Joos S, Scherthan H, Speicher M R, Schlegel J, Cremer T, Lichter P
Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Hum Genet. 1993 Feb;90(6):584-9. doi: 10.1007/BF00202475.
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.
利用肿瘤细胞的基因组DNA作为复杂探针，对正常中期染色体铺展进行染色体原位抑制杂交，对“反向染色体描绘”进行了改良。这类探针中包含的扩增DNA序列显示出特定信号，揭示了这些序列所源自的正常染色体位置。作为一个模型系统，对三种含有包括原癌基因c-myc在内的扩增单元的肿瘤细胞系的基因组DNA进行了分析。最小的扩增单元约为90 kb，有16 - 24个拷贝；最大的单元大于600 kb，有16 - 32个拷贝。用每个细胞系的基因组DNA获得了与位于染色体8q24带上的不同标记的c-myc探针共定位的特定信号。在进一步的实验中，对于一名患有多形性胶质母细胞瘤（GBM）的男性患者，使用了源自原发性肿瘤材料的基因组DNA。使用定位在7p13的表皮生长因子受体基因（EGFR）探针进行Southern印迹分析表明该基因的序列发生了扩增。通过反向染色体描绘，在7p13带和12q13 - q15带上均发现了信号。值得注意的是，12q13 - q15上的信号始终更强。较弱的7p13信号与不同标记的EGFR探针的主要信号共定位。在12q13上可见该探针的一个次要信号，提示与EGFR同源的ERB3序列发生了交叉杂交。结果表明，除了7p13带的序列外，12q13 - q15带的序列也发生了共扩增。几个癌基因定位于12q13 - q15，为肿瘤相关的原癌基因扩增提供了候选基因。尽管扩增序列的性质有待阐明，但该实验证明了用肿瘤基因组DNA进行反向染色体描绘在快速确定扩增序列所源自的DNA的正常染色体定位方面的潜力。此外，始终观察到10号染色体和X染色体的染色较弱，表明这些染色体在GBM基因组中仅以一个拷贝存在。这种快速方法可用于分析无法获得肿瘤材料中期铺展的病例。它不需要对扩增序列有任何先验知识，可用于筛查大量肿瘤。
