BL Cells Research Articles

Burkitt Lymphoma Specific Zinc Finger Protein ZNF385B Is Involved in Regulation of B Cell Apoptosis

Abstract 1584Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are common types of childhood mature B-cell non-Hodgkin's lymphoma (NHL). Both are postulated to be derived from GC B-cells but they are independent disease entities of NHL; however, they have overlapping morphologic and immunophenotypic features. A characteristic translocation t(8;14) of BL also occurs in 5 to 10 percent of cases of DLBCL. It is not uncommon to have difficulty in differentiating BL from DLBCL at initial diagnosis. Therefore, the latest WHO classification of tumors of the lymphoid tissue has recognized a category with overlapping features between BL and DLBCL. Considering this background, gene-expression profiling using microarray should provide an accurate, quantitative method for distinguishing the NHL. Actually, other researchers performed gene-expression profiling of mature B-cell lymphoma at all ages and showed that BL could be reliably distinguished from DLBCL. It has also been reported that DLBCL can be classified into two categories of patients with very different five-year overall survival rates on the basis of gene-expression profile.In an attempt to investigate the molecules specifically expressed in each type of NHL, we performed the screening of genes expressed characteristically in each type of childhood hematologic malignancy by employing a microarray system using clinical specimens from pediatric patients, consisting of 3 patients with DLBCL, 16 with BL, 6 with BCP-ALL and 6 with T-ALL. Consistent with previous reports, CD40, EBI3, FGD6, LMO2, and SERPINA9 were over-expressed in DLBCL. In addition, over-expression of IL21R, STAT3, BCL6, CD58, JAK3 was observed in DLBCL. In BL, on the other hand, BMP7 and SOX11 were over-expressed as reported previously. Furthermore, we found that ZNF385B, BMP3, PEG10, MUC4, AICDA, SMAD1, C13orf15, CD24 were over-expressed in BL.In these candidate genes, ZNF385B, also called ZNF533, was expressed in vast majority of BL cases as well as BL cell lines but not in other hematopoietic malignancies, including DLBCL, B-cell precursor ALL, and mature B-cell malignancies originate from another developmental stages of B cells. Therefore, we concluded that ZNF385B is a BL-specific zinc-finger protein and can be used as a diagnostic marker for BL.Currently three isoforms (IFs) of ZNF385B have been identified and IF-1 is the longest transcript variant, which possesses 4 ZF domains, while IF-2/3 are shorter transcript variants with 3 ZF domains. Although the function and biological significance of this protein have not been clarified at all, ZNF385A has high homology with ZNF385B IF-2/3 is known to be involved in apoptosis regulation. Therefore, we intended to elucidate the functional role of ZNF385B in B cells.First we analyzed intracellular localization of ZNF385B. When ZNF385B IF-1 proteins fused with EGFP at the N-terminus were expressed transiently in DLBCL BJAB cells, we observed the nuclear localization by obtaining fluorescent images.Next, to examine the characteristics of ZNF385B, we established a protein inducible system in a tetracycline-dependent manner for both ZNF385B IF-1 and its deletion mutant ZNF385B-DEL corresponds to IF-2/3 lacking ZF domain in N-terminus in BJAB cells. Interestingly, ectopic expression of ZNF385B IF-1 induced up-regulation of PERP (p53 apoptosis effector related to PMP-22) and activation of caspase-3 and −8, resulting in apoptosis induction, whereas ZNF385B-DEL did not. Furthermore, ZNF385B-DEL inhibited apoptosis induced by CD20 cross-linking and BCR stimulation. Immunoprecipitation by anti-p53 antibody indicated the binding of ZNF385B isoform 1 with p53. Since PERP is known to be a p53 transcriptional target, these results suggest the involvement of ZNF385B in B-cell apoptosis by modulating p53 transactivation. Considering our observation of ZNF385B expression in a portion of cells in peripheral lymphoid organs, ZNF385B possibly is involved in the regulation of death and survival that specifically occurs in germinal center B cells. Disclosures:No relevant conflicts of interest to declare.

