Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation

Abstract

The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6+/-0.9 to 9.1+/-0.5 mF/m(2) 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.

Fig. 1

Flow cytometry study of DNA content histograms for HL-60 cells showing cell frequency versus DNA content from 0 to 10 h after 100 μg/ml GEN treatment.

Fig. 2

Flow cytometry results of the Annexin V assay on HL-60 cells from 0 to 6 h after 100 μg/ml GEN treatment. A significant Annexin V-positive subpopulation relative to 0 h begins to appear at 2 h.

Fig. 3

Changes in cell DEP properties following apoptotic initiation with and without the presence of zVAD-fmk. Untreated (Control) cells and cells treated with 2% DMSO are also indicated. The bars show the standard deviation.

Fig. 4

Changes in cell crossover frequencies following apoptotic initiation. The DEP crossover frequencies for necrotic cells were greatly different from normal or apoptotic cells.

Fig. 5

DEP characteristics of HL-60 cells expressed as r× f_crossover versus the suspending medium conductivity σ_med following treatment by GEN at 100 μg/ml. Straight lines show least-square linear fits to the data.

Fig. 6

SEM pictures of HL-60 cells at magnification ×2500 at 0 (A), 1 (B), 2 (C), and 4 h (D) following GEN treatment reveal the evolution of changes in cell membrane morphology as apoptosis progressed.

Fig. 7

Use of the change in DEP crossover frequency to detect apoptosis is compared with DNA histogram and Annexin V staining methods. These results show that the DEP crossover method is the most sensitive, especially at the early time points.