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Caspase-3-mediated cleavage of Rad9 during apoptosis.

Lee MW, Hirai I, Wang HG

Oncogene ( NATURE PUBLISHING GROUP )  22 ( 41 ) 6340 - 6   2003年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

The activation of caspases is a critical event for the execution phase of programmed cell death. Caspases are highly specific in their ability to activate or inhibit many crucial proteins in the cell via cleavage. In this study, we report the identification of several caspase-3-like cleavage sites in the cell-cycle checkpoint protein Rad9. We demonstrate that human Rad9 can be specifically cleaved in cells induced to enter apoptosis by both DNA damage and staurosporine treatment. Indeed, we show that human Rad9 can be effectively cleaved both in vitro and in vivo, which can be inhibited by either a pan-caspase inhibitor or a caspase-3-specific inhibitor. Additionally, no cleavage of Rad9 can be seen in the caspase-3-deficient cell line MCF-7. Site-directed mutagenesis of three of the most conserved cleavage sites dramatically abrogates cleavage of Rad9 by caspase-3 in vitro, and in intact cells after DNA damage. Expression of the cleavage-resistant mutant Rad9 DDD/AAA appears to protect the cell from DNA damage-induced apoptosis. Immunofluorescence studies of Rad9 localization before and after induction of apoptosis show a translocation of Rad9 from the nucleus to the cytosol, concomitant to the appearance of apoptotic morphology. Furthermore, analysis of a truncated Rad9 mutant that corresponds to a putative N-terminal cleavage fragment shows that the N-terminal portion of Rad9 localizes in the cytosol, binds to Bcl-XL, and induces apoptosis. These results support a dual role for cleavage of Rad9: ( 1) the liberation and translocation of the BH3 domain-containing N-terminus of Rad9 to the cytosol, as a means of promoting apoptosis via antagonism of Bcl-XL, and ( 2) the disruption of the Rad9-Rad1-Hus1 DNA damage checkpoint complex.

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Analysis of a shared pancreatic cancer antigen recognized by an HLA-A*2601-restricted cytotoxic T-lymphocyte clone.

Kashiwagi K, Ikeda H, Hirohashi Y, Yamamoto M, Idenoue S, Hirai I, Kamiguchi K, Tamura Y, Torigoe T, Wada Y, Hirata K, Sato N

Pancreas   26 ( 4 ) e81 - 8   2003年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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IgG and IgA antibody titers against human heat-shock protein (hsp60) in sera of rheumatoid arthritis and osteoarthritis patients.

Watanabe S, Takubo N, Hirai I, Hitsumoto Y

Modern rheumatology   13 ( 1 ) 22 - 26   2003年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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T-cell receptor variable gamma chain gene expression in the interaction between rat gammadelta-type T cells and heat-shock protein 70-like molecule.

Ichinohe T, Ichimiya S, Kishi A, Tamura Y, Kondo N, Ueda G, Torigoe T, Yamaguchi A, Hiratsuka H, Hirai I, Kohama GI, Sato N

Microbiology and immunology   47 ( 5 ) 351 - 7   2003年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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A role of the C-terminal region of human Rad9 (hRad9) in nuclear transport of the hRad9 checkpoint complex.

Hirai I, Wang HG

The Journal of biological chemistry ( AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC )  277 ( 28 ) 25722 - 7   2002年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Rad9, Rad1, and Hus1 are members of the Rad family of checkpoint proteins that are required for both DNA replication and DNA damage checkpoints and are thought to function as sensors in the DNA integrity checkpoint control. These proteins can interact with each other and form a stable proliferating cell nuclear antigen-related Rad9-Rad1-Hus1 heterotrimeric complex that might encircle DNA at or near the damaged sites. In this study, we demonstrate that the human Rad9 (hRad9) protein contains a predicted nuclear localization sequence (NLS) near its C terminus, which plays an essential role in the hRad9-mediated G2 checkpoint. Deletion experiments indicate that the NLS-containing region of hRad9 is critical for the nuclear transport of not only hRad9 but also human Rad1 (hRad1) and human Hus1 (hHus1), although this region is not required for hRad9-hRad1-hHus1 complex formation. In support of the role that hRad9 NI'S plays in the nuclear targeting of the hRad9-hRad1-hHus1 complex, overexpression of a deletion mutant of hRad9 lacking the NLS-containing C-terminal region can bypass the G(2) checkpoint and result in cell death after ionizing radiation or hydroxyurea treatment. Moreover, knockdown of hRad9 expression by small interfering RNA (siRNA) results in hRad1 accumulation in the cytoplasm and significantly abrogates the G, checkpoint in the presence of damaged DNA or incomplete DNA replication. Thus, the C-terminal region of human Rad9 protein is important for G(2) checkpoint control by operating the transport of the hRad9-hRad1-hHus1 checkpoint complex into the nucleus.

