Search

The association between lymph node density and survival free of lung metastases in oral squamous cell carcinoma (SCC), has not been investigated so far to our knowledge. Lymph node density ≧ 0.07 has been reported by a multicentre international study to be a significant predictor of shorter survival in patients with oral SCC who have invaded nodes. We investigated whether a lymph node density of ≧ 0.07 correlates with shorter overall survival, survival free of distant metastases, and survival free of lung metastases, in patients with oral SCC and invaded lymph nodes. Thirty-five patients with histologically-confirmed invaded lymph nodes werestudied. Their density was calculated as the ratio of the number of invaded lymph nodes:total number of nodes. A density of ≧ 0.07 correlated significantly with shorter overall survival (p

The preparation of the composite membranes has been investigated by photo-induced graft filling polymerization. The asymmetric polyimide membranes as substrate were prepared by the phase inversion process using polyimide with benzophenone structure. Graft polymerization took place by UV irradiation supplying monomer, such as methyl acrylate without adding photoinitiator like benzophenone. These graft modified membranes showed benzene permselectivity for benzene/cyclohexane mixture (50/50 v/v) on pervaporation.

Use of a monoclonal antibody against Lafora bodies for the immunocytochemical study of ground-glass inclusions in hepatocytes due to cyanamide Aims: Ground-glass inclusions (GGIs) in hepatocytes are known to be associated with cyanamide treatment in patients with alcohol dependency. The purpose of this study was to assess the reactivity of a monoclonal antibody (MAb) raised against polyglucosan and to detect early events in GGI formation. Methods and results: Formalin-fixed paraffin-embedded liver tissues from four patients treated with cyanamide were used. Sections were stained with haematoxylin and eosin and periodic acid–Schiff with and without diastase digestion, and were immunohistochemically stained with the MAb. For electron microscopic study, routinely processed liver tissue from one patient was examined with conventional and immunoelectron microscopy with use of the MAb. All specimens from the four cyanamide-treated patients contained GGIs in the cytoplasm of hepatocytes, and these GGIs reacted intensely with the MAb. Fully developed GGIs contained various organelles, whereas early ones consisted primarily of glycogen granules and dilated smooth endoplasmic reticulum. In immunoelectron microscopic preparations, gold particles were located within GGIs, and the immunolabelled organelles appeared to be glycogen granules. Conclusions: This novel MAb is useful for the detection of GGIs caused by cyanamide. Our results support the idea that GGI formation may result from specific abnormalities in glucose metabolism.

Ganglioside GM2 is expressed on the surface of neuroblastoma and glioblastoma cells, and may also be detected on lung cancer cells. We reported previously that anti-ganglioside GM2 antibody exhibited strong in vitro anti-tumor activity against adriamycin-resistant cancer cells, which overexpressed ganglioside GM2. In the present study, we examined the in vivo anti-tumor effect of the chimeric anti-ganglioside GM2 antibody, KM966, against human lung and breast carcinoma cells, SBC-3 and MCF-7, and respective adriamycinresistant clones, SBC-3/ADM and AdrR MCF-7 in BALB/c nu/nu mice. Ratios of tumor volume (T/C) between KM966treated group and control group were 0.01 for SBC-3, 0.00 for SBC-3/ADM, 0.85 for MCF-7 and 0.34 for AdrR MCF-7 cells, respectively. Nude mice, which were pretreated with antiasialo GM1 antibody to remove natural killer cells, were transplanted with 4 3 10 7 of SBC-3 and SBC-3/ADM subcutaneously. Seven days later, when tumors had grown to a diameter of over 8 mm, mice began to receive intravenous treatment of 120 mg/mouse KM966 daily. Fourteen daily treatments induced regression to less than 4-mm diameter in 4/5 SBC-3 tumors and 5/5 of SBC-3/ADM tumors. All SBC-3/ ADM tumors disappeared completely, suggesting that KM966 exerts a strong in vivo anti-tumor effect on ganglioside GM2-expressing cancer cells. In KM966-treated mice, the surface of the tumor cells stained positive with anti-human IgG. In addition, numerous leukocytes had infiltrated into the tumor mass. Antibody-dependent cell-mediated cytotoxicity (ADCC) of KM966 against tumor cells was examined in vitro by 51 Cr-release assay and revealed that KM966 induces ADCC activity against ganglioside GM2-expressing tumors. Our results suggest that immunotherapy using KM966 may be useful for the treatment of ganglioside GM2-expressing solid tumors. Int. J. Cancer 82:759‐764, 1999. r 1999 Wiley-Liss, Inc.

Vapor-deposited films of poly(N-vinylcarbazole) (PVCz) were used as hole transport layers in organic electroluminescent devices. Hole transporting ability of the vapor-deposited PVCz films in the double-layered cell remarkably depended on their film morphology. Light emission was obtained only when a thin, homogeneous PVCz film (10–15 nm in thickness) was deposited on an anode substrate which was kept at 100°C during deposition. When the PVCz film was overcoated on a low molecular hole transporting layer, the EL performance of the mutilayered device was retained for a longer time as compared with that without the PVCz deposition. This improved durability was ascribed to the PVCz layer which prevents the low molecular layer from being degraded by aggregation.

The α-trimethylsilyl conjugated carbonyl derivatives 1 easily react with organometallics 2 (M = Al, Mg, Zn) and allow preparation of functional vinylsilanes 3 or 4 , in regioselective manner: R'-CH C(SiMe 3 )-C(OH)(R')(R) 3 ; (R')(R)CH-C(SiMe 3 )C(R')(OSiMe 3 ) 4 .