Liprin-α4 Is Required for Nickel Induced Receptor Protein Tyrosine Phosphatase-Leukocyte Antigen Related Receptor F (RPTP-LAR) Activity

Liprin-α4 was strongly induced following nickel (II) chloride exposure in a variety of cell types including BEAS-2B, A549, BEP2D and BL41 cells. Liprin-α4, a member of the Liprin alpha family, has seven isoforms but only three of these variants were detected in BEAS-2B cells (004, 201 and 202). The level of Liprin-α4 variants 201 and 004 were highly increased in BEAS-2B cells in response to nickel. We showed that Liprin-α4 bound directly to the cytoplasmic region of RPTP-LAR (receptor protein tyrosine phosphatase-leukocyte antigen-related receptor F). The cytoplasmic region of RPTP-LAR contains two phosphatase domains but only the first domain shows activity. The second domain interacts with other proteins. The phosphatase activity was increased both following nickel treatment and also in the presence of nickel ions in cell extracts. Liprin-α4 knock-down lines with decreased expression of Liprin-α4 variants 004 and 201 exhibited greater nickel toxicity compared to controls. The RPTP-LAR phosphatase activity was only slightly increased in a Liprin-α4 knock-down line. Liprin-α4 appeared necessary for the nickel induced tyrosine phosphatase activity. The presence of Liprin-α4 and nickel increased tyrosine phosphatase activity that reduced the global levels of tyrosine phosphorylation in the cell.

Comparative Proteomic Analysis Implicates COMMD Proteins as Epstein–Barr Virus Targets in the BL41 Burkitt’s Lymphoma Cell Line

Epstein-Barr virus (EBV) infection is a major health problem associated with a variety of diseases, including Burkitt's lymphoma. EBV promotes its effects through the activation of multiple signaling pathways, with NF-κB mediated transcription being a major target. We have undertaken a comparative proteomic approach using 2D-electrophoresis and mass spectrometry to identify EBV-regulated proteins in the BL41 Burkitt's lymphoma cell line. Many of the proteins differentially regulated were previously known mediators of EBV action. Among the novel proteins identified, three members of the conserved COMMD family were found to be down-regulated. Further analysis of this family at the mRNA level, using reverse-transcriptase or real-time PCR, showed that 7 out of 10 COMMD members were affected in EBV-transformed BL41 cells. Since COMMD family proteins have been implicated as negative regulators of the NF-κB transcription factor, our data are consistent with a hypothesis that EBV down-regulates COMMD proteins in order to enhance NF-κB mediated transcriptional events and B-lymphocyte transformation.

TH-C-220-10: Limit of Setup Accuracy Using Bioluminescence Imaging to Guide Focal Pre-Clinical Irradiation

Purpose: Cone beam CT (CBCT) on-board new small animal radiation research platforms (SARRPs) is inadequate for guiding focal irradiation of targets in soft tissue. In this study, we examined the setup uncertainty using the more sensitive bioluminescence imaging (BLI) and BL tomography (BLT) for off-line guided irradiation. Methods: A small glass bulb containing BL cells was implanted in six euthanized mice These were handled free-hand while five others were immobilized. Surface BLI was acquired and BLT reconstructed of each carcass using a Caliper BLI system. An ink-dot marked the “visual” centroid of the surface BL image. The carcasses were then transported to a SARRP for CBCT localization. In the visual setup, the animal was positioned to align the ink-dot with the central axis of the SARRP. In the CBCT-aided setup, the pose of each carcass was adjusted to recapitulate its pose at the time of BLI. The 2D radial offset of the “true” bulb centroid in CBCT from the central axis of the SARRP was measured. In the 3D setup, the 3D contours of CBCT and BLT contours were registered and the 3D radial offset between the 2 centroids was calculated. Results: The results are virtually identical between free-hand and immobilized setup, as well as using visual and CBCT-aided 2D setup. The onset of rigor mortis rendered immobilization un-necessary. The 2D offset from the “true” position of the BL bulb was 2.3 +/− 1.3 mm. The 3D radial offset was 1.5 +/− 0.9 mm, approaching the documented uncertainty in BLT reconstruction. Conclusions: With the stiff carcasses, the results represent the best that can be achieved with off-line 2D surface BLI and 3D BLT localization. An on-board approach with further improvement in BLT using prior information of anatomic and optical properties, localization uncertainty less than 1 mm is possible. Funded in part by a sponsored research agreement from Xstrahl Ltd. Xstrahl Ltd. has a technology transfer Agreement with Johns Hopkins University.

Ubiquitin-mediated protein degradation and methylation-induced gene silencing cooperate in the inactivation of the INK4/ARF locus in Burkitt lymphoma cell lines

