Friend spleen focus-forming virus (SFFV) causes a rapid, multi-stage erythroleukemia in mice due to expression of its unique envelope glycoprotein. It provides an important model for understanding the molecular changes that result in the hyperplastic, blast crisis and metastatic phases of leukemia and for testing therapeutic strategies to block each stage. Our previous studies demonstrated that during the first stage of SFFV-induced disease, the viral envelope protein, SFFV gp55, forms a complex with the erythropoietin receptor (EpoR) and a short form of the receptor tyrosine kinase Stk (sf-Stk), causing constitutive activation of various components of the Epo signal transduction pathway and Epo-independent erythroid cell proliferation, survival and differentiation. We further discovered that co-expression of SFFV gp55 and sf-Stk is sufficient to transform rodent fibroblasts. Our recent studies indicate that sf-Stk expression is required for maintenance of the transformed phenotype of SFFV gp55-expressing fibroblasts, and that PU.1, which is essential for transformation of erythroid cells by SFFV, plays no role in transformation of fibroblasts by SFFV gp55/sf-Stk. We also took advantage of the SFFV gp55/sf-Stk-transformed fibroblasts to examine the activation of signal transduction pathways by SFFV gp55 in the absence of the EpoR. Like SFFV-infected erythroid cells, SFFV gp55/sf-Stk- transformed fibroblasts express high levels of phosphorylated MEK, ERK, PI 3-kinase, Gab1/2, Akt, JNK and STAT3, but unlike virus-infected erythroid cells they fail to express phosphorylated STATs 1 and 5, which may require involvement of the EpoR. In addition, the p38 MAPK stress response is suppressed in the transformed fibroblasts. We further showed that the signal transducing molecules activated in fibroblasts by SFFV-activated sf-Stk can be targeted with small molecule inhibitors to modulate proliferation and/or transformation, and that it may be possible to target sf-Stk directly with the flavonoid luteolin. Our results indicate that sf-Stk is a molecular effector of transformation that could be targeted directly or with agents against its downstream signal transducers. 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 indicates that 13/28 human cancer cell lines examined express sf-RON, 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. Unlike its mouse counterpart sf-Stk, sf-RON does not transform rodent fibroblasts, with or without SFFV gp55. Studies are in progress to determine if blocking sf-RON in human cancer cell lines will their transformed phenotype. In addition to studying the effects of SFFV gp55 on erythroid cell proliferation and differentiation, we also study the second stage of SFFV-induced leukemia for molecular changes associated with transformation and metastasis. Transformation of erythroid cells by SFFV is associated with activation of the transcription factor PU.1 by retroviral integration, and our studies suggest that this results in high expression of the hematopoietic phosphatase SHP-1. This subsequently leads to a specific block in the phosphorylation of STAT1, a transcription factor important for erythroid cell differentiation that is activated by SFFV in the first stage of the disease. We recently demonstrated that SFFV-transformed erythroleukemia cells can also undergo further molecular changes which allow them to metastasize to the bone marrow. Due to their failure to differentiate, they proliferate to such high levels in the bones of the skull and vertebrae that they breech the bone and enter into the meninges of the brain and spinal cord, causing meningeal leukemia associated with hind limb paralysis. Since meningeal leukemia is a common complication of human leukemia, we are developing this as an animal model to study the mechanisms by which leukemic cells metastasize to the central nervous system and to test therapies to block these events. The SFFV-transformed erythroleukemia cells were shown to secrete high levels of vascular endothelial growth factor (VEGF) and could preferentially adhere in vitro to fibronectin. Gene prolifing showed changes in expression of genes encoding angiogenic growth factors, oncoproteins and adhesion molecules as well as genes involved in the synthesis or degradation of extracellular matrix components. Excessive and pathological angiogenesis could be seen in the bone marrow of diseased mice compared with controls. Studies are in progress to test anti-angiogenesis drugs as well as other pharmacological agents for their ability to block metastasis of the SFFV transformed erythroleukemia cells to the bone marrow and the development of meningeal leukemia. We have also been using the SFFV-transformed erythroleukemia cell lines to isolate putative leukemic stem cells by growing the cells under hypoxic conditions. We are currently testing several hypoxia-resistant clones for stem cell-like properties PVC-211 murine leukemia virus (MuLV), a variant of an erythroleukemia-inducing retrovirus, causes a rapid neurodegenerative disease when injected into newborn rats. This is an important model for understanding the molecular changes that result in neurodegeneration and for testing therapeutic strategies to block the disease. Our previous studies showed that the envelope protein of PVC-211 MuLV had undergone subtle changes in its receptor binding domain that allow it to enter the central nervous system by efficiently infecting brain capillary endothelial cells (BCEC). These changes created a unique heparin-binding domain in the viral envelope protein that may allow the virus to bind strongly to the surface of BCEC via heparin-like molecules, increasing the probability that the virus will bind to its cell surface receptor on these cells and efficiently infect them. Our more recent studies have concentrated on understanding the molecular events that occur after PVC-211 MuLV infects BCEC. We previously showed that virus-infected BCEC express high levels of inducible nitric oxide synthase and the chemokine LIX and show evidence of nitric oxide production. Our recent studies demonstrate that early in the course of the disease, before neuronal damage has occurred, the number of blood vessels increases in the cerebellum and brain stem, the sites of later spongiform neurodegeneration; the vessels in the brain become leaky; and the cerebellum expresses high levels of vascular endothelial growth factor (VEGF), suggesting brain hypoxia is occurring. At the same time, microglia in the virus-infected brain become activated and the brain and blood express elevated levels of MIP-1 alpha, a chemokine known to be secreted from activated microglia. Once the pathogenic changes can be detected in the cerebellum of virus-infected rats, one can detect elevated levels of tissue plasminogen activator (tPA), which could be responsible for neuronal death. We are also continuing studies using NMR spectroscopy to determine if elevated levels of any potentially neurotoxic brain meta [summary truncated at 7800 characters]
