Antitumor effect of novel anti-podoplanin antibody NZ-12 against malignant pleural mesothelioma in an orthotopic xenograft model.

Podoplanin (aggrus) is highly expressed in several types of cancers, including malignant pleural mesothelioma (MPM). Previously, we developed a rat anti-human podoplanin mAb, NZ-1, and a rat-human chimeric anti-human podoplanin antibody, NZ-8, derived from NZ-1, which induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against podoplanin-positive MPM cell lines. In this study, we showed the antitumor effect of NZ-1, NZ-8, and NZ-12, a novel rat-human chimeric anti-human podoplanin antibody derived from NZ-1, in an MPM orthotopic xenograft SCID mouse model. Treatment with NZ-1 and rat NK (CD161a(+) ) cells inhibited the growth of tumors and the production of pleural effusion in NCI-H290/PDPN or NCI-H226 orthotopic xenograft mouse models. NZ-8 and human natural killer (NK) (CD56(+) ) cells also inhibited tumor growth and pleural effusion in MPM orthotopic xenograft mice. Furthermore, NZ-12 induced potent ADCC mediated by human MNC, compared with either NZ-1 or NZ-8. Antitumor effects were observed following treatment with NZ-12 and human NK (CD56(+) ) cells in MPM orthotopic xenograft mice. In addition, combined immunotherapy using the ADCC activity of NZ-12 mediated by human NK (CD56(+) ) cells with pemetrexed, led to enhanced antitumor effects in MPM orthotopic xenograft mice. These results strongly suggest that combination therapy with podoplanin-targeting immunotherapy using both NZ-12 and pemetrexed might provide an efficacious therapeutic strategy for the treatment of MPM.

A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody.

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a(+)) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

Generation of a novel monoclonal antibody WMab-1 specific for IDH2-R172W mutation.

Isocitrate dehydrogenase 2 (IDH2) mutations have been detected in gliomas, cartilaginous tumors, and acute myeloid leukemias. IDH2 mutations are specific to a single codon in the conserved and functionally important Arginine 172 (R172) or Arginine 140 (R140). To date, we have established specific monoclonal antibodies (mAbs) against IDH2-R172K and IDH2-R172M. However, specific mAbs against IDH2-R172W have not been reported. To establish IDH2-R172W-specific mAbs, we immunized rats with IDH2-R172W peptides. Western-blot analysis showed that WMab-1 reacted with the IDH2-R172W recombinant protein, not with IDH2-wild type (WT) or other IDH2 mutants, indicating that WMab-1 is IDH2-R172W-specific. Furthermore, WMab-1 specifically stained the IDH2-R172W-expressing cells in immunocytochemistry, but did not stain IDH2-WT and IDH2-R172M-containing cells. WMab-1 also specifically stained the IDH2-R172W-expressing glioma cells in immunohistochemistry. This is the first report to establish an anti-IDH2-R172W-specific mAb, which could be useful in the diagnosis of IDH2-R172W-bearing tumors.

A novel monoclonal antibody GMab-m1 specifically recognizes IDH1-R132G mutation.

Isocitrate dehydrogenase 1 (IDH1) mutations occur in gliomas, acute myeloid leukemias, and cartilaginous tumors. While IDH1 catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate in cytosol, mutated IDH1 proteins possess the ability to change α-ketoglutarate into onco-metabolite R(-)-2-hydroxyglutarate (2-HG). To data, two monoclonal antibodies (mAbs), which are specific for IDH1 mutations have been established: clone HMab-1 against IDH1-R132H and clone SMab-1 against IDH1-R132S. However, specific mAbs against IDH1-R132G, which are useful for immunohistochemical analysis, have not been reported. To establish IDH1-R132G-specific mAbs, we immunized mice with IDH1-R132G-containing peptides. Established mAb GMab-m1 reacted with the IDH1-R132G peptide, but not with IDH1-wild type (WT) in ELISA. Western-blot analysis also showed that GMab-m1 reacted with the IDH1-R132G recombinant proteins, not with IDH1-WT or other IDH1 mutants, indicating that GMab-m1 is IDH1-R132G-specific. Furthermore, GMab-m1 specifically stained the IDH1-R132G-expressing glioma cells in immunohistochemistry. This is the first report to establish anti-IDH1-R132G-specific mAbs, which is useful in immunohistochemistry of IDH1-R132G-bearing tumors.

Establishment of novel monoclonal antibodies KMab-1 and MMab-1 specific for IDH2 mutations.

