Search

Small interfering RNAs (siRNAs) are potential tools for gene-silencing therapy, but their instability is one of the obstacles in the development of siRNA-based drugs. To improve siRNA stability, we synthesised a double-stranded RNA-binding cationic oligodiaminogalactose 4mer (ODAGal4) and investigated here its characteristics for siRNA stabilisation in vitro. ODAGal4 improved the resistance of various siRNAs against serum degradation. The effect of ODAGal4 on siRNA stabilisation was further amplified by introduction of modified nucleotides into the siRNA. In particular, a combination of ODAGal4 and incorporation of phosphorothioate linkages into the siRNA prominently prevented degradation by serum. The half-lives of fully phosphorothioate-modified RNA duplexes with ODAGal4 were more than 15 times longer than those of unmodified siRNAs without ODAGal4; this improvement in serum stability was superior to that observed for other chemical modifications. Serum degradation assays of RNAs with multiple chemical modifications showed that ODAGal4 preferentially improves the stability of RNAs with phosphorothioate modification among chemical modifications. Furthermore, melting temperature analysis showed that ODAGal4 greatly increases the thermal stability of phosphorothioate RNAs. Importantly, ODAGal4 did not interrupt gene-silencing activity of all the RNAs tested. Collectively, these findings demonstrate that ODAGal4 is a potent stabiliser of siRNAs, particularly nucleotides with phosphorothioate linkages, representing a promising tool in the development of gene-silencing therapies.

Supplemental Digital Content is available in the text., We previously found that heat-shock protein 70 (HSP70) is expressed on hepatocellular carcinoma cells and developed an HSP70 mRNA-transfected dendritic cell therapy for treating unresectable or recurrent hepatocellular carcinoma. The phase I trial was completed successfully. The purpose of this study is to identify a promiscuous epitope peptide derived from HSP70 for the purpose of developing a novel cancer peptide vaccine. Using a computational algorithm to analyze the specificity of previously reported major histocompatibility complex class I–binding peptides, we selected candidates that bound to >2 of the 3 HLA types. Twenty-nine HSP70-derived peptides (9-mers) that bound to HLA-class I was selected. The peptides were prioritized based on the results of peptide binding experiments. Using dendritic cells stimulated with the candidate peptide described previously as stimulators and CD8+ T cells as effectors, an ELISPOT assay was performed. Cytotoxicity of CD8 lymphocytes stimulated with the candidate peptides toward HSP70-expressing cancer cells was analyzed using an xCELLigence System. Peptides were administered to HLA-A 24 transgenic mice as vaccines, and peptide-specific T-cell induction was measured in vivo. We identified a multi-HLA-class I–binding epitope peptide that bound to HLA-A*02:01, *02:06, and *24:02 in vitro using an interferon-γ ELISPOT immune response induction assay. Cytotoxicity was confirmed in vitro, and safety and immune response induction were confirmed in vivo using HLA-A 24 transgenic mice. Our study demonstrated that the promiscuous HSP70-derived peptide is applicable to cancer immunotherapy in patients with HLA-A*24:02-positive, *02:01-positive, and *02:06-positive HSP70-expressing cancers.

Homozygous HLA-DR4/I-E transgenic mice (tgm) spontaneously developed colitis similar to human ulcerative colitis. We explored whether endoplasmic reticulum stress in colonic epithelial cells due to overexpression of HLA-DR4/I-E was involved in the pathogenesis of colitis.Major histocompatibility complex class II transactivator-knockout (CIITAKO) background tgm were established to test the involvement of HLA-DR4/I-E expression in the pathogenesis of colitis. Histological and cellular analyses were performed and the effect of oral administration of the molecular chaperone tauroursodeoxycholic acid (TUDCA) and antibiotics were investigated. IgA content of feces and serum and presence of IgA-coated fecal bacteria were also investigated.Aberrantly accumulated HLA-DR4/I-E molecules in colonic epithelial cells were observed only in the colitic homozygous tgm, which was accompanied by upregulation of the endoplasmic reticulum stress marker Binding immunoglobulin protein (BiP) and reduced mucus. Homozygous tgm with CIITAKO, and thus absent of HLA-DR4/I-E expression, did not develop colitis. Oral administration of TUDCA to homozygotes reduced HLA-DR4/I-E and BiP expression in colonic epithelial cells and restored the barrier function of the intestinal tract. The IgA content of feces and serum, and numbers of IgA-coated fecal bacteria were higher in the colitic tgm, and antibiotic administration suppressed the expression of HLA-DR4/I-E and colitis.The pathogenesis of the colitis observed in the homozygous tgm was likely due to endoplasmic reticulum stress, resulting in goblet cell damage and compromised mucus production in the colonic epithelial cells in which HLA-DR4/I-E molecules were heavily accumulated. Commensal bacteria seemed to be involved in the accumulation of HLA-DR4/I-E, leading to development of the colitis.

Purpose: The anaphylatoxin C5a is a chemoattractant that induces leukocyte migration via C5a receptor (C5aR). There is emerging evidence that C5a is generated in the cancer microenvironment. We therefore sought C5aR expression and a direct influence of the C5a–C5aR axis on cancer cells. Experimental Design: C5aR expression was investigated in human cancer tissues and cell lines. Effects of C5a stimulation on cancer cells were studied by cytoskeletal rearrangement, time-lapse analysis, Matrigel chamber assay, and invasion in nude mouse in a comparison of C5aR-expressing cancer cells with control cells. Results: C5aR was aberrantly expressed in various human cancers. Several cancer cell lines also expressed C5aR. C5a triggered cytoskeletal rearrangement and enhanced cell motility three-fold and invasiveness 13-fold of C5aR-expressing cancer cells. Such enhancement by C5a was not observed in control cells. Cancer cell invasion was still enhanced in the absence of C5a concentration gradient and even after the removal of C5a stimulation, suggesting that random cell locomotion plays an important role in C5a-triggered cancer cell invasion. C5a increased the release of matrix metalloproteinases (MMP) from cancer cells by two- to 11-fold, and inhibition of MMP activity abolished the C5a-enhancing effect on cancer cell invasion. Compared with control cells, C5aR-expressing cells spread 1.8-fold more broadly at implanted nude mouse skin sites only when stimulated with C5a. Conclusions: These results illustrate a novel activity of the C5a–C5aR axis that promotes cancer cell invasion through motility activation and MMP release. Targeting this signaling pathway may provide a useful therapeutic option for cancer treatment. Clin Cancer Res; 19(8); 2004–13. ©2013 AACR.

To study the role of TNF-α in tongue cancer metastasis, we made highly metastatic cells from a human oral squamous cell carcinoma cell line (SAS) by repeating the passage in which the cells were injected into a nude mouse tongue and harvested from metastasized cervical lymph nodes. Cancer cells after 5 passages (GSAS/N5) increased invasive activity 7-fold in a TNF-α receptor 1 (TNFR1)-dependent manner and enhanced mRNA expression of TNF-α and TNFR1. In the highly metastatic cells, NF-κB activation was upregulated via elevated phosphorylation of Akt and Ikkα/β in the signaling pathway and secretion of TNF-α, active MMP-2 and MMP-9 increased. Suppression of increase of TNF-α mRNA expression and MMP secretion by NF-κB inhibitor NBD peptide suggested a positive feedback loop in GSAS/N5 cells; TNF-α activates NF-κB and activated NF-κB induces further TNF-α secretion, leading to increase of active MMP release and promotion of invasion and metastasis of the cells. GSAS/N5 cells that had been injected into the nude mouse tongue and harvested from metastasized lungs multiplied angiopoietin-like 4 (angptl4) expression with enhanced migration activity, which indicated a possible involvement of angptl4 in lung metastasis of the cells. These results suggest that TNF-α and angptl4 promote metastasis of the oral cancer cells, thus, these molecules may be therapeutic targets for patients with tongue cancer.