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Identification of germline mutation of PTEN gene and analysis of apoptosis resistance of the lymphocytes in a patient with Cowden disease.

Kanaseki T, Torigoe T, Hirohashi Y, Hirai I, Himi T, Sato N

Pathobiology : journal of immunopathology, molecular and cellular biology ( KARGER )  70 ( 1 ) 34 - 9   2002年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Mutations of the tumor suppressor gene PTEN have been reported in patients with Cowden disease (CD) and in several malignant tumors. We analyzed a germline mutation of the PTEN gene in a patient with CD and identified a 4-bp deletion in exon 8 of the PTEN gene. The same germline mutation was detected in 3 members of her family. The mutated gene was predicted to encode a C-terminal truncated PTEN protein. Immunoblotting analysis revealed that the expression level of the wildtype PTEN protein in the patient's lymphocytes was reduced to almost half the level of the control lymphocytes, and the predicted truncated mutant PTEN could not be detected. Since PTEN is known to function as a negative regulator of the phosphatidylinositol-3-kinase signal pathway that promotes cell survival, the patient's lymphocytes were tested for the resistance against the apoptotic stimulus. It was shown that the patient's lymphocytes were more resistant to apoptosis induced by calcium ionophore than the healthy control lymphocytes. These results indicate that the germline mutation of the PTEN gene and the consequent loss of heterozygous expression may lead to an increase in the survival potential of cells, thereby elucidating a role of PTEN in the pathogenesis of tumor generation and hyperplasia of lymphoid tissue in CID. Copyright (C) 2002 S. Karger AG, Basel.

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Survival-factor-induced phosphorylation of Bad results in its dissociation from Bcl-X-L but not Bcl-2

Hirai, I, HG Wang

BIOCHEMICAL JOURNAL ( PORTLAND PRESS )  359 ( Pt 2 ) 345 - 352   2001年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The pro-apoptotic Bcl-2-family protein Bad heterodimerizes with Bcl-2 and Bel-x(L) in the outer mitochondrial membranes, nullifying their anti-apoptotic activities and promoting cell death. We report that interleukin-3 (IL-3) stimulation induces Bad phosphorylation and triggers its translocation from mitochondria to cytoplasm in cells expressing Bcl-x(L) but not Bcl-2. Over-expression of Bad sensitized Bcl-x(L)-expressing FL5.12 cells to apoptosis induced by IL-3 deprivation, but had no effect on the viability of cells expressing Bcl-2. IL-3 stimulation induced Bad phosphorylation at Ser-112, impairing its binding to Bcl-x(L) and resulting in its association with 14-3-3 proteins in the cytosol. However, Ser-112 phosphorylation could not trigger Bad dissociation from mitochondria in FL5.12 cells expressing Bcl-2. In 293T cells expressing Bcl-x(L), Bad was phosphorylated at three serines, 112, 136 and 155, and was largely localized in the cytosolic fraction. In contrast, overexpression of Bcl-2 prevented phosphorylation of Bad at Ser-136 and Ser-155, sequestering this protein in the mitochondrial membranes. When the N-terminal regions of Bcl-2 and Bel-x(L) were swapped with each other, the Bcl-x(L)(N)-Bcl-2 chimaeric protein (containing the N-terminal region of Bel-x,) failed to prevent Bad phosphorylation in cells and was unable to block the cytosolic distribution of this proapoptotic protein. Additional experiments with the Bcl-2(N)Bcl-x(L) chimaeric protein (containing the N-terminal region of Bcl-2) indicated that, although the N-terminal region of Bcl-2 is necessary, it is not sufficient for sequestering Bad in the mitochondrial membranes. These observations suggest that growth-factor-mediated phosphorylation of Bad contributes to the cytoprotective function of Bcl-x(L) but not Bcl-2.