The expression of a single glycosyltransferase, GD3 synthase, caused cholinergic differentiation with neurite sprouting. The cells that expressed GD3 were established from Neuro2a cells by transfection of a mammalian expression vector into which were carried a cDNA encoding GD3 synthase and the blasticidin-S-deaminase gene with a SV40 promoter, followed by selection with blasticidin-S-hydrochloride. The blasticidin-S-hydrochloride-resistant colonies derived from the cells transfected with the cDNA encoding GD3 synthase and the clonal cells (N2a-GD3) were spontaneously sprouting neurites but not those derived from cells transfected with only the vector without the cDNA encoding GD3 synthase (N2a-bsr). GD3 expression by N2a-GD3 was confirmed by immunostaining of the cells using the anti-GD3 monoclonal antibody, KM643. N2a-GD3 expressed not only GD3 but also GQ1b, one of the b-series gangliosides, whereas N2a-bsr did not express these gangliosides. Cell proliferation of N2a-GD3 was greatly reduced, as compared with that of N2a-bsr, and, after several passages, it completely stopped. In addition, N2a-GD3 expressed acetylcholine esterase, indicating that the differentiation of Neuro2a cells was induced by expression of GD3 synthase and subsequent modification of the biosynthesis and expression of gangliosides. These results strongly suggest that the de novo synthesis and expression of GD3 and/or b-series gangliosides induce neurite outgrowth and differentiation of Neuro2a cells. Exogenous GM1 stimulated the neuritogenesis of N2a-bsr but not differentiated N2a-GD3, indicating that the mechanism of neurite sprouting in this system may be overlapped en route with that of exogenous GM1.

We previously reported that activated platelets stimulated neutrophils and monocytes to produce superoxide anion (O2-) through the interaction between P-selectin and its carbohydrate ligand, sialyl Lewis X (sLeX) (Nagata, K., Tsuji, T., Todoroki, N., Katagiri, Y., Tanoue, K., Yamazaki, H., Hanai N., and Irimura, T. (1993) J. Immunol. 151, 3267-3273). In the present study, we investigated the role of cell surface carbohydrate chains of leukocytes in this process. Glycoconjugate-specific hydrolytic enzymes and inhibitors of glycosylation processing were applied. Granulocyte-like differentiated HL-60 (gHL-60) cells released an increased amount of O2- in response to activated platelets in a P-selectin-dependent manner. When HL-60 cells were differentiated in the presence of benzyl-alpha-N-acetylgalactosaminide (Bzl-alpha-GalNAc), an inhibitor of chain elongation of O-linked carbohydrates, the enhanced generation of O2- was abrogated in parallel with decrease in the expression of sLex structure and in the adhesion capacity to activated platelets. In contrast, treatment with swainsonine or 1-deoxymannojirimycin, inhibitors of processing of N-linked carbohydrate chains, did not show such effects. O-Sialoglycoprotease treatment of gHL-60 cells decreased the activated platelet-induced O2- production with concomitant reduction of cell surface sLex expression. Treatment of these cells with N-glycanase did not affect the O2- production. These results strongly suggested that serine/threonine-linked carbohydrate chains containing sLex played an essential role in the P-selectin-mediated leukocyte activation. By Western blotting analysis of gHL-60 cell lysates, we identified two glycoproteins which carried sLex structures and were sensitive to Bzl-alpha-GalNAc treatment.

A cDNA encoding a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing GD3 using an anti-GD3 antibody and a fluorescence-activated cell sorter. Selection of B-cell line Namalwa cells expressing transfected cDNAs in the presence of anti-GD3 monoclonal antibody KM641 gave a cDNA (pAMo-GD3) encoding a protein with a type II transmembrane topology as found for mammalian glycosyltransferases. The following evidence confirms that the cDNA encodes an alpha-2,8-sialyltransferase, which specifically converts GM3 to GD3. (i) Transfection of pAMo-GD3 into Namalwa KJM-1 cells leads to the appearance of GD3 and a GD3 synthase activity. (ii) Northern blot analysis revealed a correlation between the expression of this gene and GD3 in several cell lines. (iii) The putative COOH-terminal active domain of this cloned enzyme fused with protein A has been purified with IgG-Sepharose beads and has been shown to possess GD3-synthesizing activity, excluding the possibility that the cloned cDNA encodes a transacting factor inducing a GD3 synthase. The deduced primary sequence also contains the "sialyl motif" conserved among all the sialyltransferases cloned to date. The polymerase chain reaction analysis reveals that this gene is located on chromosome 12.

The sialyl Lewis x (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)Glc-NAc) determinants serve as ligands in the selectin-mediated adhesion of leukocytes to activated endothelium or platelets. In our efforts to identify glycosyltransferases involved in the biosynthesis of those ligands, we achieved expression cloning of a novel human alpha 1,3-fucosyltransferase termed Fuc-TVII from a THP-1 cDNA library by enrichment of the Namalwa cells highly expressing that determinant with a fluorescence-activated cell sorter. Expression of the COOH-terminal catalytic domain of Fuc-TVII showed an alpha 1,3-fucosyltransferase activity for a type II oligosaccharide with a terminal alpha 2,3-linked sialic acid among various acceptors, consistent with that in vivo acceptor specificity. Alignment of the primary sequences of five alpha 1,3-fucosyltransferases and assignment of the chromosomal location of Fuc-TVII gene, together with that acceptor specificity, indicate that Fuc-TVII consists of a unique class of the alpha 1,3-fucosyltransferase family. Determination of the expression levels of these alpha 1,3-fucosyltransferases in various cells revealed that both Fuc-TVII and a myeloid fucosyltransferase Fuc-TIV were significantly expressed in myeloid lineage cells. Fuc-TVII-transfected Namalwa cells exhibited significant binding to E-selectin in contrast to little binding of the Fuc-TIV-transfected cells. These results suggest that Fuc-TVII may participate in the biosynthesis of the selectin ligands.

