The role of B3GNT3 as an oncogene in the growth, invasion and migration of esophageal cancer cells

Purpose: To investigate the role and mechanism of ß1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) in esophageal cancer (ESCA). Methods: The starBase database was used to evaluate the expression of B3GNT3. B3GNT3 function was measured using KYSE-30 and KYSE-410 cells of esophageal squamous cell carcinoma (ESCC) cell lines. The mRNA levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8, clone formation assay and transwell assay were used to detect the changes of proliferation, invasion and migration. Results: B3GNT3 expression was higher in ESCA tissues than in normal tissues. The overall survival rate of ESCA patients with high B3GNT3 expression was lower than that of ESCA patients with low B3GNT3 expression. In vitro functional experiments showed that the proliferation ability, migration and invasion ability of KYSE-30 and KYSE-410 cells with B3GNT3 interference were lower than those of the control, and the overexpression of B3GNT3 had the opposite effect. After silencing B3GNT3 expression in ESCC cell lines, the growth of both cell lines was inhibited and the invasiveness was decreased. Knockdown of B3GNT3 reduced the growth rate and Ki-67 expression level. Conclusion: B3GNT3, as an oncogene, may promote the growth, invasion and migration of ESCC cell.

Immunopathological analysis of the expression of glomerular exostosin 1 and exostosin 2 in Japanese patients with lupus nephritis.

Exostosin 1 and exostosin 2 (EXT1/EXT2) on glomerular basement membrane (GBM) were recently reported as novel putative antigens in secondary membranous nephropathy with autoimmune disease. However, the clinical significance of glomerular EXT1/EXT2 remains elusive in patients with lupus nephritis (LN). The immunofluorescence staining pattern of glomerular EXT1/EXT2 is also undetermined in membranous LN (MLN) or proliferative LN (PLN). We cross-sectionally analyzed patients with MLN (pure class V, n = 11) and PLN (class III, IV, and mixed class III/IV + V, n = 22) who underwent renal biopsies between 2010 and 2020 at Showa University Hospital. Glomerular EXT1/EXT2 expressions were evaluated by immunofluorescence. T-helper (Th) cell-related serum inflammatory cytokines were measured using enzyme-linked immunosorbent assay. The positivity for both EXT1/EXT2 was higher in patients with MLN than PLN (90.9% vs 63.6%, P = 0.212). MLN showed global and bright granular EXT1/EXT2 expressions along GBM, while PLN showed segmental and moderate expressions on GBM. Additionally, glomerular EXT1/EXT2 positivity was not associated with the degree of proteinuria or renal function in MLN and PLN patients, but the levels of serum anti-dsDNA antibody and circulating immune complexes were lower in patients with EXT1/EXT2-positive MLN than EXT1/EXT2-negative PLN. Moreover, serum complement levels and IL-4/IFN-γ ratios were elevated in EXT1/EXT2-positive MLN than EXT1/EXT2-negative PLN. Collectively, immunofluorescence staining for glomerular EXT1/EXT2 had characteristic patterns between MLN and PLN. Glomerular EXT1/EXT2 expressions tended to be high in Th2-dominant MLN patients without severe hypocomplementemia and elevated autoantibodies. Thus, EXT1/EXT2 might be involved in the unique developmental mechanism of MLN.

REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway.

Persistent acinar to ductal metaplasia (ADM) is a recently recognized precursor of pancreatic ductal adenocarcinoma (PDAC). Here we show that the ADM area of human pancreas tissue adjacent to PDAC expresses significantly higher levels of regenerating protein 3A (REG3A). Exogenous REG3A and its mouse homolog REG3B induce ADM in the 3D culture of primary human and murine acinar cells, respectively. Both Reg3b transgenic mice and REG3B-treated mice with caerulein-induced pancreatitis develop and sustain ADM. Two out of five Reg3b transgenic mice with caerulein-induced pancreatitis show progression from ADM to pancreatic intraepithelial neoplasia (PanIN). Both in vitro and in vivo ADM models demonstrate activation of the RAS-RAF-MEK-ERK signaling pathway. Exostosin-like glycosyltransferase 3 (EXTL3) functions as the receptor for REG3B and mediates the activation of downstream signaling proteins. Our data indicates that REG3A/REG3B promotes persistent ADM through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway. Targeting REG3A/REG3B, its receptor EXTL3, or other downstream molecules could interrupt the ADM process and prevent early PDAC carcinogenesis.

