We have continued our studies to understand the molecular basis for the erythroleukemia induced in mice by the Friend spleen focus-forming virus (SFFV) and neurodegeneration induced in rats by PVC-211 murine leukemia virus (MuLV) and are now using the information gained to design experiments to understand the biological effects of the highly related human retrovirus xenotropic MuLV-related virus (XMRV). Friend spleen focus-forming virus (SFFV) causes a rapid, multi-stage erythroleukemia in mice and is a good model for understanding the molecular basis of the various stages of leukemia. We previously demonstrated that in the first stage of SFFV-induced leukemia, the viral envelope protein interacts with and activates the erythropoietin (Epo) receptor and the receptor tyrosine kinase sf-Stk in erythroid cells, causing their Epo-independent proliferation, differentiation and survival. In the second stage, SFFV integration into host DNA activates the transcription factor PU.1, leading to a block in erythroid cell differentiation and the outgrowth of transformed erythroleukemia (SFFV-MEL) cells. We recently showed that SFFV-MEL cells can metastasize to the meninges, causing central nervous system (CNS) failure. The leukemic cells proliferated to high numbers in the bone marrow (BM), where they caused destruction of the bones of the cranium and vertebrae, invaded the meninges of the brain and spinal cord, and caused meningeal leukemia. TRAP staining of bone sections showed evidence of osteoclastogenesis at putative invasion sites into the meninges. Gene expression profiling of SFFV-MEL cells relative to non-transformed, SFFV-infected spleen cells revealed up-regulation of genes involved in extracellular matrix processing as well as oncogenes, angiogenic factors and a BM adhesion integrin. Downregulated genes included those encoding anti-angiogenic proteins. Consistent with a gene expression profile that should favor angiogenesis, animals that developed meningeal leukemia showed evidence of pathological angiogenesis in the BM, and SFFV-MEL cells were shown to secrete high levels of vascular endothelial cell growth factor (VEGF). We further demonstrated that SFFV-MEL cells preferentially adhere to fibronectin via the fibronectin receptor integrin alpha 5- beta 1. This unique animal model for meningeal leukemia should facilitate studies of engraftment and proliferation of leukemic cells in the bone marrow and their invasion of the CNS as well a pre-clinical evaluation of experimental therapeutics for CNS-associated leukemias. As part of our effort to identify drugs that block the growth of SFFV-MEL cells, we have been collaborating with the CCR laboratory of Larry Keefer to test the nitric oxide (NO) prodrug JS-K, a promising anti-cancer agent. JS-K consists of a diazeniumdiolate group necessary for the release of NO as well as an arylating ring. To understand the mechanism of cytotoxicity of JS-K against SFFV-MEL cells and determine the roles of NO and arylation in this process, we compared the effects of JS-K with CDNB, which contains an arylating ring analogous to that of JS-K without the diazeniumdiolate group necessary to release NO. Our studies indicate that both JS-K and CDNB inhibit proliferation of SFFV-MEL cells with low IC-50 values and induce caspase-associated apoptosis as well as cell cycle arrest within 24 hrs. Both JS-K and CDNB blocked activation of the PI3-kinase/Akt pathway and MAP kinase pathways in these cells and this was associated with dephosphorylation and activation of the tumor suppressor FoxO3a and the subsequent upregulation of the cyclin-dependent kinase inhibitor p27-Kip1. Since we obtained the same results with JS-K and CDNB, our data suggests that the arylating capability of JS-K may be sufficient for inducing these biological effects. Studies are in progress to test the effects of JS-K in vivo against meningeal leukemia induced by SFFV-MEL cells. In addition to studying retrovirus-induced leukemia, we have also continued our studies on a retrovirus-induced animal model for neurodegeneration. PVC-211 MuLV causes a rapid neurodegenerative disease in rat and is a good model for understanding the molecular basis for similar neurodegenerative diseases in humans. We previously demonstrated that subtle changes in the envelope gene of the virus altered its host range from that of its leukemia-inducing parent, enabling it to gain access to the central nervous system by infecting brain capillary endothelial cells (BCEC). By comparing BCEC from uninfected and PVC-211 MuLV-infected rats, we found that virus-infected BCEC express high levels of inducible nitric oxide synthase and show evidence of nitric oxide (NO) production. We recently demonstrated that PVC-211 MuLV infection of BCEC results in the production of VEGF and the chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha), leading to vascular leakage and activation of microglia. Further studies demonstrated that depletion of microglia from rat brains blocks neurodegeneration induced by PVC-211 MuLV and that treatment with antiserum to MIP-1 alpha or splenectomy, both of which reduce the number of activated microglia in the brain, can delay disease, clearly demonstrating the importance of activated microglia in the development of PVC-211 MuLV-induced neurodegeneration. Studies are in progress to determine how viral infection of BCEC results in the activation of VEGF and to test pharmacological inhibitors of VEGF, MIP-1 alpha or microglia activation for their ability to block PVC-211 MuLV induced disease. Finally, we are applying our knowledge of rodent retrovirus-induced diseases to understand the biological effects of the highly related human retrovirus XMRV, which has been associated with prostate cancer and chronic fatigue syndrome. We showed by co-immunoprecipitation that the XMRV envelope (Env) protein can interact with Met-related receptor tyrosine kinases that are often overexpressed in human malignancies, including prostate cancer. We are currently examining activation of signal transduction pathways in human cells co-expressing XMRV Env and various tyrosine kinases. Because XMRV is a potential human pathogen, we analyzed the commonly used human cancer cell lines that represent the NCI-60 panel for expression of XMRV-related sequences and gene products. Only one cell line was positive: the lung cancer cell line EKVX, which was also shown to contain an infectious retrovirus. Further analysis of the retrovirus in this cell line indicated that it was more related to the mouse xenotropic murine leukemia virus than to XMRV. Finally, we had previously initiated studies to determine if there is a viral etiology to bronchioloalveolar adenocarcinoma (BAC) of the lung and have extended those studies to determine if XMRV is associated with this human disease. To date, XMRV sequences have been reproducibly detected from one tumor. Further studies with a larger sample set will help us to determine if there is an association of XMRV with BAC