Ribonucleotide reductase (RNR) consists of two non-identical subunits, R1 and R2 and is one of the key enzymes involved in DNA biosynthesis. RNR activity is considerably higher in malignant tumors than in normal tissues in the rat suggesting that RNR may play an important role in the pathogenesis of human tumors. In order to obtain immunological reagents to study the localization and level of expression of RNR in various human tissues, a synthetic peptide containing sequences corresponding to the COOH-terminal region of the human R2 subunit was used to generate rat monoclonal antibodies. The generated rat monoclonal antibodies (IgG) inhibited RNR enzymatic activity purified from murine P388 leukemia cells. These antibodies were used to immunohistochemically examine the distribution of RNR in a small panel of 8 malignant and 4 benign human breast tumors. Positive immunostaining for RNR was observed in the cytoplasm of human breast carcinoma cells in which a specific 44 kDa specific band of R2 subunit was also detected by Western blot analysis. The immunostaining was blocked by preabsorption of the antibody with an excess amount of the synthetic peptide immunogen. In 8 of 8 breast carcinomas, positive immunostaining for the R2 subunit was observed whereas noninvolved, adjacent breast tissue showed no staining with this antibody. In addition, few of the benign breast lesions exhibited staining with this antibody. These data indicate that these antibodies can immunohistochemically detect RNR in frozen or formalin-fixed, paraffin- embedded tissues and that there is a differential expression of RNR between breast tumors and non-involved breast tissue. Immunohistochemical detection of RNR using these antibodies may therefore be useful for the diagnosis of human breast tumors.

Activated platelets expressing P-selectin in their surfaces are known to adhere to monocytes and neutrophils. We examined the possibility that the leukocytes are functionally modified by their adhesion to activated platelets. We used human peripheral blood monocytes and neutrophils and measured superoxide anion generation by these cells cultured with platelets. The levels of superoxide anion production were found to be markedly elevated when thrombin-activated platelets were used. The extent of this enhancement was much smaller when leukocytes were cultured with resting platelets than activated platelets. The increase depended on incubation time and platelet concentration. The membranes prepared from activated platelets also induced superoxide anion production, but the culture supernatant of activated platelets did not. The enhanced superoxide anion production was inhibited by anti-P-selectin antibody, anti-sialyl-Lewis X antibody, or a soluble recombinant P-selectin fusion protein. These results indicate that the adhesion of activated platelets to the leukocytes through P-selectin was a crucial step for the activation of leukocyte function, and support the idea that activated platelets are actively involved in inflammation processes.

Mouse Mx1 protein is an interferon-inducible nuclear protein and confers resistance to influenza virus infection. The Mx1 protein purified from interferon-induced A2G mouse liver exhibited GTPase activity as did the Mx1 protein purified from the Mx1 cDNA-expressing Escherichia coli (Nakayama, M., Nagata, K., Kato, A., and Ishihama, A. (1991) J. Biol. Chem. 266, 21404-21408; Nakayama, M., Nagata, K., and Ishihama, A. (1992) Virus Res. 22, 227-234). The Mx1 protein purified from both mouse liver and Mx1-cDNA expressing E. coli was found to exist as assembled polymeric states judged from gel filtration pattern. By making a set of deletion derivatives of the Mx1 cDNA, the main motif for self-assembly of the Mx1 protein was mapped between amino acid residues 51-99. This motif is highly conserved not only in the Mx family of proteins but also in Mx-related proteins. The polymeric form of Mx1 from E. coli was observed as "horseshoe"-like structure by negative staining microscopy. When the Mx1 protein was incubated with GTP, this horseshoe structure was transformed to larger and tightly stacked helical forms. Electron microscopic analysis of immunostained liver of the interferon-induced mice indicated that the Mx1 protein exists in nuclei, forming giant complexes of about half the size of nucleoli.

Receptor activity modifying proteins (RAMPs) act as receptor modulators that determine the ligand specificity of receptors for the calcitonin (CT) family. The purpose of this study was to analyze the expression of RAMPs in osteoclast-like cells using the laser capture microdissection (LCM) technique. Mouse bone marrow and spleen cells were co-cultured on a film designed for LCM. After 10 days, 250 osteoclast-like cells were captured using the LCM system. Total RNA from these cells was used to synthesize cDNA and RT-PCR analysis was performed. Osteoclast-like cells expressed CT receptor (CTR), CT receptor-like receptor (CRLR) and RAMP2, but did not express RAMP1 or RAMP3. These results indicated (1) that a pure population of osteoclast-like cells can be prepared by LCM and gene expression of this population can be analyzed by RT-PCR and (2) that RT-PCR shows that osteoclast-like cells express RAMP2, CTR and CRLR, suggesting the potential for adrenomedullin binding to osteoclast-like cells. This is the first report that osteoclast-like cells express RAMP2.

The chimeric antiganglioside GM2 monoclonal antibody (MAb) KM966, which showed high effector functions such as complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), potently suppressed growth and metastases of GM2-positive human cancer cells inoculated into mice. To further improve the therapeutic efficacy of the anti-GM2 MAb in humans, we constructed a humanized anti-GM2 MAb, KM8969. The humanized KM8969 was more efficient in supporting ADCC against GM2-positive human cancer cell lines than the chimeric KM966, whereas complement-dependent cytotoxicity was slightly reduced in the humanized KM8969. In addition, the humanized KM8969 was shown to exert potent ADCC mediated by both lymphocytes and monocytes. To investigate the effect of the humanized KM8969 on the biological function of GM2 in the condition physiologically mimicking formation and growth of cancer masses, the heterospheroids composed of normal human dermal fibroblasts and GM2-positive human lung cancer cells were developed. Interestingly, the humanized KM8969 gave rise to growth inhibition of heterospheroids without dependence of the effector functions. Morphological and immunocytochemical analysis suggested that the inhibitory effect was due to the apoptosis of GM2-positive cancer cells in the heterospheroids. The result indicates that GM2 captured by the antibody on the cell surface loses its physiological function that plays a critical role in maintaining the three-dimensional growth of cancer cells in contact with its own cells or other type of cells in a microenvironment. The humanized KM8969, which can destroy the cancer cells via blocking functional GM2 on the cell surface as well as the effector functions, would have extraordinary potential in human cancer therapy.

Panosialins A and B were isolated as inhibitors of an alpha1,3-fucosyltransferase, Fuc-TVII, which is a key enzyme in the biosynthesis of selectin ligands, from culture broth of Streptomyces sp. Panosialins A and B inhibited the Fuc-TVII activity with IC50 values of 4.8 and 5.3 microg/ml, respectively. Panosialin A suppressed expression of selectin ligands on U937 cells, and inhibited the cell adhesion to immobilized E-selectin-immunoglobulin. Panosialins are the first reported Fuc-TVII inhibitors which can suppress the biosynthesis of selectin ligands and then inhibit selectin-mediated cell adhesion.

