Hideki Yagi

CD44 Variant Regulates Redox Status in Cancer Cells by Stabilizing the xCT Subunit of System xc− and Thereby Promotes Tumor Growth

CD44 is an adhesion molecule expressed in cancer stem-like cells. Here, we show that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH). Human gastrointestinal cancer cells with a high level of CD44 expression showed an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS). Ablation of CD44 induced loss of xCT from the cell surface and suppressed tumor growth in a transgenic mouse model of gastric cancer. It also induced activation of p38(MAPK), a downstream target of ROS, and expression of the gene for the cell cycle inhibitor p21(CIP1/WAF1). These findings establish a function for CD44v in regulation of ROS defense and tumor growth.

Specific niches for lung-resident memory CD8+ T cells at the site of tissue regeneration enable CD69-independent maintenance.

Takamura et al. show that most lung CD8+ TRM cells are not maintained in the inducible bronchus-associated lymphoid tissue (iBALT) but are maintained in specific niches created at the site of tissue regeneration, which are termed as repair-associated memory depots (RAMDs).

Production and characterization of highly tumor-specific rat monoclonal antibodies recognizing the extracellular domain of human l-type amino-acid transporter 1

l‐type large amino acid transporter (LAT) 1, the first light chain (lc) of cluster of differentiation 98 (CD98) to be identified, is associated with the heavy chain (hc) of CD98 and expressed on the surface of various tumor cells irrespective of their origin. Because LAT1 is a 12‐pass membrane protein and its possible immunogenic extracellular region is very small, specific monoclonal antibodies (mAb) had not been developed. We report the successful preparation and characterization of mAb recognizing the extracellular domain of human LAT1 protein. Two mAb were selected from hybridoma clones established by fusing mouse myeloma cells and spleen cells from rats immunized against RH7777 rat hepatoma cells expressing recombinant green fluorescent protein fused to human LAT1 protein. Designated SOL22 and SOL69, these mAb specifically reacted with the extracellular domain of LAT1 on cells transfected with cDNA of LAT1, but not with cells transfected with cDNA of other CD98 lc, namely, LAT2, y+LAT1, y+LAT2, and xCT amino acid transporters. These mAb immunoprecipitated 35‐ and 90‐kDa proteins under reducing conditions in extracts prepared from human HeLa tumor cells, indicating the existence of intermolecular disulfide bonds between cysteine residues in the 90‐kDa hc and 35‐kDa lc (LAT1). SOL22 and SOL69 mAb reacted with a wide variety of living unfixed human tumor cell lines, but were only weakly reactive with HEK293F human embryonic kidney cells and human peripheral blood cells. Comparative immunohistochemical analyses of normal human tissues with anti‐CD98 hc and anti‐LAT1 revealed LAT1 to be an excellent molecular target for antibody therapy, possibly even superior to CD98 hc. (Cancer Sci 2008; 99: 1000–1007)

Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy.

L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4kb. We established five homozygous LAT1-disrupted (LAT1(-/-)) cell clones, derived from a heterozygous LAT1(+/-) clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1(-/-) DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1(-/-) cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1(-/-) DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1(-/-) DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1(+/-) DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

Anti-tumor effect against human cancer xenografts by a fully human monoclonal antibody to a variant 8-epitope of CD44R1 expressed on cancer stem cells

Background CD44 is a major cellular receptor for hyaluronic acids. The stem structure of CD44 encoded by ten normal exons can be enlarged by ten variant exons (v1-v10) by alternative splicing. We have succeeded in preparing MV5 fully human IgM and its class-switched GV5 IgG monoclonal antibody (mAb) recognizing the extracellular domain of a CD44R1 isoform that contains the inserted region coded by variant (v8, v9 and v10) exons and is expressed on the surface of various human epithelial cancer cells. Methods and Principal Findings We demonstrated the growth inhibition of human cancer xenografts by a GV5 IgG mAb reshaped from an MV5 IgM. The epitope recognized by MV5 and GV5 was identified to a v8-coding region by the analysis of mAb binding to various recombinant CD44 proteins by enzyme-linked immunosorbent assay. GV5 showed preferential reactivity against various malignant human cells versus normal human cells assessed by flow cytometry and immunohistological analysis. When ME180 human uterine cervix carcinoma cells were subcutaneously inoculated to athymic mice with GV5, significant inhibition of tumor formation was observed. Furthermore, intraperitoneal injections of GV5markedly inhibited the growth of visible established tumors from HSC-3 human larynx carcinoma cells that had been subcutaneously transplanted one week before the first treatment with GV5. From in vitro experiments, antibody-dependent cellular cytotoxicity and internalization of CD44R1 seemed to be possible mechanisms for in vivo anti-tumor activity by GV5. Conclusions CD44R1 is an excellent molecular target for mAb therapy of cancer, possibly superior to molecules targeted by existing therapeutic mAb, such as Trastuzumab and Cetuximab recognizing human epidermal growth factor receptor family.

