Quantitative analysis of the neu oncogene in normal and transformed epithelial breast cells by fluorescence in situ hybridization and laser scanning microscopy.
In this study we evaluated indirect fluorescence in situ hybridization of the neu oncogene in combination with laser scanning microscopy for the detection and quantification of fluorescence signals in single cells. Cell lines from human tumor tissue with neu oncogene amplification and one nonneoplastic human epithelial cell line from a lactating breast with a neu single copy as the reference were used. After quantitative fluorescence analysis of three mixed cell populations (neoplastic cells and reference cells), we evaluated the cell morphology and location of the fluorescence signal by laser scanning microscopy. Finally, the number of oncogene copies in the tumor cell population could be estimated from the ratio of its mean background-corrected integrated fluorescence intensities to those of the reference cells. In all mixed cultures the ratio of fluorescence intensities was within the limits of oncogene copy numbers known from the literature and evaluated by southern blot analysis. The lowest oncogene copy number possible to quantify was four to eight genomic copies.
Heinrichs
M
M
Department of Pathology, Technical University of Aachen, Aachen, Germany.
Striepecke
E
E
Böcking
A
A
D001943:Breast Neoplasms / Q000235:genetics*; D002277:Carcinoma / Q000235:genetics*; D005260:Female; D005784:Gene Amplification; D018734:Genes, erbB-2 / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007834:Lasers; D008856:Microscopy, Fluorescence; D014407:Tumor Cells, Cultured