Necessity of interleukin-1β converting enzyme cascade in taxotere-initiated death signaling

Abstract

Taxotere is a new type chemotherapeutic agent which targets tubulin. In the present study, we investigated the molecular machinery of taxotere-initiated death signaling. Taxotere induced cell death in mouse fibroblast L929 cells. Cell morphological analysis revealed that this effect showed characteristics of apoptotic and necrotic cell death. To further examine taxotere-induced cell death, we investigated the direct involvement of caspase. When cells were pretreated with the synthesized tetrapeptide inhibitor of caspase, YVAD-CHO (Ac–Tyr–Val–Ala–Asp-aldehyde: inhibitor of interleukin-1β converting enzyme (ICE) subfamily) or DEVD-CHO (Ac–Asp–Glu–Val–Asp-aldehyde: inhibitor of CPP32 subfamily), taxotere-induced cell death was prevented. In addition, time course experiments demonstrated that activation of the ICE subfamily preceded activation of the CPP32 subfamily in taxotere-initiated death signaling, suggesting the direct involvement of the ICE cascade in taxotere-initiated death signaling. On the basis of these results, we suggest that taxotere causes the initiation of ICE cascade in its death signaling pathway and that the down-stream site of taxotere-initiated death signaling is the same as that of other chemotherapeutic agents.

Introduction

Programmed cell death is an essential process for cell homeostasis and is encountered in various physiological phenomena and disease states (Wyllie et al., 1980; Nagata and Golstein, 1995; Suzuki et al., 1996a, Suzuki et al., 1996b). Two morphologically distinct processes have been identified, namely `apoptotic cell death' and `necrotic cell death' (Wyllie et al., 1980). Apoptotic cell death is characterized by condensation and fragmentation of nuclei, loss of plasma membrane microvilli, condensation of cytoplasm and fragmentation of chromosomal DNAs into 180 bp oligomers, while necrotic cell death is predominantly characterized by cell membrane destruction, such as abnormal cell membrane permeability (Wyllie et al., 1980; Shimizu et al., 1996; Suzuki, 1997). Cell death is induced by various factors and conditions, and a number of drugs, including many chemotherapeutic agents, also initiate death signaling in target cells.

The CED-3 death gene, identified from Caenorhabditis elegans, shows high similarity to interleukin-1β converting enzyme (ICE) (Yuan et al., 1993). The important role of ICE/CED-3 as the death mediator in intracellular death signaling has been demonstrated (Miura et al., 1993). Recently, 10 genes showing a close similarity to ICE/CED-3 (ICE/CED-3 homologue) have been identified and termed caspase (Alnemri et al., 1996). There are three subfamilies of caspase, termed ICE-, CPP32- and ICH-1-subfamily. The involvement of the ICE cascade in death receptor Fas-initiated death signaling has been reported (Enari et al., 1996). Both ICE and CPP32 subfamilies are activated in the process of Fas-initiated death signaling, with activation of the ICE subfamily preceding that of the CPP32 subfamily (Enari et al., 1996). Because the ICE or CPP32 subfamily catalyze the proteolysis of pro-interleukin-1β (IL-1β) or poly (ADP-ribose) polymerase (PARP), the aldehyde-conjugated synthesized tetrapeptide which codes the cleavage site of pro IL-1β (Ac–Tyr–Val–Ala–Asp-aldehyde: YVAD-CHO) or PARP (Ac–Asp–Glu–Val–Asp-aldehyde: DEVD-CHO) can be used as a specific inhibitor of the ICE or CPP32 subfamily (Fernandes-Alnemri et al., 1994; Enari et al., 1995; Nicholson et al., 1995; Hasegawa et al., 1996).

We and others have reported that caspase plays a dominant role in cell death induced by chemotherapeutic agents, such as camptothecin and its derivative CPT-11, VP-16 and adriamycin (Mashima et al., 1995; Suzuki and Kato, 1996; Suzuki et al., 1996c, Suzuki et al., 1997a). These reagents induce cell death by inhibition of type I or II topoisomerase or by insertion into DNA (Glisson and Ross, 1987; Mashima et al., 1995; Suzuki and Kato, 1996; Suzuki et al., 1996c, Suzuki et al., 1997a). In contrast, the complex plant alkaloid taxol has a unique taxane ring structure which appears to affect microtubule binding to the polymeric microtubule form of tubulin and has been developed as a chemotherapeutic agent (Woods et al., 1995). Taxol induces cell death by triggering machinery different from that of other chemotherapeutic agents. We therefore investigated the molecular machinery of another tubulin-targeting chemotherapeutic agent taxotere.