The presence of at least two different H-blood-group-related beta-D-gal alpha-2-L-fucosyltransferases in human serum and the genetics of blood group H substances.

Sera from H normal, secretors and nonsecretors (H/-, Se/- and H/-, se/se), as well as from H-deficient secretors (h/h, Se/- or Bombay secretors) contain enzyme(s) for the transfer of L-fucose in the alpha-configuration to the 2-position of suitable beta-D-galactopyranosyl units. Sera from H-deficient nonsecretors (h/h, se/se; i.e., Bombay nonsecretors) are devoid of such beta-D-Gal alpha-2-L-fucosyltransferase(s). In order to study these enzymes, a comparison was made of the kinetic properties of the enzymes present in the sera of H-normal nonsecretors (H/-, se/se) with those of H-deficient secretors (h/h, Se/se) with those of H-deficient secretors (h/h, Se/-). These studies revealed a clear difference between the two sources of enzyme: (1) the apparent Km for GDP-fucose was four times lower with the H-normal nonsecretor serum (0.008 mM) than with the H-deficient secretor serum (0.028 mM); (2) acceptors with a type 1 or type 3 chain proved to be better than acceptors with a type 2 chain or than phenyl-beta-D-galactopyranoside for the enzyme present in the serum of H-deficient secretor individuals. Indeed, the synthetic type 2 compound, betaDGal (1-->4)-3-deoxy-beta-DGlcNAc-1-OCH3, which cannot act as an acceptor of beta DGlcNAc alpha-3/4-L-fucosyltransferases, remained unchanged in the serum of an H-deficient secretor but was a good acceptor in the serum of an H-normal nonsecretor, and (3) the alpha-2-L fucosyltransferease activity of the H-deficient secretor serum was more sensitive to heat inactivation than that of the H-normal nonsecretor serum (t1/2 at 46 degrees C were 10 min and 75 min, respectively). These results show that at least two distinct alpha-2-L-fucosyltransferases are present in human serum. It is concluded that the enzymatic activity found in the H-deficient secretor serum (h/h, Se/-) could be the product of the Se gene and the enzymatic activity found in the H-normal nonsecretor serum (H/-, se/se) could be the product of the H gene. This conclusion correlates well with the finding that H and Se genes are closely linked and might have derived by gene duplication in the course of evolution.     

Distribution of H type 1-4 chains of the ABO(H) system in different cell types of human respiratory epithelium.

We used three anti-H monoclonal antibodies (MAbs) specific for H Type 1, H Type 2, and H Type 3/4 antigens to investigate the distribution of H Type 1-H Type 4 chains of the ABO(H) histo-blood group in the human respiratory system. Strong staining of H Type 1 chain and weak staining of H Type 2 chain were observed in mucous cells of submucosal glands of bronchial epithelium, which were dependent on the secretor status. No H Type 3/4 chains were detected in mucous cells. Serous cells of submucosal glands of respiratory system showed no staining by three anti-H antibodies. H Type 1 and H Type 3/4 antigens were detected heterogeneously in apical surfaces of bronchial epithelium from secretors but not from nonsecretors. In contrast, basal cells of bronchial epithelium expressed H Type 2 irrespective of the secretor status, probably regulated by the H gene. Some alveolar Type II cells contained only H Types 3/4, which were dependent on the secretor status, whereas alveolar Type I cells had no H antigens. Our results indicated that different cell types in respiratory epithelium expressed different types of carbohydrate chains of histo-blood group antigens under the control of the H or the Se gene.     

Expression of Nucleophosmin/NPM1 correlates with migration and invasiveness of colon cancer cells

Background

We aimed to examine the expression level of Nucleophosmin (NPM1) protein in colon cancer tissues and to investigate the potential role of NPM1 in the regulation of cell migration and invasiveness.

Methods

Immunohistochemical assay was performed to examine the expression pattern of NPM1 in 31 groups of colonic carcinoma samples, including colon tumors, adjacent normal tissues, and matched metastatic lymph nodes from the same patients. Small interfering RNA technique and exogenous expression of wild type NPM1 methods were used to further verify the function of NPM1.

Results

High-expression of NPM1 correlates with lymph node metastasis (P = 0.0003) and poor survival rate of human colon cancer patients (P = 0.017). SiRNA-mediated reduction of NPM1 was also shown to inhibit the migration and invasiveness of metastatic colon cancer HCT116 cell line. In addition, the exogenous expression of NPM1 in HT29 cells, a NPM1 low expression and low invasive colon cancer cell line, enhanced cell migration and invasiveness along with increased cell proliferation.