Isocitrate dehydrogenase 1/2 (IDH1/2) mutations have been detected in gliomas, cartilaginous tumors, and leukemias. IDH1/2 mutations are early and frequent genetic alterations, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1 and Arginine 172 (R172) in IDH2. We previously established several monoclonal antibodies (mAbs), which are specific for IDH1 mutations: clones IMab-1 or HMab-1 against IDH1-R132H or clone SMab-1 against IDH1-R132S. However, specific mAbs against IDH2 mutations have not been reported. To establish IDH2-mutation-specific mAbs, we immunized mice or rats with each mutation-containing IDH2 peptides including IDH2-R172K and IDH2-R172M. After cell fusion, IDH2 mutation-specific mAbs were screened in Enzyme-Linked Immunosorbent Assay (ELISA). Established mAbs KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M peptides, respectively, but not with IDH2-wild type (WT) in ELISA. Western-blot analysis also showed that KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M recombinant proteins, respectively, not with IDH2-WT or other IDH2 mutants, indicating that KMab-1 and MMab-1 are IDH2-mutation-specific. Furthermore, MMab-1 specifically stained the IDH2-R172M-expressing cells in immunocytochemistry, but did not stain IDH2-WT and other IDH2-mutation-containing cells. In immunohistochemical analysis, MMab-1 specifically stained IDH2-R172M-expressing glioma. This is the first report to establish anti-IDH2-mutation-specific mAbs, which could be useful in diagnosis of mutation-bearing tumors.

Chimeric anti-podoplanin antibody suppresses tumor metastasis through neutralization and antibody-dependent cellular cytotoxicity.

Podoplanin is a platelet aggregation-inducing factor associated with tumor metastasis, malignant progression, and cancer stem cells. We produced a rat-human chimeric anti-podoplanin mAb, NZ-8, from rat anti-podoplanin mAb (NZ-1). Although both NZ-1 and NZ-8 possess high binding affinities and high neutralizing activities of platelet aggregation, the antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity of NZ-8 were much higher than NZ-1. Furthermore, both NZ-1 and NZ-8 inhibited the growth of podoplanin-expressing tumors in vivo. Both NZ-1 and NZ-8 also suppressed hematogenous metastasis of podoplanin-expressing tumors. These results suggest that anti-podoplanin mAbs suppressed hematogenous metastasis by both neutralization and antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity activities. Targeting therapy to podoplanin-expressing tumors should be useful as a novel immunotherapy.

Immunohistochemical detection of IDH1 mutation, p53, and internexin as prognostic factors of glial tumors.

Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary glioblastomas, are specific to arginine 132 (R132). Recently, we established monoclonal antibodies (mAbs) against IDH1 mutations: anti-IDH1-R132H and anti-IDH1-R132S. However, the importance of immunohistochemistry using the combination of those mAbs has not been elucidated. For this study, 164 cases of glioma were evaluated immunohistochemically for IDH1 mutations (R132H and R132S) using anti-IDH1 mAbs (HMab-1 and SMab-1). IDH1 mutation was detected, respectively, in 9.7%, 63.6%, 51.7%, and 77.8% of primary grade IV, secondary grade IV, grade III, and grade II gliomas. For each grade of glioma, prognostic factors for progression-free survival and overall survival were evaluated using clinical and pathological parameters in addition to IDH1 immunohistochemistry. IDH1 mutation, p53 overexpression, and internexin expression, as evaluated using immunohistochemistry with clinical parameters such as degree of surgical removal and preoperative Karnofsky Performance Status (KPS), might be of greater prognostic significance than histological grading alone in grade III as well as IDH1 mutation in grade IV gliomas.

Establishment of a novel monoclonal antibody SMab-1 specific for IDH1-R132S mutation.

Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in gliomas, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1. We earlier established a monoclonal antibody (mAb), IMab-1, which is specific for R132H-containing IDH1 (IDH1-R132H), the most frequent IDH1 mutation in gliomas. To establish IDH1-R132S-specific mAb, we immunized mice with R132S-containing IDH1 (IDH1-R132S) peptide. After cell fusion using Sendai virus envelope, IDH1-R132S-specific mAbs were screened in ELISA. One mAb, SMab-1, reacted with the IDH1-R132S peptide, but not with other IDH1 mutants. Western-blot analysis showed that SMab-1 reacted only with the IDH1-R132S protein, not with IDH1-WT protein or IDH1 mutants, indicating that SMab-1 is IDH1-R132S-specific. Furthermore, SMab-1 specifically stained the IDH1-R132S-expressing glioblastoma cells in immunocytochemistry and immunohistochemistry, but did not react with IDH1-WT or IDH1-R132H-containing glioblastoma cells. We newly established an anti-IDH1-R132S-specific mAb SMab-1 for use in diagnosis of mutation-bearing gliomas.

