Friend spleen focus-forming virus (SFFV) causes a rapid, multi-stage leukemia and provides an important model for understanding the molecular changes that result in various phases of leukemia and for testing therapeutic strategies to block each stage. In the first stage, SFFV-infected erythroid cells proliferate and differentiate in the absence of the erythroid hormone erythropoietin (Epo), leading to erythroid hyperplasia and polycythemia. This is due to interaction of the SFFV envelope protein with a unique receptor tyrosine kinase, sf-Stk, which is preferentially expressed in erythroid cells, resulting in activation of the kinase and various components of the Epo signal transduction pathway. The second stage of disease is characterized by the outgrowth of a rare, malignant erythroid cell in which SFFV has activated the transcription factor PU.1 to block erythroid cell differentiation. We demonstrated that activation of PU.1 in erythroid cells results in high expression of the hematopoietic phosphatase SHP-1 and subsequently to a specific block in the phosphorylation of STAT1, a transcription factor important for erythroid cell differentiation. More recently we demonstrated that SFFV-transformed erythroleukemia cells can metastasize to the bone marrow, where they are retained and subsequently cause meningeal leukemia, a common neurological complication of human leukemia. Studies are in progress to understand why the erythroleukemia cells are retained in the bone marrow and to isolate a putative leukemic stem cell in the SFFV-induced erythroleukemia cell population from which meningeal leukemia may originate. Studies are also in progress to test specific therapeutic strategies to block multi-stage leukemia using our retrovirus-induced mouse model. In collaboration with Larry Keefer and members of his laboratory, we have shown that SFFV-induced erythroleukemia cells, but not normal hematopoietic cells, are killed by the nitric oxide prodrug JS-K, and studies are in progress to determine the target of NO in these cells and to test JS-K for its efficacy in treating the various stages of SFFV-induced erythroleukemia. <P>Several years ago we made the novel observation that sf-Stk, the tyrosine kinase activated by SFFV in erythroid cells, can also be activated by the virus when it is expressed in non-erythroid cells, resulting in transformation of fibroblasts in vitro and non-erythroid malignancies in mice. All of the signal transducing molecules activated by SFFV in erythroid cells were also activated in SFFV/sf-Stk-transformed fibroblasts except for STATs 1 and 5, whose activation appears to require the Epo receptor. We are currently utilizing the SFFV/sf-Stk-transformed fibroblasts to screen potential small molecule inhibitors for sf-Stk. Since our studies on sf-Stk indicate that its activation can lead to transformation of various cell types, we are now extending our studies to determine if the human counterpart of sf-Stk, sf-RON, is activated in any human malignancies. Our data indicate that sf-RON is expressed in certain types of human cancers, with ovarian and prostate cancers showing the highest percentages. We have developed a quantitative RT-PCR assay to detect sf-RON in RNA from ovarian cancer cell lines, with the goal of using this as a high throughput screen for sf-RON in human tumor tissues. Studies are in progress using shRNA to determine if blocking sf-RON in cancer cell lines in which it is expressed will alter their transformed phenotype. <P>PVC-211 murine leukemia virus (MuLV) is a variant of the erythroleukemia-inducing Friend MuLV that causes a rapid neurodegenerative disease in rodents and provides an important model for understanding the molecular changes that result in neurodegeneration and for testing therapeutic strategies to target these changes. We previously demonstrated that PVC-211 MuLV, due to subtle changes in its envelope protein, can efficiently infect brain capillary endothelial cells (BCEC), allowing it to be expressed at sufficient levels in the neonatal rodent brain to cause a rapid neurodegenerative disease. To clarify the mechanism by which PVC-211 MuLV expression in BCEC induces neurological disease, we examined virus-infected rats at various times during neurological disease progression for vascular and inflammatory changes. Our studies indicate that early in the course of disease, morphologically abnormal and leaky blood vessels can be observed in the regions of the brain where neurodegeneration later occurs. This is likely the result of increased production of vascular endothelial growth factor (VEGF), which we detect in the brain 1-2 weeks after virus infection and after in vitro infection of primary BCEC cultures with PVC-211 MuLV. Furthermore, we showed that the brain and serum of rats injected 2 weeks previously with PVC-211 MuLV express high levels of macrophage inflammatory protein-1α (MIP-1α), a chemokine involved in recruitment of microglia to the brain. This was followed 3 weeks after virus infection by a marked accumulation of microglia in the diseased areas of the brain. Late in the course of the disease, when animals are clearly paralyzed, brain tissues show elevated levels of tissue plasminogen activator (tPA), a product of activated microglia that has been implicated in neurodegeneration. Finally, we demonstrated that depletion of microglia from rat brains blocks neurodegeneration and paralysis induced by PVC-211 MuLV, clearly demonstrating the importance of activated microglia in the development of PVC-211 MuLV-induced neurodegeneration. Our current studies on PVC-211 MuLV-induced neurodegeneration focus on using pharmacological interventions to block or mitigate PVC-211 MuLV-induced neurodegeneration. To determine the importance of MIP-1α, we treated rats with antiserum to this chemokine before and after virus infection and the treatment resulted in considerably fewer activated microglia in the diseased areas of the brain and a significant delay in disease progression. Importantly, treatment with anti-MIP-1αantibodies did not alter expression of the virus in the spleen or the brain. To determine whether the activated microglia are recruited from resident microglia in the brain or from macrophages/microglia in the periphery, rats were splenectomized 4 days after virus infection. Splenectomized rats showed a significant delay in disease development that paralleled a decrease in the number of activated microglia in the brain, suggesting that the major source of activated microglia in the brains of PVC-211 MuLV-infected rats is from macrophages in the spleen or other peripheral organs, rather than resident brain microglia. Splenectomy did not alter expression of the virus in the bone marrow or the brain. Taken together, our data suggest that the chemokine MIP-1αis directly involved in the neurodegeneration induced in rats by PVC-211 MuLV by recruiting macrophages/microglia into the diseased areas of the brain, and further indicate that the source of the activated microglia may be primarily from splenic macrophages/microglia recruited from the periphery. Studies are in progress to determine if a combination of splenectomy and treatment with MIP-1αantibodies will further delay or even block neurodegeneration induced by PVC-211 MuLV. We have also initiated studies to assess the effects on PVC-211 MuLV-induced neurodegeneration of blocking the receptor for MIP-1αas well as blocking [summary truncated at 7800 characters]
