PURPOSE: This study was conducted to identify and to functionally characterize genetic variants in ST3GAL5 and ST8SIA1 in Korean patients with thyroid-associated ophthalmopathy (TAO). MATERIALS AND METHODS: Genetic analyses were conducted using DNA samples from TAO patients (n=50) and healthy subjects (n=48) to identify TAO-specific genetic variants of ST3GAL5 or ST8SIA1. The effect of each genetic variant on the transcription or expression of these genes was examined. Additionally, correlations between functional haplotypes of ST3GAL5 or ST8SIA1 and clinical characteristics of the patients were investigated. RESULTS: Six promoter variants and one nonsynonymous variant of ST3GAL5 were identified, and four major promoter haplotypes were assembled. Additionally, three promoter variants and two major haplotypes of ST8SIA1 were identified. All ST3GAL5 and ST8SIA1 variants identified in TAO patients were also found in healthy controls. Promoter activity was significantly decreased in three promoter haplotypes of ST3GAL5 and increased in one promoter haplotype of ST8SIA1. Transcription factors activating protein-1, NKX3.1, and specificity protein 1 were revealed as having roles in transcriptional regulation of these haplotypes. The nonsynonymous variant of ST3GAL5, H104R, did not alter the expression of ST3GAL5. While no differences in clinical characteristics were detected in patients possessing the functional promoter haplotypes of ST3GAL5, exophthalmic values were significantly lower in patients with the ST8SIA1 haplotype, which showed a significant increase in promoter activity. CONCLUSION: These results from genotype-phenotype analysis might suggest a possible link between the ST8SIA1 functional promoter haplotype and the clinical severity of TAO. However, further studies with larger sample sizes are warranted.
DNA ; Exophthalmos ; Graves Ophthalmopathy* ; Haplotypes ; Healthy Volunteers ; Humans ; Korea ; Polymorphism, Single Nucleotide ; Sample Size ; Sensitivity and Specificity ; Sialyltransferases ; Transcription Factors ; Troleandomycin

Hepatitis B virus MHBst and HBx fragments were amplified to construct eukaryotic expression vector pCDNA3.1-MHBst and pCDNA3.1-HBx. ST3GalI promoter region was obtained by the method of PCR and GFP report plasmid pEGFP-N1-Psial was constructed. pCDNA3.1-MHBst or pCDNA3.1-HBx with pEGFP-N1-Psial were transiently co-transfected into QGY-7701 cells using calcium phosphate-DNA co-precipitation, respectively. The expressions of Psial-directed GFP were analyzed by FAC-Scalibur. It was found that MHBst/HBx could upregulate ST3GalI promoter activity by 35.2% and 43.8%, respectively. We report the regulation of ST3GalI by MHBst and HBx transactivators. It would be helpful to further investigate the relation between hepatitis B virus infection and sialyltransferase expression.
Gene Expression Regulation, Enzymologic ; Hepatitis B Surface Antigens ; genetics ; physiology ; Hepatitis B virus ; Promoter Regions, Genetic ; Sialyltransferases ; genetics ; Trans-Activators ; genetics ; physiology ; Transfection

