In Vitro and in Vivo Activation of B-Lymphocytes: A Flow Cytometric Study of Chromatin Structure Employing 7-Aminoactinomycin D1

The chromatin structure of a diploid precursor B-cell line (REH), in >>/fro-stimulated normal B-lymphocytes, and reactive and malignant lymph node B-lymphocytes was studied by staining formaldehyde-fixed, permeabilized cells with the DNA-specific ftuorophore 7-aminoactinomycin D (7-AMD) and measuring single-cell fluorescence by flow cytometry. Resting peripheral blood Band T-lymphocytes (Go cells) bound low amounts of 7-AMD (7-AMIr phenotype), while (., REH cells and purified B-cells stimulated with anti-M + B-cell growth factor bound nearly twice as much 7-AMD (7-AMD* phenotype). 7-AMD binding increased up to threefold and the differences in binding between Go and Gì cells were nearly abolished when nuclei were isolated prior to fixation or when fixed whole cells were treated with DNase 1. 7-AMD binding increased in parallel with autofluorescence and ap proximately linearly with time during the Go-Gi transition of in vitro stimulated B-cells, as was determined by simultaneous measurements of 7-AMD fluorescence and autofluorescence or fluorescence of fluorescein isothiocyanate-labeled antibodies to the early activation antigen 4F2 and to the transferrin receptor. In cell suspensions from lymph node biopsies, the 7-AMD* phenotype was a property of tumor cells in patients with high grade non-Hodgkin's lymphoma (H-NHL, Kiel classification, 5/5); cells with this phenotype were only found in one of nine low grade non-Hodgkin's lymphoma samples (L-NHL, 1/9). The other (8/9) L-NHL samples and the reactive lymph node contained only 7-AMD" cells. All tumors were diploid. The correlation observed between 7-AMD binding and DNase 1 susceptibility of DNA in chromatin (/' < 0.001) suggests that 7-AMD binding is a marker of general transcriptional activity. Surprisingly, the percentage of tumor cells in S phase did not correlate significantly with 7-AMD stainability (/" = 0.07), while the light scattering (cell size) of <„,/(., cells was highly correlated to 7-AMD binding (P < 0.001).