Extracellular acidification augments sclerostin and osteoprotegerin production by Ocy454 mouse osteocytes.

Osteocytes sense the microenvironmental stimuli, including mechanical stress, and regulate bone resorption by osteoclasts and bone formation by osteoblasts. Diabetes and cancer metastasis to bone raise l-lactic acid in the bone tissue, causing acidification. Here, we investigated the effects of l-lactic acid and extracellular acidification on the function of mouse Ocy454 osteocytes. L- and d-lactic acid with low chiral selectivity and acidification of the medium raised the production of sclerostin and osteoprotegerin by Ocy454 cells. The mRNA expression of their genes increased after either treatment of L- and d-lactic acid or acidification of the medium. Furthermore, the conditioned medium of Ocy454 cells cultured in an acidic environment suppressed the induction of alkaline phosphatase activity in MC3T3-E1 cells, which was recovered by the anti-sclerostin antibody. While it is reported that HDAC5 inhibits the transcription of the sclerostin gene, extracellular acidification reduced the nuclear localization of HDAC5 in Ocy454 cells. While calmodulin kinase II (CaMKII) is known to phosphorylate and induce extranuclear translocation of HDAC5, KN-62, an inhibitor of CaMKII lowered the expression of the sclerostin gene in Ocy454 cells. Collectively, extracellular acidification is a microenvironmental factor that modulates osteocyte functions.

Establishment of Down's syndrome periodontal ligament cells by transfection with SV40T-Ag and hTERT.

Down's syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down's syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down's syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down's syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down's syndrome patients.

Risk factors for unsuccessful removal of central venous access ports implanted in the forearm of adult oncologic patients.

PURPOSE: To evaluate the risk factors for unsuccessful removal of a central venous access port (CV port) implanted in the forearm of adult oncologic patients. MATERIALS AND METHODS: This study included 97 adult oncologic patients (51 males, 46 females; age range, 30-88 years; mean age, 63.7 years) in whom removal of a CV port implanted in the forearm was attempted at our hospital between January 2015 and May 2021. Gender, age at removal, body mass index, and diagnosis were examined as patient characteristics; and indwelling period, indwelling side, and indication for removal were examined as factors associated with removal of a CV port. These variables were compared between successful and unsuccessful cases using univariate analysis. Then, multivariate analysis was performed to identify independent risk factors for unsuccessful removal of a CV port using variables with a significant difference in the univariate analysis. A receiver-operating characteristics (ROC) curve was drawn for significant risk factors in the multivariate analysis and the Youden index was used to determine the optimum cut-off value for predicting unsuccessful removal of a CV port. RESULTS: Removal of CV ports was successful in 79 cases (81.4%), but unsuccessful in 18 cases (18.6%) due to fixation of the catheter to the vessel wall. Multivariate logistic regression analysis showed that the indwelling period (odds ratio 1.048; 95% confidence interval 1.026-1.070; P < 0.0001) was a significant independent risk factor for unsuccessful removal of a CV port. ROC analysis showed that the cut-off value for successful removal was 41 months, and 54% of cases with an indwelling period > 60 months had unsuccessful removal. CONCLUSION: The indwelling period is an independent risk factor for unsuccessful removal of a CV port implanted in the forearm of adult oncologic patients, with a cut-off of 41 months.

Phorbol-12-myristate 13-acetate inhibits Nephronectin gene expression via Protein kinase C alpha and c-Jun/c-Fos transcription factors.

Nephronectin (Npnt) is an extracellular matrix protein and ligand of integrin α8ß1 known to promote differentiation of osteoblasts. A search for factors that regulate Npnt gene expression in osteoblasts revealed that phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), had a strong effect to suppress that expression. Research was then conducted to elucidate the signaling pathway responsible for regulation of Npnt gene expression by PMA in osteoblasts. Treatment of MC3T3-E1 cells with PMA suppressed cell differentiation and Npnt gene expression. Effects were noted at a low concentration of PMA, and were time- and dose-dependent. Furthermore, treatment with the PKC signal inhibitor Gö6983 inhibited down-regulation of Npnt expression, while transfection with small interfering RNA (siRNA) of PKCα, c-Jun, and c-Fos suppressed that down-regulation. The present results suggest regulation of Npnt gene expression via the PKCα and c-Jun/c-Fos pathway.

High-throughput N-glycan screening method for therapeutic antibodies using a microchip-based DNA analyzer: a promising methodology for monitoring monoclonal antibody N-glycosylation.

