Zinc Inhibits UV Radiation-induced Apoptosis but Fails to Prevent Subsequent Cell Death

Abstract

Zinc ions inhibit the morphological and DNA fragmentation features of apoptosis in a number of systems. HL-60 cells pretreated with zinc and exposed to UV irradiation maintained their normal morphology for up to 8 h, whereas non-zinc-treated cells underwent extensive apoptosis. Zinc pretreatment also inhibited both single and double-stranded DNA fragmentation, which is characteristic of apoptosis. The most effective zinc concentration that blocked apoptosis over short incubation periods (up to 8 h) was also the most toxic over extended time periods (> or = 12 h). The mechanism of cell death at these longer time periods was akin to necrosis, but occurred in the absence of any DNA fragmentation. The effects of the nuclease inhibitor aurintricarboxylic acid (ATA) was also examined on UV-induced apoptosis in HL-60 cells. ATA had no toxic effects over the concentration range tested, but also failed to prevent DNA fragmentation in whole cells. Further analysis showed that it effectively inhibited DNA fragmentation in isolated nuclei.

Reactive oxygen intermediate(s) (ROI): Common mediator(s) of poly(ADP‐ribose)polymerase (PARP) cleavage and apoptosis

“…Furthermore, a number of antioxidant compounds prevented DNA fragmentation and apoptosis in these cell lines [23,24]. As mentioned above, PARP cleavage has been linked to endonuclease activity and DNA fragmentation.…”

“…Apoptotic cells were identified by examining the changes in forward and side scatter using a FACScan flow cytometer (Becton-Dickinson, USA) equipped with Lysis II software [23]. …”

“…Apoptosis was induced in HL-60 and U937 cells by exposure to UV irradiation as previously described [23]. Briefly, cells (106/ml) were seeded in polystyrene 24-well plates (Nunc, Roskilde Denmark) and exposed from below to a 302 nm UV transilluminator source at a distance of 2.5 crn for 10 min at room temperature.…”

“…As a positive control for PARP cleavage, Jurkat T-cells were treated with 1 ~tg/ml antiFas antibody for 2 h (anti-Fas CH-11 IgM, Medical and Biological Laboratories, USA). Anti-oxidants were prepared as previously described [22][23][24] and the ones chosen were zinc chloride, 1 M in 0.15 saline, pyrrolidine dithiocarbamate (PDTC), 1 M in distilled water, butylated hydroxyanisole (BHA) and phenanthroline (Phen. ), 1 M in ethanol, 2,2,6,6-tetramethylpiperidinoxyl (Tempo.)…”

“…To establish further that PARP cleavage and DNA fragmentation follow the same common pathway to apoptosis, protease inhibitors were tested against both PARP cleavage and DNA fragmentation. Given the protection observed with anti-oxidants against DNA fragmentation and apoptosis in these cells [22][23][24], we screened a range of anti-oxidants for the ability to prevent PARP cleavage. Our results indicate that both of these events (PARP cleavage and DNA fragmentation) occur following ROI production and suggest that ROI may act as common mediators, for PARP cleavage, DNA fragmentation and the eventual apoptosis of these leukaemic cells.…”