Burkitt lymphoma is one of the most aggressive tumors affecting humans. Together with the characteristic chromosomal translocation that constitutively activates the c-Myc oncogene, alterations in cellular tumor suppressor pathways are additionally required in order to allow the cells to overcome anti-oncogenic barriers and proliferate in an uncontrolled manner. The INK4a/ARF locus on chromosome 9p21 is considered a safeguard locus since it encodes the two important tumor suppressor proteins, p14ARF and p16INK4a. By regulating the p53 and Rb pathways p14ARF and p16INK4a respectively act as pro-apoptotic and cell cycle inhibitor proteins. The importance of the INK4a/ARF locus has been well documented in several human tumors as well as in Burkitt lymphoma. Although the mechanisms responsible for the transcriptional regulation of the INK4a/ARF locus have been thoroughly characterized, less is known about its posttranscriptional control. In this study we found that p16INK4a and p14Arf are concurrently inactivated in a panel of BL cell lines. We demonstrate that along with the epigenetic silencing of the p16INK4a gene, the complete inactivation of the locus is achieved by the improper turnover of INK4/ARF proteins by the ubiquitin-proteasome system (UPS), as the proteasome inhibitor MG-132 blocks p14ARF degradation and induces a dramatic stabilization of the p16INK4a protein. We establish that the simultaneous deregulation of both DNA methylation patterns and the ubiquitin-dependent proteolysis system is required to completely inactive the INK4/ARF locus, opening new prospects for the understanding and treatment of Burkitt lymphoma.

Comparative Genomic Identification of Unique Signal Transduction Pathways and Targets In Pediatric Burkitt Lymphoma (PBL)

AbstractAbstract 631 Background:The prognosis of PBL has dramatically improved over the past 25 years (> 80% 5 yrs EFS) (Cairo et al BJH 2009). This success, however, has come at the cost of the high toxicity associated with intensive multi-agent chemotherapy. A need to identify less toxic targeted therapy exists. Our recent studies show that while almost 100% of childhood BL (238 children) is characterized by an 8q24 (c-myc) gene rearrangement, 70% of the patients also had a secondary chromosomal aberration (Cairo et al Blood 2007), and those children with BL who had a 13q abnormality had a significantly poorer outcome (20% reduction in EFS; Poirel/Cairo et al Leukemia, 2009). Loss of 13q14.3 was associated with a significant decrease in OS (Nelson/Cairo/Perkins/Sanger et al BJH 2010). DLEU1, proven as a Burkitt specific classifier and a cMYC target gene (Dave/Staudt et al NEJM 2006), is located within the region of 13q14.3. DLEU1 is recognized to interact with c-Myc, TUBB2C, UBR1, and other proteins. When DLEU1 is down-regulated by a DLEU1 siRNA, the spontaneous apoptotic rate was decreased with concomitant significantly reduced levels of UBR1 and TUBB2C gene expression (Day/Cairo et al SIOP 2008). Our hypothesis is that deletion of 13q14.3, which contains DLEU1, in pediatric BL may confer a phenotype of chemoimmunotherapy resistance. Objective:To identify potential drug targets using comparative genomic microarray analysis of specimens in patients with PBL. Methods:PBL data from three different research groups were used; i.e. COG ANHL01P1 by our group (n=11), NCBI's GEO GSE10172 and GSE4475 (Klapper et al) (n=16), and GSE4732 (Dave/Staudt et al) (n=15). For comparison at equal weighted level, ANHL01P1 samples were validated by building a prediction model with Support Vector Machines using Klapper PBL as training database. RNAs were subjected to microarray studies (Affymetrix U133A_2) and analyzed by Agilent GeneSpring or Partek. Functional interpretation of the identified PBL genes was analyzed by Ingenuity Pathways Analysis. One-way ANOVA followed by Tukey test was used. To test our hypothesis, we knocked down DLEU1 and investigated cytoxan (CY) and rituximab effect on apoptotic rate of Ramos. Ramos BL cell lines were transiently transfected (24 hrs) with DLEU1 siRNA (5′-AUACUUGGCAUGAAUGAACUUAUGU-3′ and 3′-UAUGAACCGUACUUACUUGAAUACA-5′) (Day/Cairo SIOP 2008). The siRNA transfected cells were then treated with CY (0, 89.5, 895, 8950 nM) or rituximab (0, 4, 40, 400 mg/mL) for additional 4 hrs. Cells were evaluated for percent apoptosis using Annexin V-FITC and Propidium Iodide followed by FACS using BD LSRII. Results:1565 genes were identified (p<0.05), among which 376 genes showed no significant difference among three groups. High expression of cMYC (27F) and DLEU1 (9.9F) were detected. Pathway analysis indicates that these genes areinvolved in Toll-like receptor signaling (16 genes; p<0.01), including IRAK1 (22.9F), IFNAR2 (5.4F), NFKBIA (15.3F), and STAT1 (9.2F); JAK-STAT signaling (16 genes; p<0.01), including PTPN11 (25.6F), PTPN6 (8.8F), PIM1 (6.4F), and IL21R (2.9F); and MAPK signaling (19 genes; p<0.01) including MAP2K1 (11.8F), MAP3K7 (6.3F), MAPK9 (6.9F), and RAF1 (9.2F). There was a significant reduction in apoptosis in the CY-treated BL transfected DLEU1 siRNA vs mock control cells (89.5 nM CY: 10.26+0.23% reduction, p<0.05 to negative control; 895 nM CY, 10.86+0.67% reduction, p<0.01; 8950 nM, 9.85+0.32% reduction, p<0.05. There was a similar significant reduction in rituximab-induced apoptosis in the BL transfected DLEU1 siRNA vs mock control cells (4 mg/mL Rituximab: 25.45+2.55% reduction, p<0.01 to negative control; 40 mg/mL Rituximab, 18.31+5.13% reduction, p<0.04; 400 mg/mL rituximab, 32.33+1.77% reduction, p<0.02). Conclusions:Our pathway analysis of the 376 BL signature indicates that Toll-like receptor, JAK-STAT, and MAPK signaling pathways play important roles in gene regulation, anti-apoptosis, and proliferation, respectively, in PBL. The siRNA knock down of DLEU1 resulted in significantly less apoptosis on Ramos with CY or rituximab treatment. These data suggest that DLEU1 may in part play an important role in regulation of programmed cell death in BL and suggest a mechanism whereby BL with a 13q- abnormality may have a more aggressive clinical course. As CY or rituximab does not induce apoptosis in DLEU1 knockdown BL, the absence of DLEU1 may, in part, lead to drug resistance. Disclosures:No relevant conflicts of interest to declare.

