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 ce lls. Cell lines from human tumor tissue with neu oncogene amplificatio n and one nonneoplastic human epithelial cell line from a lactating br east with a neu single copy as the reference were used. After quantita tive fluorescence analysis of three mixed cell populations (neoplastic cells and reference cells), sire evaluated the cell morphology and lo cation of the fluorescence signal by Inset scanning microscopy. Finall y, 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 eons within the limits of oncogene copy numbers known from the literature and evaluated by s outhern blot analysis. The lowest oncogene copy number possible to qua ntify was four to eight genomic copies.