ABSTRACT
SCC12F cells are a line of keratinocytes that retain the capacity for terminal differentiation in vitro. We showed previously that the Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein 1 (LMP1) altered SCC12F morphology in vitro, downregulated cell-cell-adhesion molecule expression and promoted cell motility. In organotypic raft culture, LMP1-expressing cells failed to stratify and formed poorly organized structures which displayed impaired terminal differentiation. To understand better the mechanism(s) by which LMP1 induces these effects, we generated SCC12F cells in which LMP1 expression is inducible. Following induction, these cells exhibited phenotypic changes similar to those observed previously and allowed us to investigate the effects of LMP1 expression on cellular pathways associated with growth, differentiation and morphology. Using microarrays and a number of confirmatory techniques, we identified sets of differentially expressed genes that are characteristically expressed in inflammatory and hyperproliferative epidermis, including chemokines, cytokines and their receptors, growth factors involved in promoting epithelial cell motility and proliferation and signalling molecules that regulate actin filament reorganization and cell movement. Among the genes whose expression was differentially induced significantly by LMP1, the induction of IL-1beta and IL-1alpha was of particular interest, as many of the LMP1-regulated genes identified are established targets of these cytokines. Our findings suggest that alterations in the IL-1 signalling network may be responsible for many of the changes in host-cell gene expression induced in response to LMP1. Identification of these LMP1-regulated genes helps to define the mechanism(s) by which this oncoprotein influences cellular pathways that regulate terminal differentiation, cell motility and inflammation.
Subject(s)
Herpesvirus 4, Human/pathogenicity , Keratinocytes/physiology , Viral Matrix Proteins/adverse effects , Antibodies, Viral , Cell Differentiation , Cell Division/genetics , Cell Line , Cell Movement , Flow Cytometry , Gene Expression Regulation , Humans , Inflammation/genetics , Keratinocytes/pathology , Nucleic Acid Hybridization , RNA, Viral/genetics , RNA, Viral/isolation & purification , Wounds and Injuries/virologyABSTRACT
CD40, a tumour necrosis factor (TNF) receptor family member, is expressed in a variety of cell types, including B lymphocytes, macrophages, fibroblasts, endothelial and epithelial cells, and this widespread expression is likely to account for its central role in normal physiology and disease pathogenesis. In this study, we provide evidence to support a role for constitutive CD40 signalling in cell transformation. We show that the ligand for CD40 (CD40L/CD154) is expressed in CD40-positive human breast tumour biopsies, suggesting that the constitutive activation of the CD40 receptor in vivo may contribute to the oncogenic process. Coexpression of CD40 and CD40L confers oncogenic effects on immortalized human epithelial cells in vitro, increasing their proliferation, motility and invasion. Expression of LMP:CD40, a hybrid molecule comprising the N-terminus and transmembrane domains of the Epstein-Barr virus-encoded latent membrane protein-1 (LMP1) fused to the cytoplasmic tail of CD40, mimics a constitutively active CD40 receptor and promotes the transformation of immortalized rodent fibroblasts in vitro and their oncogenicity in vivo. The observed effects of aberrant CD40 activation on cell transformation are largely diminished upon suppression of the oncogenic NF-kappaB signalling pathway. Taken together, our results suggest a role for the constitutive engagement of the CD40L/CD40/NF-kappaB activation pathway in cell transformation and neoplastic growth. Strategies that neutralize this pathway may therefore be useful in cancer treatment and prevention.
