Kazuya Hirano

A Multifunctional Shuttling Protein Nucleolin Is a Macrophage Receptor for Apoptotic Cells

Early apoptotic Jurkat T cells undergo capping of CD43, and its polylactosaminyl saccharide chains serve as ligands for phagocytosis by macrophages. This suggests the presence of a polylactosaminoglycan-binding receptor on macrophages. Here we show that this receptor is nucleolin, a multifunctional shuttling protein present in nucleus, cytoplasm, and on the surface of some types of cells. Nucleolin was detected at the surface of macrophages, and anti-nucleolin antibody inhibited the binding of the early apoptotic cells to macrophages. Nucleolin-transfected HEK293 cells expressed nucleolin on the cell surface and bound the early apoptotic cells but not phosphatidylserine-exposing late apoptotic cells. This binding was inhibited by anti-nucleolin antibody, by polylactosamine-containing oligosaccharides, and by anti-CD43 antibody. Deletion of the antibody binding region of nucleolin resulted in loss of the apoptotic cell-binding ability. Moreover, truncated recombinant nucleolin in solution containing this region blocked the apoptotic cell binding to macrophages, and the blocking effect was cancelled by the oligosaccharides. These results indicate that nucleolin is a macrophage receptor for apoptotic cells.

Cleavage and phosphorylation of XRCC4 protein induced by X-irradiation

We report the p35 and p60 forms of XRCC4 protein, appearing in human leukemia MOLT‐4 or U937 cells following X‐irradiation or hyperthermia. p35 appeared in conjunction with the cleavage of DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) and the fragmentation of internucleosomal DNA, and was suppressed by Ac‐DEVD‐CHO. p35 was also produced in vitro by treating MOLT‐4 cell lysate with recombinant caspases, suggesting that p35 was a caspase‐cleaved fragment of XRCC4 in apoptotic cell death. p60 was sensitive to treatment with phosphatase or wortmannin and was undetectable in M059J cells deficient in DNA‐PKcs. However, p60 was found in ataxia‐telangiectasia cells after irradiation. These results indicated p60 as a phosphorylated form of XRCC4, requiring DNA‐PKcs but not ataxia‐telangiectasia mutated (ATM).

Nucleolin as cell surface receptor for tumor necrosis factor-α inducing protein: a carcinogenic factor of Helicobacter pylori

PurposeTumor necrosis factor-α inducing protein (Tipα) is a unique carcinogenic factor released from Helicobacter pylori (H. pylori). Tipα specifically binds to cells and is incorporated into cytosol and nucleus, where it strongly induces expression of TNF-α and chemokine genes mediated through NF-κB activation, resulting in tumor development. To elucidate mechanism of action of Tipα, we studied a binding protein of Tipα in gastric epithelial cells.MethodsTipα binding protein was found in cell lysates of mouse gastric cancer cell line MGT-40 by FLAG-pull down assay and identified to be cell surface nucleolin by flow cytometry using anti-nucleolin antibody. Incorporation of Tipα into the cells was determined by Western blotting and expression of TNF-α gene was quantified by RT-PCR.ResultsNucleolin was co-precipitated with Tipα-FLAG, but not with del-Tipα-FLAG (an inactive mutant). After treatment with Tipα-FLAG, incorporated Tipα was co-immunoprecipitated with endogenous nucleolin using anti-nucleolin antibody. The direct binding of Tipα to recombinant His-tagged nucleolin fragment (284–710) was also confirmed. Although nucleolin is an abundant non-ribosomal protein of the nucleolus, we found that nucleolin is present on the cell surface of MGT-40 cells. Pretreatment with anti-nucleolin antibody enhanced Tipα-incorporation into the cells through nucleolin internalization. In addition, pretreatment with tunicamycin, an inhibitor of N-glycosylation, decreased the amounts of cell surface nucleolin and inhibited both internalization of Tipα and expression of TNF-α gene.ConclusionsAll the results indicate that nucleolin acts as a receptor for Tipα and shuttles Tipα from cell surface to cytosol and nuclei. These findings provide a new mechanistic insight into gastric cancer development with Tipα.

