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

Abstract

The chromatin structure of a diploid precursor B-cell line (REH), in vitro-stimulated normal B-lymphocytes, and reactive and malignant lymph node B-lymphocytes was studied by staining formaldehyde-fixed, permeabilized cells with the DNA-specific fluorophore 7-aminoactinomycin D (7-AMD) and measuring single-cell fluorescence by flow cytometry. Resting peripheral blood B- and T-lymphocytes (G₀ cells) bound low amounts of 7-AMD (7-AMD^- phenotype), while G₁ REH cells and purified B-cells stimulated with anti-µ + 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 G₀ and G₁ 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 approximately linearly with time during the G₀-G₁ 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 (P < 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 (P = 0.07), while the light scattering (cell size) of G₀/G₁ cells was highly correlated to 7-AMD binding (P < 0.001).