Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe.
A modification of "reverse chromosome painting" was carried out using genomic DNA from tumor cells as a complex probe for chromosomal in situ suppression hybridization to normal metaphase chromosome spreads. Amplified DNA sequences contained in such probes showed specific signals, revealing the normal chromosome positions from which these sequences were derived. As a model system, genomic DNAs were analyzed from three tumor cell lines with amplification units including the proto-oncogene c-myc. The smallest amplification unit was about 90 kb and was present in 16-24 copies; the largest unit was bigger than 600 kb and was present in 16-32 copies. Specific signals that co-localized with a differently labeled c-myc probe on chromosome band 8q24 were obtained with genomic DNA from each cell line. In further experiments, genomic DNA derived from primary tumor material was used in the case of a male patient with glioblastoma multiforme (GBM). Southern blot analysis using an epidermal growth factor receptor gene (EGFR) probe that maps to 7p13 indicated the amplification of sequences from this gene. Using reverse chromosome painting, signals were found both on band 7p13 and bands 12q13-q15. Notably, the signal on 12q13-q15 was consistently stronger. The weaker 7p13 signal showed co-localization with the major signal of the differently labeled EGFR probe. A minor signal of this probe was seen on 12q13, suggesting cross-hybridization to ERB3 sequences homologous to EGFR. The results indicate co-amplification of sequences from bands 12q13-q15, in addition to sequences from band 7p13. Several oncogenes map to 12q13-q15 providing candidate genes for a tumor-associated proto-oncogene amplification. Although the nature of the amplified sequences needs to be clarified, this experiment demonstrates the potential of reverse chromosome painting with genomic tumor DNA for rapidly mapping the normal chromosomal localization of the DNA from which the amplified sequences were derived. In addition, a weaker staining of chromosomes 10 and X was consistently observed indicating that these chromosomes were present in only one copy in the GBM genome. This rapid approach can be used to analyze cases where no metaphase spreads from the tumor material are available. It does not require any preknowledge of amplified sequences and can be applied to screen large numbers of tumors.
Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
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Joos
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Scherthan
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Speicher
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Schlegel
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Cremer
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Lichter
D002869:Chromosome Aberrations; D002874:Chromosome Mapping / Q000379:methods*; D002879:Chromosomes, Human, Pair 10; D002881:Chromosomes, Human, Pair 12; D002897:Chromosomes, Human, Pair 7; D002898:Chromosomes, Human, Pair 8; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics; D005784:Gene Amplification; D016259:Genes, myc; D005909:Glioblastoma / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D000090063:Proto-Oncogene Mas; D011519:Proto-Oncogenes; D017422:Sequence Analysis, DNA / Q000379:methods*; D013489:Suppression, Genetic
D015342:DNA Probes; D004273:DNA, Neoplasm; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D066246:ErbB Receptors