The present study attempted to identify a useful tumor-associated antigen (TAA) for lung cancer immunotherapy and potential immunogenic peptides derived from the TAA. We focused on cell division cycle 45-like (CDC45L), which has a critical role in the initiation and elongation steps of DNA replication, as a novel candidate TAA for immunotherapy based on a genome-wide cDNA microarray analysis of lung cancer. The CDC45L was overexpressed in the majority of lung cancer tissues, but not in the adjacent non-cancerous tissues or in many normal adult tissues. We examined the in vitro and in vivo anti-tumor effects of cytotoxic T-lymphocytes (CTL) specific to CDC45L-derived peptides induced from HLA-A24 (A*24:02)-positive donors. We identified three CDC45L-derived peptides that could reproducibly induce CDC45L-specific and HLA-A24-restricted CTL from both healthy donors and lung cancer patients. The CTL could effectively lyse lung cancer cells that endogenously expressed both CDC45L and HLA-A24. In addition, we found that CDC45L (556) KFLDALISL(564) was eminent in that it induced not only HLA-A24 but also HLA-A2 (A*02:01)-restricted antigen specific CTL. Furthermore, the adoptive transfer of the CDC45L-specific CTL inhibited the growth of human cancer cells engrafted into immunocompromised mice. These results suggest that these three CDC45L-derived peptides are highly immunogenic epitopes and CDC45L is a novel TAA that might be a useful target for lung cancer immunotherapy.

Background: Identification of tumour-associated antigens (TAAs) that induce cytotoxic T lymphocytes (CTLs) specific to cancer cells is critical for the development of anticancer immunotherapy. In this study, we aimed at identifying a novel TAA of pancreatic cancer for immunotherapy. Methods: On the basis of the genome-wide cDNA microarray analysis, we focused on KIF20A (also known as RAB6KIFL/MKlp2) as a candidate TAA in pancreatic cancer cells. The HLA-A2 (A*02:01)-restricted CTL epitopes of KIF20A were identified using HLA-A2 transgenic mice (Tgm) and the peptides were examined to check whether they could generate human CTLs exhibiting cytotoxic responses against KIF20A+, HLA-A2+ tumour cells in vitro. Results: KIF20A was overexpressed in pancreatic cancer and in some other malignancies, but not in their non-cancerous counterparts and many normal adult tissues. We found that KIF20A-2 (p12–20, LLSDDDVVV), KIF20A-8 (p809–817, CIAEQYHTV), and KIF20A-28 (p284–293, AQPDTAPLPV) peptides could induce HLA-A2-restricted CTLs in HLA-A2 Tgm without causing autoimmunity. Peptide-reactive human CTLs were generated from peripheral blood mononuclear cells of HLA-A2+ healthy donors by in vitro stimulation with the three peptides, and those CTLs successfully exhibited cytotoxic responses to cancer cells expressing both KIF20A and HLA-A2. Conclusion: KIF20A is a novel promising candidate for anticancer immunotherapeutic target for pancreatic cancers.

Insulin-like growth factor-II mRNA binding protein 3 (IMP-3) is an oncofetal protein expressed in various malignancies including lung cancer. This study aimed to identify immunogenic peptides derived from IMP-3 that can induce tumor-reactive and human leukocyte antigen (HLA)-A2 (A*02:01)-restricted cytotoxic T lymphocytes (CTL) for lung cancer immunotherapy. Forty human IMP-3-derived peptides predicted to bind to HLA-A2 were analyzed to determine their capacity to induce HLA-A2-restricted T cells in HLA-A2.1 (HHD) transgenic mice (Tgm). We found that three IMP-3 peptides primed HLA-A2-restricted CTL in the HLA-A2.1 Tgm. Among them, human CTL lines reactive to IMP-3 (515) NLSSAEVVV(523) were reproducibly established from HLA-A2-positive healthy donors and lung cancer patients. On the other hand, IMP-3 (199) RLLVPTQFV(207) reproducibly induced IMP-3-specific and HLA-A2-restricted CTL from healthy donors, but did not sensitize CTL in the HLA-A2.1 Tgm. Importantly, these two IMP-3 peptide-specific CTL generated from healthy donors and cancer patients effectively killed the cancer cells naturally expressing both IMP-3 and HLA-A2. Cytotoxicity was significantly inhibited by anti-HLA class I and anti-HLA-A2 monoclonal antibodies, but not by the anti-HLA-class II monoclonal antibody. In addition, natural processing of these two epitopes derived from the IMP-3 protein was confirmed by specific killing of HLA-A2-positive IMP-3-transfectants but not the parental IMP-negative cell line by peptide-induced CTL. This suggests that these two IMP-3-derived peptides represent highly immunogenic CTL epitopes that may be attractive targets for lung cancer immunotherapy.

MS-based quantitative proteomics is widely used for large scale identification of proteins. However, an integrated approach that offers comprehensive proteome coverage, a tool for the quick categorization of the identified proteins, and a standardized biological study method is needed for helping the researcher focus on investigating the proteins with biologically important functions. In this study, we utilized isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative differential LC/MS/MS, functional annotation with a proprietary gene ontology tool (Molecular Annotation by Gene Ontology (MANGO)), and standard biochemical methods to identify proteins related to neuronal differentiation in nerve growth factor-treated rat pheochromocytoma (PC12) cells, which serve as a representative model system for studying neuronal biological processes. We performed MS analysis by using both nano-LC-MALDI-MS/MS and nano-LC-ESI-MS/MS for maximal proteome coverage. Of 1,482 non-redundant proteins semiquantitatively identified, 72 were differentially expressed with 39 up- and 33 down-regulated, including 64 novel nerve growth factor-responsive PC12 proteins. Gene ontology analysis of the differentially expressed proteins by MANGO indicated with statistical significance that the up-regulated proteins were mostly related to the biological processes of cell morphogenesis, apoptosis/survival, and cell differentiation. Some of the up-regulated proteins of unknown function, such as PAIRBP1, translationally controlled tumor protein, prothymosin alpha, and MAGED1, were further analyzed to validate their significant functions in neuronal differentiation by immunoblotting and immunocytochemistry using each antibody combined with a specific short interfering RNA technique. Knockdown of these proteins caused abnormal cell morphological changes, inhibition of neurite formation, and cell death during each course of the differentiation, confirming their important roles in neurite formation and survival of PC12 cells. These results show that our iTRAQ-MANGO-biological analysis framework, which integrates a number of standard proteomics strategies, is effective for targeting and elucidating the functions of proteins involved in the cellular biological process being studied.

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, a negative regulator of Ras. Neurofibromin is implicated in the neuronal abnormality of NF1 patients; however, the precise cellular function of neurofibromin has yet to be clarified. Using proteomic strategies, we identified a set of neurofibromin-associating cellular proteins, including axon regulator CRMP-2 (Collapsin response mediator protein-2). CRMP-2 directly bound to the C-terminal domain of neurofibromin, and this association was regulated by the manner of CRMP-2 phosphorylation. In nerve growth factor-stimulated PC12 cells, neurofibromin and CRMP-2 co-localized particularly on the distal tips and branches of extended neurites. Suppression of neurofibromin using NF1 small interfering RNA significantly inhibited this neurite outgrowth and up-regulated a series of CRMP-2 phosphorylations by kinases identified as CDK5, GSK-3b, and Rho kinase. Overexpression of the NF1-RAS-GAP-related domain rescued these NF1 small interfering RNA-induced events. Our results suggest that neurofibromin regulates neuronal differentiation by performing one or more complementary roles. First, neurofibromin directly regulates CRMP-2 phosphorylation accessibility through the complex formation. Also, neurofibromin appears to indirectly regulate CRMP-2 activity by suppressing CRMP-2-phosphorylating kinase cascades via its Ras-GAP function. Our study demonstrates that the functional association of neurofibromin and CRMP-2 is essential for neuronal cell differentiation and that lack of expression or abnormal regulation of neurofibromin can result in impaired function of neuronal cells, which is likely a factor in NF1-related pathogenesis.