Fibroblast growth factor (FGF) 8, also known as androgen-induced growth factor, was originally isolated from an androgen-dependent mouse mammary Shionogi carcinoma SC-3 cell line, in which it was shown to have androgen-regulated properties. We previously demonstrated that Fgf 8 transcripts were detected in several human prostate and breast cancer cell lines and that recombinant FGF 8 was mitogenic to an androgen-sensitive prostate cancer LNCaP cell line. In this study, to characterize the roles of FGF 8 in clinical hormone-responsive cancers, we established a monoclonal antibody against FGF 8. In Western blots, this antibody specifically interacted with a FGF 8b isoform that was identical between mouse and human but was not identical to other murine 8a and 8c isoforms. In a cell growth assay using SC-3 cells, the newly established anti-FGF 8 antibody blocked androgen- and FGF 8-stimulated growth but not basic FGF-stimulated growth. Immunohistochemical analyses by use of the established anti-FGF 8 antibody demonstrated that FGF 8 was frequently expressed in human prostate cancers, appearing in 40 of 43 cases (93%), whereas both prostatic hyperplasia specimens and normal prostate tissues included in biopsy specimens were negative for FGF 8 expression. On the other hand, FGF 8 was detected in normal ductal and lobular epithelial cells in breast tissues. FGF 8 was also frequently expressed in various breast diseases, including fibroadenomas (5 of 5 cases, 100%), intraductal papillomas (3 of 3 cases, 100%), ductal hyperplasias (3 of 6 cases, 50%), and breast cancers (8 of 12 cases, 67%). Androgen receptors were also immunohistochemically detected in FGF 8-positive prostate cancers (40 of 40 cases, 100%) and FGF 8-positive breast diseases (17 of 19 cases, 89%). These findings strongly suggest that FGF 8 is involved in hormone-related tumorigenesis of the prostate and breast.

The human interleukin-5 receptor (hIL-5R) consists of a unique alpha subunit (hIL-5Ralpha) and a common beta subunit (betac) that activate two Janus kinases (JAK1 and JAK2) and a signal transducer and activator of transcription (STAT5). The precise stoichiometry of the hIL-5R subunits and the role of JAK kinases used in IL-5 signaling were investigated. We analyzed the interaction between hIL-5Ralpha and betac by immunoprecipitation using anti-hIL-5Ralpha and anti-betac monoclonal antibodies. The binding of JAK1 and JAK2 to each hIL-5R subunit was also evaluated in the hIL-5-responsive cell line, TF-h5Ralpha. It was observed that IL-5 stimulation induced the recruitment of betac to hIL-5Ralpha, although in the absence of IL-5 the subunits remain independent. In the absence of IL-5, JAK2 and JAK1 were associated with hIL-5Ralpha and betac, respectively. IL-5 stimulation resulted in tyrosine phosphorylation of JAK2, JAK1, betac, and STAT5. Moreover, IL-5-induced dimerization of IL-5R subunits caused JAK2 activation and betac phosphorylation even in the absence of JAK1 activation. Furthermore, tyrosine phosphorylation of JAK1 was dependent on the activation of JAK2. Detailed study of the C-terminal truncated cytoplasmic domain of hIL-5Ralpha revealed that the cytoplasmic stretch at position 346-387, containing the proline-rich region, is necessary for JAK2 binding. These observations suggest that activation of hIL-5Ralpha-associated JAK2 is indispensable for the IL-5 signaling event.

The stage-specific embryonic Ag-1 (SSEA-1) is a carbohydrate Ag and regarded as an onco-developmental Ag. Sialyl SSEA-1 Ag, the sialylated form of SSEA-1, is frequently expressed in human cancer cells as well as in murine cancer cells. A mAb, FH-6, was shown to specifically recognize the Ag. We have generated five anti-Id Abs directed to the paratope-related idiotopes of the FH-6 Ab. One of these anti-Id Abs, Id-F2, increased the survival of host mice that were inoculated with Meth-A cells expressing the sialyl SSEA-1 Ag. To clarify the exact mechanism underlying the antitumor effect of the anti-Id Ab, we established a T cell line that recognized Id-F2 in association with MHC class II molecules. The T cell line was CD4+V beta 8+, and produced IL-2, exhibiting helper activity for B cells. The VH CDR2 region of the Id-F2 amino acid sequences turned out to be strongly immunogenic to T cells. When the immune complexes, consisting of the sialyl SSEA-1 Ag, FH-6, and Id-F2, were formed at the Meth-A cell-surface, the T cell line showed a strong proliferative response. The possible roles played by such T cell subsets in the anti-tumor effect are discussed.

The nm23 gene [encoding nucleoside diphosphate kinase (NDPK)] may act as a metastasis suppressor in certain tumor cells. We investigated the role of NDPK isoforms (alpha and beta) in the metastatic processes, using rat mammary-adenocarcinoma cell lines of poor (MTC) and high (MTLn3) spontaneous metastatic potential respectively. In these cell lines, as in most rat tissues, the alpha isoform (nm23-H2 homolog) was more highly expressed than the beta isoform (nm23-H1 homolog) at the mRNA and protein levels. When examined by Northern- and Western-blot analyses, expression of the 2 isoforms was reduced in highly metastatic MTLn3 cells compared with poorly metastatic MTC cells. The reduced expression was also associated with diminished NDPK-enzyme activity in the cell extracts. Southern-blot and RT-PCR-SSCP analyses suggested that the 2 genes were not grossly altered or mutated in their translation regions. MTLn3 cell clones transfected with NDPKalpha or NDPKbeta cDNA were all tumorigenic when implanted into the mammary fat pad of syngeneic rats. Among those, only clones transfected with the NDPKalpha gene exhibited reduced lung metastasis in a spontaneous metastasis assay.