X-box binding protein 1 induces the expression of the lytic cycle transactivator of Kaposi's sarcoma-associated herpesvirus but not Epstein-Barr virus in co-infected primary effusion lymphoma.

Cells of primary effusion lymphoma (PEL), a B-cell non-Hodgkin's lymphoma, are latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV), with about 80 % of PEL also co-infected with Epstein–Barr virus (EBV). Both viruses can be reactivated into their lytic replication cycle in PEL by chemical inducers. However, simultaneous activation of both lytic cascades leads to mutual lytic cycle co-repression. The plasma cell-differentiation factor X-box binding protein 1 (XBP-1) transactivates the KSHV immediate–early promoter leading to the production of the replication and transcription activator protein (RTA), and reactivation of KSHV from latency. XBP-1 has been reported to act similarly on the EBV immediate–early promoter Zp, leading to the production of the lytic-cycle transactivator protein BZLF1. Here we show that activated B-cell terminal-differentiation transcription factor X-box binding protein 1 (XBP-1s) does not induce EBV BZLF1 and BRLF1 expression in PEL and BL cell lines, despite inducing lytic reactivation of KSHV in PEL. We show that XBP-1s transactivates the KSHV RTA promoter but does not transactivate the EBV BZLF1 promoter in non-B-cells by using a luciferase assay. Co-expression of activated protein kinase D, which can phosphorylate and inactivate class II histone deacetylases (HDACs), does not rescue XBP-1 activity on Zp nor does it induce BZLF1 and BRLF1 expression in PEL. Finally, chemical inducers of KSHV and EBV lytic replication in PEL, including HDAC inhibitors, do not lead to XBP-1 activation. We conclude that XBP-1 specifically reactivates the KSHV lytic cycle in dually infected PELs.

Interleukin 10 decreases MICA expression on melanoma cell surface

Natural-killer group 2, member D (NKG2D) binds to a variety of ligands, including the major histocompatibility complex (MHC) class I chain-related proteins (MIC) and UL16-binding proteins (ULBP). It is regarded as a co-activating receptor on NK cells, having an important role in the cell-mediated immune response to tumours. We studied the influence of interleukin (IL)-10 on the regulation of MIC and ULBP expression on melanoma cells, and its effect on the cytotoxic function of NK cells in vitro. Here, we show that, in the presence of IL-10, FMS mel and BL mel cell lines decreased MICA and ULBP2 surface expression, whereas MHC class I did not change substantially on the cell surface. MICA mRNA levels decreased in IL-10-treated FMS and IL-10-transduced BL cell lines. Interestingly, we observed that MICB surface expression and its mRNA levels increased upon IL-10 treatment in a melanoma cell line. These changes in NKG2D ligands surface expression patterns owing to IL-10 treatment resulted in an effect on lysis susceptibility mediated by lymphocyte-activated killer cells, as tumour cell lines that displayed a higher decrease of MICA on their surface had lower levels of lysis. In addition, expression of CD107a was downregulated on the surface of NK cells following stimulation with IL-10-treated FMS cells. Our results suggest a novel function for IL-10 in the modulation of NKG2D ligand expression and in the control of cytotoxicity mediated by NKG2D/NKG2D ligand axis.

