Different sensitivity of chromatin to acid denaturation in quiescent and cycling cells as revealed by flow cytometry

Abstract

The properties of DNA in situ as reflected by its staining with acridine orange are different in quiescent nonstimulated lymphocytes as compared with interphase lymphocytes that have entered the cell cycle after stimulation by mitogens. The difference is seen after cell treatment with buffers at pH 1.5 (1.3-1.9 range) followed by staining with acridine orange at pH 2.6 (2.3-2.9). Under these conditions the red metachromatic fluorescence of the acridine orange-DNA complex is higher in quiescent cells than in the cycling lymphocytes while the orthochromatic green fluorescence is higher in the cycling, interphase cells. The results suggest that DNA in condensed chromatin of quiescent lymphocytes (as in metaphase chromosomes) is more sensitive to acid-denaturation than DNA in dispersed chromatin of the cycling interphase cells. The phenomenon is used for flow cytometric differentiation between G0 and G1 cells and between G2 and M cells. In contrast to normal lymphocytes the method applied to neoplastic cells indicates the presence of cell subpopulations with condensed chromatin but with DNA content characteristic not only of G1 but also of S and G2 cells. The possibility that these cells represent quiescent (resting) subpopulations, arrested in G1, S and/or G2, is discussed.