BACKGROUND: Thymocyte apoptosis has been shown to be regulated by intracellular levels of cations. Elevation of [Ca2+]i can induce cell death by apoptosis, whereas, Zn2+ prevents it. EXPERIMENTAL DESIGN: A membrane 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 apoptosis that were assessed included: chromatin degradation into oligosomal-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 established by competition with exogenous cations. FACS analysis was employed to determine the phenotype of the TPEN-sensitive thymocyte populations. RESULTS: TPEN induced DNA fragmentation within 4 hours of exposure before the onset of cell death (6 hours). Addition of equimolar exogenous Zn2+ or Cu2+, but not Mn2+ or Fe2+, at the initiation of culture prevented TPEN-induced apoptosis. A membrane impermeable chelator, diethylenetriaminepentaacetic acid, did not induce thymocyte apoptosis indicating that chelation of intracellular Zn2+ was required to trigger DNA fragmentation. The identity of the critical intracellular Zn(2+)-binding site(s) is currently unknown, but it appears that increased thymocyte [Ca2+]i may displace Zn2+ from these intracellular sites. TPEN treatment resulted in the depletion of thymocytes having a mature phenotype 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 or selection pathway during thymopoiesis.
BACKGROUND: Thymocyte apoptosis has been shown to be regulated by intracellular levels of cations. Elevation of [Ca2+]i can induce cell death by apoptosis, whereas, Zn2+ prevents it. EXPERIMENTAL DESIGN: A membrane 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 apoptosis that were assessed included: chromatin degradation into oligosomal-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 established by competition with exogenous cations. FACS analysis was employed to determine the phenotype of the TPEN-sensitive thymocyte populations. RESULTS:TPEN induced DNA fragmentation within 4 hours of exposure before the onset of cell death (6 hours). Addition of equimolar exogenous Zn2+ or Cu2+, but not Mn2+ or Fe2+, at the initiation of culture prevented TPEN-induced apoptosis. A membrane impermeable chelator, diethylenetriaminepentaacetic acid, did not induce thymocyte apoptosis indicating that chelation of intracellular Zn2+ was required to trigger DNA fragmentation. The identity of the critical intracellular Zn(2+)-binding site(s) is currently unknown, but it appears that increased thymocyte [Ca2+]i may displace Zn2+ from these intracellular sites. TPEN treatment resulted in the depletion of thymocytes having a mature phenotype 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 or selection pathway during thymopoiesis.
Authors: A Takahashi; E S Alnemri; Y A Lazebnik; T Fernandes-Alnemri; G Litwack; R D Moir; R D Goldman; G G Poirier; S H Kaufmann; W C Earnshaw Journal: Proc Natl Acad Sci U S A Date: 1996-08-06 Impact factor: 11.205