Astrocytoma is the most common primary neoplasm of the human central nervous system, and current therapy for this devastating disease is unsatisfactory. The overall objective of research proposed in this application is to investigate the molecular mechanism by which signaling interactions between interferon-gamma (IFN(gamma)) and transforming growth factor beta-1 (TGF(beta1)) occur in human astrocytoma cells. This approach was selected because TGF(beta1) and IFN(gamma) are present within glial tumor tissue and evoke biologically meaningful responses from astrocytoma cells. Detailed mechanistic understanding of interactions between counter-regulatory cytokines such as IFN(gamma) and TGF(beta1) may therefore be significant for elucidating the biology and treatment of glioma. We showed that TGF(beta1) inhibited expression of two IFN(gamma)- inducible genes, HLA-DRA and IDO, in a human astrocytoma cell line. The patterns of IFN(gamma)-inducible expressions and regulatory cis elements of these two genes were different, indicating that HLA-DRA and IDO can be used as distinct model systems to investigate crosstalk between TGF(beta1) and IFN(gamma).
The specific Aims are: 1. To investigate the mechanism by which IFN(gamma)-induced HLA-DRA transcription is impaired in TGF(beta1)-treated astrocytoma cells. We will extend our current studies by addressing protein factors binding to the HLA-DRA promoter in IFN(gamma)- and TGF(beta1)-treated cells. We will determine the cis-element needed for inhibiting transcription of the invariant chain (INV) gene, which is co-regulated with HLA-DRA, and evaluate TGF(beta1) effects on factor-binding to this element. Factors required for HLA-DRA transcription will be cloned, using a novel strategy based on retroviral expression of cDNAs encoding genetic suppressor elements (GSE) and immunoselection against HLA-DRA antigen expression. 2. To investigate TGF(beta1) inhibition of the IFN(gamma)-inducible IDO gene. We will use transfection studies to determine the cis-element required for TGF(beta1) inhibition of the 2,3-indoleamine dioxygenase (IDO) gene, and band-shifts to evaluate the effect of TGF(beta1) on factor-binding to these regulatory elements. Experiments proposed in this application will provide significant novel insight into signaling in astrocytoma cells by biologically important cytokines.
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