Neuronal apoptosis induced by amyloid beta-peptide (A beta) plays an important role in the pathophysiology of Alzheimer's disease (AD). However, the molecular mechanism underlying A beta-induced apoptosis remains undetermined. The disialoganglioside GD3 involves ceramide-, Fas- and TNF-alpha-mediated apoptosis in lymphoid cells and hepatocytes. Although the implication of GD3 has been suggested, the precise role of GD3 in A beta-induced apoptosis is still unclear. Here, we investsigated the changes of GD3 metabolism and characterized the distribution and trafficking of GD3 during A beta-induced apoptosis using human brain-derived TE671 cells. Extracellular A beta induced apoptosis in a mitochondrial-dependent manner. GD3 level was negligible in the basal condition. However, in response to extracellular A beta, both the expression of GD3 synthase mRNA and the intracellular GD3 level were dramatically increased. Neosynthesized GD3 rapidly accumulated in cell surface lipid microdomains, and was then translocated to mitochondria to execute the apoptosis. Disruption of membrane lipid microdomains with methyl-beta-cyclodextrin significantly prevented both GD3 accumulation in cell surface and A beta-induced apoptosis. Our data suggest that rapidly accumulated GD3 in plasma membrane lipid microdomains prior to mitochondrial translocation is one of the key events in A beta-induced apoptosis.
Amyloid beta-Peptides/*pharmacology ; *Apoptosis ; Cell Line ; Gangliosides/*metabolism/physiology ; Humans ; Membrane Microdomains/*metabolism ; Mitochondria/*metabolism ; Sialyltransferases/genetics/metabolism ; beta-Cyclodextrins/pharmacology

In this study, we have shown that gene expression of human GD3 synthase (hST8Sia I) is suppressed by triptolide (TPL) in human melanoma SK-MEL-2 cells. To elucidate the mechanism underlying the downregulation of hST8Sia I gene expression in TPL-treated SK-MEL-2 cells, we characterized the TPL-inducible promoter region within the hST8Sia I gene using luciferase constructs carrying 5'-deletions of the hST8Sia I promoter. Functional analysis of the 5'-flanking region of the hST8Sia I gene demonstrated that the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-kappaB, functions as the TPL-inducible promoter of hST8Sia I in SK-MEL-2 cells. Site-directed mutagenesis and ChIP analysis indicated that the NF-kappaB binding site at -731 to -722 is crucial for TPL-induced suppression of hST8Sia I in SK-MEL-2 cells. This suggests that TPL induces down-regulation of hST8Sia I gene expression through NF-kappaB activation in human melanoma cells.
Cell Proliferation/drug effects ; Diterpenes/*pharmacology ; Down-Regulation ; Epoxy Compounds/pharmacology ; Genes, Reporter ; Humans ; NF-kappa B/metabolism ; Phenanthrenes/*pharmacology ; Promoter Regions, Genetic ; Sialyltransferases/*biosynthesis/genetics ; Tumor Cells, Cultured

Abnormal glycosylation due to dysregulated glycosyltransferases and glycosidases is a key phenomenon of many malignancies, including colorectal cancer (CRC). In particular, increased ST6 Gal I (beta-galactoside alpha 2, 6 sialyltransferase) and subsequently elevated levels of cell-surface alpha 2, 6-linked sialic acids have been associated with metastasis and therapeutic failure in CRC. As many CRC patients experience metastasis to the liver or lung and fail to respond to curative therapies, intensive research efforts have sought to identify the molecular changes underlying CRC metastasis. ST6 Gal I has been shown to facilitate CRC metastasis, and we believe that additional investigations into the involvement of ST6 Gal I in CRC could facilitate the development of new diagnostic and therapeutic targets. This review summarizes how ST6 Gal I has been implicated in the altered expression of sialylated glycoproteins, which have been linked to CRC metastasis, radioresistance, and chemoresistance.
Antigens, CD/*metabolism ; Colorectal Neoplasms/*metabolism/pathology/*therapy ; Drug Resistance, Neoplasm ; Glycoproteins/*metabolism ; Humans ; Liver Neoplasms/secondary ; Lung Neoplasms/secondary ; Radiation Tolerance ; Receptor, Epidermal Growth Factor/metabolism ; Sialic Acids/*metabolism ; Sialyltransferases/*metabolism

OBJECTIVETo investigate the effect of cell surface sialic acid and its linkage on the cell-cell and cell-matrix adhesion of mammary carcinoma cells MD-MB-435.