Twenty-three primary and 27 metastatic melanoma lesions and 17 pigmented nevi lesions were tested utilizing the immunoperoxidase reaction with anti-sialyl Lewis(a) (sLea) and anti-sLex mAbs.sLea was expressed in 9, 25, and 5 and sLex was expressed in 6, 11, and 2 of these lesions, respectively. Expression of sLea in melanocytic tumors is associated with tumor progression. Serum levels of sLea and sLex were analyzed by a sandwich assay using mAbs in 25 melanoma patients. Only 2 patients at stage 4 showed higher levels of sLea and sLex than did normal control subjects. Moreover, sLea and sLex were expressed in 1 and 2 of 5 human melanoma cell lines, respectively, and expression of sLex and sLex was not modulated by cytokines. These findings suggest that the expression of sLea in melanocytic tumors is correlated with disease progression.

The expression of a single glycosyltransferase, GD3 synthase, caused cholinergic differentiation with neurite sprouting. The cells that expressed GD3 were established from Neuro2a cells by transfection of a mammalian expression vector into which were carried a cDNA encoding GD3 synthase and the blasticidin-S-deaminase gene with a SV40 promoter, followed by selection with blasticidin-S-hydrochloride. The blasticidin-S-hydrochloride-resistant colonies derived from the cells transfected with the cDNA encoding GD3 synthase and the clonal cells (N2a-GD3) were spontaneously sprouting neurites but not those derived from cells transfected with only the vector without the cDNA encoding GD3 synthase (N2a-bsr). GD3 expression by N2a-GD3 was confirmed by immunostaining of the cells using the anti-GD3 monoclonal antibody, KM643. N2a-GD3 expressed not only GD3 but also GQ1b, one of the b-series gangliosides, whereas N2a-bsr did not express these gangliosides. Cell proliferation of N2a-GD3 was greatly reduced, as compared with that of N2a-bsr, and, after several passages, it completely stopped. In addition, N2a-GD3 expressed acetylcholine esterase, indicating that the differentiation of Neuro2a cells was induced by expression of GD3 synthase and subsequent modification of the biosynthesis and expression of gangliosides. These results strongly suggest that the de novo synthesis and expression of GD3 and/or b-series gangliosides induce neurite outgrowth and differentiation of Neuro2a cells. Exogenous GM1 stimulated the neuritogenesis of N2a-bsr but not differentiated N2a-GD3, indicating that the mechanism of neurite sprouting in this system may be overlapped en route with that of exogenous GM1.

With the aim of investigating the passive immunotherapy of brain tumors, we examined the binding of a mouse/human chimeric anti-ganglioside GM2 antibody KM966 to various organs and brain tumors. Frozen sections of 51 surgically resected brain tumors were stained with antibody KM966. Fourteen gliomas out of 16 were stained positively with antibody KM966. Eleven positive sections demonstrated homogenous staining. No specific binding to normal gray matter and white matter was observed. Some cases of meningiomas, neurinomas and metastatic brain tumors were stained with KM966 but with less frequency and intensity than gliomas. In addition, KM966 demonstrated strong antibody-dependent cellular cytotoxicity against human malignant glioma cells. These results showed that antibody KM966 will be useful for passive immunotherapy of malignant gliomas.

A cDNA encoding a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing GD3 using an anti-GD3 antibody and a fluorescence-activated cell sorter. Selection of B-cell line Namalwa cells expressing transfected cDNAs in the presence of anti-GD3 monoclonal antibody KM641 gave a cDNA (pAMo-GD3) encoding a protein with a type II transmembrane topology as found for mammalian glycosyltransferases. The following evidence confirms that the cDNA encodes an alpha-2,8-sialyltransferase, which specifically converts GM3 to GD3. (i) Transfection of pAMo-GD3 into Namalwa KJM-1 cells leads to the appearance of GD3 and a GD3 synthase activity. (ii) Northern blot analysis revealed a correlation between the expression of this gene and GD3 in several cell lines. (iii) The putative COOH-terminal active domain of this cloned enzyme fused with protein A has been purified with IgG-Sepharose beads and has been shown to possess GD3-synthesizing activity, excluding the possibility that the cloned cDNA encodes a transacting factor inducing a GD3 synthase. The deduced primary sequence also contains the "sialyl motif" conserved among all the sialyltransferases cloned to date. The polymerase chain reaction analysis reveals that this gene is located on chromosome 12.

Ganglioside GM2, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin, has been focused on as a target molecule for passive immunotherapy. GM2 is thought to be one of the T-cell-independent antigens and to elicit only IgM antibody responses in rodents and humans. We have previously established two murine anti-GM2 monoclonal antibodies with high specificity and strong binding activity, KM696 and KM697, both of which are of the IgM class. Variable heavy and light chain complementary DNAs of these two murine monoclonal antibodies were cloned and used in the construction of mouse/human IgG1 chimeric antibodies, KM966 and KM967, respectively, in this study. One of the chimeric antibodies, KM966, retained strong and specific reactivity with GM2 and showed the similarity of the binding activity with tumor cell lines to that of the original murine monoclonal antibody. Indirect immunofluorescence staining of tumor cell lines with the chimeric KM966 revealed that the antigen was expressed in substantial amounts on pulmonary tumor cells and leukemia cells as well as neuroectodermal origin tumor cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, respectively, chimeric KM966 was fully effective in killing GM2-expressing tumor cells. In addition, i.v. injection of chimeric KM966 markedly suppressed the establishment of human tumor xenografts in nude mice. Taken together, chimeric KM966 is the first antibody of the human IgG class to ganglioside GM2 and has strong antitumor activity both in vitro and in vivo. It is likely that chimeric KM966 will be a useful agent for passive immunotherapy of human cancer.