RANDAM-2, a type-I transmembrane antigen constitutively expressed on the neuronal cell lineage during mouse neurogenesis, shows the highest expression level between embryonic day 8.5 (E8.5) and E10.5. As the period well overlaps with the proliferating stages of neural stem cells (NSCs), it is conceivable that NSCs are efficiently separable based on the expression level of RANDAM-2. In this paper, we show that NSCs can be efficiently enriched as RANDAM-2 high+ cells by fluorescence-activated cell sorting. Many cells in the RANDAM-2 high+ cells had the characteristics of the self-renewal capability and potential for multilineage differentiation into neural cells. In contrast, almost all of the RANDAM-2 low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. These two subpopulations also differed from each other in terms of the expression level of molecules associated with neural differentiation. These findings demonstrate that RANDAM-2 can be regarded as a useful marker for enrichment of NSCs. © 2006 Elsevier Ireland Ltd. All rights reserved.

Members of protein kinase D (PKD) family serine/threonine kinases (PKD1, PKD2 and PKD3) are expressed in wide range of cells and regulate various cellular responses including immune responses. We have previously shown that PKD is involved in the signaling pathways of a human CD4(+) T cell clone stimulated with its cognate antigen. Contrary to foregoing publications, PKD1 mRNA was not detected in human T cells, Jurkat cells and mouse thymocytes and splenocytes. Instead, mass-spectrometric and reverse transcription-PCR analyses revealed that PKD2 was predominant in T cells. To investigate the roles of PKD2, wild-type (WT) and constitutively active (CA) PKD2 were expressed in Jurkat cells together with IL-2 promoter-driven reporter gene. Expression of WT-PKD2 enhanced IL-2 promoter activity upon stimulation with anti-CD3 mAb, while expression of CA-PKD2 inhibited IL-2 promoter activity and induced cell death. Although the cell death was suppressed by the treatment with caspase inhibitor, the IL-2 promoter activity was rarely recovered in CA-PKD2-expressing cells upon TCR stimulation. WT-PKD2 localized mainly in the cytoplasm translocated into the nucleus after TCR stimulation, while CA-PKD2 was present in both the cytoplasm and the nuclei before and after stimulation. Proteomic analyses revealed that CA-PKD2 enhanced the amount of phosphorylated SET protein, a histone chaperon that regulates histone acetylation, in Jurkat cells and the recombinant SET protein was phosphorylated by CA-PKD2 in vitro. The data provide a renewing insight into the subset of PKD family kinases expressed in T cells and suggest that PKD2 is involved in IL-2 promoter regulation and cell death depending on its activity upon TCR stimulation.

The interactions between peptide/MHC complexes and their cognate TCR are essential for various T cell responses. However, the relationship between the avidity of TCR ligand and the subsequent intracellular signaling through the TCR is still unclear. To investigate the effects of TCR ligand avidity on TCR-mediated signaling, we established L cells expressing HLA-DR4 molecules covalently linked with agonistic peptide (high-affinity ligand) or altered peptide ligand (APL; low-affinity ligand) at various densities as APC for a cognate human CD4(+) T cell clone. Using this system, we demonstrated that the T cell clone stimulated with APL/HLA-DR4 complexes presented at an excessive density provoked the up-regulation of CD69, IL-2 production and proliferation, but no detectable phosphorylation of ZAP-70/LAT/SLP-76. Furthermore, in contrast to the high-affinity stimulation, the low-affinity stimulation evoked delayed and sustained activation of the B-Raf/extracellular signal-regulated kinase (ERK) pathway without Raf-1 activation. The strength and duration of B-Raf/ERK activations closely correlated with the density of the TCR ligand. A knockdown approach confirmed that B-Raf activation was indispensable for the APL-induced T cell responses. These observations suggest that the differences in TCR-peptide/MHC interactions reflect the strength and duration of B-Raf/Raf-1/ERK activation in the human CD4(+) T cells.

Although genetic studies have suggested that heparan sulfate (HS) is involved in bone morphogenetic protein (BMP)-mediated embryonic morphogenesis, it is unclear whether HS is directly involved in BMP-mediated signaling. Here, we investigate the involvement of HS in BMP-7 signaling. We show that HS and heparin chains specifically bind to BMP-7. Digestion of cell-surface HS with heparitinase interferes with BMP-7-mediated Smad phosphorylation in ROS 17/2.8 osteoblastic cells. Inhibiting sulfation of cell-surface HS with chlorate also causes interruption of Smad phosphorylation. Addition of exogenous heparin to ROS 17/2.8 cells prevents BMP-7-mediated Smad phosphorylation rather than enhances the BMP-7 signal, suggesting that HS should be anchored on the plasma membrane for BMP signaling. Moreover, BMP-7 binding to ROS 17/2.8 cells is inhibited by chlorate treatment and exogenous application of heparin. These results demonstrate that BMP-7 specifically binds to cell-surface HS and the BMP-7-HS interaction is required for BMP-7 signaling.

Altered peptide ligands (APL) induce T cell responses different from those induced by the original agonistic peptide. As shown for CD4(+) T cells, partial agonists induce partial T cell activation without proliferation because of lower affinities and higher off rates to TCR than those of agonists. To determine whether overexpression of partially agonistic TCR ligands on antigen-presenting cells provides high-avidity TCR ligands, we generated L cell transfectants expressing various numbers of HLA-DR4 covalently linked with APL derived from a streptococcal peptide and observed responses of the cognate T cells. Some overexpressed HLA-DR4/partially agonistic APL complexes induced T cell proliferation in a density-dependent manner. However, tyrosine phosphorylation of zeta-associated protein-70 (ZAP-70) and linker for activation of T cells and kinase activity of ZAP-70 were not detectable. T cell proliferation stimulated with L cell transfectants was sensitive to the PKC inhibitor Gö6976, but to a lesser extent to Gö6983, suggesting the involvement of mu isotype of PKC (PKCmu). In vitro kinase assays revealed that PKCmu activity was up-regulated only in T cells stimulated with L cell transfectants that induced T cell proliferation. Our data suggest the presence of a unique signaling pathway coupling TCR ligation with T cell proliferation associated with PKCmu activation and impaired ZAP-70 activation.

Heparan sulfate (HS), a structurally diverse molecule comprising distinct sequences of sulfated disaccharide units, is abundant in the developing brain and binds to axon guidance molecules. Addition of HS to the developing Xenopus optic pathway causes severe targeting errors yet it is not known how the structural diversity of this molecule relates to its role in axon guidance. We have used an in vivo brain assay to identify the structural characteristics of HS that induce aberrant axon targeting. Inhibiting sulfation of endogenous HS with chlorate causes axons to bypass their target, the tectum, and treatment with chemically modified heparins reveals that 2-O- and 6-O-sulfate groups have potent bypass-inducing activity. Experiments with purified heparin saccharides show that bypass-inducing activity correlates with distinct structures, particularly those containing a combination of 2-O- and 6-O-sulfate groups. Taken together the results indicate that specific sequences, rather than gross structural composition, are critical for activity. In situ hybridisation revealed that HS 6-O-sulfotransferase is regionally expressed along the border of the dorsal optic tract whereas 2-O-sulfotransferase is expressed broadly. Our results demonstrate that specific HS sequences are essential for regulating retinotectal axon targeting and suggest that regionalised biosynthesis of specific HS structures is important for guiding axons into the tectum.

We recently identified a novel cancer-testis antigen, cell division cycle associated 1 (CDCA1) using genome-wide cDNA microarray analysis, and CDCA1-derived cytotoxic T lymphocyte (CTL)-epitopes. In this study, we attempted to identify CDCA1-derived long peptides (LPs) that induce both CD4+ helper T (Th) cells and CTLs. We combined information from a recently developed computer algorithm predicting HLA class II-binding peptides with CDCA1-derived CTL-epitope sequences presented by HLA-A2 (A*02:01) or HLA-A24 (A*24:02) to select candidate CDCA1-LPs encompassing both Th cell epitopes and CTL-epitopes. We studied the immunogenicity of CDCA1-LPs and the cross-priming potential of LPs bearing CTL-epitopes in both human in vitro and HLA-class I transgenic mice in vivo. Then we analyzed the Th cell response to CDCA1 in head-and-neck cancer (HNC) patients before and after vaccination with a CDCA1-derived CTL-epitope peptide using IFN-γ enzyme-linked immunospot assays. We identified two CDCA1-LPs, CDCA1(39–64)-LP and CDCA1(55–78)-LP, which encompass naturally processed epitopes recognized by Th cells and CTLs. CDCA1-specific CTLs were induced through cross-presentation of CDCA1-LPs in vitro and in vivo. In addition, CDCA1-specific Th cells enhanced induction of CDCA1-specific CTLs. Furthermore, significant frequencies of CDCA1-specific Th cell responses were detected after short-term in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with CDCA1-LPs in HNC patients (CDCA1(39–64)-LP, 74%; CDCA1(55–78)-LP, 68%), but not in healthy donors. These are the first results demonstrating the presence of CDCA1-specific Th cell responses in HNC patients and underline the possible utility of CDCA1-LPs for propagation of both CDCA1-specific Th cells and CTLs.

