A deletion mutation within the ligand binding domain is responsible for activation of epidermal growth factor receptor gene in human brain tumors
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H Yamazaki1, Y Ohba, N Tamaoki
1Institute of Medical Science, University of Tokyo.
Abstract:
Two transplantable cell lines of human glioblastoma multiforme GL-3 and GL-5 carried an amplification and overexpression of structurally altered epidermal growth factor (EGF) receptor gene: the 140 kilodalton EGF receptors in these cases exhibited a constitutively expressed tyrosine kinase activity without the ligand. Here, we isolated the abnormal EGF receptor cDNA from GL-5 cell line, and demonstrated that this cDNA bears a single large intramolecular deletion mutation 801 base pairs long within the ligand binding domain of EGF receptor. In other regions no amino acid substitution was observed. At the level of genomic DNA, this deletion appeared to start from the 1st intron and terminate in the 6th intron of the EGF receptor gene. However, in the two lines of glioblastoma, GL-3 and GL-5, the positions of the start or the end of the deletion mutation in these introns were not identical, suggesting an involvement of a unique recombination mechanism in the formation of deletion mutation. A weak but ligand-independent transforming activity was observed in the deletion-carrying EGF receptor cDNA.
Insights
Human glioblastoma cells GL-3 and GL-5 have altered epidermal growth factor (EGF) receptors with a large deletion mutation. This mutation causes ligand-independent tyrosine kinase activity, suggesting a novel mechanism in glioblastoma development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Human glioblastoma multiforme cell lines GL-3 and GL-5 exhibit amplification and overexpression of a structurally altered epidermal growth factor (EGF) receptor gene.
- The altered 140 kDa EGF receptors display ligand-independent tyrosine kinase activity.
Purpose of the Study:
- To isolate and characterize the abnormal EGF receptor cDNA from the GL-5 glioblastoma cell line.
- To investigate the nature of the mutation and its functional consequences.
Main Methods:
- Isolation of abnormal EGF receptor cDNA from GL-5 cell line.
- Analysis of the cDNA for mutations, including deletions and amino acid substitutions.
- Genomic DNA analysis to determine the extent of the deletion within the EGF receptor gene introns.
- Assessment of the transforming activity of the mutated EGF receptor.
Main Results:
- A single large intramolecular deletion mutation, 801 base pairs long, was identified within the ligand-binding domain of the EGF receptor cDNA.
- No amino acid substitutions were observed in other regions of the receptor.
- The deletion spanned from the 1st to the 6th intron of the EGF receptor gene, with variations in precise locations between GL-3 and GL-5 cell lines.
- The deletion-carrying EGF receptor cDNA exhibited weak, ligand-independent transforming activity.
Conclusions:
- The identified deletion mutation in the EGF receptor is responsible for its constitutive tyrosine kinase activity in glioblastoma cells.
- The distinct deletion endpoints in different cell lines suggest a unique recombination mechanism in glioblastoma formation.
- The abnormal EGF receptor possesses transforming potential, contributing to glioblastoma pathogenesis.