Involvement of SAPK/JNK pathway in X-ray-induced rapid cell death of human T-cell leukemia cell line MOLT-4

We found that SAPK/JNK was phosphorylated during X-ray-induced rapid cell death of MOLT-4 cells and that acid Sphingomyelinase inhibitor D609 suppressed the rapid cell death as well as phosphorylation of SAPK/JNK. Also C2-ceramide caused phosphorylation of SAPK/JNK, followed by rapid cell death. Further we isolated X-ray-resistant radiation-hybrid clones from MOLT-4 and 50 Gy irradiated mouse FM3A cells by repeated selections with 3 Gy irradiation. One of them named Rh-1a was found resistant to X-ray- as well as C2-ceramide-induced rapid cell death. Rh-1a cells had mouse DNA but no increase in either mouse or human Bcl-2 determined by Western blotting. Accumulation of p53 after X-irradiation was similarly observed in both parental MOLT-4 and Rh-1a cells. However, contrasting to prolonged and prominent phosphorylated status of SAPK/JNK in MOLT-4 cells, Rh-1a cells exhibited short transient increase and FM3A cells showed no increase of phosphorylated status SAPK/JNK after X-irradiation. Therefore, SAPK/JNK activation is considered important in X-ray-induced rapid cell death or apoptosis of MOLT-4 cells.

Wortmannin-Enhanced X-Ray-Induced Apoptosis of Human T-Cell Leukemia MOLT-4 Cells Possibly through the JNK/SAPK Pathway

Abstract Tomita, M., Suzuki, N., Matsumoto, Y., Enomoto, A., Yin, H. L., Hosoi, Y., Hirano, K. and Sakai, K. Wortmannin-Enhanced X-Ray-Induced Apoptosis of Human T-Cell Leukemia MOLT-4 Cells Possibly through the JNK/SAPK Pathway. Radiat. Res. 160, 467–477 (2003). We demonstrated that enhancement of X-ray-induced apoptosis/rapid cell death by wortmannin accompanied by increased activation of JNK/SAPK in human leukemia MOLT-4 cells. Rapid cell death/apoptosis was determined either by the dye exclusion test or by the appearance of Annexin V-positive cells and cleaved PARP fragments. Enhancement was observed only at higher concentrations of wortmannin, i.e. 1 μM or more. At these high concentrations, both DNA-PK and ATM were inhibited. X-ray-induced phosphorylation of Ser 15 of p53/TP53, accumulation of both p53/TP53 and p21/WAF1/CDKN1A, and phosphorylation of XRCC4 were all suppressed. The enhancement of apoptosis/rapid cell death by wortmannin was prevented by addition of caspase inhibitors, Z-VAD-FMK or Ac-DEVD-CHO, or by transfection and overexpression of mouse Bcl2, which is known as an anti-apoptosis protein. The requirement for a high concentration of wortmannin, i.e. 1 μM or more, indicates that inhibition of both DNA-PK and ATM was necessary for the enhanced apoptosis/rapid cell death. Phosphorylation of AKT/PKB was completely suppressed at a much lower concentration, i.e. 0.1 μM wortmannin, where no enhancement of X-ray-induced apoptosis/rapid cell death was observed. On the other hand, X-ray-induced phosphorylation of JNK and its kinase activity as well as apoptosis/rapid cell death were all significantly enhanced only at high concentrations of wortmannin, i.e. 1 μM or more. Furthermore, the extent of enhancement of both JNK phosphorylation and of apoptosis/rapid cell death by wortmannin was less in Rh1a cells, which are ceramide- and radiation-resistant variant cells compared to the parental MOLT-4 cells. Therefore, activation of the JNK pathway was considered important for the enhancement of X-ray-induced apoptosis/rapid cell death of MOLT-4 cells by wortmannin, because of the requirement for a higher concentration of wortmannin than that required for inhibition of AKT phosphorylation. The suppression of the AKT-dependent pathway by wortmannin may have some underlying role in activating the JNK pathway toward the enhancement of cell death in the current system.

Increased Expression after X‐Irradiation of MUC1 in Cultured Human Colon Carcinoma HT‐29 Cells

The effect of X‐irradiation on production of MUC1 was studied with human colon carcinoma HT‐29 cells. As evaluated by immunocytochemical staining, the percentages of MUC1‐positive cells in cells at 4 days after 6 Gy irradiation and in unirradiated control cells were 52±3.5% (n=6) and 26±2.8% (n=6), respectively. Flow‐cytometric analysis of living cells showed that MUC1 began to rise from day 1, reaching a plateau by day 4 after 6 Gy irradiation. Western blot analysis with monoclonal antibody MY.1E12 against glycosylated MUC1 (mature form) showed dose‐dependent increases of two bands (500 and 390 kDa) corresponding to two polymorphic MUC1 alleles. Premature forms of MUC1 (350 and 240 kDa) were detectable with monoclonal antibody HMFG‐2 only in irradiated cells, suggesting that new core protein synthesis had been induced. The transcriptional activity of the MUC1 gene was analyzed in terms of transient expression of MUC1‐CAT reporter plasmids containing 5′‐flanking sequences of the MUC1 gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The results of CAT assay indicate that enhanced expression of MUC1 in irradiated HT‐29 cells was due to upregulation of MUC1 transcription, and required the upstream promoter.

