MOLECULAR BASIS AND USE OF A RADIAL GLIA CELL LINE C6-R

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Radial glial cells appear transiently during neural development and play key roles in cell migration and differentiation. However, they have been difficult to study because they are unstable in culture and cell lines with radial glial properties have not been available. We have derived a stable cell line (C6-R) from the rat C6 glioma cell line by transfection with a mutant phosphatase receptor. C6-R has radial morphology and stimulates neuronal migration in culture and in vivo. Although both C6-R and C6 can proliferate rapidly in culture, C6-R is unable to form tumors in rat brain like C6. Since expression of the transfected mutant receptor in many other C6 clones can not account for the phenotypic differences between C6-R and C6, it is likely that the alteration is due to genetic disruption at a single integration site that has been detected in Southern blots of C6-R genomic DNA. We hypothesize that this integration event disrupted genes that either promote glial tumor formation or suppress gliogenesis. The goals of this proposal are to understand the cellular properties of C6-R and the molecular basis of this unique phenotype. In addition, given the ability of C6-R cells to migrate and align along white matter in mature CNS and to support neuronal migration, they will be implanted in the CNS to explore their ability to promote growth of nerves in various situations including following injury. To understand the reduced ability of C6-R to form tumors in vivo, its cellular properties, including proliferation and death, will be compared with the parental C6 cell line. Molecular analysis will be directed to identify the genetic alterations responsible for transformation C6 into C6-R cells and the resulting differences in expression that are responsible for their phenotypic differences. Plasmid rescue and genomic cloning methods will be used to recover DNA from the locus of plasmid insertion. Genes at this locus, and other identified by subtractive suppression hybridization (SSH) using mRNA from C6-R and C6 will be analyzed to determine their roles in gliogenesis and formation of gliomas. These studies will lead to a better understanding of genes that are involved in glial development and in formation of high grade tumors. Studies of the reversion of C6-R to cells that can form tumors in vivo may have implications for understanding how gliomas progress to high grade malignancies. Finally, characterization of radial glial cell lines may yield new methods for promoting nerve growth following injury.