Integrin/ligand interaction is a therapeutic target for many diseases. We previously reported that residues critical for ligand binding are clustered in N-terminal repeat 3 (in the predicted 2-3 loop) of alpha 4, alpha 5 and alpha IIb. Here we have localized residues critical for ligand binding in the alpha 3 subunit of integrin alpha 3 beta 1 with distinct ligand specificity (laminin-5). We identified an alpha 3 epitope common to several function-blocking anti-alpha 3 antibodies at the boundary between repeats 1 and 2 (residues 75-80). We found that swapping the predicted 4-1 loop (residues 153-165) at the boundary between repeats 2 and 3 with the corresponding alpha 4 sequence and mutating Thr-162 and Gly-163 residues in this predicted loop block laminin-5 binding. Thr-162 and Gly-163 and the antibody epitope are separated in the primary structure; however, they are close to each other in the proposed beta-propeller model. Mutating residues recently reported to block (Tyr-186 and Trp-188) or enhance (Asp-122) laminin-5 binding to alpha 3 beta 1 [Krukonis, E. S., Dersch, P., Eble, J. A., and Isberg, R. R.(1998) J. Biol. Chem. 273, 31837-31843] did not affect laminin-5 binding under the assay conditions used. Thr-162 and Gly-163 are not critical for adhesion to invasin, indicating that laminin-5 and invasin may use different recognition mechanisms, and that mutation of Thr-162 and Gly-163 does not drastically affect the integrity of alpha 3 beta 1. These results suggest that residues critical for ligand binding may be similarly (but not identically) located in repeat 3 of the alpha subunit regardless of ligand specificity.

N-Glycolylneuraminic acid (NeuGc) is abundantly expressed in most mammals, but it is not detectable in humans. The expression of NeuGc is controlled by cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase activity. We previously cloned a cDNA for mouse CMP-NeuAc hydroxylase and found that the human genome contains a homologue. We report here the molecular basis for the absence of NeuGc in humans. We cloned a cDNA for human CMP-NeuAc hydroxylase from a HeLa cell cDNA library. The cDNA encodes a 486-amino acid protein, and its deduced amino acid sequence lacks a domain corresponding to the N-terminal 104 amino acids of the mouse CMP-NeuAc hydroxylase protein, although the human protein is highly identical (93%) to the rest of the mouse hydroxylase protein. The N-terminal truncation of the human hydroxylase is caused by deletion of a 92-base pair-long exon in human genomic DNA. The human hydroxylase expressed in COS-7 cells exhibited no enzymatic activity, and a mouse hydroxylase mutant, which lacks the N-terminal domain, was also inactive. A chimera composed of the human hydroxylase and the N-terminal domain of the mouse hydroxylase displayed the enzyme activity. These results indicate that the human homologue of CMP-NeuAc hydroxylase is inactive because it lacks an N-terminal domain that is essential for enzyme activity. The absence of NeuGc in human glycoconjugates is due to a partial deletion in the gene that encodes CMP-NeuAc hydroxylase.

Integrins are a family of alpha/beta heterodimers of cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions. Both alpha and beta subunits have a large extracellular domain and a short cytoplasmic domain. The alpha subunit has seven sequence repeats of 60-70 residues in its N-terminal region. The beta-propeller model, in which seven four-stranded beta-sheets are arranged in a torus around a pseudosymmetry axis, has been proposed as a structural model of these seven repeats. Several predicted loops critical for ligand binding have been identified in the upper face of the proposed beta-propeller model. Several alpha subunits (e.g., alpha 2, alpha L and alpha M) have I-domains of about 200 residues inserted between their second and third repeats. These I-domains adopt a Rossman-fold structure and have major ligand and cation binding sites (the MIDAS site) on their surfaces. The beta subunit has an I-domain-like structure in its N-terminal region. This structure includes multiple sequences/conserved oxygenated residues critical for ligand binding (e.g., Asp-119 in beta 3), and non-conserved residues critical for ligand specificities. Several "activation-dependent" epitopes have been identified in the Cys-rich (stalk) region of beta 1. It has yet to be determined how these multiple ligand binding sites in the alpha and beta subunits are involved in ligand binding, and how conformational changes on activation/ligand occupancy relate to signal transduction.

Purpose: To identify long peptides (LP) derived from a novel tumor-associated antigen (TAA), kinesin family member 20A (KIF20A), which induce tumor-specific T-helper type 1 (TH1) cells and CTLs. Experimental Design: We combined information from a recently developed computer algorithm predicting HLA class II–binding peptides with KIF20A-derived CTL-epitope sequences presented by HLA-A2 (A*02:01) or HLA-A24 (A*24:02) to select candidate promiscuous TH1-cell epitopes containing CTL epitopes. Peripheral blood mononuclear cells (PBMC) derived from healthy donors or patients with head-and-neck malignant tumor (HNMT) were used to study the immunogenicity of KIF20A-LPs, and the in vitro cross-priming potential of KIF20A-LPs bearing CTL epitopes. We used HLA-A24 transgenic mice to address whether vaccination with KIF20A-LP induces efficient cross-priming of CTLs in vivo. The TH1-cell response to KIF20A-LPs in HNMT patients receiving immunotherapy with TAA-derived CTL-epitope peptides was analyzed using IFN-γ enzyme-linked immunospot assays. Results: We identified promiscuous KIF20A-LPs bearing naturally processed epitopes recognized by CD4+ T cells and CTLs. KIF20A-specific CTLs were induced by vaccination with a KIF20A-LP in vivo. KIF20A expression was detected in 55% of HNMT by immunohistochemistry, and significant frequencies of KIF20A-specific TH1 cell responses were detected after short-term in vitro stimulation of PBMCs with KIF20A-LPs in 50% of HNMT patients, but not in healthy donors. Furthermore, these responses were associated with KIF20A expression in HNMT tissues. Conclusions: These are the first results showing the presence of KIF20A-specific TH1 cell responses in HNMT patients and underline the possible utility of KIF20A-LPs for propagation of TH1 cells and CTLs. Clin Cancer Res; 19(16); 4508–20. ©2013 AACR.

Integrin alphaIIbbeta3 plays a critical role in platelet aggregation through its interaction with fibrinogen. Elucidation of the mechanisms of alphaIIbbeta3-fibrinogen interaction is critical to understanding hemostasis and thrombosis. Here we report that mutations of Gly-184, Tyr-189, Tyr-190, Phe-191, and Gly-193 within the predicted turn structure of the third amino-terminal repeat of alphaIIb significantly block binding of alphaIIbbeta3 to soluble fibrinogen. These mutations also block binding of alphaIIbbeta3 to ligand-mimetic monoclonal antibodies PAC-1, OP-G2, LJ-CP3, which have an RGD-related RYD sequence in their antigen-binding sites. These mutations do not significantly affect the expression of alphaIIbbeta3, in contrast to most of the natural alphaIIb mutations occurring in Glanzmann's thrombasthenic patients. The data suggest that these residues are critically involved in alphaIIbbeta3-ligand interactions.