Focused differential glycan analysis with the platform antibody-assisted lectin profiling for glycan-related biomarker verification.

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.

Engineering of mucin-type human glycoproteins in yeast cells.

Mucin-type O-glycans are the most typical O-glycans found in mammalian cells and assume many different biological roles. Here, we report a genetic engineered yeast strain capable of producing mucin-type sugar chains. Genes encoding Bacillus subtilis UDP-Gal/GalNAc 4-epimerase, human UDP-Gal/GalNAc transporter, human ppGalNAc-T1, and Drosophila melanogaster core1 beta1-3 GalT were introduced into Saccharomyces cerevisiae. The engineered yeast was able to produce a MUC1a peptide containing O-glycan and also a mucin-like glycoprotein, human podoplanin (hPod; also known as aggrus), which is a platelet-aggregating factor that requires a sialyl-core1 structure for activity. After in vitro sialylation, hPod from yeast could induce platelet aggregation. Interestingly, substitution of ppGalNAc-T1 for ppGalNAc-T3 caused a loss of platelet aggregation-inducing activity, despite the fact that the sialyl-core1 was detectable in both hPod proteins on a lectin microarray. Most of O-mannosylation, a common modification in yeast, to MUC1a was suppressed by the addition of a rhodanine-3-acetic acid derivative in the culture medium. The yeast system we describe here is able to produce glycoproteins modified at different glycosylation sites and has the potential for use in basic research and pharmaceutical applications.

GMab-1, a high-affinity anti-3'-isoLM1/3',6'-isoLD1 IgG monoclonal antibody, raised in lacto-series ganglioside-defective knockout mice.

The lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1 have been identified as tumor-associated antigens whose formation is initiated by the Lc3-synthase. Until now, high-affinity IgG monoclonal antibodies (mAbs) against 3'-isoLM1 and 3',6'-isoLD1, which are highly expressed in gliomas, have not been developed, although mAbs against lacto-series gangliosides are powerful tools for functional studies. We previously produced the Lc3-synthase gene beta3Gn-T5 knockout mice. In this study, we immunized beta3Gn-T5 knockout mice with 3'-isoLM1/3',6'-isoLD1 and produced the anti-3'-isoLM1/3',6'-isoLD1 mAb GMab-1, of the IgG(3) subclass, which should be useful for functional analysis of lacto-series gangliosides and for antibody-based therapy of gliomas.

Establishment of C20Mab-11, a novel anti-CD20 monoclonal antibody, for the detection of B cells.

CD20 is one of several B-lymphocyte antigens that has been shown to be an effective target for the detection and treatment of B-cell lymphomas. Sensitive and specific monoclonal antibodies (mAbs) are required for every application used for the diagnosis of B-cell lymphoma. Although many anti-CD20 mAbs have been established, the types of applications, those anti-CD20 can be used in, are limited. In this study, we aimed to establish novel anti-CD20 mAbs to be used for broad applications, such as flow cytometry, western blot, and immunohistochemical analyses, using the Cell-Based Immunization and Screening (CBIS) method. One of the established mAbs, C20Mab-11 (IgM, kappa), detected overexpression of CD20 in CHO-K1 or LN229 cell lines, indicating that C20Mab-11 is specific for CD20. In western blot analyses, C20Mab-11 detected not only overexpression of CD20 in CHO-K1 or LN229 cell lines, but also CD20 of BALL-1 and Raji cells with both sensitivity and specificity. Furthermore, C20Mab-11 strongly stained B cells of the lymph follicle and B cell lymphomas in immunohistochemical analyses. These results indicate that C20Mab-11 develped by CBIS method, is useful for the detection of CD20 in lymphoma tissues by flow cytometry, western blot, and immunohistochemical analyses and potentially could be beneficial for the treatment of B cell lymphomas.

H2Mab-19, an anti-human epidermal growth factor receptor 2 monoclonal antibody exerts antitumor activity in mouse oral cancer xenografts.