Mutational spectrum and clinical signatures in 114 families with hereditary multiple osteochondromas: insights into molecular properties of selected exostosin variants.

Hereditary multiple osteochondromas (HMO) is a rare autosomal dominant skeletal disorder, caused by heterozygous variants in either EXT1 or EXT2, which encode proteins involved in the biogenesis of heparan sulphate. Pathogenesis and genotype-phenotype correlations remain poorly understood. We studied 114 HMO families (158 affected individuals) with causative EXT1 or EXT2 variants identified by Sanger sequencing, or multiplex ligation-dependent probe amplification and qPCR. Eighty-seven disease-causative variants (55 novel and 32 known) were identified including frameshift (42%), nonsense (32%), missense (11%), splicing (10%) variants and genomic rearrangements (5%). Informative clinical features were available for 42 EXT1 and 27 EXT2 subjects. Osteochondromas were more frequent in EXT1 as compared to EXT2 patients. Anatomical distribution of lesions showed significant differences based on causative gene. Microscopy analysis for selected EXT1 and EXT2 variants verified that EXT1 and EXT2 mutants failed to co-localize each other and loss Golgi localization by surrounding the nucleus and/or assuming a diffuse intracellular distribution. In a cell viability study, cells expressing EXT1 and EXT2 mutants proliferated more slowly than cells expressing wild-type proteins. This confirms the physiological relevance of EXT1 and EXT2 Golgi co-localization and the key role of these proteins in the cell cycle. Taken together, our data expand genotype-phenotype correlations, offer further insights in the pathogenesis of HMO and open the path to future therapies.

N-Acetylglucosaminyltransferase III (GnT-III) but not N-Acetylgalactosaminyltransferase-6 and 8 are Differentially Expressed in Invasive and In Situ Ductal Carcinoma of the Breast.

Mammary carcinoma is the most common malignant tumor in women, and it is the leading cause of mortality. In tumor context, glycosylation promotes post translational modifications necessary for cell progression, emerging as a relevant tumor hallmarker. This study aimed to analyze the association between polypeptide N-acetylgalactosaminyltransferase-6 (ppGalNAc-T6), -T8, N-acetylglucosaminyltransferase III (GnT-III) expression, Phaseolus vulgaris-leucoagglutinin (PHA-L), wheat germ agglutinin (WGA) and peanut agglutinin (PNA) staining with clinic-histopathological factors from patients with pure ductal carcinoma in situ (DCIS) and DCIS with invasive ductal carcinoma (DCIS-IDC) of breast. Formalin-fixed and paraffin-embedded samples (n = 109) were analyzed. In pure DCIS samples GnT-III was over-expressed in comedo lesions (p = 0.007). In DCIS-IDC, GnT-III expression was associated with high nuclear grade tumors (p = 0.039) while the presence of PHA-L and WGA were inversely related to HER-2 expression (p = 0.001; p = 0.036, respectively). These findings pointed to possible involvement of GnT-III, ppGalNAc-T8, L-PHA and WGA as probes in prognostic evaluation of DCIS.

B3GNT3 overexpression is associated with unfavourable survival in non-small cell lung cancer.

OBJECTIVE: The aim of this study was to evaluate the expression of beta-1,3-N-acetylglucosaminyltransferase-3 (B3GNT3) in non-small cell lung cancer (NSCLC) patients and to investigate the relevance of B3GNT3 expression in tumour prognosis. METHODS: In this study, B3GNT3 expression was examined in five pairs of resectable NSCLC tissue by Western blot and in 42 pairs of resectable NSCLC tissue by quantitative real-time PCR (qRT-PCR). Immunohistochemistry and statistical analysis were performed to assess the relationship between B3GNT3 expression scores and clinicopathological parameters, as well as clinical prognosis in a retrospective cohort of 176 NSCLC patients. RESULTS: Both B3GNT3 mRNA and protein expression levels were significantly higher in NSCLC tissue than in adjacent normal tissue. In the 176 NSCLC cases, a high B3GNT3 expression level was positively correlated with lymph node metastasis (P<0.001) and advanced TNM stage (P=0.043). Kaplan-Meier analysis indicated that patients with high B3GNT3 expression had significantly lower disease-free survival (DFS) (P<0.001) and overall survival (OS) (P<0.001) than those with low B3GNT3 expression. Moreover, in the multivariate analyses, B3GNT3 expression was an independent prognostic factor for DFS (HR 0.329, 95% CI 0.213 to 0.508, P<0.001) and OS (HR 0.383, 95% CI 0.249 to 0.588, P<0.001). CONCLUSIONS: Our study demonstrated that high expression of B3GNT3 was associated with unfavourable DFS and OS in NSCLC patients, suggesting that B3GNT3 might be a potential prognostic biomarker for NSCLC.

