Rumi Ishisaka

Activation of caspase‐3‐like protease by digitonin‐treated lysosomes

Apoptosis, a naturally occurring programmed cell death or cell ‘suicide’, has been paid much attention as one of the critical mechanisms for morphogenesis and tissue remodeling. Activation of cysteine aspartases (caspases) is one of the critical steps leading to apoptosis. Although a mitochondria‐mediated pathway has been postulated to be one of the activation mechanism of caspase‐3, another subcellular compartment might be involved in the activation of the enzyme. The present study shows that the supernatant fraction of digitonin‐treated lysosomes strongly activates Ac‐DEVD‐CHO inhibitable caspase‐3‐like protease. Activation of caspase‐3‐like protease by digitonin‐treated lysosomal fractions was specifically suppressed by leupeptin and E‐64, inhibitors of cysteine protease. These results indicate that leakage of lysosomal cysteine protease(s) into the cytosolic compartment might be involved in the activation of caspase‐3‐like protease.

C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and targeted to mitochondria

To detect the posttranslational N‐myristoylation of caspase substrates, the susceptibility of the newly exposed N‐terminus of known caspase substrates to protein N‐myristoylation was evaluated by in vivo metabolic labeling with [3H]myristic acid in transfected cells using a fusion protein in which the query sequence was fused to a model protein. As a result, it was found that the N‐terminal nine residues of the newly exposed N‐terminus of the caspase‐cleavage product of cytoskeletal actin efficiently direct the protein N‐myristoylation. Metabolic labeling of COS‐1 cells transiently transfected with cDNA coding for full‐length truncated actin (tActin) revealed the efficient incorporation of [3H]myristic acid into this molecule. When COS‐1 cells transiently transfected with cDNA coding for full‐length actin were treated with staurosporine, an apoptosis‐inducing agent, an N‐myristoylated tActin was generated. Immunofluorescence staining coupled with MitoTracker or fluorescence tagged‐phalloidin staining revealed that exogenously expressed tActin colocalized with mitochondria without affecting cellular and actin morphology. Taken together, these results demonstrate that the C‐terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N‐myristoylated upon caspase‐mediated cleavage during apoptosis and targeted to mitochondria.

Mechanism of α-tocopheryl succinate-induced apoptosis of promyelocytic leukemia cells

Selective induction of apoptosis in tumor cells is important for treating patients with cancer. Because oxidative stress plays an important role in the process of apoptosis, we studied the effect of α-tocopheryl succinate (VES) on the fate of cultured human promyelocytic leukemia cells (HL-60). The presence of fairly low concentrations of VES inhibited the growth and DNA synthesis of HL-60 cells, and also induced their apoptosis via a mechanism that was inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), an inhibitor of pan-caspases. VES activated various types of caspases, including caspase-3, 6, 8, and 9, but not caspase-1. VES triggered the reaction leading to the cleavage of Bid, a member of the death agonist Bcl-2 family, and released cytochrome c (Cyt.c) from the mitochondria into the cytosol by a z-VAD-fmk-inhibitable mechanism. VES transiently increased the intracellular calcium level [Ca2+]i and stimulated the release of Cyt.c in the presence of inorganic phosphate (Pi). However, high concentrations of VES (∼100 μM) hardly induced swelling of isolated mitochondria but depolarized the mitochondrial membrane potential by a cyclosporin A (CsA)-insensitive mechanism. These results indicate that VES-induced apoptosis of HL-60 cells might be caused by activation of the caspase cascade coupled with modulation of mitochondrial membrane function.

