Tetsuo Mashima

Actin cleavage by CPP-32/apopain during the development of apoptosis

Interleukin-1β-converting enzyme (ICE)/ced-3 family proteases play key roles in apoptosis. However, cellular substrates for ICE family proteases involved in apoptosis are not well understood. We previously showed that actin is cleaved in vitro by an ICE family protease, distinct from ICE itself, which is activated during VP-16-induced apoptosis. In this report, we demonstrate that the actin-cleaving ICE-family protease in the apoptotic cell extract is the activated CPP-32/apopain. CPP-32 effectively cleaves actin protein to 15u2009kDa and 31u2009kDa fragments. Studies with an antibody raised against Gly-Gln-Val-Ile-Thr peptide, the N-terminal sequence of the cleaved 15u2009kDa actin fragment, showed that actin is also cleaved in vivo during the development of apoptosis. Moreover, Benzyloxycarbonyl-Glu-Val-Asp-CH2OC(O)-2,6,-dichlorobenzene (Z-EVD-CH2-DCB), a selective inhibitor of CPP-32(-like) protease, efficiently inhibited the cleavage of actin and the apoptosis of VP-16-treated U937 cells. Our present results indicate that actin is the substrate of CPP-32/apopain(-like) protease both in vitro and in vivo and suggest the role of actin in the control of cell growth and apoptosis.

c-Jun NH2-terminal Kinase-mediated Activation of Interleukin-1β Converting Enzyme/CED-3-like Protease during Anticancer Drug-induced Apoptosis

Upon treatment with various anticancer drugs, myeloid leukemia U937 cells undergo apoptosis. In this study, we found that either etoposide (VP-16) or camptothecin (CPT) activated c-Jun N-terminal kinase 1/stress-activated protein kinase (JNK1/SAPK), transient c-jun expression, and ICE (interleukin-1β converting enzyme)/CED-3-like proteases in U937 cells. Phorbol ester-resistant U937 variant, UT16 cells, displayed a decreased susceptibility to apoptosis induced by these drugs. The drugs did not cause JNK1 activation, c-jun expression, nor activation of ICE/CED-3-like proteases in UT16 cells. As reported previously, benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-Asp), a preferential inhibitor of ICE/CED-3-like proteases, blocked the apoptosis of U937 cells. Interestingly, however, Z-Asp did not inhibit JNK1 activation in either VP-16- or CPT-treated U937 cells. The JNK1 antisense oligonucleotides diminished protein expression of JNK1 and inhibited drug-induced apoptosis of U937 cells, whereas sense control oligonucleotides did not. Consistent with this observation, the antisense oligonucleotide-treated cells did not respond to VP-16 or CPT with Z-Asp-sensitive proteases. These results indicate that JNK1 triggers the DNA damaging drug-induced apoptosis of U937 cells by activating Z-Asp-sensitive ICE/CED-3-like proteases.

Genetically recessive mutant of human monocytic leukemia U937 resistant to tumor necrosis factor-α-induced apoptosis

Tumor necrosis factor‐α (TNF‐α) is a cytokine that induces apoptosis in various cell systems by binding to the TNF receptor (TNFR). To study TNF‐α‐induced apoptosis, we isolated and characterized a novel TNF‐α‐resistant variant, U937/TNF clone UA, from human monocytic leukemia U937 cells. The UA cells resist apoptosis induced by TNF‐α and anti‐Fas antibody but not by anticancer drugs, such as VP‐16 and Ara‐C. Somatic cell hybridization between U937 and UA showed that apoptosis resistance to TNF‐α in UA was genetically recessive. The hybridization analysis also showed that UA and another recessive mutant clone, UC, belong to different complementation groups in TNF‐α‐induced apoptosis signaling. In UA cells, TNF‐α‐induced disruption of mitochondrial membrane potential and CPP32 activation were abrogated. Expression of TNFR, Fas, and Bcl‐2 family proteins was not changed in UA cells. These results suggest that the apoptosis resistant UA cells could have a functional defect in apoptosis signaling from the TNFR to mitochondria and interleukin‐1β converting enzyme (ICE) family protease activation. UA cells could be used to study signaling linkage between cell death‐inducing receptor and mitochondria. J. Cell. Physiol. 174:179–185, 1998.

Apoptosis resistance in tumor cells.

Various antitumor agents induce apoptotic cell death in tumor cells. Since the apoptosis program in tumor cells plays a critical role in the chemotherapy-induced tumor cell killing, it is suggested that the defect in the signaling pathway of apoptosis could cause a new form of multidrug resistance in tumor cells. This article describes the recent findings concerning the mechanisms of chemotherapy-induced apoptosis and discusses the implication of apoptosis resistance in cancer chemotherapy.

Activation of Caspase Protease During Apoptosis in Ovarian Cancer Cells

As a genetically controlled program, apoptosis has important roles in a variety of biological processes. The realization that chemotherapy can also induce apoptosis in some cancer cells both in vitro and in vivo indicates apoptosis may play a very important role in cancer and cancer therapy (1,2). Many of the molecules that participate in the apoptotic cell suicide have been identified. At the heart of this pathway are a family of cysteine proteases, the caspases (3).