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Y Tsujimoto

Publications and source records attributed to Y Tsujimoto.

At least 91 records · Page 5Linked to original sources
PubMed1997

Intracellular ATP levels determine cell death fate by apoptosis or necrosis.

Although apoptosis and necrosis are morphologically distinct manifestations of cell death, apoptosis and some necroses share common features in the death signaling pathway involving functional steps of death-driving interleukin 1beta-converting enzyme family proteases and anti-cell death protein Bcl-2. One evident physiological difference in cells undergoing apoptosis versus necrosis is in intracellular levels of ATP. In this study, we specifically addressed the question of whether apoptosis depends on intracellular ATP levels, since longer incubation under ATP-depleting conditions results in necrotic cell death. Incubation of cells in glucose-free medium with an inhibitor of mitochondrial F0F1-ATPases reduces intracellular ATP levels and completely blocks Fas/Apo-1-stimulated apoptosis. ATP supplied through glycolysis or oxidative phosphorylation restores the apoptotic cell death pathway. ATP depletion also leads to a block in Fas-induced activation of CPP32/Yama(-like) proteases, and when ATP is depleted after the activation of the proteases, subsequent apoptosis is significantly blocked. Thus, ATP-dependent steps exist both upstream and downstream of CPP32/Yama(-like) protease activation in apoptotic signal transduction. Treatment with the calcium ionophore induces apoptosis under ATP-supplying conditions but induces necrotic cell death under ATP-depleting conditions, indicating that ATP levels are a determinant of manifestation of cell death.

Adenosine Triphosphate↗
PubMed1997

Gene transfection of mouse primordial germ cells in vitro and analysis of their survival and growth control.

We evaluated electroporation, liposome-mediated transfection, and the calcium phosphate (CaPO4) coprecipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to the investigation of molecular mechanisms of the germ cell development. We found that electroporation severely damaged PGCs, and the efficiency of liposome-mediated transfection was very low. In contrast, using the CaPO4 coprecipitation method, 18% of PGCs transfected with plasmid pSV-LT expressed simian virus 40 large tumor antigen (SV 40 T-Ag) transiently. However, we did not detect any effects on the proliferation and survival of PGCs obtained from the embryonic gonads at 11.5 days postcoitum (d.p.c.) during 2 days of culture after the transfection. PGCs isolated from the 11.5-d.p.c. gonads change from spread- to round-shape and exhibit growth arrest during a few days of culture, and these rounded PGCs quickly disappear from the culture. We found that the transfection and expression of Bcl-XL or adenovirus type 2 E1B 19,000-molecular-weight protein (E1B 19K) significantly promoted the survival of PGCs and retarded the disappearance of rounded PGCs from the culture system. These results suggest that the Bcl-XL or E1B 19K can prevent the apoptosis of PGCs and inhibit the cell death of the rounded PGCs in culture.

Adenovirus E1B Proteins↗
PubMed1997

Overexpressed Bcl-x(L) prevents bacterial superantigen-induced apoptosis of thymocytes in vitro.

bcl-x, a homologous gene of bcl-2, has an anti-apoptotic function and appears to play a critical role in the development of lymphoid systems. To investigate the effect of overexpressed Bcl-x(L) on the development of T lymphocytes, we established two lines of transgenic mice by using Emu-chicken bcl-x(L) (cbcl-x(L)) transgene, where the cBcl-x(L) protein was expressed mainly in lymphoid cells. Although thymocytes and splenocytes from cbcl-x(L) transgenic mice are resistant to apoptosis in vitro, clonal deletion of thymocytes, recognizing endogenous self-superantigens in the thymus, still normally proceeded and no self-reactive T cells were found in the spleen of the transgenic mice. To dissect clonal deletion, we utilized two in vitro models, thymocytes/antigen presenting cells co-culture system and fetal thymus organ culture system. In both, bacterial superantigen staphylococcus aureus enterotoxin B (SEB) induces apoptosis of T cells with Vbeta8+ T cell receptor (TCR) reacting to SEB, which mimics clonal deletion of self-reactive thymocytes in vivo. SEB-induced depletion of Vbeta8+ T cells from thymocytes when taken from the transgenic mice was effectively inhibited. The data might raise the possibility that cell death process involved in clonal deletion in the thymus is a form of apoptosis inhibited by Bcl-x(L).