Human epidermal growth factor receptor 2 (HER2) is reported to be overexpressed in breast cancers and is associated with poor clinical outcome. Trastuzumab is a humanized anti-HER2 antibody that offers significant survival benefits to patients with HER2-overexpressing breast cancer. In this study, a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, kappa) was developed. Antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antitumor activity of H2Mab-19 were investigated using both breast cancer and oral cancer cell lines. H2Mab-19 demonstrated cytotoxicity in BT-474 (a human breast cancer cell line) and HSC-2 or SAS (human oral cancer cell lines). H2Mab-19 also possessed both ADCC and CDC activity against BT-474, HSC-2, and SAS cell lines. In comparison to control mouse IgG, H2Mab-19 significantly reduced tumor development in BT-474, HSC-2, and SAS xenografts. Collectively, these results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing breast and oral cancers.

A novel anti-EGFR monoclonal antibody (EMab-17) exerts antitumor activity against oral squamous cell carcinomas via antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity.

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases; it is a transmembrane receptor involved in cell growth and differentiation. EGFR homodimers or heterodimers in combination with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many types of cancer, including oral squamous cell carcinoma (OSCC). The present study produced novel anti-EGFR monoclonal antibodies (mAbs) possessing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and investigated antitumor activity. Mice were immunized with an EGFR-overexpressed glioblastoma cell line, LN229 (LN229/EGFR), after which ELISA was performed using recombinant EGFR. mAbs were subsequently selected according to their efficacy for LN229/EGFR, as determined via flow cytometry. After determining the subclass of mAbs, the EMab-17 (IgG2a, kappa) clone exhibited ADCC and CDC activities against two OSCC cell lines, HSC-2 and SAS. Furthermore, EMab-17 exerted antitumor activities against mouse xenograft models using HSC-2 and SAS, indicating that EMab-17 may be used in an antibody-based therapy for EGFR-expressing OSCC.

Anti­EGFR monoclonal antibody 134­mG2a exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma.

The epidermal growth factor receptor (EGFR), a transmembrane receptor and member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases, is a critical mediator of cell growth and differentiation. EGFR forms homo­ or heterodimers with other HER family members to activate downstream signaling cascades in a number of cancer cells. In a previous study, the authors established an anti­EGFR monoclonal antibody (mAb), EMab­134, by immunizing mice with the ectodomain of human EGFR. EMab­134 binds specifically to endogenous EGFR and can be used to detect receptor on oral cancer cell lines by flow cytometry and western blot analysis; this antibody is also effective for the immunohistochemical evaluation of oral cancer tissues. In the present study, the subclass of EMab­134 was converted from IgG1 to IgG2a (134­mG2a) to facilitate antibody­dependent cellular cytotoxicity (ADCC) and complement­dependent cytotoxicity (CDC). The dissociation constants (KDs) of EMab­134 and 134­mG2a against EGFR­expressing CHO­K1 (CHO/EGFR) cells were determined by flow cytometry to be 3.2x10­9 M and 2.1x10­9 M, respectively; these results indicate that 134­mG2a has a higher binding affinity than EMab­134. The 134­mG2a antibody was more sensitive than EMab­134 with respect to antigen detection in oral cancer cells in both western blot analysis and immunohistochemistry applications. Analysis in vitro revealed that 134­mG2a contributed to high levels of ADCC and CDC in experiments targeting CHO/EGFR, HSC­2, and SAS cells. Moreover, the in vivo administration of 134­mG2a significantly inhibited the development of CHO/EGFR, HSC­2, and SAS mouse xenografts in comparison to the results observed in response to EMab­134. Taken together, the findings of the present study demonstrate that the newly­formulated 134­mG2a is useful for detecting EGFR by flow cytometry, western blot analysis and immunohistochemistry. Furthermore, the in vivo results suggested that it may also be useful as part of a therapeutic regimen for patients with EGFR­expressing oral cancer.

Caenorhabditis elegans galectins LEC-1-LEC-11: structural features and sugar-binding properties.

Galectins form a large family of beta-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-2-5 and 8-11) from a lambdaZAP cDNA library. Among them, lec-2-5 were found to encode 31-35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8-11 were found to encode 16-27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1-4, -6, and 8-10 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various beta-galactosides, i.e., N-acetyllactosamine (Galbeta1-4GlcNAc), lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), and asialofetuin, demonstrated that LEC-1-4, -6, and -10 have a significant affinity for beta-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galbeta1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.