Reports have shown that activation of tumor-specific CD4(+) helper T (Th) cells is crucial for effective anti-tumor immunity and identification of Th-cell epitopes is critical for peptide vaccine-based cancer immunotherapy. Although computer algorithms are available to predict peptides with high binding affinity to a specific HLA class II molecule, the ability of those peptides to induce Th-cell responses must be evaluated. We have established HLA-DR4 (HLA-DRA*01:01/HLA-DRB1*04:05) transgenic mice (Tgm), since this HLA-DR allele is most frequent (13.6%) in Japanese population, to evaluate HLA-DR4-restricted Th-cell responses to tumor-associated antigen (TAA)-derived peptides predicted to bind to HLA-DR4. To avoid weak binding between mouse CD4 and HLA-DR4, Tgm were designed to express chimeric HLA-DR4/I-E(d), where I-E(d) α1 and β1 domains were replaced with those from HLA-DR4. Th cells isolated from Tgm immunized with adjuvant and HLA-DR4-binding cytomegalovirus-derived peptide proliferated when stimulated with peptide-pulsed HLA-DR4-transduced mouse L cells, indicating chimeric HLA-DR4/I-E(d) has equivalent antigen presenting capacity to HLA-DR4. Immunization with CDCA155-78 peptide, a computer algorithm-predicted HLA-DR4-binding peptide derived from TAA CDCA1, successfully induced Th-cell responses in Tgm, while immunization of HLA-DR4-binding Wilms' tumor 1 antigen-derived peptide with identical amino acid sequence to mouse ortholog failed. This was overcome by using peptide-pulsed syngeneic bone marrow-derived dendritic cells (BM-DC) followed by immunization with peptide/CFA booster. BM-DC-based immunization of KIF20A494-517 peptide from another TAA KIF20A, with an almost identical HLA-binding core amino acid sequence to mouse ortholog, successfully induced Th-cell responses in Tgm. Notably, both CDCA155-78 and KIF20A494-517 peptides induced human Th-cell responses in PBMCs from HLA-DR4-positive donors. Finally, an HLA-DR4 binding DEPDC1191-213 peptide from a new TAA DEPDC1 overexpressed in bladder cancer induced strong Th-cell responses both in Tgm and in PBMCs from an HLA-DR4-positive donor. Thus, the HLA-DR4 Tgm combined with computer algorithm was useful for preliminary screening of candidate peptides for vaccination.

Integrin alpha 4 beta 1 is a receptor for vascular cell adhesion molecule (VCAM)-1 and fibronectin (CS-1). The alpha 4 beta 1-ligand interaction is involved in the pathogenesis of diseases and is, therefore, a therapeutic target. Here, we identified critical residues of alpha 4 for ligand binding using alanine-scanning mutagenesis of the previously localized putative ligand binding sites (residues 108-268). Among 43 mutations tested, mutations of Tyr187, Trp188 and Gly190 significantly inhibited cell adhesion to both VCAM-1 and CS-1. This inhibition was not due to any gross structural changes of alpha 4 beta 1. These critical residues are clustered in a predicted beta-turn structure (residues 181-190) of the third N-terminal repeat in alpha 4. The repeat does not contain divalent cation binding motifs. Notably, the mutations within the corresponding region of alpha 5 significantly reduced fibronectin-alpha 5 beta 1 interaction. These findings suggest that the predicted beta-turn structure could be ubiquitously involved in ligand binding of non-I domain integrins.

Prostaglandin (PG) E receptor EP3D is coupled to both Gi and Gs. To examine the roles of the interaction of alpha-carboxylic acid of PGE2 and its putative binding site, the arginine residue in the seventh transmembrane domain of EP3D, in receptor-G protein coupling, we have mutated the arginine residue to the noncharged glutamine. PGE2 with a negatively charged alpha-carboxylic acid and sulprostone, an EP3 agonist with a noncharged modified alpha-carboxylic acid, inhibited the forskolin-stimulated adenylate cyclase activity via Gi activation in the EP3D receptor in the same concentration-dependent manner. In contrast, the adenylate cyclase stimulation via Gs activation by sulprostone was much lower than that by PGE2. On the other hand, both PGE2 and sulprostone showed potent Gi activity but failed to show Gs activity in the mutant receptor. EP3D receptor showed a high affinity binding for PGE2 in the form coupled to either Gi or Gs. Although the mutant receptor showed high affinity binding when coupled to Gi, it lost high affinity binding in the condition of Gs coupling. Furthermore, sulprostone bound to the Gi-coupled EP3D receptor with higher affinity than the Gs-coupled receptor. Among various EP3 agonists, alpha-carboxylic acid-unmodified agonists showed both Gi and Gs activities, but the modified agonists showed only Gi activity. These findings suggest that the interaction between the alpha-carboxylic acid of PGE2 and the arginine residue of the receptor regulates the selectivity of the G protein coupling.

Neutral glycosphingolipids were purified from non-cataractous lenses of Sprague-Dawley rats by a combination of solvent extraction, Folch's partition, and column chromatography using DEAE-Sephadex and Iatrobeads. Six major GSLs from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis and glycosidase digestion. Structural relationships among the six neutral glycosphingolipids revealed metabolic pathways leading to the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide (IV3Gal alpha nLc4), instead of a Lewis(x) glycolipid (Gal beta 1- 4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide, III3FucnLc4), from neolactotetraosylceramide (nLc4), together with isoglobotriaosylceramide (iGb3). The alpha-galactosyl epitope, Gal alpha 1-3Gal-R, is evolutionarily conserved in many types of cells of non-primate mammals, prosimians and New World monkeys, but not in those of Old World monkeys or humans. This evolution-related difference in carbohydrate epitopes suggests different cell-to-cell attachments, which may be mediated through cell surface glycosphingolipids, between rat and human lenses.

Three isoforms of the mouse prostaglandin-E-receptor EP3 subtype (EP3), EP3 alpha, EP3 beta and EP3 gamma, with different C-termini, which are produced through alternative splicing, showed different efficiencies with respect to heterotrimeric GTP-binding protein activation and adenylate cyclase inhibition [Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, T., Honda, A., Watabe, A., Hirata, M., Narumiya, S.Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718; Irie, A., Sugimoto, Y., Namba, T., Harazono, A., Honda, A., Watabe, A., Negishi, M., Narumiya, S.Ichikawa, A. (1993) Eur. J. Biochem. 217, 313-318]. To assess the role of the C-terminus in GTP-binding protein coupling, we truncated the C-terminus of EP3 at an alternative splicing site and expressed the mutant receptor. The truncated receptor retained the ability to physically associate with Gi2, forming an agonist/receptor/Gi2 ternary complex, and to undergo the characteristic conversion of its agonist-binding affinity, mediated by a guanine nucleotide from a low-affinity state to a high-affinity state. However, sulprostone, an EP3 agonist, failed not only to inhibit the forskolin-induced cAMP accumulation in the mutant receptor-expressing cells but also to stimulate the GTPase activity in the mutant receptor-expressing cell membrane. These results indicated that the C-terminus of EP3 is essential for the activation of GTP-binding protein.

Nuclear factor‐κB (NF‐κB) activation contributes to the development of metastasis, thus leading to a poor prognosis in many cancers, including OSCC. However, little in vivo experimental data are available about the effects of NF‐κB inhibition on OSCC metastasis. OSCC sublines were established from a GFP‐expressing parental cell line, GSAS, and designated GSAS/N3 and N5 according to the in vivo passage number after cervical lymph node metastasis by a serial orthotopic transplantation model. In vitro migration and invasion were assessed in these cells, and the NF‐κB activities and expression of NF‐κB‐regulated metastasis‐related molecules were also examined. In in vivo experiments, the metastasis and survival of tumor‐engrafted mice were monitored. Furthermore, the effects of a selective NF‐κB inhibitor, NEMO‐binding domain (NBD) peptide, on metastasis in GSAS/N5‐engrafted mice were assessed, and engrafted tongue tumors were immunohistochemically examined. Highly metastatic GSAS/N3 and N5 cells showed an enhanced NF‐κB activity, thus contributing to increased migration, invasion, and a poor prognosis compared with the parent cells. Furthermore, the expression levels of NF‐κB‐regulated metastasis‐related molecules, such as fibronectin, β1 integrin, MMP‐1, ‐2, ‐9, and ‐14, and VEGF‐C, were upregulated in the highly metastatic cells. The NBD peptide suppressed metastasis and tongue tumor growth in GSAS/N5‐inoculated mice, and was accompanied by the downregulation of the NF‐κB‐regulated metastasis‐related molecules in engrafted tongue tumors. Our results suggest that the selective inhibition of NF‐κB activation by NBD peptide may provide an effective approach for the treatment of highly metastatic OSCC. (Cancer Sci 2012; 103: 455–463)