The surface engagement of high affinity immunoglobulin E receptor (Fc epsilon RI) of rat basophilic leukemia 2H3 (RBL-2H3) cells induced histamine secretion and leukotriene release following activation of the tyrosine kinase Lyn together with phosphatidylinositol 3-kinase (PI3-kinase). Wortmannin inhibited the activity of partially purified PI3-kinase from calf thymus, as well as the PI3-kinase activity in anti-PI3-kinase p85 immunoprecipitates from RBL-2H3 cells, at a concentration as low as 1.0 nM and with IC50 values of 3.0 nM, but did not inhibit PI4-kinase activity. The inhibition of PI3-kinase by wortmannin was irreversible. Wortmannin inhibited both Fc epsilon RI-mediated histamine secretion and leukotriene release up to 80% with IC50 values of 2.0 and 3.0 nM, respectively. Wortmannin inhibited PI3-kinase activity in intact cells up to 80% with an IC50 value of 2.0 nM, which is almost equal to those for PI3-kinase in vitro and for histamine secretion and leukotriene release. With anti-wortmannin antibody, we have shown that wortmannin binds to the 110-kDa protein, but not to PI3-kinase 85-kDa regulatory subunit both in vitro and in whole cells. Furthermore, there was a positive correlation between the potencies of wortmannin derivatives as inhibitors of PI3-kinase and as inhibitors of histamine secretion. Wortmannin had no effect on the activation of the tyrosine kinase Lyn. These results suggest that PI3-kinase is involved in the signal transduction pathway responsible for histamine secretion following stimulation of Fc epsilon RI and that wortmannin blocks these responses through direct interaction with the catalytic subunit of this enzyme.

The fate of anti-tumor monoclonal antibodies (mAbs) after binding to the cell surface is one of the important factors for application of mAbs in therapy. For mAbs to remain on the cell surface for a long time is preferable character for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). On the other hand, quick internalization of mAbs into cytoplasm is advantageous for cytotoxic effects of toxins or cytotoxic drugs conjugated with mAbs. In this study we investigated the localization of anti-carbohydrate mAbs in vitro by immunocytochemical methods at the electron microscopic level. Anti-ganglioside GM3 mAb (mouse IgM) was localized both in cytoplasmic vesicles and on the cell surface membrane of mouse melanoma cells after incubation for 30 min at 37 degrees C. Anti-ganglioside GM2 mAb (mouse IgM), anti-ganglioside GD2 mAb (mouse IgG3) anti-ganglioside GD3 mAb (mouse IgG3) mainly existed on the cell surface of human small cell lung carcinoma cells, human neuroblastoma cells and human melanoma cells, respectively, after incubation for 30 min at 37 degrees C. On the other hand, anti-sialyl Lea mAb (mouse IgG1) was intensively incorporated into the cytoplasm of human colon tumor cells under the same conditions. Anti-ganglioside GD3 mAb and anti-sialyl Lea mAb were radiolabelled with 125Iodine and traced in in vitro culture. When the cells were incubated in the presence of the 125I-mAb for 90 min on ice, the ratio of intracellular counts to surface counts was 8/92 for anti-ganglioside GD3 mAb and 31/69 for anti-sialyl Lea mAb respectively. After the subsequent incubation for 60 min at 37 degrees C, the ratio altered to 13/87 for anti-ganglioside GD3 mAb and 54/46 for anti-sialyl Lea antigen mAb. This study suggested that internalization of anti-carbohydrate mAbs after the binding to the cell surface was different among the mAbs depending on the character of antigens. In conclusion, anti-ganglioside mAbs have beneficial characters for CDC or ADCC while anti-sialyl Lea mAb is suitable for immunoconjugates.

This report describes the isolation of a cDNA encoding a novel human Gal beta (1-3/1-4)GlcNac alpha 2,3-sialyl-transferase involved in the biosynthesis of the sialyl Lewis x determinant (NeuAc alpha 2-3 Gal beta 1-4(Fuc alpha 1-3)GlcNAc). A cDNA library of the human melanoma cell line WM266-4 was constructed in an Epstein-Barr virus-based cloning vector. Selection of the B-cell line Namalwa expressing transfected cDNAs in the presence of the cytotoxic lectin Ricinus communis agglutinin 120 gave a cDNA encoding a protein with type II transmembrane topology, as found for mammalian glycosyltransferases. The use of this lectin, which is specific to galactose residues (especially the Gal beta 1-4GlcNAc structure), originates from our prediction that the modification of the Gal beta 1-4GlcNAc structure (a backbone of the sialyl Lewis x structure) by glycosyltransferases may increase the levels of resistance to this lectin. Comparison of this cDNA sequence with those of three other cloned sialyltransferases revealed two conserved regions shared by all four enzymes. Expression of the COOH-terminal catalytic domain of this protein showed alpha 2,3-sialyltransferase activity with substrate specificity different from that of CMP-N-acetylneuraminate:N-acetyllactosaminide alpha-2,3-sialyltransferase (Gal-beta 1-3(4)GlcNAc alpha 2,3-sialyltransferase, EC 2.4.99.6). Furthermore, expression of this cDNA in Namalwa cells increased the level of sialyl Lewis x antigens. The cloning approach based on lectin resistance may be useful for the isolation of cDNAs encoding other mammalian glycosyltransferases.

GM2 and GD2 have been intensely studied as tumor antigens of neuroectodermal-origin tumors. We have established several mouse or rat IgM anti-ganglioside GM2 and GD2 MoAbs and applied them in antigen-specific drug delivery. In the immunofluorescence assay, anti-GM2 MoAbs bound to neuroblastoma cells and leukemia cells, and anti-GD2 MoAb bound to neuroblastoma cells and melanoma cells. Sulfhydryl subunits of reduced IgM were directly coupled to maleimide groups on the surface of the liposomes followed by the incorporation of adriamycin by the use of Na+/K+ chemical gradient. The resultant immunoliposomes had a size of 86 nm-diameter containing approximately 400 molecules of adriamycin in its unilamellar structure and 17 molecules of the MoAb on its surface. Mouse anti-GM2 MoAb lost the binding specificity when covalently bound to the liposomes. The immunoliposome coupled with mouse anti-GD2 MoAb retained targeting activity to the antigen-positive neuroblastoma cells, IMR-32. However, it did not kill IMR-32 cells, probably because the amount of adriamycin taken up by the tumor cell was below the fatal amount. The immunoliposome coupled with rat anti-GM2 MoAbs delivered adriamycin to the neuroblastoma cells, IMR-32, and leukemia cells, TYH, in the antigen-specific manner. It also target-specifically suppressed the (3H)thymidine-uptake of the cells while the same concentration of adriamycin in the free form killed all the cell lines examined. IMR-32 cells had GM2 and GD2 in almost the same amounts, and interacted with either mouse anti-GD2 MoAb--immunoliposome or rat anti-GM2 MoAb-immunoliposome. The different cytotoxic activities of the two immunoliposomes against IMR-32 cells was probably due to the difference in the facility of internalization of the immunoliposomes after binding.

