The goal of this application is to understand how the levels of expression of GFAP (one of the proteins which constitute glial filaments) affect the growth and shape of astrocytoma cells. Such an understnading is likely to come from the determination of how glial filaments interact with other cellular components through associated proteins such as IFAP 300. The importance of these associated proteins in regulating the function of glial filaments during neoplasia is suggested by the finding that IFAP 300 is expressed in astrocytomas, but not in normal astrocytes. To extend these studies the aims are: 1) To correlate the intracellular distribution and expression levels of IFAP 300 with the malignant features of astrocytoma cells; 2) To investigate the relationship between the expression and intracellular distribution of IFAP 300 and the degree of differentiation of astrocytoma cells; 3) To characterize at the molecular level the interactions between IFAP 300, GFAP, vimentin, and the plasma membrane; 4) To determine the factors regulating the interactions between IFAP 300 and glial filaments in astrocytoma cells. For these studies they will characterize and compare using immunofluorescence and Western blot the amount and distribution pattern of IFAP 300 in astrocytoma cells. In order to gain insights into the function of IFAP 300 in astrocytoma cells, the results of the above ascribed experiments will be correlated with the growth, motility and adhesion of various astrocytoma cells. Differentiation of astrocytoma cells will be induced with exogenous factors such as interleukin-1 and by increasing the levels of GFAP expression by transfecting the cells with a GFAP cDNA. This will allow them to examine how differentiation affects the expression levels and subcellular distribution of IFAP 300. To dissect the molecular interactions between IFAP 300 and the plasma membrane, overlay assays will be performed and the proteins found to bind to IFAP 300 will be characterized by molecular cloning and/or protein sequencing. Overlay assays will also be used to determine if there is any difference in the binding sites of GFAP and vimentin to plasma membranes isolated from normal versus neoplastic astrocytes. The levels and sites of phosphorylation of IFAP 300, GFAP, and vimentin (all known phosphoproteins) will be examined in normal and neoplastic astrocytes to determine whether phosphorylation regulates the interaction of glial filaments with IFAP 300. The possibility that glial filament associated proteins other than IFAP 300 bind to glial filaments in astrocytomas cell will also be examined using a series of binding and overlay experiments.