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The cell surface-expressed HSC70-like molecule preferentially reacts with the rat T-cell receptor V delta 6 family

A Kishi, T Ichinohe, Hirai, I, K Kamiguchi, Y Tamura, M Kinebuchi, T Torigoe, S Ichimiya, N Kondo, K Ishitani, T Yoshikawa, M Kondo, A Matsuura, N Sato

IMMUNOGENETICS ( SPRINGER-VERLAG )  53 ( 5 ) 401 - 409   2001年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

We previously showed that the cell surface-expressed M-r 70,000 heat shock cognate (hsc70, a constitutively expressed member of the hsp70 family) protein-like molecule (#067 molecule) interacts with rat CD3(+), CD4(-), CD8(-), T-cell receptor (TCR)alpha beta (-), natural killer recetor-Pl(-) T cells. This 70hsc-like molecule was also suggested to present cellular peptide antigens to these T cells. In the present study, we identified the genetic structure of the TCR by establishing T-cell hybridomas between these T cells and mouse BW5147 cells. Our data indicated that these T cells preferentially used TCRs with the V delta6 family. Analysis of the nucleotide sequence of the CDR3 junctional portion showed that there are substantial diversities, with insertion of seven to nine amino acid residues. These data provide indirect evidences for our hypothesis that an hsc70-like molecule could be presented together with cellular peptide antigens to particular T cells with TCR gamma delta chains. Since the expression of this hsc70-like #067 antigen on the cell surface is usually induced along with cell transformation by activated oncogenes, T cells with the TCR V delta6 family are likely to contribute to host resistance to tumor cells.

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The expression of a novel natural killer inhibitory molecule, Cho-1, on the chorionic cytotrophoblast cells of successful pregnancy, but not of spontaneous abortion

M Nagata, T Akazawa, Y Tamura, K Kamiguchi, Hirai, I, S Ohtani, S Sagae, R Kudo, T Torigoe, N Sato

PATHOLOGY INTERNATIONAL ( BLACKWELL SCIENCE ASIA )  50 ( 10 ) 824 - 831   2000年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The regulatory mechanism of the recognition and cytotoxicity by natural killer (NK) cells in placental tissue remains unclarified. Previous reports indicated that monoclonal antibody Cho-1-defined molecule (Cho-1 molecule) may act as the negative regulator in the cytotoxicity by human NK cells. The Cho-1 molecule is composed of non-covalently asssociated cell surface molecules of approximately 200 kDa and 40 kDa. In the present study we analyzed the expression of this novel molecule in extravillous cytotrophoblast cells, which are presumed to be exposed to the cytotoxic action by maternal NK cells, from clinical cases of successful pregnancy and spontaneous abortion. By using monoclonal antibody Cho-1, our immunohistochemical data indicated that the Cho-1 molecule is clearly expressed in the cytotrophoblast cells of the early phase of successful pregnancy, but only weakly expressed in those from spontaneous abortion. The cytotrophoblast cells in the late phase (9-10 months) of pregnancy also expressed this molecule. Fluorescence-activated cell sorter analysis also showed that it is expressed on the cytotrophoblast cell surface of successful pregnancy but not on that of spontaneous abortion, suggesting that Cho-1 antigen may act as a negative regulator of the cytotoxicity by NK cells in successful pregnancy of the fetus.

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Human CD8 and CD4 T cell epitopes of epithelial cancer antigens

N Sato, Y Nabeta, H Kondo, H Sahara, Y Hirohashi, K Kashiwagi, T Kanaseki, Y Sato, S Rong, Hirai, I, K Kamiguchi, Y Tamura, A Matsuura, S Takahashi, T Torigoe, H Ikeda

