LACK OF CA2+ INVOLVEMENT IN THYMOCYTE APOPTOSIS INDUCED BY CHELATION OF INTRACELLULAR ZN2+

BACKGROUND: Chelation of intracellular Zn2+ with N, N, N', N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) triggers apoptosis predominantly in mature thymocytes. A rise in intracellular-free Ca2+ concentration ([Ca2+](i)) has been associated with induction of thymocyte apoptosis by a variety of agents. Zn2+ can affect intracellular Ca2+ homeostasis, so the aim of this study was to investigate whether TPEN-induced apoptosis is mediated by Ca2+ signalling. EXPERIMENTAL DESIGN: The possible role of Ca2+ in TPEN-induced apoptosis was investigated. Apoptotic markers used were DNA cleavage into oligonucleosomal fragments and formation of apoptotic nuclei. The change in [Ca2+](i) in thymocytes after TPEN treatment was monitored using the fluorescence Ca2+ indicator dye, fura-a. The requirement of an increase in [Ca2+](i) for TPEN-induced apoptosis was examined in thymocytes preloaded with the intracellular Ca2+ buffer, bis-(o-aminophenoxy)-ethane-N, N,N',N'-tetraacetic acid, or incubated in nominally Ca2+-free medium supplemented with EGTA. The effect of an increase in [Ca2+](i) on TPEN-induced DNA fragmentation was studied by using thapsigargin or ionomycin to elevate [Ca2+](i) in thymocytes. RESULTS: No increase in [Ca2+](i) could be detected before DNA fragmentation in thymocytes during TPEN treatment. Buffering intracellular Ca2+ with bis-(o-aminophenoxy)-ethane-N, N, N', N'-tetraacetic acid or incubating cells in nominally Ca2+-free medium with EGTA had little effect on TPEN-induced DNA fragmentation and formation of apoptotic nuclei. Increasing thymocyte [Ca2+](i) with thapsigargin or ionomycin administration during TPEN treatment resulted in an additive effect on TPEN-induced DNA fragmentation in thymocytes. CONCLUSIONS: Our study shows that TPEN induces apoptosis in thymocytes by Ca2+-independent mechanisms and that apoptosis triggered by Zn2+ chelation and [Ca2+](i) elevation affects distinct thymocyte subpopulations.