Joanna B. Wilson

Epstein-Barr Virus LMP2A Drives B Cell Development and Survival in the Absence of Normal B Cell Receptor Signals

Epstein-Barr virus (EBV) establishes a persistent latent infection in peripheral B lymphocytes in humans and is associated with a variety of malignancies and proliferative disorders. Latent membrane protein 2A (LMP2A) is one of only two viral proteins expressed in latently infected B lymphocytes in vivo. LMP2A blocks B cell receptor (BCR) signal transduction in vitro by binding the Syk and Lyn protein tyrosine kinases. To analyze the significance of LMP2A expression in vivo, transgenic mice with B cell lineage expression of LMP2A were generated. LMP2A expression results in the bypass of normal B lymphocyte developmental checkpoints allowing immunoglobulin-negative cells to colonize peripheral lymphoid organs, indicating that LMP2A possesses a constitutive signaling activity in nontransformed cells.

Expression of Epstein-Barr virus nuclear antigen-1 induces B cell neoplasia in transgenic mice.

The Epstein‐Barr virus (EBV) nuclear antigen‐1 (EBNA‐1) is a pleiotropic protein which has been characterized extensively both biochemically and functionally. It is the only one of the identified latent protein‐encoding genes to be consistently expressed in viral‐associated endemic Burkitts lymphoma cells. As such, it is the only candidate viral protein to possibly perform a maintenance function in the tumour pathology. Despite this, no oncogenic activity has been attributed to the protein in tissue culture assays. The experiments described here were initiated to explore the activity of the protein in B cells in vivo. EBNA‐1 transgenic mice were generated with transgene expression directed to the B cell compartment using the mouse Ig heavy chain intron enhancer. Transgene expression was demonstrated in the lymphoid tissues of mice of two independent lines. Transgenic positive mice of both lines succumb to B cell lymphoma. The B cell tumours are monoclonal, frequently of follicular centre cell origin and remarkably similar to those induced by transgenic c‐myc expression. These results demonstrate that EBNA‐1 is oncogenic in vivo and suggest that the gene product may play a direct role in the pathogenesis of Burkitts lymphoma and possibly other EBV‐associated malignancies.

Expression of the BNLF-1 oncogene of Epstein-Barr virus in the skin of transgenic mice induces hyperplasia and aberrant expression of keratin 6

The BNLF-1 gene of Epstein-Barr virus (EBV) encodes the latent membrane protein (LMP), one of the putative oncogene products of the virus. This gene has been expressed from two different enhancer-promoter constructs in transgenic mice, to determine its biological activity and possible contribution to oncogenesis. While transgenic mice expressing LMP in many tissues demonstrated poor viability, expression of LMP specifically in the epidermis induces a phenotype of hyperplastic dermatosis. Concomitant with the expression of LMP in this tissue (and in the esophagus) is an induction of the expression of a hyperproliferative keratin, K6, at aberrant locations within the epidermis. The epithelial hyperplastic phenotype caused by the LMP-encoding transgenes implies that the LMP plays a role in the acanthotic condition of the tongue epithelium in the human EBV- and HIV-associated syndrome oral hairy leukoplakia, as well as possibly predisposing the nasopharyngeal epithelium to carcinogenesis.

The targeting of the cyclin D1 oncogene by an Epstein-Barr virus promoter in transgenic mice causes dysplasia in the tongue, esophagus and forestomach.

Cyclin D1 in cooperation with its major catalytic partners, cyclin-dependent kinases cdk4 and cdk6, facilitates progression through the G1 phase of the eukaryotic cell cycle, in part through phosphorylation of the retinoblastoma protein. Cyclin D1s oncogenic properties have been suggested by its cooperation with ras or adenovirus E1a to transform cultured cells, as well its overexpression in transgenic mice that leads to breast cancer. Activated by a number of different mechanisms in human cancers, the cyclin D1 gene is frequently amplified in squamous epithelial cancers derived from the head/neck and esophageal regions. In order to study the functional consequences of cyclin D1 overexpression in these squamous epithelial specific sites, we have linked the Epstein-Barr virus ED-L2 promoter to the human cyclin D1 cDNA and utilized this transgene to generate founder lines. This transgene is transcribed specifically in the tongue, esophagus and forestomach, all sharing a stratified squamous epithelium. The transgene protein product localizes to the basal and suprabasal compartments of these squamous epithelial tissues, and mice from different lines develop dysplasia, a prominent precursor to carcinoma, by 16 months of age in contrast to age-matched wild-type mice. This transgenic model is useful in demonstrating cyclin D1 may be a tumor initiating event in aero-upper digestive squamous epithelial tissues.