Journal Article↗
PubMed1997

Targeted expression of ced-3 and Ice induces programmed cell death in Drosophila.

CED-3 is a cysteine protease required for programmed cell death in the nematode, Caenorhabditis elegans, and shares a sequence similarity with mammalian ICE (interleukin-1beta converting enzyme) family proteases. Both CED-3 and ICE family proteases can induce programmed cell death in mammalian cells. Structural and functional similarities between CED-3 and ICE family proteases indicate that the mechanism of cell death is evolutionarily conserved, suggesting the presence of a similar mechanism involving CED-3/ICE-like proteases in Drosophila. Here we determined whether CED-3 or ICE functions to induce programmed cell death in Drosophila. We have generated transformant lines in which ced-3 or Ice is ectopically expressed using the GAL4-UAS system. Expression of CED-3 and ICE can elicit cell death in Drosophila and the cell death was blocked by coexpressing the p35 gene which encodes a viral inhibitor of CED-3/ICE proteases. Results support the idea that the mechanism of programmed cell death controlled by CED-3/ICE is conserved among widely divergent animal species including Drosophila, and the system described provides a tool to dissect cell death mechanism downstream of CED-3/ICE proteases.

Journal Article↗
PubMed1997

Apoptosis and necrosis: intracellular ATP level as a determinant for cell death modes.

Apoptosis and necrosis are two distinct modes of cell death with respective morphological characteristics. However, apoptosis and some forms of necrosis must share common steps since both modes of cell death can be suppressed by the anti-apoptotic Bcl-2 protein and caspase inhibitors. Intracellular ATP levels have been implicated both in vitro and in vivo as a determinant of the cell's decision to die by apoptosis or necrosis.

Journal Article↗
PubMed1997

Caspase-4 and caspase-5, members of the ICE/CED-3 family of cysteine proteases, are CrmA-inhibitable proteases.

Proteases of the caspase family are implicated in mammalian apoptosis and constitute a protease cascade. We characterized caspase-4 (TX/ICH-2/ICErelII) and caspase-5 (ICErelIII/TY), which are most closely related to caspase-1 (ICE) among the caspase family. Although overexpression of caspase-4 and caspase-5 induced apoptosis, confirming previous observations, this apoptosis was not inhibited by a caspase-1-specific tetrapeptide inhibitor (Ac-YVAD-CHO), suggesting that caspase-4 and caspase-5 have different substrate specificities from caspase-1 and also that caspase-4- and caspase-5-induced apoptosis is not mediated by caspase-1. CrmA, a cowpox virus-derived caspase-1 inhibitor that prevents apoptosis induced by various stimuli, was cleaved by caspase-4 and caspase-5, and inhibited their proteolytic activity as assessed by cleavage of pro-caspase-3 (pro-CPP32/Yama/apopain). Thus, caspase-4 and caspase-5 are CrmA-inhibitable proteases like caspase-1 and might be involved in apoptosis.

Journal Article↗
PubMed1997

Essential role of active nuclear transport in apoptosis.

BACKGROUND: Apoptosis is defined by chromatin condensation, nuclear fragmentation and the formation of apoptotic bodies. Because apoptotic signals are transmitted through a common pathway that includes the target steps of death-driving ICE-family proteases and anti-cell death protein Bcl-2 in the cytoplasm, the signals must be transferred from the cytoplasm to the nucleus, at least to induce the apoptotic manifestation of the nucleus. Small signal molecules might diffuse across nuclear pores, but larger molecules are transported by active mechanisms requiring ATP and GTP hydrolysis. It is not known whether apoptotic signals are transmitted into the nucleus by the mechanisms of active nuclear transport. RESULTS: To test the possibility that active nuclear transport is involved in apoptotic signal transmission, we have analysed the effects of molecules that inhibit active nuclear transport on apoptosis. Wheat germ agglutinin (WGA), excess amounts of p10 protein, Ran-GTPgammaS complex, and anti-PTAC58 antibody, which all inhibit active nuclear transport when exogenously microinjected, prevent Fas-induced apoptotic nuclear manifestation. WGA also prevents apoptotic nuclear change promoted by microinjected active CPP32beta/Yama protease (an ICE family member), which plays an essential role in most apoptosis. CONCLUSIONS: The results presented here strongly suggest that active nuclear transport is essential for apoptotic signal transduction.