METHODSMD-MB-435 cells were sense-transfected with ST6Gal I cDNA or antisense-transfected with part of the ST6Gal I sequence inserted in pcDNA 3.1 vector, with mock transfection with pcDNA3.1 vector as the control. The cell surface alpha2, 6-linked sialylation was determined by fluorescence-activated cell sorting (FACS) using lectin SNA (Sambucus nigra agglutinin specific to alpha2, 6-linked sialic acid on N-linked glycoprotein). A significantly increased alpha2, 6-sialylation subclone in sense-transfectants and a decreased alpha2, 6-sialylation subclone in antisense-transfectants were selected for further examination of cell-cell and cell-matrix (collagen IV) adhesion. The transfectants were also treated with sialidase to compare the capacity of cell adhesion affected by cell surface sialylation.

RESULTSSense-transfection subclone showed a reduced cell-cell aggregation but enhanced cell-matrix adhesion. In contrast, the antisense-transfection subclone exhibited increased cell-cell aggregation and decreased cell-matrix adhesion. After treatment with sialidase, the cell-matrix adhesion of all the transfectants and the parental MDA-MB-435 cells were significantly reduced to the level of 31%-57% of untreated cells.

CONCLUSIONCell surface sialic acid and alpha2, 6-linked sialylation play an important role in cell-cell and cell-matrix adhesion of mammary carcinoma cell MDA-MB-435.

Antigens, CD ; genetics ; metabolism ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Adhesion ; Cell Line, Tumor ; Cell Membrane ; metabolism ; Cell-Matrix Junctions ; metabolism ; Collagen Type IV ; metabolism ; Extracellular Matrix ; metabolism ; Humans ; N-Acetylneuraminic Acid ; metabolism ; Sialyltransferases ; genetics ; metabolism ; Transfection

OBJECTIVETo study the effects of antisense oligonucleotide (ASODN) targeting ST6Gal I on cell adhesion and invasiveness of human cervical carcinoma cell line HeLa which over-expressed ST6Gal I .

METHODSASODN and sense oligonucleotide (SODN) targeting ST6Gal I were designed and constructed, and transfected into a cervical cancer cell line, HeLa, by lipofectmine 2000. HeLa cells were cultured and divided into 4 groups: blank control group, liposome group, SODN group and ASODN group. RT-PCR was used to examine the ST6Gal I mRNA expression. Flow cytometry was used to examine the amount of alpha2, 6-sialylation on the HeLa cell surface. The HeLa cell adhesion and invasiveness to extracellular matrix ( ECM) were analyzed by using CytoMatrixTM kit and cell invasion assay kit, respectively.

RESULTSThe expression of ST6Gal I mRNA in HeLa cells at 48 hrs after transfection in the ASODN group was significantly decreased in comparison with that in the blank control group, liposome group, and SODN group(P <0. 01). The amount of alpha2,6-sialylation on cell surface in ASODN group was significantly lower than that of the other 3 groups ( P <0. 05). The adhesion and invasiveness of the cells in the ASODN group decreased remarkably, both significantly lower than those of the other 3 groups ( all P < 0. 05).

CONCLUSIONSpecific ASODN targeting ST6Gal I effectively inhibits HeLa cell ST6Gal I expression, decreases the amount of alpha2,6-sialylation on cell surface and leads to a decline of cell adhesion and invasiveness to ECM. This result also established a fine base for further studying on anti-tumor treatment with antisense oligonucleotide.

Cell Adhesion ; genetics ; physiology ; Cell Movement ; genetics ; physiology ; Flow Cytometry ; Gene Silencing ; HeLa Cells ; Humans ; Neoplasm Invasiveness ; Oligodeoxyribonucleotides, Antisense ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sialyltransferases ; biosynthesis ; genetics ; Transfection

OBJECTIVETo study the effects of small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) targeting ST6Gal I on adhesion and invasiveness of human colonic carcinoma cell line SW480 over-expressing ST6Gal I.