Single Quantum Dot-Based Nanosensor for Sensitive Detection of O-GlcNAc Transferase Activity.

Protein glycosylation is a ubiquitous post-translational modification that plays crucial roles in modulating biological recognition events in development and physiology. Human O-GlcNAc transferase (OGT) is an intracellular enzyme responsible for O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, and the deregulation of OGT activity occurs in cancer, diabetes, and neurodegenerative disease. Here we develop a single quantum dot (QD)-based nanosensor for sensitive OGT assay. We design a Cy5/biotin-modified peptide with a serine hydroxyl group for sensing OGT and a protease site adjacent to the glycosylation site for proteinase cleavage, with a universal nonradioactive UDP-GlcNAc as the sugar donor and a Cy5/biotin-modified peptide as the substrate. In the presence of OGT, it catalyzes the glycosylation reaction to generate a glycosylated peptide that is a protease-protection peptide. The resultant glycosylated Cy5/biotin-modified peptides may assemble on the surface of the streptavidin-coated QD to obtain a QD-peptide-Cy5 nanostructure in which the fluorescence resonance energy transfer (FRET) from the QD to Cy5 can occur, leading to the emission of Cy5 which can be quantified by single-molecule detection. This method exhibits high sensitivity with a limit of detection of 3.47 × 10-13 M, and it is very simple and straightforward without the involvement of any enzyme purification, radioisotope-labeled sugar donors, specific antibodies, and the synthesis of fluorescent UDP-GlcNAc analogues. Moreover, this method can be used for enzyme kinetic analysis, quantitative detection of cellular OGT activity, and the screening of OGT inhibitors, holding great potential for further application in drug discovery and clinical diagnosis.

In Vitro Biochemical Assays for O-GlcNAc-Processing Enzymes.

O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are the only enzymes that regulate the dynamics of protein O-GlcNAcylation. Protein O-GlcNAcylation is an important post-translational modification (PTM) of nuclear and cytoplasmic proteins with O-linked ß-N-acetyl-glucosamine (O-GlcNAc). O-GlcNAc and its enzymes are involved in a wide variety of cellular processes and are linked to the pathological progression of chronic diseases. Considering their emerging biological significance, systematic and rapid methods to determine the activities of OGT and OGA have become essential, and several chemical/biochemical methods for measuring the activities of these enzymes have been developed. This minireview mainly focuses on the various biochemical assay methods developed to date, while also providing a description of the fundamental principles underlying the monitoring of O-GlcNAc enzyme activities.

Chondroitin sulfate synthase 1 expression is associated with malignant potential of soft tissue sarcomas with myxoid substance.

The glycosyltransferases chondroitin sulfate synthase 1 (CHSY1) and exostoses-like 3 (EXTL3) specifically function in biosynthesis of the glycans chondroitin sulfate and heparan sulfate, respectively. Although these glycans play important roles in pathogenesis of various tumors, their significance in soft tissue sarcoma remains unknown. Here, we asked whether CHSY1 or EXTL3 expression correlates with malignant potential of soft tissue sarcomas with myxoid substance. To do so, we examined 40 samples representing specific types, including 12 cases of myxoid liposarcoma, 14 of myxofibrosarcoma, 12 of malignant peripheral nerve sheath tumor, and 2 of low-grade fibromyxoid sarcoma. We performed immunohistochemistry with anti-CHSY1 and anti-EXTL3 antibodies and compared enzyme expression levels with tumor histologic grade as assessed by the Fédération Nationale des Centres de Lutte Contre le Cancer classification and with patient 5-year survival rate. CHSY1 and EXTL3 were expressed in 72.5% and 32.5% of all tumors, respectively. Notably, CHSY1 was strongly expressed in myxofibrosarcoma and malignant peripheral nerve sheath tumor compared with other tumors and significantly associated with higher- rather than lower-grade tumors (P < .01). High expression of CHSY1 was also significantly associated with poorer patient outcomes (P = .031) and higher stages assessed by American Joint Committee on Cancer staging system (P = .004). By contrast, EXTL3 expression was not correlated with histologic grade or patient prognosis. We conclude that CHSY1 expression is closely associated with malignant potential of soft tissue sarcomas with myxoid substance.

