Features of DNA oligonucleosomal fragmentation in human tumor cell lines and its detection by flow cytometry: utility and limitations.

Cultured HL-60, HeLa S3 and WiDr cells were treated with various doses of ethanol, then subjected to flow cytometry and gel electrophoresis of cellular DNA. On electrophoresis of DNA from HL-60 cells treated with 0.5 or 1.0 mM ethanol, a ladder pattern was recognized after 3 h. At higher doses of ethanol (2.0 and 5.0 mM), a smear pattern resulted. On flow cytometry, however, A0 cells (lower fluorescence level than G0+G1 cells) were noted from 0.5 to 5.0 mM ethanol. The observation of A0 cells at higher doses indicated loss of DNA after random DNA degradation. HeLa S3 and WiDr cells were partially detached from flasks after administration of ethanol and separated into adherent and non-adherent categories. In DNA from non-adherent HeLa S3 cells treated with 0.5 mM ethanol, a ladder pattern was observed after 24 h. On flow cytometry, prior to the appearance of A0 cells, an accumulation in the G2 + M-phase became obvious after 3 h. Increased mitotic indices indicated that this phenomenon was due to M-phase arrest. Adherent HeLa S3 cells showed no DNA oligonucleosomal fragmentation or A0 cells. These findings indicate that detection of A0 cells by flow cytometry is not proof of cell death by DNA oligonucleosomal fragmentation.