METHODSsiRNA and ASOs targeting ST6Gal I were constructed and transfected into SW480 cells via lipofectmine 2000. SW480 cells were cultured and divided into 7 groups, namely the blank control group, liposome group, siRNA group (transfected with ST6Gal I siRNA), ASO(1) group (transfected with ST6Gal I ASO whose target site is different from the siRNA), ASO(2) group (transfected with ST6Gal I ASO targeting the same site as siRNA), siRNA+ASO(1) group (transfected with siRNA and ASO(1)), siRNA+ASO(2) group (transfected with siRNA and ASO(2)). RT-PCR was used to examine ST6Gal I mRNA expression following the treatment. Flow cytometry was used to examine the amount of alpha2,6-sialylation on SW480 cell surface. SW480 cell adhesion and invasiveness to the extracellular matrix (ECM) were analyzed using CytoMatrix kit and cell invasion assay kit, respectively.

RESULTSThe expression of ST6Gal I mRNA, the amount of alpha2,6-sialylation on the cell surface and cell adhesion and invasion to ECM decreased remarkably in groups siRNA, ASO(1), ASO(2), siRNA+ASO(1) and siRNA+ASO(2), all significantly lower than those of the blank control and liposome groups (all P<0.05), especially in siRNA+ASO(1) group. Significant difference was noted between siRNA+ASO(1) and siRNA groups (P<0.05), but not between siRNA+ASO(2) and siRNA groups, or between blank control and liposome groups (all P>0.05).

CONCLUSIONChemically synthesized specific siRNA targeting ST6Gal I effectively inhibits SW480 cell ST6Gal I expression and leads to diminished cell adhesion and invasiveness to ECM, suggesting a combined effect of siRNA and ASO with different targeting sites.

Cell Adhesion ; Cell Movement ; Colonic Neoplasms ; genetics ; pathology ; Flow Cytometry ; Gene Silencing ; Humans ; Neoplasm Invasiveness ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sialyltransferases ; genetics ; metabolism ; Transfection

In order to compare the prognoses of patients with diffuse malignant lymphomas on the basis of histology and immunophenotypes, we retrospectively studied 62 cases of diffuse lymphoma arising in lymph nodes. We also evaluated the reactivity patterns of monoclonal antibodies (MoAb) LN1, LN2 and LN3 to determine the criteria for making a differential diagnosis in B cell lymphomas. The immunologic phenotypes were determined by the avidin biotin peroxidase complex method, using frozen or paraffin fixed tissues. The majority (66.3%) were B cell with the remaining 20.9% being T cell and 12.9% were non-B, non-T cell lineage. Immunological heterogeneity was found especially in the mixed small and large cell and the immunoblastic lymphomas. There was no significant difference between B- and T-cell lymphomas with respect to survival and death (P > 0.05). Histologically 79% (49/62) of the lymphoma was large cell and 21% (13/62), small cell lymphoma. There was a difference in prognosis between low, intermediate and high-grade of lymphomas. However there were no significant differences among the subtypes of the diffuse aggressive lymphomas. Factors associated with poor prognosis were advanced stages (P < 0.025) and histology of the malignant lymphomas. MoAb LN1, LN2 and LN3 gave positive staining in 83.3%, 91.7% and 60% of B cell lymphomas, respectively. The most common phenotypic pattern in B cell lymphomas was LN1+, LN2+, LN3+/-, suggestive of follicular center cell origin. As a panel, phenotypic patterns of MoAb LN1, LN2 and LN3 may be useful in differentiation of follicular center cell lymphoma from others.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD/biosynthesis ; Antigens, Differentiation, B-Lymphocyte/biosynthesis ; Child ; Female ; Follow-Up Studies ; Histocompatibility Antigens Class II/biosynthesis ; Humans ; Immunoenzyme Techniques ; Immunophenotyping ; Lymphoma, B-Cell/immunology ; Lymphoma, Non-Hodgkin/immunology/*pathology ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis ; Sialyltransferases/biosynthesis