Detection of Core2 ß-1,6-N-Acetylglucosaminyltransferase in Post-Digital Rectal Examination Urine Is a Reliable Indicator for Extracapsular Extension of Prostate Cancer.

To identify appropriate candidates for aggressive treatment such as radical prostatectomy or radiation therapy of localized prostate cancer (PCa), novel predictive biomarkers of PCa aggressiveness are essential. Core2 ß-1,6-N-acetylglucosaminyltransferase-1 (GCNT1) is a key enzyme that forms core 2-branched O-glycans. Its expression is associated with the progression of several cancers. We established a mouse IgG monoclonal antibody (mAb) against GCNT1 and examined the relationship of GCNT1 expression to the clinicopathological status of PCa. Paraffin-embedded PCa specimens were analyzed by immunohistochemistry for GCNT1 expression using a newly established mouse anti-GCNT1 mAb by ourselves. GCNT1-positive tumor showed significantly higher Gleason score and larger tumor volume. The number of GCNT1-positive cases was significantly lower in cases of organ-confined disease than in cases of extracapsular extension. GCNT1-negative tumors were associated with significantly better prostate-specific antigen (PSA)-free survival compared with GCNT1-positive tumors. Multivariate analysis revealed that detection of GCNT1 expression was an independent risk factor for PSA recurrence. We established new methods for GCNT1 detection from PCa specimens. Immunoblotting was used to examine post-digital rectal examination (DRE) urine from PCa patients. Over 90% of GCNT1-positive PCa patients with high concentrations of PSA showed extracapsular extension. In conclusion, GCNT1 expression closely associates with the aggressive potential of PCa. Further research aims to develop GCNT1 detection in post-DRE urine as a marker for PCa aggressiveness.

Gene and protein expression of O-GlcNAc-cycling enzymes in human laryngeal cancer.

Aberrant protein O-GlcNAcylation may contribute to the development and malignant behavior of many cancers. This modification is controlled by O-linked ß-N-acetylglucosamine transferase (OGT) and O-GlcNAcase (OGA). The aim of this study was to determine the expression of O-GlcNAc cycling enzymes mRNA/protein and to investigate their relationship with clinicopathological parameters in laryngeal cancer. The mRNA levels of OGT and MGEA5 genes were determined in 106 squamous cell laryngeal cancer (SCLC) cases and 73 non-cancerous adjacent laryngeal mucosa (NCLM) controls using quantitative real-time PCR. The level of OGT and OGA proteins was analyzed by Western blot. A positive expression of OGT and MGEA5 transcripts and OGT and OGA proteins was confirmed in 75.5 and 68.9 % and in 43.7 and 59.4 % samples of SCLC, respectively. Higher levels of mRNA/protein for both OGT and OGA as well as significant increases of 60 % in total protein O-GlcNAcylation levels were noted in SCLC compared with NCLM (p < 0.05). As a result, an increased level of OGT and MGEA5 mRNA was related to larger tumor size, nodal metastases, higher grade and tumor behavior according to TFG scale, as well as incidence of disease recurrence (p < 0.05). An inverse association between OGT and MGEA5 transcripts was determined with regard to prognosis (p < 0.05). In addition, the highest OGT and OGA protein levels were observed in poorly differentiated tumors (p < 0.05). No correlations with other parameters were noted, but the results showed a trend of more advanced tumors to be more frequently OGT and OGA positive. The results suggest that increased O-GlcNAcylation may have an effect on tumor aggressiveness and prognosis in laryngeal cancer.

Lectin histochemistry reveals SNA as a prognostic carbohydrate-dependent probe for invasive ductal carcinoma of the breast: a clinicopathological and immunohistochemical auxiliary tool.

