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.


METHODS
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-2. 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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-independent mechanisms and that apoptosis triggered by Zn2+ chelation and [Ca2+]i elevation affects distinct thymocyte subpopulations.
Jiang
S
S
Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
Chow
S C
SC
McCabe
M J
MJ
Orrenius
S
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D005029:Ethylenediamines / Q000494:pharmacology*; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013601:T-Lymphocytes / Q000187:drug effects / Q000502:physiology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D015032:Zinc / Q000378:metabolism*
D002614:Chelating Agents; D005029:Ethylenediamines; D013729:Terpenes; D015759:Ionomycin; D019284:Thapsigargin; D004247:DNA; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D002118:Calcium