A search for genetic changes in human glial tumors has demonstrated that the epidermal growth factor (EGF) receptor gene is amplified in 40% of glioblastoma multiforme tumors and that these amplified genes are frequently rearranged. In nearly all the cases examined so far, the extracytoplasmic domain of the receptor has been the target of these alterations. These findings suggest that the EGF receptor plays a role in the development of a subset of tumors. The research plan centers around characterizing the structure of these alterations and their effect on several functions. For structural analysis, a large number of glial tumors will be screened in order to categorize the various kinds of changes that can occur and their frequency. This will be done using Southern blotting to evaluate gene structure, Northern blotting to examine transcript size, and sequence analysis of cDNA fragments amplified by the polymerase chain reaction to deduce the amino acid sequence. To facilitate these experiments and to understand the range of combinations possible, the intron/exon boundaries corresponding to the extracytoplasmic domain will be determined by sequencing. RNase protection experiments will be used to quantitate the relative levels of mutant and normal transcripts. To evaluate possible autocrine stimulation, the levels of TGF-alpha will be measured. To test the hypothesis that these mutations were selected for because they have higher transforming or tumorigenic potential and that they are ligand independent, the EGF receptor will be mutated to match the alterations characterized and cloned into an expression vector. These constructs will be transfected into the NIH 3T3 cell line and quantitated in the presence and absence of EGF for their ability to: induce focus formation, anchorage independence, and tumorigenicity in nude mice. It will be determined if these parameters correlate with increased DNA synthesis and cell growth. Two glial cells lines that are not tumorigenic nor have amplified, rearranged genes, but were derived from biopsies that did, will be transfected to see if this restores tumorigenicity. To determine whether these effects are the result of constitutive activation of the receptor or independence from normal regulatory control, the transfected cell lines will be assayed for their EGF and TGF-alpha binding affinity, and the effect of EGF on: receptor phosphorylation of proteins, receptor internalization, and induction and duration of c-fos transcription. These studies will add to our understanding of these tumors. A clinically useful application resulting from this research will be to use the information about the abnormal sequences to derive tumor specific monoclonal antibodies.