Increased sialylation and ß1,6-branched oligosaccharides has been associated with a variety of structural changes in cell surface carbohydrates, most notably in tumorigenesis. Lectins are defined as proteins that preferentially recognize and bind carbohydrate complexes protruding from glycolipids and glycoproteins. This interaction with carbohydrates can be as specific as the interaction between antigen and antibody. Due to this type of interaction lectins have been used as experimental auxiliary tools in histopathological diagnosis of cancer. This study was designed to evaluate the differential expression of sialic acids and ß1,6-N-acetylglucosaminyltransferase V (MGAT5) in invasive (IDC) and in situ (DCIS) ductal carcinoma of the breast and its possible application as prognostic biomarkers. A possible transition between pre-malign and malign lesions was evaluated using DCIS samples. Biopsies were analyzed regarding the expression of MUC1, p53, Ki-67, estrogen receptor, progesterone receptor, HER-2 and MGAT5. α2,6-linked sialic acids residues recognized by SNA lectin was overexpressed in 33.3% of IDC samples and it was related with Ki-67 (p=0.042), PR (p=0.029), lymphnodes status (p=0.017) and death (p=0.011). Regarding survival analysis SNA was the only lectin able to correlate with specific-disease survival and disease-free survival (p=0.024 and p=0.041, respectively), besides, it presents itself as an independent variable by Cox Regression analysis (p= 0.004). Comparing IDC and DCIS cases, only SNA showed different staining pattern (p=0.034). The presence of sialic acids on tumor cell surface can be an indicative of poor prognosis and our study provides further evidence that SNA lectin can be used as a prognostic probe in IDC and DCIS patients.

Correlation of deregulated like-acetylglucosaminyl transferase and aberrant α-dystroglycan expression with human tongue cancer metastasis.

PURPOSE: The present study examined the correlation of α-dystroglycan (α-DG) expression and like-acetylglucosaminyl transferase (LARGE) with metastasis of human tongue cancer. MATERIALS AND METHODS: Fifty human tongue cancer tissues and 2 tongue squamous cell carcinoma cell lines (CAL27 and SCC4) were involved. Immunohistochemistry was used to detect the expression of α-DG and LARGE. Methylation-specific polymerase chain reaction was performed to assess the methylation status of the LARGE gene promoter. CAL27 and SCC4 cells were transfected with exogenous LARGE and treated with 5-aza-2'-deoxycytidine (Aza-dC), respectively. Glycol sites of α-DG were detected by western blotting. In addition, the laminin overlay assay, cell adhesion assay, and invasion assay were performed. RESULTS: Immunohistochemical results showed that decreased expression of VIA4-1 and IIH6 (antibodies that recognize the glycol sites of α-DG) were correlated with the lymph node metastasis of tongue cancer (n = 50; P = .016 and .025, respectively). Decreased LARGE expression and hypermethylation of the LARGE gene promoter were correlated with lymph node metastasis and α-DG glycosylation in human tongue cancer (n = 50; P = .043 and .015 respectively). In addition, LARGE overexpression and Aza-dC treatment actively led to restoration of functional α-DG expression, elevation of laminin binding, and decrease of migratory ability in cancer cells. CONCLUSION: The results suggested that absent α-DG expression and LARGE deregulation were closely associated with nodal metastasis of tongue cancer. Aberrant α-DG expression and glycosylation were attributed at least in part to the abnormal epigenetic modification of LARGE, especially the hypermethylation of its promoter.

Expression of N-acetylglucosaminyltransferase V in the subserosal layer correlates with postsurgical survival of pathological tumor stage 2 carcinoma of the gallbladder.

BACKGROUND: N-Acetylglucosaminyltransferase V (GnT-V), an enzyme that catalyzes the ß1-6 branching of N-acetylglucosamine on asparagine-linked oligosaccharides of cellular proteins, enhances the malignant behaviors of carcinoma cells in experimental models. The aim of this study was to determine clinical significance of GnT-V expression in human pT2 gallbladder carcinoma with simple in vitro experiments. METHODS: Ninety patients with pT2 gallbladder carcinoma were included for this study. The in vitro and in vivo biological effects of GnT-V were investigated using gallbladder carcinoma cells with variable GnT-V expression levels induced by a small interfering RNA. RESULTS: Of the 90 cases, 57 showed positive staining and the remaining 33 demonstrated negative staining, the subcellular localization in the 57 cases was classified into the granular-type in 31 cases and the diffuse-type in 26 cases. In 76 cases with curative resection, postsurgical survival was significantly poorer in those showing positive staining than in those showing negative staining (P = 0.028). In all of the 76 cases, postsurgical recurrence was significantly more frequent in those showing diffuse-type localization than in those showing negative staining. Experimental analyses demonstrated that the down-regulation of GnT-V expression in gallbladder carcinoma cells induced suppression of cell growth in vitro. The expression levels of GnT-V in the cells were highly correlated with the rapid in vivo growth coupled with the enhanced angiogenesis, and the tendency to form liver metastasis. CONCLUSIONS: GnT-V expression in the subserosal layer of pT2 gallbladder carcinoma is correlated with the aggressiveness of the disease.

