Increased uptake of vital dye Hoechst 33342 during S phase in synchronized HeLa S3 cells.

Abstract

Uptake of the vital dye Hoechst 33342 (HO342) was studied in HeLa S3 cells synchronized with hydroxyurea or thymidine. Cell monolayers were incubated with 0.05-0.5 micrograms/ml HO342 for 1-10 min at 37 degrees C, and the fluorescence intensity of DNA-bound intracellular dye was then measured in a flow cytometer. Modal fluorescence intensity normalized to DNA content was used as a measure of the dye uptake. No efflux of the dye from vitally stained cells was observed during 2 hr at 37 degrees C in a monolayer or at 20 degrees C in a suspension. HO342 uptake increased within in 1 hr after the renewal of progression (early S phase), achieved a maximum level in 2 hr (mid-S phase) and returned to the initial level in 4 hr (late S-G2 phase). The pattern of increased uptake of HO342 was similar in cells synchronized with either hydroxyurea or with thymidine. To estimate the relationship between fluorescence and dye uptake, the amount of dye released by Triton X-100 after vital staining was measured. The method for this measurement was based on the simultaneous addition of chicken erythrocytes ("indicator cells") with Triton X-100. Fluorescence intensity of chicken erythrocytes was significantly higher in the mixture containing mid-S phase than in the mixture containing asynchronous or late S-G2 cells. Thus the fluorescence after vital staining with HO342 reflected HO342 uptake and, possibly, fluctuations of membrane permeability. The period of maximal HO342 uptake in synchronized cells was also characterized by a a high sensitivity of cytotoxic effects of adriamycin. Uptake of HO342 at nonsaturating concentrations may be used as a marker of cell sensitivity and resistance to a number of cytotoxic compounds.