CANCER CHEMOTHERAPY AND PHARMACOLOGY ( SPRINGER-VERLAG )  46   S86 - S90   2000年06月

掲載種別: 研究論文（学術雑誌）

　概要を見る

Recent human tumor immunology research has identified several genes coding immunogenic peptides recognized by CD8 cytotoxic T lymphocytes (CTLs) in melanoma tumors. Very recently, CD4 T cell antigenic epitopes were also determined in certain melanoma tumors. The use of these peptides in conjunction with human immunotherapy could prove to be of great benefit. However, such peptides in clinically common tumors of epithelial cell origin, such as of the stomach, colon, lung, etc., have not yet been determined extensively. We describe for the first time an HLA-A31 (A*31012)-restricted natural antigenic peptide recognized by the CD8 CTL TcHST-2 of gastric signet ring cell carcinoma cell line HST-2. We also identified the HLA-DRB1*08032-restricted peptide recognized by the CD4 T cell line TcOSC-20 of squamous cell carcinoma OSC-20 derived from the oral cavity. The antigenic peptide of HST-2, designated F4.2, is composed of 10 amino acid residues with two anchor motif residues necessary for binding to HLA-A31 molecules. The synthetic F4.2 peptide enhanced the reactivity of TcHST-2 against HST-2 cells. Furthermore, introduction of an expression minigene coding F4.2 peptide to HLA-A31(+) cells conferred cytotoxic susceptibility to TcHST-2 on the cells. Some stomach cancer lines into which the HLA-A31 gene had been introduced, such as MKN28-A31-2, were lysed by TcHST-2, suggesting the presence of F4.2 peptide in at least some HLA-A31(+) stomach cancers. Furthermore, F4.2 peptide induced an F4.2 peptide-specific CTL response in at least 30-40% of HLA-A31(+) peripheral blood lymphocytes from gastric cancer patients, suggesting that F4.2 peptide could be used as a cancer vaccine for gastric tumors. The natural antigenic peptide of OSC-20 was also determined using acid extraction and biochemical separation and by mass spectrometry. Consequently, OSC-20 peptide was designated as the 6-1-5 peptide, an HLA-DRB1*08032-restricted 16-mer peptide with two possible anchor motifs. It has an amino acid sequence identical to that of human alpha-enolase, suggesting that it was derived from the processed parental alpha-enolase protein. We are presently attempting to determine the genes that code tumor rejection antigens recognized by HLA-A24- and A26-restricted T cells, including those of pulmonary and pancreatic carcinomas. The search for these antigenic peptides may lead to the identification of immunogenic peptide antigens that would be suitable for clinical use in commonly occurring epithelial cancers.

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Immunosuppressive effect on T cell activation by interleukin-16-cDNA-transfected human squamous cell line

T Fujita, Y Matsumoto, Hirai, I, K Ezoe, T Saito, A Yagihashi, T Torigoe, K Homma, S Takahashi, WW Cruikshank, K Jimbow, N Sato

CELLULAR IMMUNOLOGY ( ACADEMIC PRESS INC )  202 ( 1 ) 54 - 60   2000年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

It is well known that it is difficult to induce an immunotolerance with allogeneic skin transplantation. We attempted to find the immunosuppressive protocol for prolonging skin allograft rejection by using interleukin-16 because IL-16 is considered one of the natural ligands to CD4 molecules. First we examined whether synergistic immunosuppressive effects of recombinant IL-16 plus anti-CD4 mAbs are induced in mixed lymphocyte reaction (MLR). Next we used IL-16-cDNA-transfected OSC-20 (human oral squamous cell carcinoma cell line) as an in vitro model of the epidermal keratinocyte equivalent and examined whether this transfectant could inhibit the activation of allogeneic T cells. Our data indicated that IL-16 clearly inhibited human MLR and that IL-16 increased synergistically the immunosuppressive effect of anti-CD4 mAb. We also used IL-16 transfectant and this produced more than 50 ng/ml of IL-16 in the supernatant by which human MLR was significantly inhibited. Furthermore, this transfectant also inhibited the activation of allogeneic lymphocytes stimulated directly with transfectant cells. These results indicated that the IL-16-producing allogeneic skin graft might have a local immunosuppressive action that would prolong graft survival. (C) 2000 Academic Press.

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Molecular cloning of rat NK target structure - The possibility of CD44 involvement in NK cell-mediated lysis

K Kanki, T Torigoe, Hirai, I, H Sahara, K Kamiguchi, Y Tamura, A Yagihashi, N Sato

MICROBIOLOGY AND IMMUNOLOGY ( CENTER ACADEMIC PUBL JAPAN )  44 ( 12 ) 1051 - 1061   2000年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

The nature of target molecules of natural killer (NK) cell-mediated lysis remains to be elucidated. As we previously reported, mAb 109 recognizes one of the tumor-associated antigens, designated as 109 antigen (Ag), expressed on the cell surface of rat fibrosarcomas W31 and W14, which are transformants of WFB (rat fetal fibroblast cell line) with H-ras oncogene, 109Ag was thought to be a target structure of NK cells since mAb 109 inhibited NK cell-mediated lysis against W31 and W14, Here, we demonstrate by molecular cloning that 109Ag is identical to rat CD44, Immunoprecipitation and immunoblotting studies also showed that mAb 109 and anti-rat CD44 mAb OX-50 recognize the same protein of W31 cell lysates with an 86 kDa molecular size. CD44 was suggested to be a target structure of NK cell-mediated lysis; however, rat CD44 cDNA transfection alone into CD44 null cell lines did not result in up-regulation of target cell susceptibility to NK cell-mediated lysis, Our results therefore indicated that CD44 may play a crucial role as one of the target structures in our rat fibrosarcoma system though the cell surface expression of CD44 alone does not affect NK susceptibility of the target cells.

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