Antibodies↗
PubMed1997

Cell ablation by ectopic expression of cell death genes, ced-3 and Ice, in Drosophila.

We have developed a system for killing specific cells in Drosophila using ectopic expression of cell death genes. CED-3 and ICE (caspase-1) are proteins required for programmed cell death in the nematode Caenorhabditis elegans and in mammals, respectively. Our previous study has shown that both ced-3 and Ice can elicit cell death in Drosophila. By expressing ced-3 or Ice in several kinds of cells using a GAL4-UAS system and examining the resulting morphological defects, we show that these abnormalities are thought to be caused by the action of ced-3 or Ice genes. As cells are killed by apoptosis in our system, we could eliminate the possibility of harmful effects on the neighboring cells. Our system provides an alternative and novel cell ablation method to elucidate mechanisms of cell differentiation and cell-cell interactions during development in Drosophila.

Animals↗
PubMed1997

Susceptibility of cerebellar granule neurons derived from Bcl-2-deficient and transgenic mice to cell death.

Overproduced Bcl-2 oncoprotein has been shown to suppress cell death induced by a variety of stimuli in many cell types, including neuronal cells. Because bcl-2 is expressed in the nervous system where massive cell death is observed during development, endogenous Bcl-2 is likely to be involved in regulating neuronal cell death. Here we examined the possible role of endogenous Bcl-2 in the regulation of neuronal cell survival in the central nervous system using primary cultured cerebellar granule neurons from bcl-2-deficient, wild-type and NSE-bcl-2-transgenic mice. Cerebellar granule neurons from bcl-2-deficient mice were more susceptible than those from normal littermates to death induced by reducing the K+ concentration of the medium from high (25 mM) to low (5 mM), and neurons from bcl-2-transgenic mice were least susceptible. Similar results were obtained when cell death was induced by serum withdrawal under high K+ conditions or by the presence of etoposide, A23187 or nimodipine. Consistently, bcl-2 deficiency reduced the number of cerebellar granule neurons per mouse. These results indicate that Bcl-2 impedes neuronal cell death induced by various stimuli in a dose-dependent manner, and that endogenous levels of Bcl-2 are able to regulate neuronal cell survival in the central nervous system.

Animals↗
PubMed1997

A functional role for death proteases in s-Myc- and c-Myc-mediated apoptosis.

Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade. However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli such as Myc and p53. In this study, we provide lines of evidence that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc were induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate, Ac-DEVD-MCA, appeared in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death was effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Furthermore, exposing the cells to a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), also significantly inhibited s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, AEBSF did not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the AEBSF-sensitive step in the signaling pathway of Myc-mediated apoptosis.

Animals↗
PubMed1997

Renal pelvic cancer with tumor thrombus in the vena cava inferior. A case report and review of the literature.

Renal pelvic cancer with a large vein tumor thrombus is very rare. We report a case of transitional cell carcinoma of the right renal pelvis with tumor thrombus in the inferior vena cava. Based on the radiological findings, the patient was diagnosed having renal cell carcinoma with tumor thrombus in the vena cava preoperatively. Radical nephrectomy with thrombectomy was performed. A literature review of 17 reported cases revealed that 13 cases (76.5%) were diagnosed as renal cancer preoperatively. Prognosis of transitional cell carcinoma with vena cava tumor thrombus is relatively poor compared to that of renal cell carcinoma with a large vein tumor thrombus.

Carcinoma, Renal Cell↗
PubMed1997

[Molecular biology of apoptosis].

