  
GM Lima ◽  
CJ Eaves
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Attachment ◽  
Attachment Site ◽  
Receptor Antibodies

Abstract Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

scholarly journals Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1667
Author(s):  
Laura Abaandou ◽  
David Quan ◽  
Joseph Shiloach
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Hek293 Cell ◽  
Chinese Hamster ◽  
Production Performance ◽  
Cellular Processes ◽  
Hek293 Cell Line ◽  
Global Engineering ◽  
Genomic Tools ◽  
Serum Free Suspension

The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line’s most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering.

scholarly journals Underutilized Mexican Plants: Screening of Antioxidant and Antiproliferative Properties of Mexican Cactus Fruit Juices

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 368
Author(s):  
Elda M. Melchor Martínez ◽  
Luisaldo Sandate-Flores ◽  
José Rodríguez-Rodríguez ◽  
Magdalena Rostro-Alanis ◽  
Lizeth Parra-Arroyo ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Liver Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
In Vitro Assays ◽  
Total Phenolic ◽  
Liver Cancer Cell ◽  
Antiproliferative Activities

Cacti fruits are known to possess antioxidant and antiproliferative activities among other health benefits. The following paper evaluated the antioxidant capacity and bioactivity of five clarified juices from different cacti fruits (Stenocereus spp., Opuntia spp. and M. geomettizans) on four cancer cell lines as well as one normal cell line. Their antioxidant compositions were measured by three different protocols. Their phenolic compositions were quantified through high performance liquid chromatography and the percentages of cell proliferation of fibroblasts as well as breast, prostate, colorectal, and liver cancer cell lines were evaluated though in vitro assays. The results were further processed by principal component analysis. The clarified juice from M. geomettizans fruit showed the highest concentration of total phenolic compounds and induced cell death in liver and colorectal cancer cells lines as well as fibroblasts. The clarified juice extracted from yellow Opuntia ficus-indica fruit displayed antioxidant activity as well as a selective cytotoxic effect on a liver cancer cell line with no toxic effect on fibroblasts. In conclusion, the work supplies evidence on the antioxidant and antiproliferative activities that cacti juices possess, presenting potential as cancer cell proliferation preventing agents.

scholarly journals Sirtuin 2 in Endometrial Cancer: A Potential Regulator for Cell Proliferation, Apoptosis and RAS/ERK Pathway

2020 ◽  
Vol 19 ◽  
pp. 153303382098078
Author(s):  
Yanjuan Guo ◽  
Nannan Zhao ◽  
Jianli Zhou ◽  
Jianxin Dong ◽  
Xing Wang
Keyword(s):  
Cell Proliferation ◽  
Endometrial Cancer ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Erk Pathway ◽  
Uterine Epithelial Cell ◽  
Knock Down ◽  
Ishikawa Cells ◽  
And Control

Objective: The present study aimed to explore the function of sirtuin 2 (SIRT2) on cell proliferation, apoptosis, rat sarcoma virus (RAS)/ extracellular signal-regulated kinase (ERK) pathway in endometrial cancer (EC). Methods: SIRT2 expression in human EC cell lines and human endometrial (uterine) epithelial cell (HEEC) line was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. SIRT2 knock-down and control knock-down plasmids were transfected into HEC1A cells, respectively; SIRT2 overexpression and control overexpression plasmids were transfected into Ishikawa cells, respectively. After transfection, SIRT2, HRas proto-oncogene, GTPase (HRAS) expressions were evaluated by RT-qPCR and western blot. ERK and phosphorylated ERK (pERK) expressions were evaluated by western blot. Meanwhile, cell proliferation and cell apoptosis were measured. Results: Compared to normal HEEC cell line, SIRT2 mRNA and protein expressions were increased in most human EC cell lines (including HEC1A, RL952 and AN3CA), while were similar in Ishikawa cell line. In HEC1A cells, SIRT2 knock-down decreased cell proliferation but increased apoptosis. In Ishikawa cells, SIRT2 overexpression induced cell proliferation but inhibited apoptosis. For RAS/ERK pathway, SIRT2 knock-down reduced HRAS and inactivated pERK in HEC1A cells, whereas SIRT2 overexpression increased HRAS and activated pERK in Ishikawa cells, suggesting that SIRT2 was implicated in the regulation of RAS/ERK pathway in EC cells. Conclusion: SIRT2 contributes to the EC tumorigenesis, which appears as a potential therapeutic target.

scholarly journals Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Lisa Grossman ◽  
Chris Chang ◽  
Joanne Dai ◽  
Pavel A. Nikitin ◽  
Dereje D. Jima ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Cellular Aggregation ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out. Epstein-Barr virus (EBV), an oncogenic herpesvirus, infects and transforms primary B cells into immortal lymphoblastoid cell lines (LCLs), providing a model for EBV-mediated tumorigenesis. EBV transformation stimulates robust homotypic aggregation, indicating that EBV induces molecules that mediate cell-cell adhesion. We report that EBV potently induced expression of the adhesion molecule CD226, which is not normally expressed on B cells. We found that early after infection of primary B cells, EBV promoted an increase in CD226 mRNA and protein expression. CD226 levels increased further from early proliferating EBV-positive B cells to LCLs. We found that CD226 expression on B cells was independent of B-cell activation as CpG DNA failed to induce CD226 to the extent of EBV infection. CD226 expression was high in EBV-infected B cells expressing the latency III growth program, but low in EBV-negative and EBV latency I-infected B-lymphoma cell lines. We validated this correlation by demonstrating that the latency III characteristic EBV NF-κB activator, latent membrane protein 1 (LMP1), was sufficient for CD226 upregulation and that CD226 was more highly expressed in lymphomas with increased NF-κB activity. Finally, we found that CD226 was not important for LCL steady-state growth, survival in response to apoptotic stress, homotypic aggregation, or adhesion to activated endothelial cells. These findings collectively suggest that EBV induces expression of a cell adhesion molecule on primary B cells that may play a role in the tumor microenvironment of EBV-associated B-cell malignancies or facilitate adhesion in the establishment of latency in vivo. IMPORTANCE Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out.

scholarly journals Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

1987 ◽  
Vol 7 (1) ◽  
pp. 450-457 ◽  
Author(s):  
E H Brown ◽  
M A Iqbal ◽  
S Stuart ◽  
K S Hatton ◽  
J Valinsky ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Gene Cluster ◽  
Cell Lines ◽  
Heavy Chain ◽  
Dna Content ◽  
Immunoglobulin Heavy Chain ◽  
S Phase ◽  
C Value ◽  
Murine Erythroleukemia

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

scholarly journals Integrative analysis of mRNA and miRNA profiles identifies miR-193 and miR-210 as potential regulatory biomarkers in different molecular subtypes of breast cancer

2020 ◽  
Author(s):  
Adriane Feijo Evangelista ◽  
Renato J Oliveira ◽  
Viviane A O Silva ◽  
Rene A D C Vieira ◽  
Rui M Reis ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Cell Line ◽  
Cell Lines ◽  
Breast Cancer Cell ◽  
Cancer Progression ◽  
Triple Negative ◽  
Molecular Subtypes ◽  
Breast Cancer Cell Lines ◽  
Mcf 7

Abstract Introduction: Breast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated. Methods: The miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex, flow cytometry and transwell assays were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively. Results: The most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a mediator on invasion and metastasis, and the tumor suppressor gene RUNX3. Conclusion: In summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers.

Sign in / Sign up

Export Citation Format

Share Document