Biomedical subjects

Itaru Hirai

Publications and source records attributed to Itaru Hirai.

8 recordsLinked to original sources

PubMed2005

Loss of RPA1 induces Chk2 phosphorylation through a caffeine-sensitive pathway.

RPA is an important component of DNA replication, repair and recombination, but its involvement in the signaling of cell-cycle checkpoints is not well understood. In this study, we show that knockdown of RPA1 by siRNA duplexes induces ATM (Ser1981) and Chk2 (Thr68), but not Chk1 (Ser345) phosphorylation and results in p21 upregulation in HeLa cells. However, the induction of Chk2 (Thr68) phosphorylation and p21 expression by RPA1 siRNA transfection can be completely blocked by the ATM inhibitor caffeine. Moreover, transfection of siRNAs targeting ATM dramatically reduces Chk2 (Thr68) phosphorylation in RPA1 knockdown cells. Taken together, these results suggest that loss of RPA1 activates the Chk2 signaling pathway in an ATM-dependent manner.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

Human hRad1 but not hRad9 protects hHus1 from ubiquitin-proteasomal degradation.

Three of the Rad family proteins, Rad9, Rad1, and Hus1, can interact with each other and form a heterotrimeric complex that is thought to play a role in the sensing step of the DNA integrity checkpoint pathways, but the nature of the Rad9-Rad1-Hus1 complex assembly remains enigmatic. Here, we demonstrate that the human hRad1 protein plays a significant role as molecular chaperone in the process of the hRad9-hRad1-hHus1 heterotrimeric complex formation. In contrast to hRad1, hHus1 is an unstable protein that is actively degraded via the ubiquitin-proteasome pathway. We show that treating cells with proteasome-specific inhibitors stabilizes hHus1 expression. Moreover, hRad1 can associate with hHus1 in the absence of hRad9 and protect hHus1 from ubiquitination and degradation in the cytoplasm. Importantly, genotoxic stress induces hRad1 expression and stabilizes the hHus1 protein. Taken together, these findings suggest a novel role of hRad1 as a potential intrinsic chaperone in the stabilization of hHus1 for the hRad9-hRad1-hHus1 checkpoint complex formation.

Cell Cycle↗

PubMed2004

Tumor-derived heat shock protein 70-pulsed dendritic cells elicit tumor-specific cytotoxic T lymphocytes (CTLs) and tumor immunity.

Vaccination with autologous tumor-derived heat shock proteins (Hsp), such as Hsp70, Hsp90 and gp96, has been demonstrated to elicit specific immune responses against the tumor from which the Hsps were isolated. The effect of Hsp immunization is wholly dependent on the presence of functional antigen-presenting cells (APCs) in the immunized host, and Hsp receptors on APCs have recently been identified. Here we show that bone marrow-derived dendritic cells (DCs) are able to internalize HSP-peptide complex and that peptides are re-presented by DCs via the major histocompatibility complex (MHC) class I presentation pathway. In addition, immunization with tumor-derived HSP-pulsed DCs induces strong cytotoxic T cell (CTL) responses against multiple antigenic peptides in a transporter-associated antigen processing (TAP)-dependent manner. The results of the present study provide strong evidence of an efficient cross-priming activity of Hsp70, which could be exploited in the development of new and more effective immunotherapeutic strategies for cancer patients.

Animals↗

PubMed2003

Caspase-3-mediated cleavage of Rad9 during apoptosis.

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.

Apoptosis↗

PubMed2003

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

INTRODUCTION: We have generated HLA-A*2601-restricted CD8+ CTL clones against an autologous pancreatic cancer cell line. AIMS: To characterize the antigen expressed on the cancer cells. METHODOLOGY: We assessed cytotoxic activities and cytokine production of these CTL clones reacting against cancer cell lines that stably or transiently expressed the HLA-A*2601 gene. RESULTS: These CTL clones recognized 4 of 10 allogeneic pancreatic cancer cell lines and a gallbladder cancer cell line in the context of HLA-A*2601. However, the CTL clones did not recognize three hepatocellular carcinoma cell lines, two esophageal squamous cell carcinoma cell lines, or a lung adenocarcinoma cell line. CONCLUSIONS: Thus, the CTL clones may recognize a shared, but not ubiquitously expressed, tumor antigen on pancreatic and gallbladder cancer cells.

Antigens, Neoplasm↗

PubMed2003

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

We previously reported that rat T-cell receptor (TCR) Vdelta6 of T-cell hybridomas was preferentially involved in recognition of the cell surface-expressed 70 kDa rat heat-shock cognate (hsc70, a constitutively expressed member of the hsp 70 family) protein-like molecule (#067 molecule). In the present study, we analyzed usage of the TCR Vgamma family of #067-restricted T-cell hybridomas. Our data indicated that most of these hybridomas expressed transcripts of TCR Vgamma1 and/or Vgamma2. However, some of the Vgamma2 transcripts were out-of-frame, suggesting that the TCR Vgamma1 family may be important for the recognition of #067-defined molecules. TCR Vgamma1 transcripts were detected in not only #067-restricted T-cell hybridomas, but #067-non restricted ones as well. However, V-J nucleotide sequences of #067-restricted and #067-non restricted T-cell hybridomas suggested that #067-restricted T-cell hybridomas showed limited insertion of nucleotide stretch as compared with #067-non restricted ones. In terms of amino acids, only one amino acid was added in #067-restricted T-cell hybridomas, whereas two or three amino acids were added in #067-non restricted ones. These data suggest that the heterodimer of the TCR relatively short stretch form of Vgamma1 molecule and TCR Vdelta6 may participate in recognition of the #067 molecule.

Animals↗

PubMed2002

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

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 G(2) 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 NLS 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(2) 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.

Cell Cycle Proteins↗

PubMed2002

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

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 wild-type 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 CD.

Apoptosis↗