Abstract
Amplification of the epidermal growth factor receptor (EGFR) gene is found in about 40% of glioblastomas (GBMs) but is rarely detected in GBM cell lines. We confirmed that the exceptional SKMG-3 GBM cell line retained amplified 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-grade astrocytic tumors: extra-chromosomal amplification of the cyclin-dependent kinase-4 (CDK4) gene and a homozygous mutation within the PTEN tumor suppressor gene. Immunoblots revealed very high levels of EGFR, moderately increased expression of CDK4, and no detectable PTEN protein. The over-expressed SKMG-3 EGFRs responded to exogenous ligand and resembled normal rather than mutant receptors. A heterozygous mutation of the p53 gene (p53R282W) correlated with failure of radiation to induce the expression of cyclin-dependent kinase inhibitor p21waf1 or an early G1 cell cycle arrest. Although each of these gene mutations occurs in GBMs, SKMG-3 cells had an unusual genotype in that a p53 gene mutation co-existed with amplified EGFR genes. Nonetheless, the SKMG-3 cell line can be exploited as a model to study how oncogenic EGFR signals in GBM cells interact with over-expressed CDK4 and loss of PTEN to confer the malignant phenotype.
Original language | English (US) |
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Pages (from-to) | 605-615 |
Number of pages | 11 |
Journal | Acta Neuropathologica |
Volume | 101 |
Issue number | 6 |
DOIs | |
State | Published - 2001 |
Funding
tion and have defective p53-dependent responses to radiation. ÍÍA Autoradiograph of DNA sequence gel of region adjacent to codon 282 in exon 5 of the p53 genes. The arrow indicates heterozygous mutation in the first base of codon 282 in the SKMG-3 but not in control cells. B Immunoblot for expression of p53 and p21waf1 in SKMG-3 and control RKO colon cancer cells at different times after a single exposure to 4 Gy gamma radiation. T98G cells (T98G) express mutant p53 (Topo topoisomerase as loading control). C DNA flow cytometry of DNAs from SKMG-3 and RKO cells collected at the indicated time points following radiation. The arrow denotes impaired exit of RKO cells from G1 into S phase (see text); x axis number of cells; y axis DNA content as measured by propidium iodide fluorescence. D Changes in the G1/S ratios at 0 h as compared to 12 h after radiation Acknowledgements. We thank Michael Chouinard for his technical assistance and Melody Stallings-Mann for technical assistance and help in preparation of the manuscript. This work was made possible by grants from the Mayo Foundation (C.T.) and NIH – CA77715 (M.K.), CA88357 (C.D.J), CA64928 (P.B.), and CA50905 (R.J.).
Keywords
- Epidermal growth factor receptor
- Glioblastoma
- PTEN
- Tumor cells
- p53 gene
ASJC Scopus subject areas
- Clinical Neurology
- Cellular and Molecular Neuroscience
- Pathology and Forensic Medicine