We recently identified four isoforms of bovine prostaglandin E receptor EP3 subtype, which are coupled to different signaling pathways; EP3A is coupled to inhibition of adenylate cyclase, while EP3B and EP3C are coupled to its stimulation and EP3D is coupled to phosphatidylinositol turnover, in addition to the adenylate cyclase system (Namba, T., Sugimoto, Y., Negishi, M., Irie, A., Ushikubi, F., Kakizuka, Ito, S., A., Ichikawa, A., and Narumiya, S. (1993) Nature 365, 166-170). We examined here the identity of coupled G proteins and their regulation by one of the isoforms, EP3C, in the membranes of EP3C cDNA-transfected Chinese hamster ovary cells. M&B 28767, an EP3 agonist, stimulated the GTPase activity in the pertussis toxin (PT)-treated cell membrane, but inhibited it in the cholera toxin (CT)-treated cell membrane, while the agonist neither stimulated nor inhibited it in the both PT- and CT-treated cell membrane. In the PT- and CT-treated cell membrane reconstituted with various G proteins, M&B 28767 inhibited the GTPase activity of G(o), but stimulated that of Gs. On the other hand, M&B 28767 did not affect the GTPase activity of Gi1, Gi2, or Gi3. M&B 28767 increased the apparent affinity of G(o) for GDP without any change in that for GTP, as assessed by displacement of [35S]GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) binding to G(o). In contrast, M&B 28767 increased the apparent affinity of Gs for GTP but decreased that for GDP. These results demonstrated that the EP3 receptor isoform is coupled to two different G proteins, and oppositely regulates their activities, inhibition of G(o), and stimulation of Gs.

Severe acute respiratory syndrome (SARS) spread during the winter of 2003, and attempt have been made to develop vaccines against SARS corona virus (SARS-CoV). The present study provides a strategy to rapidly identify SARS-CoV-derived antigenic peptides recognized by HLA-A2-restricted cytotoxic T lymphocytes (CTLs). Forty-three candidate peptides having HLA-A2-binding motifs were selected in silico and HLA-A2/Db chimeric MHC class I-transgenic mice were immunized with these peptides and a new derivative of muramyl dipeptide that can induce upregulation of HLA-DR, CD80, CD86, and CD40 in human CD14+ antigen presenting cells, was administered as an adjuvant. Six HLA-A2-restricted mouse CTL epitopes were identified, including two new epitopes which have never been reported before. One of the novel peptides was naturally processed and successfully induced HLA-A2-restricted specific CTLs in both HLA transgenic mice and healthy donors. The method was useful, convenient and efficient for rapid identification of CTL epitopes derived from SARS-CoV proteins and will be possibly applicable for other pathogens to develop a peptide-based vaccine.

To establish efficient anticancer immunotherary, it is important to identify tumor-associated antigens (TAAs) directing the immune system to attack cancer. A genome-wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA-A24 (A*2402)-restricted and SPARC-derived CTL epitopes. We previously identified H-2K(d)-restricted and SPARC-derived CTL epitope peptides in BALB/c mice, of which H-2K(d)-binding peptide motif is comparable with that of HLA-A24 binding peptides. By using these peptides, we tried to induce HLA-A24 (A*2402)-restricted and SPARC-reactive human CTLs and demonstrated an antitumor immune response. The SPARC-A24-1(143-151) (DYIGPCKYI) and SPARC-A24-4(225-234) (MYIFPVHWQF) peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA-A24 (A*2402). Furthermore, the adoptive transfer of the SPARC-specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA-A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy.

A binding protein which exhibits high affinity to [3H]glycyrrhetinic-acid in the rat liver microsomal fraction was solubilized with 0.2% Triton DF-18 and then purified to homogeneity. The equilibrium dissociation constant of the [3H]glycyrrhetinic-acid binding reaction and the maximal concentration for the binding of the purified protein, as determined by Scatchard plot analysis, were 27.6 nM and 7.79 nmol/mg protein, respectively. The molecular mass of the subunit (34 kDa) and 30 amino acids of N-terminal sequence of the purified protein were entirely the same as those of the reported 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). In each purification step, the recovery and purification (fold) of the glycyrrhetinic-acid binding activity corresponded to the values of 11 beta-HSD activity. These results show that the purified [3H]glycyrrhetinic-acid binding protein is 11 beta-HSD. From the molecular mass of 11 beta-HSD (135 kDa) and the maximal concentration of the binding site, it was calculated that one glycyrrhetinic acid molecule binds to one 11 beta-HSD molecule. The inhibitory effects of various glycyrrhetinic-acid derivatives on [3H]glycyrrhetinic acid binding and 11 beta-HSD activity indicate that the C30-carboxyl and C11-carbonyl groups of glycyrrhetinic acid are the principal structures for the 11 beta-HSD inhibition.

The present study attempted to identify a target antigen for immunotherapy for cholangiocarcinoma. Forkhead box M1 (FOXM1) was selected as a candidate antigen based on the data of previous cDNA microarray analysis of clinical samples of cholangiocarcinoma. The level of FOXM1 mRNA was more than 4 times higher in cancer cells in comparison to adjacent normal epithelial cells, in all of 24 samples of cholangiocarcinoma tissues. An immunohistochemical analysis also detected FOXM1 protein in the cancer cells but not in the normal cells. Twenty‐three human FOXM1‐derived peptides predicted to bind to HLA‐A2 were analyzed to determine their ability to induce HLA‐A2‐restricted T cells in HLA‐A2 transgenic mice. FOXM1362‐370 (YLVPIQFPV), FOXM1373‐382 (SLVLQPSVKV), and FOXM1640‐649 (GLMDLSTTPL) peptides primed HLA‐A2‐restricted cytotoxic T lymphocytes (CTLs) in the HLA‐A2 transgenic mice. Human CTL lines reactive to these 3 peptides could also be established from HLA‐A2‐positive healthy donors and cancer patients. Natural processing of the 3 epitopes from FOXM1 protein was confirmed by specific killing of HLA‐A2‐positive FOXM1‐transfectants by peptide‐induced CTLs. FOXM1 is expressed in various types of cancers and it is also functionally involved in oncogenic transformation and the survival of cancer cells. Therefore, FOXM1 may be a suitable target for immunotherapy against various cancers including cholangiocarcinoma.

In vivo assay to evaluate anti-cancer immunotherapy at the pre-clinical phase is eagerly needed. We currently established xenotransplantation-based method to analyze in vivo priming of cancer-antigen-specific human cytotoxic T lymphocytes (CTLs). We transplanted human peripheral T cells and analyzed priming of CTLs in NOG mice. Half of the mice engrafted with bulk lymphocytes including CD4(+) T cells died before analysis probably due to xenoreactive graft versus host disease. All of the mice engrafted with purified CD8(+) T cells survived until the analysis, and successful engraftment was observed in 80% of recipient mice. Thus, transfer of purified CD8(+) T cells is sufficient and safer than that of bulk lymphocytes. To add antigenic stimulation to the CD8(+) T cells in vivo, injection of antigenic peptide-loaded and monocyte-derived autologous dendritic cells (DCs) was simultaneously done and repeated 7 days later. The DC-based vaccinization resulted in efficient priming of HLA class I-restricted and MART1, WT1 or CMV peptides-specific CTLs in the recipient mice. This system may be useful to evaluate the stimulation of antigen-specific human CTLs in vivo.