N-Glycosylation of therapeutic antibodies is a critical quality attribute (CQA), and the micro-heterogeneity affects the biological and physicochemical properties of antibodies. Therefore, the profiling of N-glycans on antibodies is essential for controlling the manufacturing process and ensuring the efficacy and safety of the therapeutic antibodies. To monitor N-glycosylation in recombinant proteins, a high-throughput (HTP) methodology for glycan analysis is required to handle bulk samples in various stages of the manufacturing process. In this study, we focused on the HTP methodology for N-glycan analysis using a commercial microchip electrophoresis-based DNA analyzer and demonstrated the feasibility of the workflow consisting of sample preparation and electrophoretic separation. Even if there is a demand to analyze up to 96 samples, the present workflow can be completed in a day without expensive instruments and reagent kits for sample preparation, and it will be a promising methodology for cost-effective and facile HTP N-glycosylation analysis while optimizing the manufacturing process and development for therapeutic antibodies.

Ultraselective transcatheter arterial embolization with small-sized microcoils for acute lower gastrointestinal bleeding.

PURPOSE: To evaluate the clinical outcome of ultraselective transcatheter arterial embolization (TAE) with small-sized microcoils for acute lower gastrointestinal bleeding (LGIB). MATERIALS AND METHODS: The subjects were 17 consecutive patients (mean age, 69 years) with LGIB who were treated with ultraselective TAE using small-sized microcoils between December 2013 and December 2019. Ultraselective TAE was defined as embolization of one or both of the long or short branches of the vasa recta. The etiologies of bleeding were colonic diverticulosis in 16 patients (94%) and malignancy in one patient (6%). The bleeding foci were in the ascending colon in 11 patients (65%), transverse colon in 2 patients (12%), and sigmoid colon in 4 patients (23%). A total of 18 branches (diameter: range 0.5-1.5 mm, mean 1.1 mm) of the vasa recta in 17 patients were embolized with small-sized microcoils (size range 1-3 mm, mean combined lengths of all microcoils 7.6 cm). The mean follow-up period was 19 months (range 1-80 months). The technical and clinical success rate, recurrent bleeding rate, major complications and long-term clinical outcomes were retrospectively evaluated. RESULTS: Technical and clinical success was achieved in all patients (17/17). The rates of early recurrent bleeding (within 30 days of TAE) and major complications were 0% (0/17). Recurrent bleeding occurred in one patient at 2 months after TAE, but was stopped with conservative treatment. There were no other bleeding episodes or complications in the follow-up period. CONCLUSION: Ultraselective TAE with small-sized microcoils is a highly effective and safe treatment modality for LGIB.

Zoledronate promotes inflammatory cytokine expression in human CD14-positive monocytes among peripheral mononuclear cells in the presence of γδ T cells.

Bisphosphonates distributed to bone exert toxic effects specifically towards osteoclasts. On the other hand, intravenous administration of a nitrogen-containing bisphosphonate (N-BP) such as zoledronate induces acute-phase reactions (APRs), including influenza-like fever 1 day later, indicating an interaction with immunocompetent cells circulating blood. Although it has been reported that activation of γδ T cells is pivotal to induce an APR following treatment with zoledronate, downstream events, including the production of inflammatory cytokines after activation of γδ T cells, remain obscure. We investigated the effects of zoledronate on inflammatory cytokine expression in human peripheral blood mononuclear cells (PBMCs) in vitro. While zoledronate induced mRNA expressions of tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 and interferon-γ (IFN-γ) in PBMC, depletion of γδ T cells abolished that zoledronate-induced expression of those cytokines, indicating the necessity of γδ T cells for expression induction by zoledronate. However, which types of cells were responsible for the production of those cytokines in blood remained unclear. As it is generally accepted that monocytes and macrophages are primary sources of inflammatory cytokines, CD14+ cells from PBMC were exposed to zoledronate in the presence of PBMC, which resulted in induced expression of mRNAs for IL-1ß, IL-6 and IFN-γ, but not for TNF-α. These results indicate that CD14+ cells are responsible, at least in part, for the production of IL-1ß, IL-6 and IFN-γ in blood exposed to zoledronate. This suggests that CD14+ cells play an essential role in the occurrence of APRs following N-BP administration.

Analysis of Minor Acidic N-Glycans in Human Serum.

