THE ROLE OF PROTEOLYSIS IN T-CELL APOPTOSIS TRIGGERED BY CHELATION OFINTRACELLULAR ZN2+

Our previous work showed that chelation of intracellular Zn2+ with N,N ,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) induces apopt osis in rat thymocytes. The molecular mechanism involved in TPEN-trigg ered apoptosis remains unknown, except that it is a Ca2+-independent p rocess. In the present study, we show that TPEN is unable to induce DN A fragmentation when added to isolated thymocyte nuclei, indicating th at activation of a cytoplasmic component is essential for TPEN-induced apoptosis, Since cytosolic proteases related to interleukin-1 beta-co nverting enzyme (ICE) are implicated as key activators of apoptos is i n many different systems, we investigated the possible involvement of such proteases in TPEN-induced apoptosis, We found that treatment of t hymocytes with TPEN caused an early degradation of nuclear poly(ADP-ri bose) polymerase (PARP) and lamin prior to DNA cleavage. This could be inhibited by Z-Val-Ala-Asp-chloromethylketone (VADcmk), an inhibitor of ICE-like proteases, but not by an inhibitor of Ca2+-regulated serin e protease, Jurkat T cells also underwent extensive DNA fragmentation when incubated with TPEN, A cytosolic fraction, prepared from TPEN-tre ated Jurkat cells, produced extensive DNA fragmentation when applied t o isolated thymocyte nuclei, whereas the cytoplasmic extract from untr eated cells was ineffective either alone or together with TPEN, The ap optosis-inducing activity in cytosolic fraction from TPEN-treated Jurk at cells was blocked by incubating cells in the presence of VADcmk or another inhibitor of ICE-like proteases, Ac-Asp-Glu-Val-Asp-aldehyde ( DEVD-CHO), which has been found to competitively inhibit CPP32/apopain . An increase in enzyme activity that cleaves Ac-Asp-Glu-Val-Asp-7-ami no-4-methylcoumarin (DEVD-AMC), a fluorogenic substrate of CPP32/apopa in and Mch3 alpha, was detected in TPEN-treated thymocytes and Jurkat cells, In addition, the proteolytic cleavage of CPP32 resulting in the formation of two active fragments (p17 and p12) was observed in cytos olic extracts from TPEN-treated Jurkat cells, but not in extracts whic h were prepared from cells treated with TPEN in the presence of VADcmk or DEVD-CHO. Our results suggest that activation of cytosolic ICE-lik e proteases is an essential step in TPEN-induced apoptosis, and that C PP32/apopain is critically involved in this process.