Molecular analysis of the pathophysiological binding of the platelet aggregation-inducing factor podoplanin to the C-type lectin-like receptor CLEC-2.

The mucin-type sialoglycoprotein podoplanin (aggrus) is involved in tumor cell-induced platelet aggregation and tumor metastasis. C-type lectin-like receptor-2 (CLEC-2) was recently identified as an endogenous receptor of podoplanin on platelets. However, the pathophysiological importance and function of CLEC-2 have not been elucidated. Here we clarified the pathophysiological interaction between podoplanin and CLEC-2 in vitro and in vivo. Using several deletion mutants of CLEC-2 expressed as Fc chimeras, we first identified an important podoplanin-recognition domain in CLEC-2. Furthermore, the podoplanin-CLEC-2 interaction was confirmed using several deletion mutants of podoplanin expressed as Fc chimeras. Not only the disialyl-core1-attached glycopeptide but also the stereostructure of the podoplanin protein was found to be critical for the CLEC-2-binding activity of podoplanin. We next synthesized various glycopeptides of podoplanin that included both the platelet aggregation-stimulating domain and O-glycan on Thr52. Interestingly, a disialyl-core1-attached glycopeptide was recognized specifically by CLEC-2. Moreover, the anti-podoplanin monoclonal antibody NZ-1 suppressed both the podoplanin-CLEC-2 interaction and podoplanin-induced pulmonary metastasis, suggesting that CLEC-2 is the first pathophysiological receptor of podoplanin to be identified. In summary, we clarified the molecular interaction in vitro and in vivo between a platelet aggregation-inducing factor, podoplanin, and its specific pathophysiological receptor on platelets, CLEC-2. Podoplanin and CLEC-2 might represent promising therapeutic targets in cancer metastasis.

Induction of podoplanin by transforming growth factor-beta in human fibrosarcoma.

Podoplanin/aggrus is increased in tumors and its expression was associated with tumor malignancy. Podoplanin on cancer cells serves as a platelet-aggregating factor, which is associated with the metastatic potential. However, regulators of podoplanin remain to be determined. Transforming growth factor-beta (TGF-beta) regulates many physiological events, including tumorigenesis. Here, we found that TGF-beta induced podoplanin in human fibrosarcoma HT1080 cells and enhanced the platelet-aggregating-ability of HT1080. TGF-beta type I receptor inhibitor (SB431542) and short hairpin RNAs for Smad4 inhibited the podoplanin induction by TGF-beta. These results suggest that TGF-beta is a physiological regulator of podoplanin in tumor cells.

Expression of highly sulfated keratan sulfate synthesized in human glioblastoma cells.

Keratan sulfate (KS) proteoglycan is expressed in the extracellular matrix or cell surface in numerous tissues, predominantly in those of the cornea, cartilage, and brain. However, its structure, function, and regulation remain poorly understood. Our investigation of KS expression in glioblastoma cell lines using Western-blot and flow cytometry with anti-KS antibody (5D4) revealed that LN229 glioblastoma cell highly expresses KS on a cell surface. Real-time PCR analysis showed that LN229 expresses a high level of keratan sulfate Gal-6-sulfotransferase. Results of this study also demonstrate that recombinant 5D4-reactive aggrecan is produced in LN229. Taken together, these results suggest that LN229 produces 5D4-reactive highly sulfated KS and is useful to investigate the KS structure and function in glioblastoma.

Podocalyxin expression in malignant astrocytic tumors.

Podocalyxin is an anti-adhesive mucin-like transmembrane sialoglycoprotein that has been implicated in the development of aggressive forms of cancer. Podocalyxin is also known as keratan sulfate (KS) proteoglycan. Recently, we revealed that highly sulfated KS or another mucin-like transmembrane sialoglycoprotein podoplanin/aggrus is upregulated in malignant astrocytic tumors. The aim of this study is to examine the relationship between podocalyxin expression and malignant progression of astrocytic tumors. In this study, 51 astrocytic tumors were investigated for podocalyxin expression using immunohistochemistry, Western blot analysis, and quantitative real-time PCR. Immunohistochemistry detected podocalyxin on the surface of tumor cells in six of 14 anaplastic astrocytomas (42.9%) and in 17 of 31 glioblastomas (54.8%), especially around proliferating endothelial cells. In diffuse astrocytoma, podocalyxin expression was observed only in vascular endothelial cells. Podocalyxin might be associated with the malignant progression of astrocytic tumors, and be a useful prognostic marker for astrocytic tumors.