Prior investigations by our research group focused on the method development for the simultaneous analysis of sulfated and phosphorylated glycans. Herein, the developed method was applied to analyze minor acidic N-glycans including sulfated and phosphorylated N-glycans in human serum. First, 2-aminobenzoic acid-labeled minor acidic N-glycans were enriched from the serum using a serotonin-immobilized column and were then separated into groups using hydrophilic interaction liquid chromatography, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phosphorylated hybrid-type and sulfated bi-antennary N-glycans were detected in the serum. In addition, we observed that multiple types of glucuronidated N-glycans were present. These results indicate that the developed method is applicable to the analysis of glucuronidated as well as sulfated and phosphorylated N-glycans. It was also applied to the sera obtained from 17 healthy subjects and 15 pancreatic cancer patients, and the profiles of sulfated, phosphorylated, and glucuronidated N-glycans were compared. The expressed amount of glucuronidated N-glycans was significantly decreased in some pancreatic cancer patients. Numerous examples of the N-glycan analysis in human serum were reported, but phosphorylated and glucuronidated glycans were not investigated. The methods described herein allow the analysis of minor acidic glycans that are typically difficult to detect.

Preliminary clinical assessment of dynamic 18F-fluorodeoxyglucose positron emission tomography/computed tomography for evaluating lymph node metastasis in patients with lung cancer: a prospective study.

OBJECTIVE: We assessed the diagnostic capacity of dynamic fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) and dual-time-point (DTP) PET/CT to explore the optimal scan timing for nodal staging in lung cancer. METHODS: Thirty-four patients with lung cancer underwent dynamic and consecutive DTP PET/CT scans. Two readers visually evaluated FDG uptake within each lymph node (LN) and pulmonary artery (metastatic LN: n = 10; nonmetastatic LN: n = 121). For each dynamic and DTP scan, we compared the maximum standardized uptake value (SUVmax) and the retention index of the SUVmax (RI-SUVmax) between metastatic and nonmetastatic LNs. We compared the diagnostic capacity of the dynamic and DTP scans using receiver operating characteristic (ROC) analyses. RESULTS: In the visual analyses of LN metastases, a sensitivity of 20.0-60.0% and specificity of 97.5-100.0% were identified for the first to third dynamic scans. The sensitivity of the 1-h early and 2-h delayed scans was 80.0% and 90.0%, respectively, whereas the specificity was 66.9% and 47.9%, respectively. The visual analysis of the dynamic second phase had the highest accuracy. Semiquantitative analyses revealed that the SUVmax was significantly higher for metastatic LNs than for nonmetastatic LNs in the dynamic second and third phases and the 1-h early and 2-h delayed phases (p < 0.05 for all). The RI-SUVmax was higher in metastatic LNs than in nonmetastatic LNs for the dynamic scan (p = 0.004) and the DTP scan (p = 0.002). The ROC analyses showed that SUV2 and SUV3 had higher performances with high specificity, high negative predictive value, and high accuracy than the other parameters. The area under the ROC curve of the RI-SUV-dual-time-point had the highest value (0.794) without any significant differences between the area under the ROC curves for all parameters (p > 0.05 for all). CONCLUSIONS: Based on the visual and semiquantitative analyses, 18F-FDG dynamic PET/CT exhibited excellent performance with extremely high specificity in the dynamic second phase.

Utility of the Virtual Liver Parenchymal Perfusion Area Using a Commercially Available Workstation in Transarterial Chemoembolization for Hepatocellular Carcinoma.

PURPOSE: To evaluate the accuracy of the virtual liver parenchymal perfusion area using a commercially available workstation and liver analysis software in conventional transarterial chemoembolization (cTACE) for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This method was retrospectively applied to 29 treated HCCs in 23 patients. The virtual embolic area (VEA) was estimated based on cone beam computed tomography during hepatic arteriography using a commercially available workstation and liver analysis software by two observer groups (group A: experts; group B: semi-experts). The real embolic area (REA) was defined as the area where iodized oil accumulated on computed tomography at 1 week after cTACE. The REA was estimated by each of the two groups, and the mean REA between the groups (mREA) was used as a standard reference. Agreement of volume and cross-sectional area in three orthogonal planes between the VEA and mREA were analyzed using intraclass correlation coefficients (ICCs) and Bland-Altman plots. RESULTS: The ICCs for volume between VEA and mREA were 0.97 and 0.88 for groups A and B, respectively, and those for cross-sectional area were 0.94 and 0.88 for the axial plane, 0.95 and 0.83 for the coronal plane, and 0.87 and 0.74 for the sagittal plane, respectively. Thus, the overall agreement was excellent, except for the sagittal imaging plane in group B. CONCLUSION: This method using a commercially available workstation and liver analysis software can be useful for estimating the embolic area in cTACE.

Simultaneous Analysis of Sulfated and Phosphorylated Glycans by Serotonin-Immobilized Column Enrichment and Hydrophilic Interaction Chromatography.