Thyroxine enhancement and the role of reactive oxygen species in tadpole tail apoptosis

Our objective is to clarify the role of reactive oxygen species (ROS) in the atrophying tail of anuran tadpoles (tail apoptosis). Changes in catalase, superoxide dismutase (SOD) and caspase activity, genomic DNA, and nitric oxide (NO) generation were investigated biochemically using Rana japonica tadpole tails undergoing regression during thyroid hormone enhancement. DNA fragmentation and ladder formation with concomitant shortening of tadpole tail were induced by DL-thyroxine (T4) in culture medium. Catalase activity was also decreased by T4 treatment. T4 was also found to increase NO synthase (NOS) activity in cultured tadpole tail with concomitant increase in the concentration of NO2- plus NO3- (NOx) in the culture medium. Additional treatment with N-monomethyl-L-arginine (NMMA), a potent inhibitor of NOS, suppressed the enhancing effects of T4 on tail shortening and catalase activity reduction. It was also found that treatment with isosorbide dinitrate (ISDN), a NO generating drug, alone also had an enhancing effect on tail shortening and catalase activity reduction similar to that seen with T4. Both NO and an NO donor (ISDN) strongly suppressed catalase activity. Kinetic analysis revealed that catalase activity decreased and caspase-3-like activity increased during normal tadpole tail atrophy (apoptosis). These results suggested that T4 enhances NO generation, thereby strongly inhibiting catalase activity, resulting in an increase in hydrogen peroxide, and that the oxidative stress elicited by excess hydrogen peroxide might activate cysteine-dependent aspartate-directed protease-3 (caspase-3-like protease), which is thought to cause DNA fragmentation, leading to apoptosis.

Suppression of T3- and fatty acid-induced membrane permeability transition by l-carnitine

Cytochrome c (Cyt. c) is known to be released from the mitochondria into the cytosol by means of the membrane permeability transition (MPT) mechanism, thereby activating caspase cascade activity, and inducing cell apoptosis. Recently we reported that L-carnitine suppressed palmitoyl-CoA-induced MPT as well as apoptosis in some cell types (Biochem. Pharmacol, in press). In the present study T(3) was found to induce MPT and Cyt. c release, while cyclosporin A (CsA), bovine serum albumin (BSA) and L-carnitine were found to inhibit this action in a concentration-dependent manner. Similarly, long chain fatty acid (LCFA) also induced MPT and Cyt. c release, which was then inhibited by CsA, BSA and L-carnitine. From these results the authors postulate that T(3)-induced MPT is in part regulated by fatty acid metabolism through a dynamic balance between LCFAs and L-carnitine.

Transmembrane TNF (pro-TNF) is palmitoylated

Human transmembrane tumor necrosis factor (pro‐TNF) was examined for protein acylation. The cDNA encoding pro‐TNF was expressed in both COS‐1 cells and Sf9 cells and metabolic labeling with [3H]myristic or [3H]palmitic acid was attempted. The 17 kDa mature TNF secreted from the transfected cells was not labeled, whereas the 26 kDa pro‐TNF was specifically labeled with [3H]palmitic acid. The [3H]palmitic acid labeling of pro‐TNF was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation of a cysteine residue via a thioester bond. Site‐directed mutagenesis of the two cysteine residues residing in the leader sequence of pro‐TNF demonstrated that palmitoylation of pro‐TNF occurs solely at Cys‐47, located at the boundary between the transmembrane and cytoplasmic domains of pro‐TNF. Thus, pro‐TNF interacts with the plasma membrane via both its proteinaceous transmembrane domain and a lipid anchor.

Effects of a magnetic fields on the various functions of subcellular organelles and cells.

Magnetic fields (MF) are widely distributed in environment and their effects are increasing by the development of electrical machines. Several investigators reported that the MF might affect various functions of cells. However, an acceptable hypothesis has not yet been proposed. Thus, we studied the effects of weak MFs on various biological functions of cells, such as mitochondrial functions, stimulation dependent signal transduction of neutrophils, cell growth and transformation of HL-60 cells, H(2)O(2)-induced apoptosis and the expression of apoptotic genes in HL-60 cells. As a result of the study, a weak MF has scarcely any effects on various biological functions of cells. We also studied the direct effect of a static strong MF (SSMF, 600-2000 G) on the functions of cells or on Fe(2+)-induced lipid peroxidation and on reactive oxygen species (ROS) generation in oral polymorphonuclear leukocytes (OPMN) without stimulation using Ferrite magnets. The generation of ROS from OPMN was slightly inhibited but Fe(2+)-induced lipid peroxidation of biological membrane was slightly stimulated by exposure to the SSMF. At present, however, conclusive results have been neither obtained experimentally nor any acceptable idea proposed.

