Epstein-Barr virus (EBV) has been associated with several human malignancies including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and immunoblastic B-cell lymphoma. The Epstein-Barr virus nuclear antigen-1 (EBNA-1) is an EBV protein that plays a key role in the initiation of DNA replication and viral genome maintenance in latently-infected, EBV-positive cells. Recent studies have shown that EBNA-1 is required for the survival of EBV-positive Burkitt's lymphoma cells. EBNA-1 inhibits apoptosis (programmed cell death) in B cells, and may thereby contribute to the development of EBV-positive lymphomas. Latent Membrane Protein 1 (LMP1) and Latent Membrane Protein 2A (LMP2A) allow EBV to exploit the signal transduction pathways normally activated within B cells by antigen and T cells. In vivo, EBV infects naïve B cells, driving the activation and proliferation of these cells. This is done by substituting for an activated B-cell receptor and signal provided by T cells, transforming the cells from B cells to resting EBV-positive memory cells. Mammary acini are the glandular epithelial cells of breast tissue where fluid and milk are produced. Mature mammary acini have distinguishing histological features including a polarized structure surrounding a hollow lumen. Apoptosis is essential for the formation of this hollow lumen. The development and maintenance of the mammary acinar's structure is critical for the normal function of mammary tissue. MCF10A cells, epithelial cells derived from normal human breast tissue, form acinar-like structures containing a polarized epithelium surrounding a hollow lumen when placed in a three-dimensional culture system containing basement membrane proteins (Matrigel). Expression of various oncogenes in MCF10A cells has been previously shown to alter the formation of these acinar-like structures and recapitulate features of oncogenic transformation. The purpose of this study is to determine whether EBNA-1 and LMP2A, either by themselves or in combination, can alter the growth and morphogenesis of MCF10A cells grown in three-dimensional basement membrane cultures. Three cell lines will be prepared for this study from the MCF10A parental cell line: one cell line engineered to express LMP2A, one cell line expressing EBNA-1, and one cell line expressing both LMP2A and EBNA-1. All of the cell lines will be cultured in the three-dimensional Matrigel system. Immunoflorescence and DNA staining will be used to monitor polarization and luminal formation during growth in Matrigel. Loss of polarity and/or failure to create the luminal space will support a role for EBNA-1 and/or LMP2A in the development of EBV-associated carcinomas. This study was supported with funding from the Simons Foundation.