O-linked mannose ß-1,2-N-acetylglucosaminyltransferase 1 correlated with the malignancy in glioma.

O-linked mannose ß-1,2-N-acetylglucosaminyltransferase 1 (PomGnT1) constitutes one third of the O-linked glycoproteins in brain tissue. However, its functions have been seldom investigated in brain cancers. In this study, immunohistochemistry was used for the detection of the PomGnT1 protein in 133 cases of glioma tissues. Spearman correlation analysis was used for the relationship between PomGnT1 staining and the glioma grade. Receiver operating characteristic curve was used to measure the diagnostic value of PomGnT1 protein in the degree of glioma malignance. We found that PomGnT1 expression was correlated with glioma grade, and it could be used as a marker to distinguish low- and high-grade gliomas. Stably transfected U87 cells were constructed to overexpress short hairpin RNA of PomGnT1. Immunofluorescence test detected that this protein also could restrain the generation of U87 cells' pseudopodia. Western blotting further showed that the PomGnT1 protein had an impact on the c-myc protein level. In conclusion, our data suggest that PomGnT1 protein was correlated with the malignance of glioma progression, the mechanism involved in glioma cell's pseudopodium formation, and the expression of c-myc protein.

Synthesis and evaluation of a radioiodinated trisaccharide derivative as a synthetic substrate for a sensitive N-acetylglucosaminyltransferase V radioassay.

N-acetylglucosaminyltransferase V (GnT-V) is one of the most relevant glycosyltransferases to tumor invasion and metastasis. Based on previous findings of molecular recognition between GnT-V and synthetic substrates, we designed and synthesized a p-iodophenyl-derivatized trisaccharide, 2-(4-iodophenyl)ethyl 6-O-[2-O-(2-acetamido-2-deoxy-ß-D-glucopyranosyl)-α-d-mannopyranosyl]-ß-D-glucopyranoside (IPGMG, 1) and its radiolabeled form, [(125)I]IPGMG ([(125)I]1), for use in assays of GnT-V activity in vitro. The tributyltin derivative, 2-[4-(n-tributylstannyl)phenyl]ethyl 6-O-[2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-ß-D-glucopyranosyl)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl]-2,3,4-tri-O-acetyl-ß-D-glucopyranoside (21), was synthesized as a precursor for the preparation of [(125)I]1. The iododestannylation of 21 using hydrogen peroxide as an oxidant followed by deacetylation yielded [(125)I]1. When [(125)I]1 was incubated in GnT-V-expressing cells with a UDP-GlcNAc donor, the production of ß1-6GlcNAc-bearing IPGMG (IPGGMG, 2) was confirmed by radio-HPLC. In kinetic analysis, 1 was found to be a good substrate with a K(m) of 23.7 µM and a V(max) of 159 pmol/h. µg protein. [(125)I]1 would therefore be a useful synthetic substrate for the quantitative determination of GnT-V activity.

Benign cartilaginous tumors of bone: from morphology to somatic and germ-line genetics.

Benign cartilaginous tumors of bones, intrinsic to their name, are tumors forming cartilaginous matrix with a clinically benign behavior. In this group, we recognize osteochondromas, (en)chondromas, chondroblastomas, and chondromyxoid fibromas. This group includes common tumors, that is, osteochondroma and (en)chondroma as well as rare tumors such as chondroblastoma and chondromyxoid fibroma. Several benign and malignant tumors may mimic benign cartilaginous tumors of bones. We reviewed the main morphologic features and the differential diagnosis is discussed. The genetics of these tumors is intriguing ranging from single gene event (ie, EXT mutation in multiple osteochondromas) to heterogeneous rearrangements with no recurrent involved chromosomal regions such as in chondroblastoma. The main genetic findings are hereby reviewed.

The molecular and cellular basis of exostosis formation in hereditary multiple exostoses.

