Bettina Kempkes

B-cell proliferation and induction of early G1-regulating proteins by Epstein-Barr virus mutants conditional for EBNA2.

Infection of primary B‐lymphocytes by Epstein‐Barr virus (EBV) leads to growth transformation of these B‐cells in vitro. EBV nuclear antigen 2 (EBNA2), one of the first genes expressed after EBV infection of B‐cells, is a transcriptional activator of viral and cellular genes and is essential for the transforming potential of the virus. We generated conditional EBV mutants by expressing EBNA2 as chimeric fusion protein with the hormone binding domain of the estrogen receptor on the genetic background of the virus. Growth transformation of primary normal B‐cells by mutant virus resulted in estrogen‐dependent lymphoblastoid cell lines expressing the chimeric EBNA2 protein. In the absence of estrogen about half of the cells enter a quiescent non‐proliferative state whereas the others die by apoptosis. EBNA2 is thus required not only for initiation but also for maintenance of transformation. Growth arrest occurred at G1 and G2 stages of the cell cycle, indicating that functional EBNA2 is required at different restriction points of the cell cycle. Growth arrest is reversible for G1/G0 cells as indicated by the sequential accumulation and modification of cell cycle regulating proteins. EBV induces the same cell cycle regulating proteins as polyclonal stimuli in primary B‐cells. These data suggest that EBV is using a common pathway for B‐cell activation bypassing the requirement for antigen, T‐cell signals and growth factors.

RBPJκ-Dependent Signaling Is Essential for Long-Term Maintenance of Neural Stem Cells in the Adult Hippocampus

The generation of new neurons from neural stem cells in the adult hippocampal dentate gyrus contributes to learning and mood regulation. To sustain hippocampal neurogenesis throughout life, maintenance of the neural stem cell pool has to be tightly controlled. We found that the Notch/RBPJκ-signaling pathway is highly active in neural stem cells of the adult mouse hippocampus. Conditional inactivation of RBPJκ in neural stem cells in vivo resulted in increased neuronal differentiation of neural stem cells in the adult hippocampus at an early time point and depletion of the Sox2-positive neural stem cell pool and suppression of hippocampal neurogenesis at a later time point. Moreover, RBPJκ-deficient neural stem cells displayed impaired self-renewal in vitro and loss of expression of the transcription factor Sox2. Interestingly, we found that Notch signaling increases Sox2 promoter activity and Sox2 expression in adult neural stem cells. In addition, activated Notch and RBPJκ were highly enriched on the Sox2 promoter in adult hippocampal neural stem cells, thus identifying Sox2 as a direct target of Notch/RBPJκ signaling. Finally, we found that overexpression of Sox2 can rescue the self-renewal defect in RBPJκ-deficient neural stem cells. These results identify RBPJκ-dependent pathways as essential regulators of adult neural stem cell maintenance and suggest that the actions of RBPJκ are, at least in part, mediated by control of Sox2 expression.

Macrophage Polarisation: an Immunohistochemical Approach for Identifying M1 and M2 Macrophages

Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity. M2 macrophages contribute to tissue repair and promote Th2 responses. CD68 and CD163 are used to identify macrophages in tissue sections. However, characterisation of polarised macrophages in situ has remained difficult. Macrophage polarisation is regulated by transcription factors, pSTAT1 and RBP-J for M1, and CMAF for M2. We reasoned that double-labelling immunohistochemistry for the detection of macrophage markers together with transcription factors may be suitable to characterise macrophage polarisation in situ. To test this hypothesis, we have studied conditions associated with Th1- and Th2-predominant immune responses: infectious mononucleosis and Crohn’s disease for Th1 and allergic nasal polyps, oxyuriasis, wound healing and foreign body granulomas for predominant Th2 response. In all situations, CD163+ cells usually outnumbered CD68+ cells. Moreover, CD163+ cells, usually considered as M2 macrophages, co-expressing pSTAT1 and RBP-J were found in all conditions examined. The numbers of putative M1 macrophages were higher in Th1- than in Th2-associated diseases, while more M2 macrophages were seen in Th2- than in Th1 related disorders. In most Th1-related diseases, the balance of M1 over M2 cells was shifted towards M1 cells, while the reverse was observed for Th2-related conditions. Hierarchical cluster analysis revealed two distinct clusters: cluster I included Th1 diseases together with cases with high numbers of CD163+pSTAT1+, CD68+pSTAT1+, CD163+RBP-J+ and CD68+RBP-J+ macrophages; cluster II comprised Th2 conditions together with cases displaying high numbers of CD163+CMAF+ and CD68+CMAF+ macrophages. These results suggest that the detection of pSTAT1, RBP-J, and CMAF in the context of CD68 or CD163 expression is a suitable tool for the characterisation of macrophage polarisation in situ. Furthermore, CD163 cannot be considered a reliable M2 marker when used on its own.