Abstract 561: Role of nucleolin in activation of NF-κB signaling with Tip≤, a carcinogenic factor of H. pylori

TNF-α inducing protein (Tip≤), a carcinogenic factor secreted from H. pylori, enters the cell through binding to nucleolin, a receptor of Tip≤ on the cell surface. Internalized Tip≤ induces NF-κB activation and TNF-α gene expression and finally induces tumor promotion in the stomach. Nucleolin is a major protein in nucleoli, but significant amounts of nucleolin localize on the cell surface of several gastric cancer cell lines. We investigate the relationship between induction of both NF-κB activation and TNF-α gene expression and the presence of nucleolin, the latter of which acts as a shuttling protein of various molecules, such as Tip≤, lactoferrin and AS1411, from cell surface to cytosol or nuclei. Lactoferrin, iron-binding glycoprotein with 80 kDa, shows anti-cancer activity and preventive activity of H. pylori infection. AS1411 is a nucleolin targeted DNA aptamer associated with a potent anti-cancer activity. First, we studied the effects of lactoferrin, AS1411, and anti-NUC295 (an antibody recognizing cell surface nucleolin) on Tip≤ incorporation into the cells and Tip≤-induced TNF-α gene expression. Pretreatment of mouse gastric cancer cells (MGT-40) with lactoferrin significantly inhibited the incorporation of Tip≤ and Tip≤-induced TNF-α gene expression. However, pretreatment with AS1411 enhanced the incorporation of Tip≤, and inhibited Tip≤-induced TNF-α gene expression. It is of interest to note that Anti-NUC295 enhanced both the incorporation of Tip≤ and Tip≤-induced TNF-α gene expression. These results suggest that both lactoferrin and AS1411 inhibit tumor promoting activity of Tip≤ mediated through inhibition of NF-κB signaling. Thus we think that nucleolin acts as not only a shuttling protein but also a direct regulator of NF-κB signaling. We discuss how lactoferrin and AS1411 inhibit NF-κB activation induced by Tip≤-nucleolin complex, and how nucleolin regulates the NF-κB signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 561. doi:1538-7445.AM2012-561

Abstract 1934: Molecular mechanism of abnormal localization of nucelolin on cell-surface of gastric cancer cells, with regard to Ras signaling pathway

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Our intensive study of TNF-α inducing protein (Tipα), a carcinogenic factor secreted from H. pylori, revealed that nucleolin acts as a receptor of Tipα in a mouse gastric cancer cell line (MGT-40). Although nucleolin is a major nucleolar protein, nucleolin was found in the cell membrane by cell fractionation and on the cell-surface by flow cytometrical analysis. We further found that nucleolin shuttles Tipα from cell surface to the cytosol, resulting in expression of TNF-α gene, an endogenous tumor promoter. Moreover, a large amount of nucleolin was found in the membrane fraction of MGT-40 cells, but not in that of mouse normal glandular stomach. These results suggest that abnormal localization of nucleolin occures in gastric cancer, as an associated process of carcinogenesis. In the light of this evidence, we think that the abnormal localization of nucleolin on cell surface produces the optimal carcinogenic environment for Tipα binding in gastric cancer development by H. pylori infection. Experimentally we found that large amounts of nucleolin were expressed on the surface of four human gastric cancer cell lines (MKN-45, MKN-74, AGS, and KATOIII), with smaller amounts on that of another cell line (MKN-1), although the total amounts of nucleolin were almost the same among these five cell lines. It is important to note that the amounts of cell-surface nucleolin are correlated well with those of Ras-GTP form, the active form of Ras protein in these cell lines, and we confirmed that MKN-1 cells had the lowest amounts of Ras-GTP form among the five lines. Moreover, the treatment of AGS cells with okadaic acid, a tumor promoter and inhibitor of protein phosphatases 1 and 2A, reduced the amount of nucleolin localized on cell-surface, indicating that abnormal localization of nucleolin on cell surface can be induced by Ras signaling pathway and post-translational modifications. Thus, we discuss the relationship between the abnormal localization of nucleolin and the disorder in the Ras signaling pathway in gastric cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1934. doi:10.1158/1538-7445.AM2011-1934