S1P lyase in thymic perivascular spaces promotes egress of mature thymocytes via up-regulation of S1P receptor 1

Sphingosine 1-phosphate (S1P) and S1P receptor 1 (S1P1) play an important role in the egress of mature CD4 or CD8 single-positive (SP) thymocytes from the thymus. Fingolimod hydrochloride (FTY720), an S1P1 functional antagonist, induced significant accumulation of CD62L(high)CD69(low) mature SP thymocytes in the thymic medulla. Immunohistochemical staining using anti-S1P1 antibody revealed that S1P1 is predominantly expressed on thymocytes in the thymic medulla and is strongly down-regulated even at 3h after FTY720 administration. 2-Acetyl-4-tetrahydroxybutylimidazole (THI), an S1P lyase inhibitor, also induced accumulation of mature SP thymocytes in the thymic medulla with an enlargement of the perivascular spaces (PVS). At 6h after THI administration, S1P1-expressing thymocytes reduced partially as if to form clusters and hardly existed in the proximity of CD31-expressing blood vessels in the thymic medulla, suggesting S1P lyase expression in the cells constructing thymic medullary PVS. To determine the cells expressing S1P lyase in the thymus, we newly established a mAb (YK19-2) specific for mouse S1P lyase. Immunohistochemical staining with YK19-2 revealed that S1P lyase is predominantly expressed in non-lymphoid thymic stromal cells in the thymic medulla. In the thymic medullary PVS, S1P lyase was expressed in ER-TR7-positive cells (reticular fibroblasts and pericytes) and CD31-positive vascular endothelial cells. Our findings suggest that S1P lyase expressed in the thymic medullary PVS keeps the tissue S1P concentration low around the vessels and promotes thymic egress via up-regulation of S1P1.

Towards therapeutic antibodies to membrane oncoproteins by a robust strategy using rats immunized with transfectants expressing target molecules fused to green fluorescent protein.

Cell‐surface molecules containing growth factor receptors, adhesion molecules and transporter proteins are often over‐expressed in various cancer cells, and could be regarded as suitable targets for therapeutic monoclonal antibodies (mAb). Anti‐cancer therapeutic mAb are claimed to bind these cell‐surface molecules on viable cancer cells: therefore, it is necessary to produce mAb recognizing epitopes on the extracellular domains of native but not denatured proteins. We have experienced difficulty in obtaining mAb bound to viable cancer cells using synthetic peptides or recombinant proteins produced in bacteria as immunogens, although these immunogens are relatively easy to prepare. In this context, we have concluded that viable cancer cells or cells transfected with cDNA encoding target proteins are suitable immunogens for the production of anti‐cancer therapeutic mAb. Furthermore, we selected rats as the immunized animals, because of their excellent capacity to generate diverse antibodies. Because many target candidates are multi‐pass (type IV) membrane proteins, such as 7‐pass G protein‐coupled receptors and 12‐pass transporter proteins belonging to the solute carrier family, and their possible immunogenic extracellular regions are very small, production of specific mAb was extremely difficult. In this review, we summarize the successful preparation and characterization of rat mAb immunized against the extracellular domain of type I, type II and type IV membrane oncoproteins fused to green fluorescent protein as an approach using reverse genetics, and also introduce the discovery of cell‐death‐inducing antibodies as an approach using forward genetics and a strategy to produce reshaped antibodies using mimotope peptides as the immunogen. (Cancer Sci 2011; 102: 25–35)

Kupffer cells support extramedullary erythropoiesis induced by nitrogen-containing bisphosphonate in splenectomized mice.