Conclusions

The current study uncovered the critical role of NPM1 in the regulation of colon cancer cells migration and invasion, and NPM1 may serve as a potential marker for the prognosis of colon cancer patients.     

Aberrant secretors in donors and patients with duodenal ulcer

Results of the screening of aberrant (paradoxal) secretors in 14372 healthy donors and 614 patients with ulcus duodeni are presented. An extremely high frequency of aberrant secretors was established (12.6%) in patients with ulcus duodeni in comparison to donors (0.10%). A new type of aberrant secretors was registered in patients of A1 and A1B blood groups. Their salivas normally inhibited anti-A sera, but the expected inhibition of anti-A1 reagents (dolichos biflorus) could not be observed. The data of comparative quantitative investigations of the inhibiting strength of the ABH antigen in salivas of normal secretors, nonsecretors and aberrant secretors are presented. Various theoretical explanations of aberrant secretors are discussed.     

Immunohistochemical localization of collagen type XI alpha1 and alpha2 chains in human colon tissue.

In previous studies, collagen XI mRNA has been detected in colon cancer, but its location in human colon tissue has not been determined. The heterotrimeric collagen XI consists of three alpha chains. While it is known that collagen XI plays a regulatory role in collagen fibril formation, its function in the colon is unknown. The characterization of normal human colon tissue will allow a better understanding of the variance of collagen XI in abnormal tissues. Grossly normal and malignant human colon tissue was obtained from pathology archives. Immunohistochemical staining with a 58K Golgi marker and alpha1(XI) and alpha2(XI) antisera was used to specifically locate their presence in normal colon tissue. A comparative bright field microscopic analysis showed the presence of collagen XI in human colon. The juxtanuclear, dot-like collagen XI staining in the Golgi apparatus of goblet cells in normal tissue paralleled the staining of the 58K Golgi marker. Ultra light microscopy verified these results. Staining was also confirmed in malignant colon tissue. This study is the first to show that collagen XI is present in the Golgi apparatus of normal human colon goblet cells and localizes collagen XI in both normal and malignant tissue. Although the function of collagen XI in the colon is unknown, our immunohistochemical characterization provides the foundation for future immunohistopathology studies of the colon.     

Novel blood group H glycolipid antigens exclusively expressed in blood group A and AB erythrocytes (type 3 chain H). II. Differential conversion of different H substrates by A1 and A2 enzymes, and type 3 chain H expression in relation to secretor status

The novel A-associated H antigen (type 3 chain H), described in the accompanying paper (Clausen, H., Levery, S.B., Kannagi, R., and Hakomori, S. (1986) J. Biol. Chem. 261, 1380-1387), as well as globo-H were found to be present in greater quantity in A2 erythrocytes than in A1 erythrocytes. A1 erythrocytes contain the repetitive A epitope (type 3 chain A) (Clausen, H., Levery, S.B., Nudelman, E., Tsuchiya, S., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1199-1203), which is defined by A1-specific monoclonal antibody TH-1, in addition to globo-A. The ability of alpha-GalNAc transferase from A1 and A2 serum to catalyze the conversion of type 2 chain H, type 3 chain H, and globo-H to type 2 chain A, type 3 chain A, and globo-A, respectively, was compared. The conversion to type 3 chain A and globo-A occurred to a minimal degree in the presence of the A2 enzyme as compared with the A1 enzyme, particularly at low substrate concentration. Although a lower conversion from type 2 chain H to type 2 chain A was also observed in the presence of the A2 enzyme than in the presence of the A1 enzyme, the conversion of type 2 chain H to type 2 chain A was less restricted than the type 3 chain conversion catalyzed by the A2 enzyme, particularly at low substrate concentration. The conversion from globo-H to globo-A was essentially absent in the presence of the A2 enzyme. Since the expression of type 1 chain H in erythrocytes is dependent on secretor status, the distribution of type 3 chain H and globo-H in erythrocytes from secretors and non-secretors was compared. These antigens appeared to be present in the same quantity in erythrocytes of secretors and nonsecretors.     

Presence of H type 3/4 chains of ABO histo-blood group system in serous cells of human submandibular gland and regulation of their expression by the secretor gene (FUT2).