When apoptosis is initiated by manganese (II) loading, hyperthermia or thapsigargin treatment, human HL-60 and AsPC-1 cells initiate de novo synthesis of the C5a receptor (C5aR) and generation of its ligand, the ribosomal protein S19 (RP S19) homodimer. The ligand-receptor interaction, in an autocrine/paracrine fashion, promotes apoptosis, which can be bypassed by exogenous administration of C5a, another ligand. The proapoptotic function of the RP S19 dimer is reproduced by a C5a/RPS19 chimera that contains the body of C5a and the C-terminal region (Ile134-His145) of RP S19. The RP S19 dimer or C5a/RPS19 and C5a inversely regulate the expression of Regulator of G protein Signaling 3 (RGS3) gene in the apoptosis-initiated cells. Namely, the RP S19-type proteins upregulate RGS3 expression, whereas the C5a reduce it. Transformation of HL-60 cells to overexpress RGS3 promotes apoptosis in association with the downregulation of the Extracellular signal-Regulated Kinase (ERK) signal, and vice versa in the RGS3 knocked-down cells. Consistent with this result, an inhibitor of ERK phosphorylation effectively enhances the apoptotic rate in wild-type HL-60 cells. Moreover, a dominant negative effect on the RP S19 dimer production encourages apoptosis-initiated HL-60 cells with a longer lifespan in mouse than the natural effect. Our data indicate that, in apoptosis-initiated cells, the ligand-dependent C5aR-mediated dual signal affects the fate of cells, either apoptosis execution or survival, through regulation of RGS3 gene expression and subsequent modulation of ERK signal.

By using the recently established culture system that reproduces the terminal differentiation process of connective tissue-type mast cells, we found significant transcriptional induction of CD44. As CD44 is a primary receptor for hyaluronan (HA), which is one of the major extracellular matrix components, we investigated the role of CD44 in cutaneous mast cells. When co-cultured with fibroblasts, mouse bone marrow-derived cultured mast cells (BMMCs) were found to form clusters in an HA-dependent manner. As compared with BMMCs derived from the wild-type mice, those from the CD44(-/-) mice exhibited impaired growth during the co-cultured period. Furthermore, in the peritoneal cavities and ear tissues, mature mast cells were fewer in number in the CD44(-/-) mice than in the wild-type mice. We investigated roles of CD44 in mast cell proliferation by reconstituting BMMCs into the tissues of mast cell-deficient, Kit(W)/Kit(W-v) mice, and found that the number of metachromatic cells upon acidic toluidine blue staining in the tissues transplanted with CD44(-/-) BMMCs was not significantly changed for 10 weeks, whereas that in the tissues transplanted with the CD44(+/+) BMMCs was significantly increased. These results suggest that CD44 plays a crucial role in the regulation of the cutaneous mast cell number.

We previously reported that the secreted protein acidic and rich in cystein (SPARC) was overexpressed in melanoma in humans, and the serum SPARC level was useful as a novel tumor marker for melanoma. SPARC was also reported to be overexpressed in various human cancers. In this study, we asked whether SPARC-specific cytotoxic T lymphocytes (CTL) could induce antitumor immunity to SPARC-expressing tumor in mice or not as a preclinical study of SPARC-directed anticancer immunotherapy. Because of similarities in the structural motifs of major histocompatibility complex-binding peptides between H2-Kd and HLA-A24 (A*2402), the most common human leukocyte antigen class I allele in the Japanese population, we attempted to identify the H2-Kd-restricted SPARC epitope for CTL in BALB/c mice and we found that the mouse SPARC143–151 (DYIGPCKYI) and SPARC225–234 (MYIFPVHWQF) peptides could induce peptide-reactive CTL in BALB/c mice without causing autoimmune diseases. The immunization of mice with SPARC225–234 peptide-pulsed bone marrow-derived dendritic cells (BMDC) inhibited the growth of s.c. inoculated mouse mammary cancer cell line, N2C, expressing SPARC and these mice lived longer than the mice immunized with peptide-unpulsed BMDC. In conclusion, our study indicated that SPARC peptide-based cancer immunotherapy was effective and safe at least in a mouse tumor prevention model. (Cancer Sci 2009; 100: 132–137)

Toward the development of a novel cancer immunotherapy, we have previously identified several tumor-associated antigens (TAAs) and the epitopes recognized by human histocompatibility leukocyte (HLA)-A2/A24-restricted cytotoxic T lymphocyte (CTL). In this study, we tried to identify a TAA of lung cancer (LC) and its HLA-A2 restricted CTL epitopes to provide a target antigen useful for cancer immunotherapy of LC. We identified a novel cancer testis antigen, cell division cycle associated gene 1 (CDCA1), overexpressed in nonsmall cell LC using a cDNA microarray analysis. The expression levels of CDCA1 were also increased in the majority of small cell LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers. We used HLA-A2.1 transgenic mice to identify the HLA-A2 (A*0201)-restricted CDCA1 epitopes recognized by mouse CTL, and we investigated whether these peptides could induce CDCA1-reactive CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA-A2-positive donors and a NSCLC patient. Consequently, we found that the CDCA1(65-73) (YMMPVNSEV) peptide and CDCA1(351-359) (KLATAQFKI) peptide could induce peptide-reactive CTLs in HLA-A2.1 transgenic mice. In HLA-A2(+) donors, in vitro stimulation of PBMC with these peptides could induce peptide-reactive CTLs which killed tumor cell lines endogenously expressing both HLA-A2 and CDCA1. As a result, CDCA1 is a novel cancer-testis antigen overexpressed in LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers, and CDCA1 may therefore be an ideal TAA useful for the diagnosis and immunotherapy of these cancers.

Aeromonas sobria causes pus and edema at sites of infection. However, the mechanisms underlying these effects have not been elucidated. C5a, the amino-terminal fragment of the complement 5th component (C5), mimics these events. To investigate the involvement of C5a in the pathophysiology of A. sobria infection, we examined release of C5a from human C5 by a serine protease (ASP), a putative virulence factor secreted by this bacterium. C5 incubated with enzymatically active ASP induced neutrophil migration in a dose-dependent manner from an ASP concentration of 3 nM and in an incubation time-dependent manner in as little as 7 min, with neutrophil accumulation in guinea pigs at intradermal injection sites and neutrophil superoxide release. These effects on neutrophils were inhibited by a C5a-receptor antagonist. The ASP incubation mixture with C5 but not C3 elicited vascular leakage in a dose- and incubation time-dependent manner, which was inhibited by a histamine H1-receptor antagonist. Together with these C5a-like activities, ASP cleaved C5 to release only one C5a Ag, the m.w. of which was similar to that of C5a. Immunoblotting using an anti-C5a Ab revealed generation of a C5a-like fragment from human plasma incubated with ASP. These results suggest that ASP-elicited neutrophil migration and vascular leakage via C5a production from C5 could occur in vivo, which was supported by that ASP did not affect functions of C5a and neutrophil C5a receptor. Through C5a generation, ASP could be associated with the induction of pus and edema caused by infection with this bacterium.

The activities of the Raf kinase family proteins control extracellular signal-regulated kinase (ERK) activation in many aspects of cellular responses. However, the relative contributions of individual isozymes to cellular functions including T cell responses are still unclear. In addition to Raf-1, another Raf family kinase, B-Raf, is expressed in murine thymocytes and peripheral T cells, and its activation was induced by TCR stimulation. Here, we investigated the function of B-Raf in development of T cells by generating chimeric mice in which a T cell-compromised host was reconstituted with fetal liver-derived cells from embryonic lethal B-Raf-deficient mice. Although B-Raf was dispensable for normal T cell lineage differentiation at the CD4(-)CD8(-) double-negative stage, thymocytes in the chimeric mice derived from B-Raf(-/-) cells exhibited a drastic arrest of differentiation at the CD4(+)CD8(+) double-positive stage, suggesting that B-Raf is crucial for T cell development, especially for the transition to CD4(+) and CD8(+) single-positive thymocytes. Regarding intracellular signaling, we found that activation of ERK following TCR stimulation was impaired in the thymocytes from the chimeric mice. In conclusion, we present first evidence for the important role of B-Raf-mediated signaling in T cell development.

