CHELATION OF INTRACELLULAR ZINC TRIGGERS APOPTOSIS IN MATURE THYMOCYTES

BACKGROUND: Thymocyte apoptosis has been shown to be regulated by intr acellular levels of cations. Elevation of [Ca2+]i can induce cell deat h by apoptosis, whereas, Zn2+ prevents it. EXPERIMENTAL DESIGN: A memb rane permeable metal ion chelator, N,N,N'N',-tetrakis (2-pyridylmethyl )ethylenediamine (TPEN), was used to examine the role of intracellular zinc in thymocyte (rat and human) apoptosis. Characteristics of apopt osis that were assessed included: chromatin degradation into oligosoma l-sized fragments and nuclear condensation. The necessity for protein synthesis in TPEN-induced apoptosis was ruled out using the inhibitors , cycloheximide and emetine. Metal ion specificity for TPEN was establ ished by competition with exogenous cations. FACS analysis was employe d to determine the phenotype of the TPEN-sensitive thymocyte populatio ns. RESULTS: TPEN induced DNA fragmentation within 4 hours of exposure before the onset of cell death (6 hours). Addition of equimolar exoge nous Zn2+ or Cu2+, but not Mn2+ or Fe2+ at the initiation of culture p revented TPEN-induced apoptosis. A membrane impermeable chelator, diet hylenetriaminepentaacetic acid, did not induce thymocyte apoptosis ind icating that chelation of intracellular Zn2+ was required to trigger D NA fragmentation. The identity of the critical intracellular Zn2+-bind ing site(s) is currently unknown, but it appears that increased thymoc yte [Ca2+]i may displace Zn2+ from these intracellular sites. TPEN tre atment resulted in the depletion of thymocytes having a mature phenoty pe with respect to CD3, CD4, and CD8. Moreover, lymph node cells were more sensitive to TPEN than thymocytes. CONCLUSIONS: These experiments show that Zn2+ chelation has disparate effects on immature and mature T cells, and suggest that zinc availability controls the cell death o r selection pathway during thymopoiesis.