Biochemical Properties of Human Oral Polymorphonuclear Leukocytes

Polymorphonuclear leukocytes (PMN) isolated from the oral cavity of healthy human volunteers, spontaneously generated superoxide, nitric oxide (NO) and other reactive oxygen species (ROS) which exhibited strong luminol chemiluminescence (LCL). To understand the physiological roles of oral PMN (OPMN), biochemical properties of the cells were analyzed. Biochemical analysis revealed that OPMN were already primed under physiological conditions. Western blot analysis revealed that they strongly expressed the inducible type of NO synthase (NOS II) and exhibited the activity to catalyze tyrosine phosphorylation of various proteins including a 115 kDa protein (cbl product). OPMN also generated H2O2 and .OH by some superoxide dismutase (SOD)-sensitive mechanism and released myeloperoxidase (MPO). Kinetic analysis using specific inhibitors revealed that OCl- generated by OPMN was predominantly responsible for the enhanced LCL. During the incubation under standard culture conditions, OPMN underwent apoptosis which proceeded more rapidly than that of the circulating PMN (CPMN). Immunochemical analysis revealed that expression of apoptosis-related gene products, such as Bcl-2, Bcl-xL and Bax, was below detectable levels with both cell types. However, caspase-3 but not caspase-1 was markedly activated in OPMN. These results indicate that the primed OPMN spontaneously generate ROS and play an important role in the defense mechanism in the oral cavity and that the generated ROS activate caspase-3 thereby inducing apoptosis of the cells.

Involvement of Lysosomal Cysteine Proteases in Hydrogen Peroxide-induced Apoptosis in HL-60 Cells

Hydrogen peroxide is a well-known mediator of apoptosis. As a mechanism for H2O2-induced apoptosis, both a mitochondrial Cyt.c-dependent pathway and a lysosome-mediated pathway have been suggested. However, the relative roles of and the relation between these two pathways in H2O2-induced apoptosis remain to be discovered. In this study, to find the relative roles of the lysosomal and mitochondrial pathways, the effects of E-64-d, a cell-permeable inhibitor of lysosomal cysteine proteases, on apoptosis caused by H2O2 in HL-60 cells were investigated. It was found that the concentration of H2O2 strongly affected the inhibitory effect of E-64-d on the apoptosis in HL-60 cells: dose-dependent inhibition (up to 40%) of both DNA fragmentation and caspase-3 activation was observed when a high concentration of H2O2 (50 μM) was used to induce apoptosis, but no inhibitory effect was detected when a low concentration (10 μM) was used. Consistent with these observations, apparent lysosomal destabilization was observed only with 50 μM H2O2. The release of mitochondrial Cyt.c, in contrast, was observed at both 10 μM and 50 μM. These results indicated that the mitochondrial Cyt.c-mediated pathway predominates in the H2O2-induced apoptosis in HL-60 cells and the lysosomal mediated pathway is partially involved when high concentrations of H2O2 are used to induce apoptosis.

Effects of Milk and Colostrum on Superoxide Generation of Oral Polymorphonuclear Neutrophils and the Changes That Occur during Storage at Low Temperature

Human oral polymorphonuclear neutrophils (OPMN) generate reactive oxygen species even in the absence of stimulants. Because OPMN from newborn babies are exposed to colostrum and mature milk, the biological properties of these cells including the generation of reactive oxygen species might possibly be affected by the constituents of colostrum and milk. The present work reports the effects of colostrum and mature milk, including the effects of storage at low temperature, on superoxide generation by OPMN. Fresh colostrum and mature milk did not affect either endogenous or formyl-methionyl-leucyl-phenylalanine-induced generation of superoxide by OPMN. However, superoxide generation stimulated by phorbol myristate acetate or arachidonic acid was inhibited by colostrum and mature milk presumably due to binding of the ligands to milk proteins. During the storage of milk at 4°C, free forms of unsaturated long-chain fatty acids increased, and there was concomitant increase in the ability of milk to generate superoxide radicals in OPMN. Kinetic analysis suggested that colostrum and mature milk regulate superoxide generation by OPMN, thereby modulating the bactericidal activity of these cells in the oral cavity.