Cell cycle activation by c‐myc in a Burkitt lymphoma model cell line

The product of the proto‐oncogene c‐myc (myc) is a potent activator of cell proliferation. In Burkitt lymphoma (BL), a human B‐cell tumor, myc is consistently found to be transcriptionally activated by chromosomal translocation. The mechanisms by which myc promotes cell cycle progression in B‐cells is not known. As a model for myc activation in BL cells, we have established a human EBV‐EBNA1 positive B‐cell line, P493‐6, in which myc is expressed under the control of a tetracycline regulated promoter. If the expression of myc is switched off, P493‐6 cells arrest in G0/G1 in the presence of serum. Re‐expression of myc activates the cell cycle without inducing apoptosis. myc triggers the expression of cyclin D2, cyclin E and Cdk4, followed by the activation of cyclin E‐associated kinase and hyper‐phosphorylation of Rb. The transcription factor E2F‐1 is expressed in proliferating and arrested cells at constant levels. The Cdk inhibitors p16, p21, p27 and p57 are expressed at low or not detectable levels in proliferating cells and are not induced after repression of myc. Ectopic expression of p16 inhibits cell cycle progression. These data suggest that myc triggers proliferation of P493‐6 cells by promoting the expression of a set of cell cycle activators but not by inactivating cell cycle inhibitors. Int. J. Cancer 87:787–793, 2000.

Differential gene expression patterns of EBV infected EBNA-3A positive and negative human B lymphocytes.

The genome of Epstein-Barr virus (EBV) encodes 86 proteins, but only a limited set is expressed in EBV–growth transformed B cells, termed lymphoblastoid cell lines (LCLs). These cells proliferate via the concerted action of EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), some of which are rate limiting to establish a stable homeostasis of growth promoting and anti-apoptotic activities. We show here that EBV mutants, which lack the EBNA-3A gene, are impaired but can still initiate cell cycle entry and proliferation of primary human B cells in contrast to an EBNA-2 deficient mutant virus. Surprisingly, and in contrast to previous reports, these viral mutants are attenuated in growth transformation assays but give rise to permanently growing EBNA-3A negative B cell lines which exhibit reduced proliferation rates and elevated levels of apoptosis. Expression profiles of EBNA-3A deficient LCLs are characterized by 129 down-regulated and 167 up-regulated genes, which are significantly enriched for genes involved in apoptotic processes or cell cycle progression like the tumor suppressor gene p16/INK4A, or might contribute to essential steps of the viral life cycle in the infected host. In addition, EBNA-3A cellular target genes remarkably overlap with previously identified targets of EBNA-2. This study comprises the first genome wide expression profiles of EBNA-3A target genes generated within the complex network of viral proteins of the growth transformed B cell and permits a more detailed understanding of EBNA-3As function and contribution to viral pathogenesis.

Notch1IC Partially Replaces EBNA2 Function in B Cells Immortalized by Epstein-Barr Virus

ABSTRACT Immortalization of B cells by Epstein-Barr virus (EBV) depends on the virally encoded EBNA2 protein. Although not related by sequence, the cellular Notch protein and EBNA2 share several biochemical and functional properties, such as interaction with CBF1 and the ability to activate transcription of a number of cellular and viral genes. Whether these similarities are coincidental or exemplify EBNA2 mimicry of evolutionarily conserved cellular signaling pathways is unclear. We therefore investigated whether activated forms of Notch could substitute for EBNA2 in maintaining the immortalized phenotype of EBV-infected B cells. To address this question, we devised a transcomplementation system using EREB2.5 cells. EREB2.5 cells are immortalized by EBV expressing a conditional estrogen receptor EBNA2 fusion protein (EREBNA2), and cellular proliferation is dependent on the availability of estrogen. Withdrawal of estrogen results in inactivation of EREBNA2, leading to growth arrest and eventually to cell death. Transduction of EREB2.5 cells with a lentiviral vector expressing wild-type EBNA2 rescued EREB2.5 cells from the growth-inhibitory effects of estrogen deprivation, in contrast to transduction with the lentivirus vector alone. EREB2.5 cells were also rescued by enforced expression of human Notch1IC after estrogen starvation, but this effect was restricted to cells expressing high levels of the transcription factor. Compared to wild-type EBNA2-expressing EREB2.5 cells, the Notch-expressing cells expanded more slowly after estrogen starvation, and once established, they continued to display a lower proliferation rate. Analysis of viral and cellular gene expression from transduced EREB2.5 cells after estrogen withdrawal indicated that both wild-type EBNA2- and Notch1IC-positive cells expressed c-Myc at levels similar to those found in parental EREB2.5 cells. However, the latter cells expressed LMP-1 far less efficiently than cells transduced with the wild-type EBNA2 gene. Cells rescued by either wild-type EBNA2 or Notch1IC expressed surface CD21 and CD23 proteins, but not CD10, indicating that induction of relevant type III latency markers was maintained. The data imply that both Notch and EBNA2 activate an important subset of cellular genes associated with type III latency and B-cell growth, while EBNA2 more efficiently induces important viral genes, such as LMP-1. Thus, exploitation of conserved Notch-related signaling pathways may represent a key mechanism by which EBNA2 contributes to EBV-induced cell immortalization.