We have investigated by immunochemistry the distribution of H Type 3/4 chains of the ABO histo-blood group system in human submandibular gland using a monoclonal anti-H MBr1 antibody specific for H Type 3/4 chains, and have found the expression of H Type 3/4 chains was mainly in the serous cells. Serous cells from secretors were stained by MBr1 but not by anti-A and anti-B antibodies, whereas serous cells from nonsecretors exhibited a negative reaction with MBr1. Mucous cells were not stained by MBr1. Only a few striated duct cells showed a weak reaction with anti-H MBr1. These results suggested that the H Type 3/4 chains were distributed predominantly in the serous cells of the human submandibular gland and that secretor Type alpha(1,2)fucosyltransferase (Se enzyme) controlled the synthesis of H Type 3/4 chains in vivo. Saliva also contained H Type 3/4 chains, which were controlled by the secretor gene (FUT2). The differences in the distributions of H Type 1, H Type 2, and H Type 3/4 chains of the ABO histo blood group system in the submandibular gland are discussed.     

Molecular mechanisms of expression of Lewis b antigen and other type I Lewis antigens in human colorectal cancer.

Lewis b (Leb) antigens are gradiently expressed from the proximal to the distal colon, i.e., they are abundantly expressed in the proximal colon, but only faintly in the distal colon. In the distal colon, they begin to increase at the adenoma stage of cancer development and then increase with cancer progression. We aimed to clarify the molecular basis of Leb antigen expression in correlation with the expression of other type I Lewis antigens, such as Lewis a (Lea) and sialylated Lewis a (sLea), in colon cancer cells. Considering the Se genotype and the relative activities of the H and Se enzymes, the amounts of Leb antigens were proved to be determined by both the H and Se enzymes in noncancerous and cancerous colon tissues. But the Se enzyme made a much greater contribution to determining the Lebamounts than the H enzyme. In noncancerous colons, the Se enzyme were gradiently expressed in good correlation with the Leb expression, while the H enzyme was constantly expressed throughout the whole colon. In distal colon cancers, the H and Se enzymes were both significantly upregulated in comparison with in adjacent noncancerous tissues. In proximal colon cancers, expression of the H enzyme alone was highly augmented. The augmented expression of Leb antigens in distal colon cancers is caused mainly by upregulation of the Se enzyme and partly by the H enzymes, while it is caused by upregulation of the H enzyme alone in proximal colon cancers. The Se gene dosage profoundly influences the amounts of the Leb, Lea, and sLea antigens in whole colon tissues, regardless of whether they are noncancerous or cancerous tissues. It suggests that the Se enzyme competes with alpha2,3 sialyltransferase(s) and the Le enzyme for the type I acceptor substrates.     

The expression of human FUT1 in HT-29/M3 colon cancer cells instructs the glycosylation of MUC1 and MUC5AC apomucins

Recently, we have reported that in normal gastric epithelium, the expression of gastric apomucins MUC5AC and MUC6 is associated with the specific expression of type 1 and type 2 Lewis antigens, and FUT2 and FUT1 fucosyltransferases, respectively. Until now, there are no data demonstrating the direct implication of specific glycosyltransferases in the specific patterns of apomucin glycosylation.HT29/M3 colon cancer cell line express MUC1, MUC5AC, type 1 Lewis antigens and FUT2 but not type 2 structures and FUT1, as it occurs in the epithelial cells of the gastric superficial epithelium. These cells were transfected with the cDNA of human FUT1, the -1,2-fucosyltransferase responsible for the synthesis of type 2 Lewis antigens, to assess the implication of FUT1 in the glycosylation of MUC1 and MUC5AC.The M3-FUT1 clones obtained express high levels of type 2 Lewis antigens: H type 2 and Ley antigens. Immunoprecipitation of MUC1 and MUC5AC apomucins gives the direct evidence that FUT1 catalyses the addition of -1,2-fucose to these apomucins, supporting the hypothesis that the pattern of apomucin glycosylation is not only instructed by the mucin primary sequence but also by the set of glycosyltransferases expressed in each specific cell type.     

Expression of CYP and GST in human normal and colon tumor tissues

We investigated the immunohistochemical staining characteristics of cytochrome P450 1A1 (CYP1A1), CYPB1, CYP2E1, and glutathione S-transferase P1 (GSTP1), GSTT1, GSTO1, GSTK1 in colon tumor and surrounding normal colon tissues. Tissues were obtained from 47 patients with colon adenocarcinoma and the staining intensity of tumor and control tissues was compared. CYP1A1, CYP1B1, CYP2E1, GSTP1, GSTT1, GSTO1 and GSTK1 expressions in colon cancer cells were significantly greater than those in normal colon epithelial cells. No significant relation was found between the isoenzyme expressions and age, gender, smoking status, tumor grade and tumor stage. The higher expressions of CYP1A1, CYP1B1, CYP2E1, GSTP1, GSTO1, GSTT1 and GSTK1 in tumor than in normal colon tissues may be important for colon cancer progression and development.     