B cell receptor triggering sensitizes human B cells to TRAIL-induced apoptosis

TRAIL is known to cause death in tumor cells, but physiological regulation of its activity remains poorly characterized. We demonstrate that BCR triggering sensitizes transformed centroblast-like BL cells and peripheral blood memory B cells to TRAIL-mediated apoptosis. The sensitization correlated with surface down-regulation and intracellular retention of TRAIL-R4, along with changes in the expression of several Bcl-2 protein family members. Although enhancing FAS-mediated cell death, CD40 activation protected B cells from TRAIL-induced apoptosis. Combination of Ig cross-linking with CD40 ligation did not prevent TRAIL-R4 down-regulation but induced changes in the mitochondria-regulated pathway of apoptosis that are known to be associated with resistance to TRAIL. Human CD5(+) B cells, presumably stimulated by reactivity to self without immunological help, exhibited very high ex vivo sensitivity to TRAIL. Our results define the first B-lymphocyte-specific physiological signal that increases cellular sensitivity to TRAIL. This may be important for our understanding of TRAIL involvement in the control of B cell responses and aid in designing TRAIL-based therapies for B cell lymphomas.

Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis. Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore as a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions. The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program. In this short review we touch upon aspects which are the subject of our present work. We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells. The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.

Burkitt lymphoma-associated molecules impair HLA class II activation of CD4+ T cells (100.20)

Abstract Burkitt Lymphoma (BL) is a very fast growing form of non-Hodgkin's lymphoma. The common characteristic of all BL is the translocation and constitutive activation of the c-myc oncogene. Our present study suggests that BL and Epstein-Barr Virus-transformed B-lymphoblastoid cell lines (B-LCL) constitutively express HLA class II proteins and bind peptide ligands. However, unlike B-LCL, BL cells were not capable of activating CD4+ T cells. While BL and B-LCL possessed similar levels of HLA class II, invariant chain (Ii) and HLA-DM, BL cells expressed lower levels of co-stimulatory molecules (CD80/CD86). Biochemical and functional assays showed that addition of external co-stimulatory signals to BL cells failed to functionally deliver HLA class II antigens to CD4+ T cells. Although both BL and B-LCL were capable of binding peptides at neutral and acidic pH, BL cells were only able to activate T cells at low pH (5.5), suggesting that BL-associated inhibitory molecules were eluted at the acidic pH. BL conditioned medium, as well as acid eluent from BL cells and primary B-cell tumors, inhibited functional class II presentation by both B-LCL and mature dendritic cells, while those eluent from B-LCL and primary B-cells did not. No characteristic inhibitory cytokines were identified in the acid eluent from BL or primary B-cell tumors. Taken together, these results suggest that BL-associated molecules may inhibit HLA class II activation of CD4+ T cells.

Epstein-Barr Virus-Encoded Bcl-2 Homologue Functions as a Survival Factor in Wp-Restricted Burkitt Lymphoma Cell Line P3HR-1

Burkitt lymphoma (BL) is etiologically associated with Epstein-Barr virus (EBV). EBV-positive BL tumors display two latent forms of infection. One is referred to as latency I infection, in which EBV expresses the virus genome maintenance protein EBNA1 as the only viral protein. The other is referred to as Wp-restricted latency and was recently identified in a subset of BL tumors. In these tumors, EBV expresses EBNA1, EBNA3A, EBNA3B, EBNA3C, a truncated form of EBNA-LP, and the viral Bcl-2 homologue BHRF1, all of which are driven by the BamHI W promoter (Wp). To investigate the role of EBV in Wp-restricted BL, we conditionally expressed a dominant-negative EBNA1 (dnEBNA1) mutant which interrupts the virus genome maintenance function of EBNA1 in the P3HR-1 BL cell line. Induction of dnEBNA1 expression caused loss of the EBV genome and resulted in apoptosis of P3HR-1 cells in the absence of exogenous apoptosis inducers, indicating that P3HR-1 cells cannot survive without EBV. Stable transfection of the BHRF1 gene into P3HR-1 cells rescued the cells from the apoptosis induced by dnEBNA1 expression, whereas stable transfection of truncated EBNA-LP, EBNA3A, or EBNA3C did not. Moreover, knockdown of BHRF1 expression in P3HR-1 cells resulted in increased cell death. These results indicate that EBV is essential for the survival of P3HR-1 cells and that BHRF1 functions as a survival factor. Our finding implies a critical contribution of BHRF1 to the pathogenesis of Wp-restricted BLs.