Changes in the structures and quantities of sulfated glycans play important roles in inflammatory and neurological diseases and cancer. Therefore, sulfated glycans are expected to become diagnostic markers for a variety of diseases such as Alzheimer's disease and cancer. On the other hand, structural abnormalities in the phosphorylated glycans on lysosomal enzymes cause a number of lysosomal diseases, while novel phosphorylated glycans have been found in other proteins. As with sulfated glycans, structural and quantitative changes in these phosphorylated glycans and their associations with disease are also of interest. In this article, we introduce a new method for the simultaneous analysis of sulfated and phosphorylated glycans. We first employ a serotonin-immobilized column to enrich these glycans. Glycans obtained in this manner were sequentially subjected to other analytical techniques without desalting. We employed hydrophilic interaction chromatography to distinguish the sulfate and phosphate groups of the glycans and were able to analyze sulfated and phosphorylated N-glycans expressed on thyroglobulin, ovalbumin, and myozyme. We showed that our method not only analyzes sulfated and phosphorylated glycans, but also glycans containing the GlcNAc-HPO3 residue. We comparatively analyzed sulfated glycans, phosphorylated glycans, and GlcNAc-HPO3-residue-containing glycans expressed on MKN7 cells (well-differentiated gastric cancer cells) and MKN45 cells (poorly differentiated gastric cancer cells). To the best of our knowledge, this is the first report of a method for the simultaneous and quantitative analysis of sulfated and phosphorylated glycans.

Microchip electrophoresis utilizing an in situ photopolymerized Phos-tag binding polyacrylamide gel for specific entrapment and analysis of phosphorylated compounds.

A method was developed for the specific entrapment and separation of phosphorylated compounds using a Phos-tag polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate)-made microchip was filled with a solution comprising acrylamide, N,N-methylene-bis-acrylamide, Phos-tag acrylamide, and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with a UV LED laser beam. The fabricated Phos-tag gel (100 × 100 × 30 µm) contains ca. 20 fmol of the Phos-tag group and therefore could entrap phosphorylated compounds at the femtomolar level. The electrophoretically trapped phosphorylated compounds were released from the gel by switching the voltage to deliver high concentrations of phosphate and EDTA in a background electrolyte. The broad sample band eluted from the gel was effectively reconcentrated at the boundary of a pH junction generated by sodium ions delivered from the outlet reservoir. The reconcentrated sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the phosphorylated compounds were concentrated by a factor of 100-fold, and the peak resolution was comparable to that obtained by pinched injection. This method was successfully utilized to preconcentrate and analyze phosphorylated peptides in a complex peptide mixture.

The usefulness of cone-beam computed tomography during chemoembolization of hepatocellular carcinomas fed exclusively by the cystic artery.

PURPOSE: To evaluate the usefulness of cone-beam computed tomography (CBCT) for monitoring the transcatheter arterial chemoembolization (TACE) of hepatocellular carcinomas supplied by the cystic artery. MATERIALS AND METHODS: In seven tumors (mean diameter: 19 mm), the iodized oil distributions in the tumor and gallbladder wall were evaluated by CBCT after injecting iodized oil emulsion (LipCBCT) through the cystic artery. Gelatin sponge particles were injected to completely obstruct the tumor-feeding vessel when iodized oil deposition was seen in less than one third of the wall circumference. The following parameters were retrospectively investigated: (1) the iodized oil distribution during LipCBCT and on CT scans 1 week after TACE; (2) local tumor control; and (3) complications. RESULTS: LipCBCT showed iodized oil accumulation throughout the entire tumor in all cases, and iodized oil deposition in the gallbladder wall in three cases (43 %) (less than one third of the circumference). Therefore, gelatin sponge particles were used in all cases. CT 1 week after TACE showed an almost identical iodized oil distribution to intraoperative LipCBCT. None of the tumors recurred during follow-up, and no complications occurred. CONCLUSION: CBCT is useful for evaluating the distribution of iodized oil in TACE via the cystic artery.

Three-dimensional CT for preoperative detection of the left gastric artery and left gastric vein in laparoscopy-assisted distal gastrectomy.