Lactate threshold responses to a season of professional British youth soccer

Objective: To examine the changes in aerobic endurance performance of professional youth soccer players throughout the soccer season. Methods: Nine youth soccer players were tested at six different time points throughout the soccer season by sub-maximal blood lactate assessment, using an incremental treadmill protocol. Whole blood lactate concentration and heart frequency (Hf) were determined at each exercise stage. Running velocities at the first lactate inflection point (v-Tlac) and at a blood lactate concentration of 4 mmol l−1 (v-4mM) were determined. Results: Running velocity at the two lactate thresholds increased from the start of pre-season training to the early weeks of the competitive season, from 11.67 (0.29) to 12.96 (0.28) km h−1 for v-Tlac, and from 13.62 (0.25) to 14.67 (0.24) km h−1 for v-4mM (p<0.001). However, v-Tlac and v-4mM when expressed relative to maximum heart frequency (Hfmax) remained unchanged. The Hf to blood lactate concentration relationship was unchanged after the pre-season training period. The two expressions of lactate threshold did not reveal differences between each other. Conclusion: Running velocity at v-Tlac and v-4mM increased significantly over the pre-season period, but v-Tlac and v-4mM were unchanged when expressed relative to Hfmax. This finding may indicate that increased endurance performance may be mainly attributable to alterations in Vo2max. Although lactate assessment of soccer players is useful for determining endurance training adaptations in soccer players, additional assessment of the other two determinants of endurance performance (Vo2max and running economy) may provide more useful information for determining physiological adaptations resulting from soccer training and training interventions.

bcl-xL and RAG genes are induced and the response to IL-2 enhanced in EmuEBNA-1 transgenic mouse lymphocytes.

We have described transgenic mice expressing Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) in B-cells which show a predisposition to lymphoma. To investigate the underlying oncogenic mechanisms, we have cross bred transgenic strains of mice, examined the pre-tumour B-cell phenotype and investigated the expression levels of selected cellular genes as a response to EBNA-1 expression. We have found that bcl-xL and the recombination activating genes (RAG) 1 and 2 are induced in pre-neoplastic samples of EBNA-1 expressing mice. Induction of bcl-xL may explain the observed redundancy in lymphomagenesis between transgenic EBNA-1 and bcl-2. In addition, bone marrow cells derived from the EμEBNA-1 mice show a greater capacity for cultured growth compared to controls, particularly in the presence of IL-2. Notably, bcl-xL expression is responsive to IL-2. These data shed new light on the potential contribution of EBNA-1 to EBV associated tumorigenicity as well as to the viral life cycle and open a potential avenue for therapeutic intervention.

Epstein-Barr Virus Latent Membrane Protein 1 (CAO) Up-regulates VEGF and TGFα Concomitant with Hyperlasia, with Subsequent Up-regulation of p16 and MMP9

EBV latent membrane protein 1 (LMP1) is an oncoprotein frequently expressed in nasopharyngeal carcinoma. We have generated transgenic mice expressing the nasopharyngeal carcinoma-derived CAO strain of LMP1 and LMP1 of the B95-8 strain, using the viral ED-L2 promoter for epithelial expression. LMP1(CAO) and LMP1(B95-8) induce transforming growth factor alpha expression and epidermal hyperplasia. However, levels of total epidermal growth factor receptor (EGFR) decline with the appearance of phosphorylated EGFR products, suggesting that the negative feedback loop upon EGFR expression is intact or that there is faster turnover at these early stages of carcinogenesis. In the L2LMP1(CAO) mice, increased levels of vascular endothelial growth factor are also seen at an early stage in the skin. As the phenotype worsens, with increasing hyperplasia and vascularization leading to keratoacanthoma, p16(INK4a) and matrix metalloproteinase 9 expression is induced. The lesions can progress spontaneously to carcinoma. Carcinoma cell lines developed from these mice show high levels of total and phosphorylated EGFR. These data show that the induction of signaling through EGFR by LMP1 is an early event in carcinogenesis and that any inhibition upon EGFR expression is lifted during progression. Furthermore, expression of LMP1 is not sufficient to inhibit induction of p16(INK4a) in response to abnormal proliferation. These data are consistent with the cooperative effects seen between LMP1 and loss of the INK4a locus in transgenic mice and with the frequency of loss of this locus in EBV-associated nasopharyngeal carcinoma.

