This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Project 2-A: Analysis of Polyamine Metabolism in EBV Lymphomagenesis In the Burkitts lymphoma (BL) cell line Akata, maintenance of the malignant phenotype in vitro is dependent on retention of the EBV genome. Global gene expression analysis of paired EBV-positive and -negative Akata cell clones identified spermidine/spermine N1-acetyltransferase (SSAT) as one of a limited number of genes whose expression was significantly decreased in EBV-positive clones. SSAT is a key enzyme in the catabolism of polyamines, which are essential for cell growth. Increased polyamine levels have been detected in a number of tumors. Thus, we sought to test the hypothesis that decreased SSAT mRNA in EBV-infected cells represents viral manipulation of the polyamine metabolic pathway and a factor in EBV lymphomagenesis. We have concluded the polyamine studies, with final progress reported below. Those studies have provided the foundation for a new direction, outlined in project 2-B.Project 2-B: EBV microRNA-Mediated Epigenetic Modification of Cellular ChromatinCarcinoma cell lines infected by EBV in vitro developed subclones that lost virus but stably retained a morphological phenotypic, induced at infection, which distinguished them from the EBV-negative parental cell line. When transiently infected clones were compared by gene expression arrays to their EBV-negative parental counterparts, some 80 downregulated genes (many of which are hypermethylated in cancer) were identified in cells that had lost virus. Treatment with methyltransferase inhibitors restored the parental cell morphologic phenotype (and, in cases tested, gene expression). These preliminary observations have led us to test the hypothesis that EBV induces stable epigenetic changes that may make virus redundant with respect to continued malignant progression. Retention of a virally-induced epigenetic imprint in transiently infected cells introduces a new paradigm for how a human tumor virus might mechanistically regulate a cell in a hit-and-run fashion. We will focus on determining which EBV gene products contribute to epigenetic modification of cellular chromatin. EBV latent membrane protein 1 (LMP1) has been reported to induce DNA methyltransferase 1, 3a, and 3b and hypermethylate the E-cadherin promoter. However, the discovery of at least 17 EBV microRNAs (miRNAs), together with the ability of cellular miRNAs to target DNA for gene silencing, prompted our hypothesis that the EBV miRNAs direct methylation of specific cellular genes in a sequence specific manner. Using bioinformatic approaches, we will query which EBV miRNAs might target these cellular promoters. Transfection of the individual miRNA will provide a direct demonstration that candidate miRNAs so identified do in fact produce epigenetic silencing in EBV infection.
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