An anti lung adenocarcinoma murine monoclonal antibody (MoAb), KM195 (IgG1), was generated using mice which underwent tolerance treatment to normal lung tissues. KM195 was selected from among a number of hybridoma clones because of its advantageous reactivity such as high binding to cell membranes of lung adenocarcinoma tissues and low binding to cell membranes of major normal tissues. In a binding assay using cultured cell lines KM195 was found to bind cytoplasmic antigen in many adenocarcinoma cells. Detailed immunohistochemical analysis using paraffin-fixed tissue sections showed that many adenocarcinoma cells such as gastric cancer, colorectal cancer, pancreatic cancer, mammary cancer, ovary cancer and cervical cancer reacted positively with KM195, as well as lung adenocarcinoma cells. KM195 also positively stained a small number of normal cells found in adult and fetal tissues like lung, intestine, pancreas, liver and kidney. Western blot analysis using membrane fraction of lung adenocarcinoma tissues revealed two major KM195-positive bands which were electrophoresed nearby at molecular weights (M.W.) of 40 Kd. The protein corresponding to the two major bands was purified by immuno-affinity chromatography and sequenced. The amino-terminal 19 residues of the lower band was identified as VLEVDPNIQAVXTQEXEQI, which is identical to that of the human cytokeratin 8 (residues 77 to 95), M.W. 52Kd. The amino-terminal sequence of the upper band was blocked and not determined. To examine the ability of KM195 for tumor imaging, 125I-labeled KM195 was injected i.v. into nude mice bearing SW1116 xenografts. Significantly higher radioactivity was observed in the tumor compared with major organs at days 3 and 5. These data indicate that KM195, which recognizes cytokeratin 8-like cytoplasmic antigen, could be a potential MoAb for use in the immunohistochemical diagnosis and radioimmunodetection of adenocarcinoma.

Anti-idiotype monoclonal antibodies (MoAbs) were developed for a carbohydrate antigen, Sialyl Le(a), by immunizing mice with an anti-Sialyl Le(a) MoAb, KM231 (mouse IgG1). The anti-idiotype MoAbs inhibited the binding of KM231 to Sialyl Le(a)-positive mucin protein, but did not affect the binding of another MoAb (mouse IgG1) which recognized a peptide epitope on the same mucin protein. The selected four anti-idiotype MoAbs bound to all of the five anti-Sialyl Le(a) MoAbs examined, but not to other MoAbs developed against Sialyl Le(a)-related carbohydrate antigens such as Le(a), Le(x), and Sialyl Le(x) blood type A, B, H antigens. Immunization of rats with one of the anti-idiotype MoAbs, of which the reactivity was remarkably inhibited by Sialyl Le(a) oligosaccharide, resulted in the induction of antibody (Ab3) to purified Sialyl Le(a) glycolipid. Taken together, one of the anti-idiotype MoAbs developed in this study was the first Ab2 beta-type anti-idiotype MoAb which carried the internal image of Sialyl Le(a) antigen. The positive reaction of tumor cells expressing Sialyl Le(a) antigen with the Ab3 gave rise to the possibility that the anti-idiotype MoAb would become an effective tool for active immunotherapy in patients with Sialyl Le(a)-positive tumor.

A glycolipid detected in human gastric mucosa with anti-GM2 monoclonal antibody was characterized to be GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4GlcNAc beta 1-3Gal 1-4Glc-Cer (NGM-1), which was lost in gastric cancer tissue with complementary increase of GM2 sharing the same terminal carbohydrate structure as NGM-1 (Dohi, T., Ohta, S., Hanai, N., Yamaguchi, K., and Oshima, M. (1990) J. Biol. Chem. 265, 7880-7885). The study on differential expression of NGM-1 in gastric fundic mucosa, pyloric mucosa, gastric cancer, and various other tissues indicated that NGM-1 existed specifically in fundic mucosa. The content of GM3 and sialylparagloboside (SPG), which are the substrates for the synthesis of GM2 and NGM-1, respectively, were not significantly different in these tissues. Therefore, the presence of two kinds of beta 1,4GalNAc transferases having different substrate specificity was considered to be critical for the expression of NGM-1 and GM2. The activity of beta 1,4GalNAc transferase which synthesizes GM2 or NGM-1 was determined by detecting the products with specific monoclonal antibodies. The activity of beta 1,4GalNAc transfer to SPG was high in fundic mucosa, while it was absent in pyloric mucosa or cancer. On the other hand, the increased activity of beta 1,4GalNAc transfer to GM3 was observed in cancer tissues and cancer cell lines which were rich in GM2. Our conclusion is that the limited expression of NGM-1 in fundic mucosa and the increase of GM2 in cancer are attributed to two types of beta 1,4GalNAc transferases localized in each region with different substrate specificity; the one in fundic mucosa transfers GalNAc to SPG but not to GM3, and the other one enhanced in cancer transfers GalNAc to GM3 but not to SPG.