Heparin is a highly sulfated glycosaminoglycan and has been shown to activate osteoclastic bone resorption though how is not yet clear. Here we investigate the molecule involved in heparin-induced activation of osteoclasts using an in vitro osteoclast culture assay. The formation and activation of osteoclasts are induced by receptor activator of NFkappaB ligand (RANKL) on osteoblasts, and inhibited by osteoprotegerin (OPG), a decoy receptor of RANKL, which is secreted from osteoblasts. In a coculture of mouse bone marrow cells and osteoblasts treated with 1,25-dihydroxyvitamin D(3) and prostaglandin E(2) on dentin slices, the bone marrow cells differentiate into osteoclasts, and resorption pits are formed on the dentin slices. Addition of heparin, various glycosaminoglycans, and chemically modified heparins to the coculture reveals that heparin enhances the pit-forming activity of osteoclasts, and this effect of heparin on the activation of osteoclasts is dependent on its sugar chain structure. By contrast, mRNA expression levels of RANKL, RANK, and OPG in the coculture are not altered by heparin treatment. Furthermore, neither RANK nor RANKL binds to heparin, suggesting that heparin does not directly interact with these proteins. Instead, heparin specifically binds to OPG and prevents OPG-mediated inhibition of osteoclastic bone resorption in the coculture. Heparin treatment does not enhance osteoclastic bone resorption in a monoculture of osteoclasts derived from bone marrow cells, and in the coculture using osteoblasts from OPG-deficient mice. A (125)I-OPG binding assay showed that OPG binds to osteoblasts and that this binding is inhibited by the addition of heparin, suggesting that OPG binds to RANKL on the osteoblast membrane and that heparin blocks this interaction. These results demonstrate that heparin enhances osteoclastic bone resorption by inhibiting OPG activity.

A T cell receptor (TCR) recognizes and responds to an antigenic peptide in the context of major histocompatibility complex-encoded molecules. This provokes T cells to produce interleukin-2 (IL-2) through extracellular signal-regulated kinase (ERK) activation. We investigated the roles of B-Raf in TCR-mediated IL-2 production coupled with ERK activation in the Jurkat human T cell line. We found that TCR cross-linking could induce up-regulation of both B-Raf and Raf-1 activities, but Raf-1 activity was decreased rapidly. On the other hand, TCR-stimulated kinase activity of B-Raf was sustained. Expression of a dominant-negative mutant of B-Raf abrogated sustained but not transient TCR-mediated MEK/ERK activation. The inhibition of sustained ERK activation by either expression of a dominant-negative B-Raf or treatment with a MEK inhibitor resulted in a decrease of the TCR-stimulated nuclear factor of activated T cells (NFAT) activity and IL-2 production. Collectively, our data provide the first direct evidence that B-Raf is a positive regulator of TCR-mediated sustained ERK activation, which is required for NFAT activation and the full production of IL-2.

A membrane-surface glycoprotein, RANDAM-2, is one of the neuronal cell lineage-specific antigens involved in the neuronal differentiation of P19 embryonic carcinoma (EC) cells and the mouse central nervous system (CNS). Complementary DNA cloning of RANDAM-2 indicated that its nucleotide sequence completely matched that of PA2.26 antigen, a sialomucin-like transmembrane glycoprotein previously found on tumorigenic keratinocytes. RANDAM-2 transcripts were detectable from the embryonic stage of 6.5 days, and then the expression continued throughout the remaining embryonic stages and adulthood, with a localization restricted to the CNS. In growth factor-induced neurospheres and adult cerebrum, RANDAM-2-expressing cells coincided well not only with nestin-positive cells but also with glutamate-positive neurons, but not with γ-aminobutyric acid-positive ones. These results indicate that RANDAM-2 is one of the type I membrane surface antigens constitutively expressed on undifferentiated neuronal cells and the glutamatergic neuronal cells during mouse neurogenesis. © 2003 Wiley-Liss, Inc.

Little is known about the cues that guide retinal axons across the diencephalon en route to their midbrain target, the optic tectum. Here we show that chondroitin sulfate proteoglycans are differentially expressed within the diencephalon at a time when retinal axons are growing within the optic tract. Using exposed brain preparations, we show that the addition of exogenous chondroitin sulfate results in retinal pathfinding errors. Retinal axons disperse widely from their normal trajectory within the optic tract and extend aberrantly into inappropriate regions of the forebrain. Time-lapse analysis of retinal growth cone dynamics in vivo shows that addition of exogenous chondroitin sulfate causes intermittent stalling and increases growth cone complexity. These results suggest that chondroitin sulfate may modulate the guidance of retinal axons as they grow through the diencephalon towards the optic tectum. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 330–342, 2002

We cloned cDNAs for mouse and human CMP- N -acetylneuraminic acid (CMP-NeuAc) hydroxylases and showed that the human CMP-NeuAc hydroxylase protein is inactive because of a partial deletion in the hydroxylase gene. We report here that no other active CMP-NeuAc hydroxylases are present in humans. Southern blot analysis showed that the human homologue of the mouse CMP-NeuAc hydroxylase is one gene in the human genome and no other homologues of the mouse hydroxylase exist in human genome. The mouse and the human CMP-NeuAc hydroxylases were mapped to chromosome 13A3 and chromosome 6p22, respectively, by fluorescence in situ hybridization. The chromosomal location of the human hydroxylase is syntenic to that of the mouse hydroxylase. These results demonstrate that the human CMP-NeuAc hydroxylase is the only homologue of the mouse hydroxylase, and CMP-NeuAc hydroxylase is exclusively inactive in humans.

We recently cloned the mouse prostaglandin (PG) E receptor EP3 subtype that is coupled to adenylate cyclase inhibition through Gi and identified three isoforms which are produced through alternative splicing. In Chinese hamster ovary cells expressing each EP3 isoform, PGE2 induced an immediate increase in the intracellular Ca2+ concentration ([Ca2+]i) due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. This increase was abolished by prior treatment with pertussis toxin (PT). PGE2 also stimulated an accumulation of inositol trisphosphate (IP3) in a PT-sensitive manner. Both the PGE2-induced increase in [Ca2+]i and accumulation of IP3 were blocked by the phospholipase C inhibitor U-73122. Thus, EP3 is linked to phospholipase C activation via Gi, and this activation leads to Ca2+ mobilization from internal stores and influx from the extracellular medium.

A functional cDNA for the human prostacyclin receptor was isolated from a cDNA library of CMK cells, a human megakaryocytic leukaemia cell line. The cDNA encodes a protein consisting of 386 amino acid residues with seven putative transmembrane domains and a deduced molecular weight of 40,956. [3H]Iloprost specifically bound to the membrane of CHO cells stably expressing the cDNA with a Kd of 3.3 nM. This binding was displaced by unlabelled prostanoids in the order of iloprost = cicaprost ⪢ carbacyclin ⪢ prostaglandin E1 (PGE1) > STA2. PGE2, PGD2 and PGF2α did not inhibit it. Iloprost in a concentration-dependent manner increased the cAMP level and generated inositol trisphosphate in these cells, indicating that this human receptor can couple to multiple signal transduction pathways.

A functional cDNA clone for a third isoform of the mouse prostaglandin-E-receptor EP3 subtype, derived by alternative RNA splicing, named the EP3 gamma receptor, was obtained in addition to those for the two other isoforms, EP3 alpha and EP3 beta. The three isoforms are only different in the amino acid sequence of the putative cytoplasmic carboxy-terminal tail. When expressed, EP3 gamma shows identical ligand-binding properties to these of the other isoforms. The EP3-selective agonist, MB 28767, increased the basal cAMP level and inhibited the forskolin-induced increase in the cAMP level in EP3 gamma, while it decreased both the basal and forskolin-elevated cAMP levels in EP3 alpha and EP3 beta. The MB 28767-stimulated GTPase activity consisted of pertussis-toxin-sensitive and cholera-toxin-sensitive portions in the EP3 gamma-expressing cell membrane, suggested that EP3 gamma is coupled to both guanine nucleotide-binding inhibitory and stimulatory proteins. These results indicate that EP3 gamma is coupled to both stimulation and inhibition of adenylate cyclase, but that EP3 alpha and EP3 beta are exclusively coupled to inhibition of adenylate cyclase. Thus, alternative splicing produces a third isoform with a different carboxy-terminal tail, which differs from the other two isoforms in the specificity of coupling to a signal-transduction pathway.