Malignant astrocytomas comprise the majority of central nervous system tumors in humans and are associated with a dismal prognosis despite the application of multimodality therapy. It is hypothesized that the genesis of these tumors occurs as a multistep progression from benign astrocytoma to anaplastic astrocytoma (AA), and finally, to glioblastoma multiforme (GBM). In this transformation from benign to malignant, numerous, and as of now, poorly understood cytogenetic and biochemical changes take place. Recently it was demonstrated that the progression of astrocytes from a benign to a malignant phenotype is accompanied by a change in the RNA processing of fibroblast growth factor receptor 1 (FGFR-1) gene. The level of a high affinity form of the FGFR-1 is dramatically elevated as a result altered expression and RNA splicing. The strong correlation between altered FGFR gene expression and astrocyte malignancy underscores the importance of understanding the role of FGFR in normal and malignant astrocyte cell growth. This proposal will test the hypothesis that alterations in FGFR RNA splicing contribute to abnormal growth of malignant astrocytes. The specific objectives of this proposal are 1) to develop a model system which mimics the FGFR-1 RNA processing pathways observed in normal brain glial cells and glioblastoma, 2) to use this model to identify sequence elements within the FGFR-1 precursor RNA which act to regulate splicing, 3) to use the regulatory sequence information to identify regulatory factors acting in trans, 4) to determine the relationship between trans-acting factor expression and transformation.
Aim 1 will be accomplished by examining available astrocyte cell lines for the RNA splicing patterns of FGFR-1 transcripts. We will also express a chimeric FGFR-1 minigene in these cell lines to determine if its RNA transcripts are processed in a manner analogous to the endogenous gene.
Aim 2 will be accomplished by introducing sequence alterations, deletions, substitutions and mutations, into the FGFR-1 minigene and determining their effect on RNA processing decisions.
In Aim 3 we will develop an assay systems to be used to purify the factor(s) involved in regulation of FGFR-1 RNA splicing. Two approaches will be used: identification of RNA- binding proteins through sequence-specific interaction and use of expression cloning which functionally detects RNA splicing. Finally, in Aim 4 we will use cDNA sequence information derived from Aim 3 to measure the level of trans-acting factor expression in graded tumor samples. A further understanding of the mechanisms underlying the changes in FGFR-1 RNA processing in malignant astrocytomas may shed light on the transformation process in astrocytes and provide new targets for suppressing their growth.
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