DNA damage, cytotoxic effect and cell‐cycle perturbation of Hoechst 33342 on L1210 cells in vitro

Abstract

This study was designed to evaluate the effects of vital dye Hoechst 33342 (HO 33342), at concentrations used to obtain a good DNA histogram resolution, on DNA integrity, cell growth, and cell-cycle phase distribution of Ll210 cells. HO 33342 exposure for 2 h, at 37°C produced DNA single-strand breaks as assessed by the method of alkaline elution. DNA single-strand breaks were concentration dependent (in the range .5-5 pg/ml) and increased significantly when HO 33342 (0.5-1.5 pg/ml) was associated with exposure in a flow cytometer to U.V. laser beam illumination.HO 33342 produced a cytotoxic effect on cell growth even at the concentration of 0.5 pg/ ml-a concentration ten-fold smaller than those required to obtain a good DNA histogram resolution. HO 33342 produced a severe block of the cells in the Gz-M phase of the cell cycle already evident 24 h after stain exposure and continuing up to 144 h after start of recovery. A new polyploid cell population (with a 4 c DNA content) not present in the unstained cells was already evident 24 h after dye exposure. The data shown in the present paper would imply caution in using sorted cells stained with HO 33342 dye for biological, biomedical, and pharmacological studies.Key terms: Flow cytometry, danger in staining Among the DNA probes used in flow cytometry, the bis-benzirnide Hoechst 33342 (HO 33342) dye is of particular interest as it permits vital staining of the DNA structure of cells. HO 33342 was introduced for flow cytometry analysis of living cells by Ardnt-Jovin and Jovin (1); HO 33342 binds to DNA without intercalating, being specific for the A-T sequences (11-15). Upon binding to DNA its quantum efficiency is increased 60-fold and 97% of the fluorescent emission from dyetreated living cells originates from the nucleus (1). Studies with synthetic polynucleotides also showed that not only is a sequence of three A-T pairs necessary for a high dye-binding affinity, but their structural arrangement in a helix is also of importance (18). HO 33342 has been used in many investigations including analysis of DNA content in living cells (1,10,12), sorting of cells with respect to their fluorescence in terms of the DNA content or cell-cycle phase distribution (8,17), and in measures of cell proliferation after drug treatment or irradiation (17J9).Due to its spectral fluorescence characteristics it can be used in conjunction with rhodamine for rapid analysis of cellular DNA and proteins (3, or Despite preliminary studies which suggested that this DNA probe was suitable for vital DNA staining and sorting without affecting cellular integrity (l), it now appears that cellular toxicity and cell-cycle perturbations due to HO 33342 vary considerably from one cell line to another, and that the concentration of this dye required to obtain a good DNA histogram resolution is also cell line dependent (3,6,9,20,22,23,26).Duran and Olive have also shown that concentrations greater than 5 pM of HO 33342 were mutagenic on Chinese hamster V79 lung fibroblast cells, whereas DNA damage, a...

Tissue distribution of adoptively transferred adherent lymphokine-activated killer cells assessed by different cell labels

“…After 51Cr, H33342 was the least attractive of the labelling methods evaluated. Even low doses of H33342 have been reported to interfere with cell proliferation [6] and in our hands, spontaneous loss of H33342 from the A-LAK cells led to an underestimation of the cell recovery (Table 4). Furthermore, the extensive uptake by liver phagocytes of H33342 released from dead cells, as previously described [2], impedes the distinction between viable and dead A-LAK cells.…”

Adult Kidney Tubular Cell Population Showing Phenotypic Plasticity, Tubulogenic Capacity, and Integration Capability into Developing Kidney

“…However, it also was demonstrated previously that Hoechst 33342 dye has cytotoxic effects on cultured lymphocytes (35). This raises the possibility that Hoechst high /non-LRTC failed to form tubular structures or integrate into the developing nephron because of Hoechst toxicity.…”

A simple electronic volume cell sorter for clonogenicity assays

“…3) is very similar to that obtained by mitotic shaking (241, drug blockage (19,25), centrifugal elutriation (14,21), or DNA content analysis and sorting (13). One advantage of volume sorting over use of DNA content analysis with vital dyes is the lack of the cytotoxicity, mutagenicity, and cell cycle perturbation induced by DNA staining and laser illumination (2,4,21). The data in this article and in previous ones by our group (5,7) have demonstrated that there is essentially no toxicity associated with volume cell sorting.…”