Caspase-8-mediated cleavage of Bid and protein phosphatase 2A-mediated activation of Bax are necessary for Verotoxin-1-induced apoptosis in Burkitt's lymphoma cells

Verotoxin (VT-1) is a cytotoxin, produced by Shigella dysenteriae type 1 or by Shiga toxin-producing Escherichia coli, which binds specifically to globotriaosylceramide (Gb3). This glycosphingolipid is a B cell differentiation antigen (Gb3/CD77) strongly expressed on Burkitt's lymphoma cells. We have previously shown that, in these cells, VT-1 induces apoptosis via a caspase- and mitochondria-dependent pathway. In this report, we provide new insights into this signal transduction pathway. First, we demonstrate that VT-1-induced apoptosis requires degradation of the caspase-8 inhibitory molecule c-FLIPL and that this degradation occurs through the ubiquitin–proteasome pathway. Furthermore, we show that mitochondrial activation is mainly due to i) cleavage and activation of the pro-apoptotic Bcl-2 family member Bid by caspase-8 and ii) Bax relocalization to mitochondrial membranes which lead to cytochrome c release. However, tBid is not involved in Bax relocalization, and relocalization is most likely controlled by the extent of Bax phosphorylation: in non-treated BL cells, p38 MAPK participates in the retention of Bax in the cytoplasm in an inactive form whereas in VT-1 treated cells, protein phosphatase 2A is activated and induces Bax relocalization to mitochondria.

Activation of the JAK/STAT Pathway in Epstein Barr Virus+-Associated Posttransplant Lymphoproliferative Disease: Role of Interferon-γ

Epstein Barr virus (EBV) is associated with B-cell lymphomas in posttransplant lymphoproliferative disease (PTLD). Latent membrane protein 1 (LMP1), the major oncogenic protein of EBV, promotes tumorigenesis through activation of NF-kappaB, Erk, p38, JNK and Akt. The Jak/STAT signal transduction pathway is also constitutively active in PTLD-associated EBV(+) B-cell lymphomas. Here we determine the mechanism of Jak/STAT activation in EBV(+) B-cell lymphomas and the role of LMP1 in this process. Immunoprecipitation studies revealed no direct interaction of LMP1 and JAK3, but known associations between JAK3 and common gamma chain, and between LMP1 and TRAF3, were readily detected in EBV(+) B cell lines from patients with PTLD. An inducible LMP1 molecule expressed in EBV(-) BL41 Burkitt's cells demonstrated STAT activation only after prolonged LMP1 signaling. While LMP1 induced IFN-gamma production in BL41 cells, IFN-gamma receptor blockade and IFN-gamma neutralization prior to LMP1 activation markedly decreased STAT1 activation and expression of LMP1-driven IFN-gamma inducible genes. Understanding the mechanisms by which EBV induces cellular signal transduction pathways may facilitate development of new treatments for PTLD.

Carboxy-terminal domain of AID required for its mRNA complex formation in vivo

Activation-induced cytidine deaminase (AID) is essential for the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. Originally, AID was postulated to be an RNA-editing enzyme, because of its structural homology with a known RNA-editing enzyme, APOBEC1. In support of this idea, AID shares many of the properties of RNA-editing enzymes, including nucleocytoplasmic shuttling and a dependency on de novo protein synthesis. However, it has not been shown whether AID recognizes a specific mRNA and edits it to generate an enzyme involved in CSR or SHM. Here, we examined the association between AID and polyadenylated [poly(A)(+)] RNA in vivo, using UV cross-linking coupled with a poly(A) capture method that relies on biotinylated oligo(dT) and streptavidin-conjugated beads. We found that both exogenous AID expressed in transfected CH12 cells and endogenous AID expressed in BL2 cells were associated with poly(A)(+) RNA. Similar protein-poly(A)(+) RNA complexes were formed by APOBEC1 and APOBEC3G. However, the interactions of all of these cytidine deaminase family members, including AID, with poly(A)(+) RNA were indirect. This was expected for APOBEC1, which is known to act through an RNA-interacting cofactor, APOBEC1 complementation factor (ACF). In addition, the carboxy-terminal region of AID, which is essential for class switching, was also required for its interaction with poly(A)(+) RNA. These results suggest that the CSR activity of AID requires an ACF-like cofactor that specifically interacts with the carboxy-terminal domain of AID.

Analysis of Aurora B Kinase in Non-Hodgkin's Lymphoma.

