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Extrachromosomal amplification of the epidermal growth factor receptor gene in a human colon carcinoma cell line
1Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston.
Abstract:
In situ hybridization, using a biotinylated cDNA probe for the epidermal growth factor receptor (EGFR) gene, indicates that the amplified EGFR genes in the colon tumor cell line, DiFi, are localized in many small double minute chromosomes (dmin) of varying size and visibility. Analysis of the electrophoretic mobility of gamma-irradiated DNA from DiFi by pulsed-field gel electrophoresis and Southern blot hybridization using EGFR probe, indicates that the amplified EGFR in DiFi exists in extrachromosomal, covalently closed circular episomes, presumably equivalent to dmin. Two major and one minor species were observed which had estimated sizes of 650, 1,300, and 2,000 kb, respectively. The DiFi cell line appears to represent a unique case of extrachromosomal EGFR gene amplification in human cells.
Insights
Colon tumor cells (DiFi) show amplified epidermal growth factor receptor (EGFR) genes. These genes are found on small, extrachromosomal double minute chromosomes (dmin), indicating a unique amplification mechanism in human cancer.
Area of Science:
- Molecular Biology
- Cancer Genetics
Background:
- Epidermal growth factor receptor (EGFR) gene amplification is common in various cancers.
- Understanding gene amplification mechanisms is crucial for targeted cancer therapies.
Purpose of the Study:
- To investigate the physical state and localization of amplified EGFR genes in the DiFi colon tumor cell line.
- To characterize the nature of double minute chromosomes (dmin) associated with EGFR amplification.
Main Methods:
- In situ hybridization using a biotinylated cDNA probe for the EGFR gene.
- Pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization of gamma-irradiated DiFi DNA.
- Analysis of DNA mobility to determine the size and structure of amplified EGFR elements.
Main Results:
- Amplified EGFR genes in DiFi cells are localized on numerous small double minute chromosomes (dmin).
- EGFR amplification exists in extrachromosomal, covalently closed circular episomes, consistent with dmin.
- Three distinct episomal species (650 kb, 1,300 kb, and 2,000 kb) were identified.
Conclusions:
- The DiFi cell line exhibits a unique form of extrachromosomal EGFR gene amplification in human cells.
- These findings contribute to understanding the heterogeneity of gene amplification in cancer.
- The characterized episomal structures may serve as models for studying gene amplification dynamics.