Our previous study indicated that injecting nitrogen-containing bisphosphonate (NBP) induced the site of erythropoiesis to shift from the bone marrow (BM) to the spleen. This was due to the depletion of BM-resident macrophages, which support erythropoiesis. In this study, we examined NBP treatment-induced extramedullary hematopoiesis in splenectomized mice, focusing on hepatic hematopoiesis. NBP-treated mice did not display anemia or significant change in erythropoietin production, while megakaryopoiesis and erythropoiesis were constantly observed in the liver. Erythroblastic islands were detected in the sinusoidal lumen. Kupffer cells expressed VCAM-1 following NBP treatment, which is an important factor for erythroblast differentiation. Cl(2)MBP-liposome treatment depleted the erythroblastic islands, and decreased the number of hematopoietic cells in the liver, as determined by colony forming assays. Together, these results indicate that Kupffer cells support erythropoiesis, acting as stromal cells in the liver, and that they might act as a niche for hematopoietic precursor cells in an emergency.

Simultaneous induction of apoptotic, autophagic, and necrosis-like cell death by monoclonal antibodies recognizing chicken transferrin receptor

Programmed cell death (PCD) is categorized as apoptotic, autophagic, or necrosis-like. Although the possibility that plural (two or three) death signals could be induced by a given stimulus has been reported, the precise mechanisms regulating PCD are not well understood. Recently, we have obtained two anti-chicken transferrin receptor (TfR) monoclonal antibodies (mAbs; D18 and D19) inducing a unique cell death. Although the cell death had several features of apoptosis, autophagic and necrosis-like morphological alterations were simultaneously observed in electron microphotographs. In addition to cells with condensed chromatin and an intact plasma membrane (apoptotic cells), cells having many vacuoles in the cytoplasm (autophagic cells), and enlarged cells with ruptured plasma membranes (necrosis-like cells) were observed in DT40 cells treated with the mAbs, however, the latter two types of dead cells were not detected upon treatment with staurosporine, a typical apoptosis inducer. In autophagic cells, numerous membrane-bound vesicles occupying most of the cytoplasmic space, which frequently contained electron-dense materials from cytoplasmic fragments and organelles, were observed. The simultaneous induction of multiple death signals from a stimulus via the TfR is of great interest to those researching cell death. In addition, activation of caspases was observed in DT40 cells treated with D19, however, the cell death was not inhibited with z-VAD-fmk, a pan-caspase inhibitor, suggesting that at least in part, a caspase-independent pathway is involved in the TfR-mediated cell death.

Cell-death-inducing monoclonal antibodies raised against DT40 tumor cells: Identification of chicken transferrin receptor as a novel cell-death receptor

We obtained unique cell‐death‐inducing monoclonal antibodies (mAbs) named D18 and D19 against chicken DT40 cells. D18 and D19 caused several signs of apoptosis, such as exposed phosphatidyl serine on the cell surface, a sub G0/G1 peak, and DNA fragmentation, and inhibited the proliferation of DT40 cells. Flow cytometric and immunohistological analyses of various normal chicken tissues revealed the expression of the antigen recognized by these mAbs to be restricted to cells in lymphoid organs including bone marrow and bursa of fabricius, and to cells in some epithelial tissues. The cell death induced by the mAbs progressed through a mitochondrial pathway with loss of mitochondrial membrane potential. Apoptosis is generally characterized by cell shrinking; however, D18 and D19 elicited swelling, which preceded the cell death. We analyzed the antigen immunoprecipitated by the mAbs, and identified a 90‐ to 100‐kDa cell‐surface glycoprotein as the chicken transferrin receptor (TfR). Epitopes recognized by the two mAbs were confirmed to be different by the binding inhibition assay. The reactivity of the mAbs against DT40 cells was not inhibited by excess chicken serum, suggesting that the cell death induced by D18 and D19 was not caused by inhibition of the binding of transferrin (Tf) to chicken TfR. Since D18 and D19 have induced cell death in human embryonic kidney cells transfected with cDNA of the full‐length chicken TfR, we expect human TfR to be a promising target in antibody therapy for various human malignancies. (Cancer Sci 2008; 99: 894–900)