Aurora B kinase is one of the key regulators of mitosis and has been reported to be over-expressed in a various malignancies including acute myelogenous leukemia; however, expression of Aurora B in non-Hodgkin's lymphoma (NHL) and its role in lymphoma genesis remain to be fully elucidated. This study explored the levels of Aurora B in seventy four lymph nodes and tumor specimens excised operatively from individuals with various types of NHLs including diffuse large B cell lymphoma (n=22, DLBCL), follicular lymphoma (n=12, FL), Burkitt's lymphoma (n=10, BL), mantle cell lymphoma (n=4, MCL), mucosa associated lymphoid tissue (n=4, MALT), adult T-cell leukemia/lymphoma (n=11, ATL), peripheral T-cell lymphoma (n=8, PTCL), and Hodgkin's lymphoma (n=3, HL). Immunohistochemical examination found that DLBCL (11/22, 50 %) and BL (8/10, 80 %) cells highly (percentage of positive cells, >20 %) expressed Aurora B in their nucleus. On the other hand, FL (11/12, 92 %), MCL (1/4, 25 %), and MALT (1/4, 25 %) cells slightly (percentage of positive cells, 5–20 %) expressed Aurora B and none of them, except for one case of FL, highly expressed this protein kinase, suggesting that expression of Aurora B correlated with histological grade in B cell NHLs (p=0.0004). We next examined whether Aurora B could be a molecular target for treatment of NHLs utilizing BALM-14, -18, and -27, Daudi, Ramos, and Raji Burkitt's lymphoma/leukemia cells. Exposure of these cells to AZD1152, a selective inhibitor of Aurora B kinase, potently induced apoptosis in association with activation of caspase 3, as measured by annexin V staining and Western blot analysis, respectively. Moreover, we found that AZD1152 synergistically enhanced the effects of vincristine (VCR), a tubulin depolymerizing agent, to induce growth arrest and apoptosis of these cells. Further experiments found that VCR increased levels of the phosphorylated forms of Aurora B (p-Aurora B) via activation of c-Jun N-terminal kinase (JNK), which was blocked in the presence of AZD1152 in BALM-27 cells. Taken together, Aurora B kinase is a promising molecular target for treatment of individuals with aggressive types of NHLs. The combined administration of AZD1152 and the conventional chemotherapeutic agents to patients with NHL warrants further investigation.

An Fc-Engineered Humanized Anti-CD40 Monoclonal Antibody, XmAb5485, Exhibits Potent Activity in Vitro and in Vivo against Non-Hodgkin Lymphoma, Chronic Lymphocytic Leukemia and Multiple Myeloma

CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor family, is an attractive target for cancers of lymphoid origin since it is expressed on most mature B-cell malignancies, some early B-cell acute lymphocytic leukemias, and multiple myeloma. Finding efficient therapies for multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and rituximab-refractory Non-Hodgkin Lymphoma (NHL) represents an unmet need. Several anti-CD40 antibodies, both agonistic and antagonistic, have demonstrated objective responses in early clinical NHL trials and thus validated this antigen as a target for lymphoproliferative diseases. Here we present the characterization of a novel Fc-engineered and humanized anti-CD40 antibody, XmAb®5485, that was generated using our XmAb antibody engineering technology. This antibody is highly cytotoxic against lymphoma, leukemia and multiple myeloma cell lines as well as primary cancer cells. XmAb5485 is characterized by: i) increased affinity for Fc gamma receptors (FcgR), ii) improved effector function, and iii) significantly increased antitumor potency. We investigated several direct and indirect (Fc-mediated) mechanisms of antibody-mediated cytotoxicity in vitro. The potency (EC50) of XmAb5485 in antibody-dependent cell-mediated cytotoxicity (ADCC) increased up to 150-fold relative to the native non Fc-engineered version (anti-CD40 IgG1) of the antibody in a screen of Burkitt's lymphoma [BL], CLL and MM-derived cell lines. In the same cell lines, ADCC potency and maximal efficacy (% lysis) of XmAb5485 were also superior to that of rituximab: 74- and 1.3-fold higher in CLL, 12.5- and 1.4-fold higher in BL, and 190- and 1.9-fold higher in MM. In a MM cell line with low density of CD40 expression (~3500 per cell) XmAb5485 facilitated efficient ADCC whereas anti-CD40 IgG1 was virtually ineffective. Furthermore, using a BL cell line (Ramos) XmAb5485 displayed antibody-dependent cellular phagocytosis (ADCP) with potency and efficacy increased relative to rituximab (15- and 1.6-fold) and anti-CD40 IgG1 (5- and 1.2-fold). XmAb5485 also exhibited anti-proliferative apoptotic activity that was similar to that of rituximab. Ex vivo, XmAb5485 mediated potent ADCC of multiple primary patient-derived CLL, MCL, and plasma cell leukemia (PCL, an aggressive form of MM) cells, with substantially increased potency and efficacy relative to rituximab; in contrast, anti-CD40 IgG1 displayed minimal or no activity in these primary tumor cells. In vivo, in an established large (210–350 mm3) sc Ramos tumor xenograft model, 6 mg/kg XmAb5485 cured 80% of mice of detectable tumors and displayed statistically significant superiority over anti-CD40 IgG1. In contrast, only 7% of animals in the rituximab cohort were cured. In summary, our data suggest that XmAb5485, an anti-CD40 Fc variant antibody engineered for increased effector function, is a promising next-generation immunotherapeutic for leukemias, lymphomas, and multiple myeloma.