The different clinical entities of osteochondromas, hereditary multiple exostoses (HME) and non-familial solitary exostosis, are known to express localized exostoses in their joint metaphyseal cartilage. In the current study biopsies of osteochondromas patients were screened with respect to a number of cellular and molecular parameters. Specifically, cartilaginous biopsy samples of nine HME patients, 10 solitary exostosis patients and 10 articular cartilages of control subjects were collected and cell cultures were established. Results obtained showed that one of the two HME samples that underwent DNA sequencing analysis (HME-1) had a novel mutation for an early stop codon, which led to an aberrant protein, migrating at a lower molecular weight position. The EXT-1 mRNA and protein levels in chondrocyte cultures derived from all nine HME patients were elevated, compared with solitary exostosis patients or control subjects. Furthermore, cell cultures of HME patients had significantly decreased pericellular heparan sulphate (HS) in comparison with cultures of solitary exostosis patients or control subjects. Immunohistochemical staining of tissue sections and Western blotting of cell cultures derived from HME patients revealed higher levels of heparanase compared with solitary exostosis patients and of control subjects. Further investigations are needed to determine whether the low pericellular HS levels in HME patients stem from decreased biosynthesis of HS, increased degradation or a combination of both. In conclusion, it appears that due to a mutated glycosyltransferase, the low content of pericellular HS in HME patients leads to the anatomical deformations with exostoses formation. Hence, elevation of HS content in the pericellular regions should be a potential molecular target for correction.

Positive expressions of N-acetylglucosaminyltransferase-V (GnT-V) and beta1-6 branching N-linked oligosaccharides in human testicular germ cells diminish during malignant transformation and progression.

N-acetylglucosaminyltransferase-V (GnT-V) is an enzyme that catalyzes beta1-6 branching of N-acetylglucosamine on asparagines (N)-linked oligosaccharides of cell proteins. We examined the implication of GnT-V and beta1-6 branching N-linked oligosaccharide expression in human testicular germ cells during malignant transformation and cancer progression. We analyzed immuhistochemically orchiectomy specimens of 130 patients with testicular germ cell tumors (TGCT) using anti-GnT-V monoclonal antibody, and compared GnT-V expression with clinicopathological features. N-linked oligosaccharide structural analysis was also performed to confirm the oligosaccharide profile produced by GnT-V. GnT-V was positive in all normal testis samples. This positive incidence declined in TGCT according to clinical stage; 16/71 (22.5%) in stage I, and 3/59 (5.1%) in stage II/III (p=0.015, chi(2) test). When divided into pathological subtypes, GnT-V positive incidences in stage I seminoma, stage II/III seminoma, stage I non-seminomatous germ cell tumor (NSGCT), and stage II/III NSGCT were 3/43 (7%), 0/22 (0%), 13/28 (46.4%), and 3/37 (8.1%), respectively. In stage I NSGCT, patients with GnT-V-negative tumor samples were at a significantly higher risk of recurrence than those with GnT-V-positive tumors (p=0.015, log-rank test). N-linked oligosaccharide structural analysis revealed that a normal testis has three kinds of beta1-6 branching N-linked oligosaccharides, all of which are downregulated in TGCT tissues. These results suggest that GnT-V and beta1-6 branching N-linked oligosaccharide expressions are downregulated during carcinogenesis and progression of human TGCT. GnT-V may be a promising recurrence predictor for stage I NSGCT.

Loss of L-PHA-, PNA-, or ConA-reactive oligosaccharides is associated with a poor prognosis in human Burkitt's lymphoma.

The expression of cell surface oligosaccharides is associated with various biological phenomena. To clarify the relationship between lectin binding and the survival of patients with Burkitt's lymphoma, tumor samples from nine patients with Burkitt's lymphoma were analyzed by lectin histochemistry. Kaplan-Meier analysis showed that survival is significantly shorter for patients with negative reactivity for lectins from Phaseolus vulgaris (L-PHA), Arachis hypogaea (PNA), or Canavalia ensiformis (ConA) than for those with positive reactivity for these lectins. Immunohistochemistry for N-acetylglucosaminyltransferase V, which synthesizes beta1,6-branched oligosaccharides such as L-PHA-reactive oligosaccharides, was positive in 8 of 9 patients, but there was no correlation between its expression and that of L-PHA-reactive oligosaccharides. Collectively, a loss of L-PHA-, PNA-, or ConA-reactive oligosaccharides is closely associated with a poor prognosis in patients with Burkitt's lymphoma.