The Oncogenic Potential of Epstein-Barr Virus Nuclear Antigen 1 in Transgenic Mice

The human herpes virus, Epstein-Barr virus (EBV) is highly prevalent among all communities. Although largely asymptomatic prior to puberty it is the etiologic agent of the lymphoproliferative disorder, infectious mononucleosis. The virus is also tightly associated with two major forms of human B-cell malignancy: endemic Burkitt’s lymphoma (BL) and the lymphomas to which immunosupressed individuals are prone (for review see Epstein and Achong, 1986).

Epstein‐Barr virus nuclear antigen‐1 and Myc cooperate in lymphomagenesis

The lymphomagenic action of myc genes in conjunction with Epstein‐Barr virus nuclear antigen‐1 (EBNA‐1) have been examined using transgenic mice in several separate tests. Synergy between Myc and EBNA‐1 in lymphomagenesis was revealed in a cross breed study where co‐expression of transgenic myc and EBNA‐1 led to a tumor latency period reduced significantly in some crosses. In the resulting bitransgenic tumors, expression of the Eμ‐myc genes was not affected by EBNA‐1 expression. MoMLV was utilized as a transposon tag to activate cellular oncogenes by infection of EμEBNA‐1 mice. Rearrangement at the c‐myc locus in B cell tumors from these mice again suggests a cooperative action between myc and EBNA‐1. Tumors arising in EμEBNA‐1 mice typically showed a trisomy of chromosome 15, upon which the c‐myc locus resides. Bitransgenic tumors (EBNA‐1/c‐myc) did not show trisomy 15. This raises the possibility that amplification of c‐myc is factorial in the selection of trisomy 15 in these tumors. These data indicate that myc and EBNA‐1 act cooperatively and are not redundant in lymphomagenesis. Expression of EBNA‐1 by EBV may provide a selection pressure in addition to translocation of the c‐myc locus in the genesis of endemic Burkitts lymphoma (BL).

Lymphocyte deficiency limits Epstein-Barr virus latent membrane protein 1 induced chronic inflammation and carcinogenic pathology in vivo

BackgroundThe importance of the malignant cell environment to its growth and survival is becoming increasingly apparent, with dynamic cross talk between the neoplastic cell, the leukocyte infiltrate and the stroma. Most cancers are accompanied by leukocyte infiltration which, contrary to an anticipated immuno-protective role, could be contributing to tumour development and cancer progression. Epstein-Barr virus (EBV) associated cancers, including nasopharyngeal carcinoma and Hodgkins Disease, show a considerable leukocyte infiltration which surrounds the neoplastic cells, raising the questions as to what role these cells play in either restricting or supporting the tumour and what draws the cells into the tumour. In order to begin to address this we have studied a transgenic model of multistage carcinogenesis with epithelial expression of the EBV primary oncoprotein, latent membrane protein 1 (LMP1). LMP1 is expressed particularly in the skin, which develops a hyperplastic pathology soon after birth.ResultsThe pathology advances with time leading to erosive dermatitis which is inflamed with a mixed infiltrate involving activated CD8+ T-cells, CD4+ T-cells including CD4+/CD25+/FoxP3+ Treg cells, mast cells and neutrophils. Also significant dermal deposition of immunoglobulin-G (IgG) is observed as the pathology advances. Along with NF-kappaB activation, STAT3, a central factor in inflammation regulation, is activated in the transgenic tissue. Several inflammatory factors are subsequently upregulated, notably CD30 and its ligand CD153, also leukocyte trafficking factors including CXCL10, CXCL13, L-selectin and TGFβ1, and inflammatory cytokines including IL-1β, IL-3 and the murine IL-8 analogues CXCL1, CXCL2 and CXCL5-6, amongst others. The crucial role of mature T- and/or B-lymphocytes in the advancing pathology is demonstrated by their elimination, which precludes mast cell infiltration and limits the pathology to an early, benign stage.ConclusionsLMP1 can lead to the activation of several key factors mediating proliferation, angiogenesis and inflammation in vivo. With the initiation of an inflammatory programme, leukocyte recruitment follows which then itself contributes to the progressing pathology in these transgenic mice, with a pivotal role for B-and/or T-cells in the process. The model suggests a basis for the leukocyte infiltrate observed in EBV-associated cancer and its supporting role, as well as potential points for therapeutic intervention.