Integrative analysis and validation of dysregulated long non‐coding RNAs in colon cancer

It is an increasing evidence that long non‐coding RNAs (lncRNAs) are involved in tumour initiation and progression. Here, we analysed RNA‐sequencing data from the Cancer Genome Atlas (TCGA) datasets. Totally, 1176lncRNAs, 245miRNAs and 2081mRNAs were identified to be differentially expressed (DE) in colon cancer tissues compared with normal tissues. CASC21, a novel lncRNA located in 8q24.21 locus, was significantly overexpressed in 30 colon cancer tissues compared with matched normal tissues by qRT‐PCR assay. CASC21 tended to higher expression as the increase of the tumour‐node‐metastasis (TNM) classification. Functionally, CASC21 promoted cell proliferation by regulating cell cycle and enhanced tumour metastasis by epithelial‐mesenchymal transition (EMT) in colon cancer. Mechanism study indicated that CASC21 might be involved in activating WNT/β‐catenin pathway in colon cancer. In addition, we also built a competing endogenous RNA (ceRNNA) network by bioinformatic analysis using TCGA datasets. Together, our results not only provide novel lncRNAs as potential candidates for further study but also prove that CASC21 is an oncogenic regulator through activating WNT/β‐catenin signalling in colon cancer.     

Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon

Background

Obesity is known to increase the risk of colorectal cancer. However, mechanisms underlying the pathogenesis of obesity-induced colorectal cancer are not completely understood. The purposes of this study were to identify differentially expressed genes in the colon of mice with diet-induced obesity and to select candidate genes as early markers of obesity-associated abnormal cell growth in the colon.

Methods

C57BL/6N mice were fed normal diet (11% fat energy) or high-fat diet (40% fat energy) and were euthanized at different time points. Genome-wide expression profiles of the colon were determined at 2, 4, 8, and 12 weeks. Cluster analysis was performed using expression data of genes showing log₂ fold change of ≥1 or ≤?1 (twofold change), based on time-dependent expression patterns, followed by virtual network analysis.

Results

High-fat diet-fed mice showed significant increase in body weight and total visceral fat weight over 12 weeks. Time-course microarray analysis showed that 50, 47, 36, and 411 genes were differentially expressed at 2, 4, 8, and 12 weeks, respectively. Ten cluster profiles representing distinguishable patterns of genes differentially expressed over time were determined. Cluster 4, which consisted of genes showing the most significant alterations in expression in response to high-fat diet over 12 weeks, included Apoa4 (apolipoprotein A-IV), Ppap2b (phosphatidic acid phosphatase type 2B), Cel (carboxyl ester lipase), and Clps (colipase, pancreatic), which interacted strongly with surrounding genes associated with colorectal cancer or obesity.

Conclusions

Our data indicate that Apoa4, Ppap2b, Cel, and Clps are candidate early marker genes associated with obesity-related pathological changes in the colon. Genome-wide analyses performed in the present study provide new insights on selecting novel genes that may be associated with the development of diseases of the colon.

     

Histochemical demonstration of O-glycosidically linked,type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Summary Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo-and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with -N-acetylgalactosaminidase and -L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with -galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B₄ reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo--N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from nonsecretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(1-3)-N-acetyl-D-galactosamine1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal, normal and neoplastic gastrointestinal tract.

Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for alpha 2----6 sialylated type 2 chain) and IB9 (for the alpha 2----6 sialylated type 2 chain and glycoproteins having NeuAc alpha 2----6Gal-NAc), and 188C1 (for short- and long-chain Lex antigens) and FH2 (for the long-chain Lex antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The alpha 2----6 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Lex antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its alpha 2----6 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Lex antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc alpha 2----6GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal,normal and neoplastic gastrointestinal tract

Summary Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for 26 sialylated type 2 chain) and IB9 (for the 26 sialylated type 2 chain and glycoproteins having NeuAc26GalNAc), and 188C1 (for short- and long-chain Le^x antigens) and FH2 (for the long-chain Le^x antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The 26 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Le^x antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its 26 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Le^x antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc26GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.Abbreviations Fuc fucose - Gal galactose - GalNAc N-acetylgalactosamine - Glc glucose - GlcNAc N-acetylglucosamine - MAb monoclonal antibody - NeuAc N-acetylneuraminic acid - PBS phosphate-buffered saline