Epstein-Barr virus nuclear antigen 2 is a transcriptional suppressor of the immunoglobulin mu gene: implications for the expression of the translocated c-myc gene in Burkitt's lymphoma cells.

A conditional mutant of Epstein‐Barr virus nuclear antigen 2 (EBNA2) regulated by estrogen was employed to study the effect of EBNA2 on the cellular phenotype. Activation of EBNA2 in lymphoblastoid cell lines (LCLs) and in B cell lymphoma lines resulted in down‐regulation of cell surface IgM and Ig‐mu steady‐state RNA expression. In LCLs, activation of EBNA2 is required for maintaining proliferation, whereas in Burkitts lymphoma (BL) cell lines with t(8;14) translocations, activation of EBNA2 induces growth arrest. In these cells, Northern and nuclear run‐on analyses revealed rapid simultaneous repression of Ig‐mu and c‐myc transcription as early as 30 min after activation of EBNA2. Since c‐myc expression is under the control of the Ig heavy chain locus in BL cell lines with a t(8;14) translocation, we propose that Ig‐mu and c‐myc are down‐regulated by EBNA2 through a common mechanism.

Cellular Target Genes of Epstein-Barr Virus Nuclear Antigen 2

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines.

Neoplastic transformation by Notch is independent of transcriptional activation by RBP-J signalling.

Signalling through the transmembrane receptor Notch is triggered by ligand binding, which induces the proteolytic cleavage of the Notch protein. This cleavage generates an intracellular fragment of the Notch protein (Notch-IC), which translocates into the nucleus and modifies transcription of target genes through its association with the RBP-J protein. Thus, the isolated Notch-IC protein represents the constitutively activated receptor. We have performed a deletion analysis of Notch IC in order to identify the transferable transactivation domain of Notch-IC and the minimal domain of Notch-IC required for RBP-J dependent transactivational activation. Functionally, Notch-IC has been linked to cell fate decision in development and oncogenesis in vivo. In vitro, Notch-IC can cooperate in neoplastic transformation of baby rat kidney cells with the adenoviral E1A protein. We have defined the minimal domain of Notch-IC required for E1A cotransformation. This domain, consisting of the ankyrin repeats of Notch-IC only, can neither activate RBP-J dependent transcription nor does it carry a transactivation domain. Therefore, the ankyrin repeat domain of Notch-IC might trigger novel pathways relevant for transformation but unrelated to RBP-J signalling.

A Somatic Knockout of CBF1 in a Human B-Cell Line Reveals that Induction of CD21 and CCR7 by EBNA-2 Is Strictly CBF1 Dependent and that Downregulation of Immunoglobulin M Is Partially CBF1 Independent

ABSTRACT CBF1 is a cellular highly conserved DNA binding factor that is ubiquitously expressed in all tissues and acts as a repressor of cellular genes. In Epstein-Barr virus growth-transformed B-cell lines, CBF1 serves as a central DNA adaptor molecule for several viral proteins, including the viral transactivator Epstein-Barr virus nuclear antigen 2 (EBNA-2). EBNA-2 binds to CBF1 and thereby gains access to regulatory regions of target genes and activates transcription. We have inactivated the CBF1 gene by homologous recombination in the human B-cell line DG75 and characterized changes in cellular gene expression patterns upon loss of CBF1 and activation of EBNA-2. CBF1-negative DG75 cells were viable and proliferated at wild-type rates. Loss of CBF1 was not sufficient to release repression of the previously described EBNA-2 target genes CD21 or CCR7, whereas induction of both target genes by EBNA-2 required CBF1. In contrast, repression of immunoglobulin M by EBNA-2 was mainly CBF1 independent. CBF1-negative DG75 B cells thus provide an excellent tool to dissect CBF1-dependent and -independent functions exerted by the EBNA-2 protein in future studies.