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Requirement of caspase and p38MAPK activation in zincâinduced apoptosis in human leukemia HLâ60 cells
- Kondoh, Masuo, Tasaki, Emi, Araragi, Saeko, Takiguchi, Masufumi, Higashimoto, Minoru, Watanabe, Yoshiteru, Sato, Masao
- European journal of biochemistry 2002 v.269 no.24 pp. 6204-6211
- DNA, NAD ADP-ribosyltransferase, apoptosis, caspases, chelating agents, cytotoxicity, dose response, humans, leukemia, mitogen-activated protein kinase, permeability, phosphorylation, proteolysis, zinc
- Zinc (Zn), an endogenous regulator of apoptosis, and has abilities both to induce apoptosis and inhibit the induction of apoptosis via the modulation of caspase activity. Due to the multifunctions of Zn, the intracellular Zn level is strictly regulated by a complex system in physiological and pathological conditions. The commitment of Zn to the regulation of apoptosis is not fully understood. In the present study, we investigated the role of intracellular Zn level in the induction of apoptosis in human leukemia cells (HLâ60 cells) using a Zn ionophore [pyrithione (Py)]. Treatment of HLâ60 cells with Zn for 6 h in the presence of Py (1âÂµm) exhibited cytotoxicity in a Zn doseâdependent manner (25â200âÂµm). Necrotic cells, assayed by trypan blue permeability, increased in number in a Zn doseâdependent fashion (50â100âÂµm), but the appearance of apoptotic cells, assayed by formation of a DNA ladder and terminal deoxynucleotidyltransferaseâmediated dUTPâbiotin nick endâlabeling method, peaked at 25âÂµm, suggesting the dependence of intracellular Zn level on the execution of apoptosis. In fact, treatment with Py resulted in increases in intracellular Zn levels, and N,N,Nâ²,Nâ²âtetrakis (2âpyridylmethyl)ethylenediamine, a cellâpermeable Zn chelator, inhibited DNA ladder formation induced by Py/Zn treatment (1âÂµm Py and 25âÂµm Zn). Py/Zn treatment activated the caspases, as assessed by the proteolysis of poly(ADPâribose) polymerase (PARP), which is a substrate of caspase, and activated p38 mitogenâactivated protein kinase (p38MAPK), which is a transducer of apoptotic stimuli to the apparatus of the apoptosis execution. ZâAspâCH2âDCB, a broadâspectrum inhibitor of caspase, attenuated proteolysis of PARP and DNA ladder formation by Py/Zn, indicating that apoptosis induced by Py/Zn is mediated by caspase activation. The p38MAPKâspecific inhibitor SB203580 also inhibited induction of apoptosis by Py/Zn. Although SB203580 suppressed the proteolysis of PARP, ZâAspâCH2âDCB did not inhibit the phosphorylation of p38MAPK, raising the possibility that apoptosis triggered by Py/Zn might be mediated by the p38MAPK/caspase pathway.