Glioblastoma-related gene mutations and over-expression of functional epidermal growth factor receptors in SKMG-3 glioma cells

Amplification of the epidermal growth factor receptor (EGFR) gene is found in about 40% of glioblastomas (GBMs) but is rarely detected in GBM cell lin es. We confirmed that the exceptional SKMG-3 GBM cell line retained amplifi ed EGFR genes in vitro, and found that these sequences were concentrated on extra-chromosomal DNA particles similar to double-minute chromosomes. The cells contained two other gene mutations that are associated with high-grad e astrocytic tumors: extrachromosomal amplification of the cyclin-dependent kinase-4 (CDK4) gene and a homozygous mutation within the PTEN tumor suppr essor gene. Immunoblots revealed very high levels of EGFR, moderately incre ased expression of CDK4, and no detectable PTEN protein. The overexpressed SKMG-3 EGFRs responded to exogenous ligand and resembled normal rather than mutant receptors. A heterozygous mutation of the p53 gene (p53(R282W)) cor related with failure of radiation to induce the expression of cyclin-depend ent kinase inhibitor p21(waf1) or an early G1 cell cycle arrest. Although e ach of these gene mutations occurs in GBMs, SKMG-3 cells had an unusual gen otype in that a p53 gene mutation co-existed with amplified EGFR genes. Non etheless, the SKMG-3 cell line can be exploited as a model to study how onc ogenic EGFR signals in GBM cells interact with over-expressed CDK4 and loss of PTEN to confer the malignant phenotype.