Background: During malignant progression, tumor cells need to acquire novel characteristics that lead to uncontrolled growth and reduced immunogenicity. In the Bovine Leukemia Virus-induced ovine leukemia model, silencing of viral gene expression has been proposed as a mechanism leading to immune evasion. However, whether proviral expression in tumors is completely suppressed in vivo was not conclusively demonstrated. Therefore, we studied viral expression in two selected experimentallyinfected sheep, the virus or the disease of which had features that made it possible to distinguish tumor cells from their nontransformed counterparts.

Large-animal models for leukemia have the potential to aid in the understanding of networks that contribute to oncogenesis. Infection of cattle and sheep with bovine leukemia virus (BLV), a complex retrovirus related to human T-cell leukemia virus type 1 (HTLV-1), is associated with the development of B-cell leukemia. Whereas the natural disease in cattle is characterized by a low tumor incidence, experimental infection of sheep leads to overt leukemia in the majority of infected animals, providing a model for studying the pathogenesis associated with BLV and HTLV-1. Tax BLV , the major oncoprotein, initiates a cascade of events leading toward malignancy, although the basis of transformation is not fully understood. We have taken a cross-species ovine-to-human microarray approach to identify Tax BLV -responsive transcriptional changes in two sets of cultured ovine B cells following retroviral vector-mediated delivery of Tax BLV . Using cDNA-spotted microarrays comprising 10,336 human genes/expressed sequence tags, we identified a cohort of differentially expressed genes, including genes related to apoptosis, DNA transcription, and repair; proto-oncogenes; cell cycle regulators; transcription factors; small Rho GTPases/GTPase-binding proteins; and previously reported Tax HTLV-1 -responsive genes. Interestingly, genes known to be associated with human neoplasia, especially B-cell malignancies, were extensively represented. Others were novel or unexpected. The results suggest that Tax BLV deregulates a broad network of interrelated pathways rather than a single B-lineage-specific regulatory process. Although cross-species approaches do not permit a comprehensive analysis of gene expression patterns, they can provide initial clues for the functional roles of genes that participate in B-cell transformation and pinpoint molecular targets not identified using other methods in animal models.DNA microarray technology has facilitated the identification of a large number of genes involved in the complex deregulation of cell homeostasis taking place in cancer (25, 59). There exists, however, a significant limitation in the variety of organisms for which microarrays have been developed because of a lack of genomic sequence data. Although species-specific small-scale application-targeted arrays are useful for monitoring specific networks of genes (9, 66), they do not address the broad spectrum of genes involved in the complex deregulations taking place in cancer. Larger-scale microarrays for domestic species are currently under investigation, but these emerging tools suffer from a lack of annotated genes. A solution to this limitation is to use microarrays designed for one species to analyze RNA samples from closely related species. The assumption is that the conservation of gene sequences between species will be sufficient to generate a reasonable amount of good-quality data. While there have been relatively few previously published reports that described the use of microarrays for cross-species hybridization (8,24,26,31,47)...

Ovine leukemia/lymphoma resulting from bovine leukemia virus infection of sheep offers a large animal model for studying mechanisms underlying leukemogenesis. Silencing of viral information including Tax, the major contributor to the oncogenic potential of the virus, is critical if not mandatory for tumor progression. In this study, we have identified epigenetic mechanisms that govern the complete suppression of viral expression, using a lymphoma-derived B-cell clone carrying a silent provirus. Silencing was not relieved by injection of the malignant B cells into sheep. However, exogenous expression of Tax or treatment with either the DNA methyltransferase inhibitor 5azacytidine or the histone deacetylase (HDAC) inhibitor trichostatin A rescued viral expression, as demonstrated by in vivo infectivity trials. Comparing silent and reactivated provirus, we found mechanistic connections between chromatin conformation and tumor-associated transcriptional repression. Silencing is associated with DNA methylation and decreased accessibility of promoter sequences. HDAC1 and the transcriptional corepressor mSin3A are associated with the inactive but not the reactivated promoter. Silencing correlates with a repressed chromatin structure marked by histone H3 and H4 hypoacetylation, a loss of methylation at H3 lysine 4, and an increase of H3 lysine 9 methylation. These observations point to the critical role of epigenetic mechanisms in tumor-specific virus/oncogene silencing, a potential strategy to evade immune response and favor the propagation of the transformed cell.

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Bovine leukemia virus (BLV) is closely associated with the development of B-cell leukemia and lymphoma in cattle. BLV infection has also been studied extensively in an in vivo ovine model that provides a unique system for studying B-cell leukemogenesis. There is no evidence that BLV can directly infect ovine B cells in vitro, and there are no direct data regarding the oncogenic potential of the viral Tax transactivator in B cells. Therefore, we developed ovine B-cell culture systems to study the interaction between BLV and its natural target, the B cell. In this study, we used murine CD154 (

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