Cell cycle-related changes in chromatin structure detected by flow cytometry using multiple DNA fluorochromes

Three-color staining and sequential fluorescence analysis of the dyes bound to specific base regions and intercalating sites on DNA were used to detect alterations of chromatin organization in single cells. Two non-intercalating DNA-reactive fluorochromes, Hoechst 33342 (HO) with preferential binding to A-T and mithramycin (MI) which binds to G-C regions, were used for cell staining in combination with propidium iodide (PI), a dye that intercalates between DNA base pairs without base specificity. Blue (HO), green (MI), and red (PI) fluorescence intensities were quantitatively analyzed following sequential excitation of stained cells in a three-laser flow system (uv, 457 nm and 530 nm wavelengths). Competition for dye binding was not significant and, although energy transfer occurs, under appropriate conditions, DNA distribution profiles for exponentially growing cells exhibit relatively low coefficients of variation (CVs), G2 + M/G1 peak ratios near values of 2, and comparable percentages of cells in G1, S, and G2 + M for each of the three dyes. The fluorescence intensity of each dye is proportional to the relative number of specific base regions or intercalating sites accessible for dye binding in DNA. Electronic ratio analyses of blue to green and red to green fluorescence signals provided a measure of the correlation of A-T to G-C and intercalation to G-C sites, respectively, for dye-bound regions on the DNA. Ratio analyses were sensitive to conformational changes in chromatin structure that alter the relative proportion of different base regions in DNA accessible for dye binding. Using this method, rearrangements in chromatin structure were observed in synchronized CHO cells during progression through the cell cycle. The sensitivity of this method will make it useful in investigations on effects of various agents (i.e., drugs, irradiation, viruses) on chromatin structure in single cells. Also, the three-color DNA staining and analysis techniques have potential for improving chromosome resolution in flow karyotyping studies.