A mouse monoclonal antibody (MoAb), KM231 raised against human gastric cancer was found to recognize sialyl Lea -epitope expressed on glycoprotein and glycolipid with high affinity. KM231 reacted with many human gastrointestinal cancer tissues and could detect the antigen shed in sera of cancer patients. The present study was designed to evaluate competence of KM231 for immunotherapy of cancer. We first confirmed that KM231 could probe the cancer cells in vivo by injecting biotinylated KM231 into nude mice bearing human colorectal carcinoma cell, SW1116. Light- and electron-microscopic examination showed that the MoAb was localized in the tumor tissues and bound to the plasma membrane and cytoplasmic endosomes. Imaging studies with 125I-labeled KM231 revealed specific localization of the antibody in SW1116 tumors transplanted into nude mice. From Scatchard analysis of KM231 binding, the number of KM231 molecules bound to per SW1116 cell was calculated approximately 1.9 x 10(6) and the association constant was 1.3 x 10(8) liter/mol. We made KM231-ricin A chain immunotoxin for evaluating the tumoricidal effect of KM231. The immunotoxin exerted strong cytotoxicity toward sialyl Lea-expressing tumor cells specifically in vitro, but not toward sialyl Lea non-expressing cells. The in vivo tumoricidal effect of the immunotoxin was examined on ascites and subcutaneous xenograft tumors in nude mice. Three intraperitoneal injections of the immunotoxin (1.6 x 10(-6) mol) into nude mice bearing SW1116 ascites tumor resulted in extension of survival by 204% compared with controls. Further, repeated intraperitoneal administration of the immunotoxin (1.4 - 2 x 10(-6) mol) significantly inhibited the growth of established subcutaneous tumor (ratio of tumor inhibition = 0.7 - 0.54). These results indicated that KM231 has the ability to probe sialyl Lea-expressing tumor cells in vivo with high efficiency and to become tumoricidal drug when it conjugated with cytotoxic reagents like ricin A chain.

Four murine monoclonal antibodies (MoAbs), KM191, KM206, KM230 and KM231, raised against gastric cancer exhibited very similar reactivities to human carcinoma cells, whereas their abilities in probing the antigen shed from the cancer cells were quite different. We found in this study that the four MoAbs reacted immunologically with the same monosialo gangliosides derived from a gastric cancer cell line by two-dimensional high performance thin-layer chromatography. When the reactivities of the MoAbs to a number of purified gangliosides or oligosaccharides were examined, the carbohydrate structure of the antigen was determined as sialyl Lea. KM231 exhibited the highest binding avidity to the oligosaccharide and the ganglioside among the four MoAbs. In a comparative study of KM231 and NS19-9, which is a widely used sialyl Lea-reactive MoAb, KM231 bound to the oligosaccharide with higher affinity and detected the antigen in tissue sections with higher sensitivity. In addition, KM231 could detect a small amount of the antigen ganglioside in human gastric normal and cancerous mucosa and in gastric cancer cell lines by HPTLC-immunostaining.

The ganglioside fraction of human gastric mucosa was analyzed with a newly established anti-GM2 monoclonal antibody KM531. Using this antibody, accumulation of GM2 was observed in all of four cases of gastric carcinoma. In all ganglioside fractions extracted from normal gastric mucosa obtained from eight cases of peptic ulcer GM2 itself was not detected, but three kinds of glycolipid showing slower mobility than GM2 on thin-layer plates were detected by immunostaining with KM531. These glycolipids were assigned as NGM-1, -2, and -3. They were completely lost in all carcinoma tissues and in non-cancerous gastric mucosa from two cases of gastric cancer, and they were also not detected in the ganglioside fraction of small or large intestine. Of these glycolipids, the major one, NGM-1, was isolated from the pooled ganglioside fraction of normal gastric mucosa obtained from cases of peptic ulcer. The structure was determined by proton nuclear magnetic resonance, negative ion fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry, and treatment with exoglycosidases and mild acid hydrolysis. The structure was GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4GlcNAc beta 1----3 Gal beta 1----4Glc beta 1----1Cer, which has the same terminal sequence as GM2 but has internal neolacto series structure. This epitope was previously identified as Cad blood group antigen. The decrease of this glycolipid and the increase of GM2 was considered to be a cancer-associated change in gastric mucosa.

The distribution of a variant of blood group A antigen recognized by a murine monoclonal antibody, KM-32, generated against human squamous cell lung carcinoma was investigated in various tissues and sera. By immunoperoxidase staining, the antibody was found to react with a number of lung carcinoma tissues of squamous cell carcinoma, adenocarcinoma, and small cell carcinoma, and several other tumor tissues. Positive staining was also observed in a small number of cells of some normal tissues, such as bronchiolar epithelium, gastrointestinal glands, and convoluted tubules of the kidney. The antibody could also be used in detecting macromolecular antigens, designated KA-32, in sera of patients with lung cancer. The antigen level in serum was determined by an inhibition assay using purified KM-32. The higher level of inhibition was seen in sera from over half of patients with lung cancer and patients with benign diseases when compared with those in sera from healthy adults. Purification of the antigen in serum was performed by gel filtration chromatography, immunoaffinity chromatography, and polyacrylamide gradient gel electrophoresis. Purified antigen exhibited a glycoprotein nature, and its molecular weight was estimated at more than 500,000.

Four murine monoclonal antibodies against human lung carcinoma were generated using a novel immunization procedure. BALB/c mice were rendered neonatally tolerant to normal human lung tissues and subsequently immunized with human lung tumor tissues. The lower level of antibody-reactivity to the tolerogen was seen in the sera of mice rendered neonatally tolerant as compared with the level of reactivity in the sera of nontolerant mice. The mice which maintained a sufficiently tolerant state were selected for hybridoma production. Two monoclonal antibodies, KM-32 and KM-34, were developed from mice immunized with lung squamous cell carcinoma tissues and two other monoclonal antibodies, KM-52 and KM-93, were developed from mice immunized with lung adenocarcinoma tissues. Distribution of antigens detected by the monoclonal antibodies were investigated by enzyme-linked immunosorbent assay using membrane fractions prepared from a number of tumorous and normal tissues and various human normal and tumor cell lines. KM-32 recognized a carbohydrate antigen expressed predominantly on lung squamous cell carcinoma cells and its carbohydrate structure appeared to be associated with blood group A antigen. KM-93 recognized a sialylated carbohydrate epitope on the antigen expressed on lung adeno-carcinoma cells and a few other tumor cells. KM-34 and KM-52 detected protein or glycoprotein antigens and they showed predominant reactivity to lung squamous cell carcinoma and lung adenocarcinoma, respectively. KM-32 and KM-34 antibodies showed complement-dependent cytotoxicity against a lung tumor cell line. These results suggest that the tolerance technique is useful for efficient screening of murine monoclonal antibodies specific to human tumors. The efficacy of these four monoclonal antibodies in diagnosis and therapy of lung cancer will be the subject of a sequential study.