Differential Proteomic Expression in Glioblastoma Multiforme (GBM). Selective tissue microdissection was used to obtain pure populations of GBM cells, which were studied using two-dimensional protein gel electrophoresis (2-DGE) and protein sequencing. These molecular technique identified select target proteins that were expressed differentially among GBMs that permitted distinguishing between the 2 main categories of GBMs (primary and secondary). We isolated and sequenced 11 unique proteins that were differentially expressed in the primary and secondary GBMs and that produced 2 distinctive proteomic patterns. Thus, the 2 patterns of GBMs, primary versus secondary, previously distinguished by clinical and genetic differences, can be recognized at the protein level and may have implications for prognosis and treatment options. We are using the same approach to identify differentially expressed proteins in different stages of glioma formation, as well as within gliomas with different phenotypic expression. Nuclear Receptor Corepressor (N-CoR) Expression in GBM. We have found that N-CoR is overexpressed in GBM cells. Studies indicate that the subcellular localization and presence of N-CoR is related to the differentiation state of GBM cells. Cells expressing nuclear N-CoR do not express glial fibrillary acidic protein (GFAP), a marker of astrocytic differentiation. Cells with cytoplasmic N-CoR or loss of N-CoR express GFAP. N-CoR binds to unliganded nuclear receptors such as the retinoid acid receptor and thyroid hormone receptor. When N-CoR forms a complex with silencing mediator of retinoid and thyroid hormone receptors (SMRT), histone deacetylase 3 (HDAC3) and retinoic acid receptor, (RAR), transcription of RAR specific target genes is repressed resulting in increased cell proliferation. We have found that oxadaic acid acts synergistically with retinoic acid (RA) to inhibit GBM cell growth and increase cell differentiation. Phoshastase-1 inhibitors such as okadaic acid are known to inhibit N-CoR activity. Oxadaic acid is not specifically targeted to N-CoR and is likely to have significant side-effects. We believe these findings provide critical information for enhancing current understanding of glioma biology which may lead to new therapeutic options for patients. Therefore, we seek more specific phosphatase inhibitors from new chemical compounds that affect cell growth and differentiation in GBM by specifically targeting the NCo-R pathway, but not others. Recently a CRADA between Lixte Biotechonolgy Inc. and NINDS was executed for studying this novel therapeutic paradigm and developing new small chemical compounds. Work is currently in progress studying the inhibitory activity of a series of phosphatase inhibitors and HDAC inhibitors. LB1, a phosphatase inhibitor and LB 2, an HDAC inhibitor were developed and provided by our CRADA partner. During the last year, protocols for evaluating the new drugs and combinations of drugs for activities in an animal model of human GBM were jointly planned and developed. The results of the study show an inhibitory effect on tumor growth of the mouse xeno-grafted human GBM with both new compounds, LB1 and LB2. Recently, the chemical structure of these 2 compounds have been modified for a better penetration to the blood brain barrier. We are planning to test these compounds in a GBM animal tumor model. Identification of CNTF Receptor in Brain Tumors as Potential Diagnostic and Therapeutic Target. We used a more recently developed proteomic technique to distinguish the proteome of GBM stem cells by capillary isoelectric focusing (CIEF) with nano-reversed-phase liquid chromatography (nRPLC) peptide separation and MS/MS protein sequencing. We have found that CNTF receptor protein consistently appeared in GBM proteomes but not in normal adult brain. Subsequent studies indicated that its expression correlates with the pathologic grades of the glioma and their cell differentiation. Taking CNTF receptor as an unique expression character in GBM stem cells and tissues and its membrane localization with large portion of protein extra-cellular distribution, we further studied antibody-dependent cellular cytotoxicity (ADCC) in GBM cells. The preliminary results of the study showed an inhibition of tumor cell growth and strong cellular cytotoxicity effect. We are planning to develop a CNTF receptor antibody specifically binding to CNTF receptor in brain tumor for therapeutic use, as well as potential molecular imaging marker. Developmental Biology and Tumorigenesis of von Hippel-Lindau disease (VHL). Analagous to other tumor supressor gene syndromes, tumorigenesis in VHL is most commonly initiated by a VHL wild-type deletion in susceptible cells. Several key questions, however, remain unexplained in most, if not all, tumor suppressor gene syndromes: 1) in any organ, only one specific type of tumor occurs;2) tumorigenesis is restricted to specific sets of organs and 3) there is no obvious association between tumor suppressor gene function and tumorigenesis. Our recent studies on the histogenesis of hemangioblastomas revealed evidence that hemangioblastomas represent developmentally-arrested tissue. We have further established developmental effects of pVHL deficiency on central nervous system tissues by the discovery of numerous mesenchymal precursors that precede hemangioblastoma formation. In analogy to embryonal blood island differentiation, blood island formation in hemangioblastomas is associated with transient expression of the erythropoietin (Epo) receptor (EpoR). EpoR expression coincides with expression of Epo secondary to VHL deficiency, which we have recently proposed as a mechanism of tumor progression. This mechanism of tumor progression appears to be applicable to other VHL disease-associated tumors, including renal clear cell carcinoma and endolymphatic sac tumor. Current projects on VHL disease-associated hemangioblastomas include a) expression of developmental hemangioblast-associated proteins in hemangioblastomas, b) detailed characterization of hemangioblastoma precursor material, c) investigation of the vessel cell origin of hemangioblastoma by genetic analysis to address the critical questions whether the tumor cell participating directly to the angiogenesis. Based on the uniform expression of EPO and EPOR by VHL associated tumors and previous data indicating a potential EPO driven autocrine loop in these tumors, we attempted to establish the tumor cell line in culture conditions. The maintenance of tumor cells in culture requires an EPO-rich media. Growth of the VHL tumor cells was disrupted by addition of EPO or EPOR antibodies in the culture media. Using EPO rich culture media, we cultured the primary tumors and maintained them in culture for in vitro testing. We have characterized and expanded these tumor cells to nucleated erythrocytes and mature erythrocytic progeny by varying the cell culture conditions. These findings further support that the EPO and EPOR functional pathway in these tumors is critical in VHL tumor growth and differentiation. Further, the established culture cell lines will be useful for testing other reagents.
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