INTRODUCTION: We evaluated 3-D CT imaging for preoperative classification of the left gastric artery and vein in patients with early gastric cancer and estimated its clinical benefit. METHODS: Between April 2009 and March 2014, 279 patients underwent preoperative 3-D CT using a 64-row multi-detector CT scanner, followed by laparoscopy-assisted distal gastrectomy. The 3-D CT images of the arterial and portal phases were reconstructed and fused. The operative outcomes were compared between patients who had not undergone 3-D CT (2007-2008) and who had undergone 3-D CT (2009-2011). RESULTS: According to Adachi's classification, the numbers of type I, II, III, IV, V, and VI arterial patterns were 253, 15, 1, 3, 3, and 1, respectively. Three cases could not be classified. According to the Douglass classification, the left gastric vein flowed into the portal vein, splenic vein, junction of the portal vein and splenic vein, and left branch of the portal vein in 119, 111, 36, and 5 patients, respectively. The left gastric vein could not be visualized in six patients, and two patients could not be classified. In addition, the relation was absent for an Adachi type I vein and one of the "other" types of veins. The total operative time was significantly shorter with 3-D CT than without it (P = 0.01), and the degree of lymph-node dissection was significantly higher (P = 0.01). Inflammatory parameters and operative morbidity tended to decrease with 3-D CT. CONCLUSION: Three-dimensional CT is a useful modality to visualize the vessel anatomy around the stomach, and it improves clinical effectiveness and reduces the invasiveness of surgery.

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines : HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.

[Structural analysis of carboxymethyl cellulose used as an antiadhesive material for surgical wound healing].

Carboxymethyl cellulose (CMC) is one of the most important cellulose derivatives and used in the fields of food, pharmaceuticals, cosmetics, and paint. Fibrous CMC is used an antiadhesive material to prevent postoperative wound adhesions. The degree of substitution and distribution of the substituent (i.e., the carboxymethyl group) are the most important parameters for the function of CMC. Thus, CMC used for antiadhesive material must be carefully evaluated, because the CMC product is retained in patients' bodies over the long term. Although identification tests of CMC are defined in the Japanese Pharmacopoeia, it is difficult to evaluate its structure using those tests. In the present study, we propose improved methods for evaluating CMC products by analyzing monosaccharides after hydrolysis.

Expression of the clustered NeuAcα2-3Galß O-glycan determines the cell differentiation state of the cells.

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of >239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAcα2-3Galß O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1-60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1-60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.

Common glycoproteins expressing polylactosamine-type glycans on matched patient primary and metastatic melanoma cells show different glycan profiles.

Recently, we reported comparative analysis of glycoproteins which express cancer-specific N-glycans on various cancer cells and identified 24 glycoproteins having polylactosamine (polyLacNAc)-type N-glycans that are abundantly present in malignant cells [ Mitsui et al., J. Pharm. Biomed. Anal. 2012 , 70 , 718 - 726 ]. In the present study, we applied the technique to comparative studies on common glycoproteins present in the matched patient primary and metastatic melanoma cell lines. Metastatic melanoma cells (WM266-4) contained a large amount of polyLacNAc-type N-glycans in comparison with primary melanoma cells (WM115). To identify the glycoproteins expressing these N-glycans, glycopeptides having polyLacNAc-type N-glycans were captured by a Datura stramonium agglutinin (DSA)-immobilized agarose column. The captured glycopeptides were analyzed by LC/MS after removing N-glycans, and some glycoproteins such as basigin, lysosome-associated membrane protein-1 (LAMP-1), and chondroitin sulfate proteoglycan 4 (CSPG4) were identified in both WM115 and WM266-4 cells. The expression level of polyLacNAc of CSPG4 in WM266-4 cells was significantly higher than that in WM115 cells. In addition, sulfation patterns of chondroitin sulfate (CS) chains in CSPG4 showed dramatic changes between these cell lines. These data show that characteristic glycans attached to common proteins observed in different stages of cancer cells will be useful markers for determining degree of malignancies of tumor cells.

Comparative studies on glycoproteins expressing polylactosamine-type N-glycans in cancer cells.

In the series of our previous reports, we showed that some cancer cell lines specifically express polylactosamine-type N-glycans and such glycans were often modified with fucose and sulfate residues. To confirm the proteins expressing these glycans, glycopeptide mixture obtained by digestion of whole protein fractions with trypsin was captured by a polylactosamine-specific lectin, Datura strasmonium agglutinin (DSA). And the peptides and glycans of the captured glycopeptides after digestion with N-glycoamidase F were extensively analyzed by HPLC and MS techniques. We found that some glycoproteins such as CD107a and CD107b commonly contained polylactosamine-type glycans in all the examined cancer cells. But integrin-α5 (CD49e) and carcinoembryonic antigen-related cell adhesion molecule 5 (CD66e) having these glycans were specifically found in U937 (human T-lymphoma) and MKN45 (human gastric cancer) cells, respectively. These data clearly indicate that specific glycans attached to specific proteins will be promising markers for specific tumors with high accuracy.