Apoptosis, a mechanism involving programmed cell death, is important for normal development and maintenance of tissue homeostasis of multicellular organisms. Apoptotic cells are defined by their fragmented nuclei with condensed chromatin, fragmented or condensed cytoplasm and formation of apoptotic bodies. The apoptotic signal transducing pathways activated by a variety of stimuli, including depletion of growth factors, heat shock, cytokines, DNA damaging reagents and crosslinking of Fas receptor, finally converge into the phylogenically conserved apoptotic main machinery, consisting of death-driving ICE-family proteases and anti-cell death protein Bcl-2. Recently, we noted that necrotic cell death induced by chemical hypoxia shares at least some part of the apoptotic main machinery. Using this system, we have shown that Bcl-2 prevents the loss of the mitochondrial membrane potential observed in both apoptotic and necrotic cell death. We also showed that the ICE protease cascade operates in apoptosis and that Bcl-2 functions upstream of the ICE prolease cascade. Here, we review the signal transducing pathway of the apoptotic main machinery.

Animals↗
PubMed1997

Bcl-2 and Bcl-xL block apoptosis as well as necrosis: possible involvement of common mediators in apoptotic and necrotic signal transduction pathways.

The proto-oncogene bcl-2 and a bcl-2-related gene bcl-x prevent apoptotic cell death induced by various treatments. Although a mechanism has been proposed that involves Bcl-2 activity on reactive oxygen species (ROS), we find that expression of Bcl-2 or Bcl-xL prevents cell death induced by withdrawal of oxygen (hypoxia) and that the cell death does not involve ROS, suggesting that Bcl-2 or Bcl-xL exerts an anti-cell death function by a mechanism other than through regulation of ROS activity. Using electron microscopy, and confocal and non-confocal fluorescence microscopy, we show that hypoxia induces both necrosis and apoptosis. Overexpression of Bcl-2 or Bcl-xL blocks hypoxia-induced apoptosis and, although to a lesser extent, necrosis. The anti-apoptotic proteins Bcl-2 and Bcl-xL effectively inhibit KCN-induced cell death which is characterized by necrotic features including apparently intact chromatin, remarkable mitochondrial swelling with loss of crista structure and loss of plasma membrane integrity. The necrotic cell death is also inhibited by inhibitors of ICE (interleukin-1 beta converting enzyme)(-like) proteases, the common mediators of apoptosis. These results indicate that Bcl-2/Bcl-xL and ICE(-like) proteases modulate both apoptotic and at least some forms of necrotic cell death, suggesting that both cell death pathways involve some common mediators.

Animals↗
PubMed1996

Functional conservation of mouse Notch receptor family members.

All the known members of the mouse Notch receptor family were examined for their biochemical function by interaction with a DNA binding protein RBP-Jkappa. mNotch2, mNotch3 and int3 (= mNotch4) were shown to interact with RBP-Jkappa by the GST-fusion pull down assay and dominant negative competition with Epstein Barr virus nuclear antigen 2. Furthermore the intracellular region of int3 was shown to transactivate the Epstein Barr virus TP1 promoter. These results indicate that all mouse Notch family members have biochemical functions similar to mNotch1, which transduces proliferative signal by direct interaction with the DNA binding protein RBP-Jkappa.

Amino Acid Sequence↗
PubMed1996

Accelerated disappearance of melanocytes in bcl-2-deficient mice.

Follicular melanocytes in bcl-2(-/-) mice have been reported to turn gray during the second hair cycle. Light microscopic analysis revealed that about half of bcl-2(-/-) mouse hair shafts had no detectable melanin granules after the second hair follicle cycle, but the remaining hair appeared to be pigmented normally. After depilation to induce new anagen hair, more than 97% of the hair shafts did not have visible melanin granules in bcl-2(-/-) mice, whereas 100% of the hair shafts in bcl-2(+/+) mice were pigmented. In bcl-2(+/+) mice, dopa-positive melanocytes appeared on day 4 after depilation, whereas bcl-2(-/-) mice developed few dopa-positive melanocytes after depilation, as assessed by light and electron microscopic observation. bcl-2(-/-) mouse hair in the second hair cycle contained about 60-70% less melanin than normal mouse hair, and newly generated bcl-2(-/-) mouse hair after depilation contained a level of melanin as low as that of albino mouse hair. These observations suggest that the expression of bcl-2 might be essential for melanocyte maintenance after the second hair cycle.

Animals↗