There is an increasing body of evidence linking chronic inflammation and cancer but the mechanistic connection is poorly understood and the mediators of inflammation immensely complex. Most cancers are accompanied by leukocyte infiltration (and nasopharyngeal carcinoma is no exception), which, contrary to the anticipated immune role, could be contributing to tumour development and progression. In order to explore key carcinogenic factors in EBV associated malignancy we have studied a transgenic mouse model of epithelial carcinogenesis in which the Epstein-Barr virus oncogene LMP1 (CAO variant) is expressed. The preneoplastic skin, demonstrating hyperplasia and erosive dermatitis, was found to be inflamed with a mixed infiltrate involving T-cells (including NKT cells), mast cells and neutrophils. Several inflammatory factors were upregulated in the affected tissue, notably CD30 and CD30L, also MIP2, MIP3, CRG2/IP-10 and IL-1β and particularly the B-cell attractant CXCL13 (BLC). Immunoglobulin deposition was found to occur as the pathology worsens indicating a B-cell involvement. The role of mature T- and/or B-lymphocytes in the advancing pathology was demonstrated by their elimination, which limits the pathology to an early, benign stage. We have found that a chitinase-related protein, lacking enzymatic activity (termed YM2 or chitinase3-like4) is massively upregulated in the LMP1 expressing epidermis. We will present evidence to suggest that YM2 may be acting in concert with immunoglobulin to promote inflammation. The repeated identification of over-expressed chitinase-like proteins in models of inflammatory disorders suggests a crucial role for this protein family in inflammation. Factors which promote or sustain chronic inflammation may prove to be effective chemotherapeutic targets and their identification is therefore an important prerequisite. In this model LMP1 is the inducing factor and its effect upon and purturbation of immune system cells (of both innate and adaptive systems) leading to an inflamed state may augment its oncogenic activity in the development of EBV associated carcinoma.

Background: There is growing evidence that EBNA-1 can influence B-cell survival, but whether this is sufficient to predispose transgenic mice to lymphomagenesis is controversial. We have previously shown that two out of 12 established transgenic mouse lines generated using an EµEBNA-1 transgene expressed EBNA-1 (Wilson et al., 1996, EMBO J., 15, p3117). First, this demonstrated that in vivo expression of full length EBNA-1 from a relatively simple transgene is not efficient. Second, mice of the two expressing lines succumb to B-cell lymphoma with identical pathology, but with dramatically different penetrance and latency to onset, essentially a fast tumour line (line 26) and a slow tumour line (line 59). A further development from the “fast line” was that a spontaneous partial transgene deletion arose, giving rise to a sub-line of mice (designated 26A), which no longer showed EBNA-1 expression or developed lymphoma. Methods: In order to explore the contribution of EBNA-1 to the phenotype and to examine if there is any influence from cellular sequences at the sites of transgene insertion, we have taken three approaches: [1] To explore the phenotype of lymphocytes from both transgenic lines, prior to tumour development, where any characteristic in common between the two lines must result from EBNA-1 expression; [2] To use dominant negative EBNA-1 expression to examine the effect of EBNA-1 “withdrawal”; [3] To identify and characterise the transgene insertion sites. Results: [1] Lymphocytes explanted and cultured from mice of both transgenic lines initially show enhanced proliferation and then prolonged survival compared to non-transgenic wild-type sibling controls. This property is only evident when the cells are cultured in the presence of interleukin-2 (IL-2). [2] Transfection and expression of dominant negative forms of EBNA-1 in a cell line derived from an EBNA-1 expressing line 59 tumour (co-expressing LMP1) is not compatible with the survival of these cells, while expression in cell lines derived from LMP1-only tumours is innocuous. [3] The transgene insertion site for line 59 has been precisely mapped to murine chromosome 4 band D3. No known oncogenes lie withing a Mb region encompassing the transgene. The transgene insertion site for sub-line 26A has been precisely mapped to murine chromosome 3 band H2. No known oncogenes lie within a Mb region encompassing the transgene. The transgene insertion site for line 26 has been mapped to murine chromosome 5 band B and fine mapping is ongoing. Conclusion: EBNA-1 promotes lymphocyte survival in the transgenic system.