Role of DNA polymerases η, ι and ζ in UV resistance and UV-induced mutagenesis in a human cell line

Genes coding for DNA polymerases η, ι and ζ, or for both Pol η and Pol ι have been inactivated by homologous recombination in the Burkitt's lymphoma BL2 cell line, thus providing for the first time the total suppression of these enzymes in a human context. The UV sensitivities and UV-induced mutagenesis on an irradiated shuttle vector have been analyzed for these deficient cell lines. The double Pol η/ι deficient cell line was more UV sensitive than the Pol η-deficient cell line and mutation hotspots specific to the Pol η-deficient context appeared to require the presence of Pol ι, thus strengthening the view that Pol ι is involved in UV damage translesion synthesis and UV-induced mutagenesis. A role for Pol ζ in a damage repair process at late replicative stages is reported, which may explain the drastic UV-sensitivity phenotype observed when this polymerase is absent. A specific mutation pattern was observed for the UV-irradiated shuttle vector transfected in Pol ζ-deficient cell lines, which, in contrast to mutagenesis at the HPRT locus previously reported, strikingly resembled mutations observed in UV-induced skin cancers in humans. Finally, a Pol η PIP-box mutant (without its PCNA binding domain) could completely restore the UV resistance in a Pol η deficient cell line, in the absence of UV-induced foci, suggesting, as observed for Pol ι in a Pol η-deficient background, that TLS may occur without the accumulation of microscopically visible repair factories.

TGFβ-mediated apoptosis of Burkitt's lymphoma BL41 cells is associated with the relocation of mitochondrial BimEL

In this study, we showed that the transforming growth factor beta (TGFbeta)-mediated apoptosis of Burkitt's lymphoma BL41 cells is dependent on the BH3-only protein Bim. In contrast to what has been observed with other cell types, TGFbeta activation did not promote Bim upregulation in BL41 cells, but instead resulted in Bim release from the mitochondria. Indeed, Bim levels were high in healthy BL41 cells, in which they dimerized with the Bcl-2-like protein Mcl-1 at the mitochondrial surface. In healthy and TGFbeta-activated BL41 cells, unlike in epithelial cells or hepatocytes, Bim did not associate with Bcl-2 or Bcl-xL. TGFbeta activation of BL41 cells triggered the p38-dependent activation of caspase-8, causing the cleavage of Mcl-1 and the transfer of Bim from the mitochondria to the cytoskeleton. In addition to mitochondrial activation, this relocation of Bim may facilitate the complete demise of a cell death that is beyond the commitment point to apoptosis and may represent a hallmark of the TGFbeta-mediated apoptosis of human lymphoma B cells.

The MAPK Signaling Cascade is a Central Hub in the Regulation of Cell Cycle, Apoptosis and Cytoskeleton Remodeling by Tripeptidyl-Peptidase II

Tripeptidyl-peptidase II (TPPII) is a serine peptidase highly expressed in malignant Burkitt's lymphoma cells (BL). We have previously shown that overexpression of TPPII correlates with chromosomal instability, centrosomal and mitotic spindle abnormalities and resistance to apoptosis induced by spindle poisons. Furthermore, TPPII knockdown by RNAi was associated with endoreplication and the accumulation of polynucleated cells that failed to complete cell division, indicating a role of TPPII in the cell cycle. Here we have applied a global approach of gene expression analysis to gain insights on the mechanism by which TPPII regulates this phenotype. mRNA profiling of control and TPPII knockdown BL cells identified one hundred and eighty five differentially expressed genes. Functional categorization of these genes highlighted major physiological functions such as apoptosis, cell cycle progression, cytoskeleton remodeling, proteolysis, and signal transduction. Pathways and protein interactome analysis revealed a significant enrichment in components of MAP kinases signaling. These findings suggest that TPPII influences a wide network of signaling pathways that are regulated by MAPKs and exerts thereby a pleiotropic effect on biological processes associated with cell survival, proliferation and genomic instability.