Sulfotransferases of two specificities function in the reconstitution of high endothelial cell ligands for L-selectin.

L-selectin, a lectin-like receptor, mediates rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs by interacting with HEV ligands. These ligands consist of a complex of sialomucins, candidates for which are glycosylation- dependent cell adhesion molecule 1 (GlyCAM-1), CD34, and podocalyxin. The ligands must be sialylated, fucosylated, and sulfated for optimal recognition by L-selectin. Our previous structural characterization of GlyCAM-1 has demonstrated two sulfation modifications, Gal-6-sulfate and GlcNAc-6-sulfate in the context of sialyl Lewis x. We now report the cloning of a Gal-6-sulfotransferase and a GlcNAc-6-sulfotransferase, which can modify GlyCAM-1 and CD34. The Gal-6-sulfotransferase shows a wide tissue distribution. In contrast, the GlcNAc-6-sulfotransferase is highly restricted to HEVs, as revealed by Northern analysis and in situ hybridization. Expression of either enzyme in Chinese hamster ovary cells, along with CD34 and fucosyltransferase VII, results in ligand activity, as detected by binding of an L-selectin/IgM chimera. When coexpressed, the two sulfotransferases synergize to produce strongly enhanced chimera binding.

A glycolipid antigen associated with Burkitt lymphoma defined by a monoclonal antibody.

The antigen defined by a rat monoclonal antibody directed to a Burkitt lymphoma cell line was identified as globotriaosylceramide [Gal alpha (1 leads to 4)-Gal beta (1 leads to 4)-Glc beta (1 leads to 1)-ceramide]. The antibody demonstrated a strict steric specificity since it did not react with globoisotriaosylceramide [Gal alpha (1 leads to 3)-Gal beta (1 leads to 4)-Glc beta (1 leads to 1)-ceramide], the positional isomer of the antigen associated with the Burkitt lymphoma. Chemical analysis of various Burkitt lymphoma cell lines revealed that the Burkitt lymphoma cells contained more than 100 times as much of the glycolipid antigen as was found in other human lymphoma and leukemia cell lines.

Impairment of selectin-mediated leukocyte adhesion to venular endothelium in spontaneously hypertensive rats.

The present study was designed to elucidate whether molecular mechanisms for leukocyte adhesion to microvascular endothelium may differ between spontaneously hypertensive rats and Wistar Kyoto rats. Leukocyte rolling and adhesion were investigated while monitoring venular wall shear rates in the mesenteric microcirculation stimulated with histamine or tert-butyl hydroperoxide in the two strains. In Wistar Kyoto rats, 10 microM histamine as well as 500 microM tertbutyl hydroperoxide promoted a significant reduction of venular leukocyte rolling velocity and subsequent adhesion. These changes in leukocyte behavior were blocked by monoclonal antibodies against P-selectin (PB 1.3) and against sialyl Lewis X-like carbohydrates (2H5). However, spontaneously hypertensive rats exhibited a blunted response of the stimulus-elicited leukocyte rolling, which was associated with impairment of venular P-selectin expression as well as a decrease in the expression of sialyl Lewis X-like carbohydrates on circulating neutrophils. No significant differences were detected between the two strains not only in the surface CD11b/CD18 expression but also in the CD18-mediated adhesivity of neutrophils to intracellular adhesion molecule-1 transfectants in vitro. These results suggest that impairment of selectin-mediated leukocyte adhesion is an event responsible for disorders of inflammatory responses in spontaneously hypertensive rats.

Alterations in gastric mucin with malignant transformation: novel pathway for mucin synthesis.

BACKGROUND: Mucins are high-molecular-weight glycoproteins produced by both normal and cancer cells. However, in cancer cells, abnormal mucins are synthesized and potentially can be used as markers for the development and progression of certain malignancies. In a previous study, we reported the production of a new monoclonal antibody directed against a mucin antigen termed F1 alpha, an O-linked oligosaccharide similar to sialyl Tn and Thomsen-Friedenreich (T) antigens, that has not been previously detected in human cancers. F1 alpha is expressed in a high percentage (80.2%; 89/111) of gastric cancers. PURPOSE: In the present study, we compared the expression of F1 alpha with that of sialyl Tn and T antigens in human gastric cancer tissues to determine how differences in the expression of these cancer-associated antigens correlated with the biological properties of cancer cells. METHODS: A total of 141 cases of gastric cancer were studied. Sections of formalin-fixed, paraffin-embedded tissue were immunostained for F1 alpha, sialyl Tn, and T antigens. The relationship between the expression of these antigens and the patient's clinicopathologic characteristics was studied. The chi-square test (two-sided) was used for statistical analyses. RESULTS: F1 alpha was expressed in a high percentage of the cases of early to advanced cancers, irrespective of the degree of malignant progression. The rate of expression of sialyl Tn antigen in early carcinoma was low, but it increased significantly as depth of invasion increased (P < .05) and was significantly higher in patients with hepatic or lymph node metastasis than in those without such metastasis (P < .01). Expression of T antigen significantly increased with depth of invasion (P < .01) and was significantly higher in patients with hepatic metastasis (P < .05), lymph node metastasis (P < .05), or peritoneal dissemination (P < .01) than in those without such metastasis or dissemination. In consecutive sections of the same specimen, the sites of staining for F1 alpha and sialyl Tn antigens seldom coincided. In many cases, F1 alpha staining was predominant, but the sialyl Tn-dominant region tended to increase as gastric cancer progressed. Regions of T-antigen staining were usually circumscribed by those of F1 alpha staining. CONCLUSION: Our findings indicate that the expression of F1 alpha begins almost at the same time as does carcinogenesis in gastric epithelial cells. Moreover, in association with progression of gastric carcinoma, synthetic pathways for sialyl Tn antigen and T antigen probably are activated independently.

Variant type of sialyl Lewis X antigen expressed on adult T cell leukemia cells is associated with skin involvement.

Expression of a variant type of sialyl Le(x) antigen defined by 2F3 monoclonal antibody on leukemia cells was studied in 15 adult T cell leukemia (ATL) patients. The expression of 2F3-defined sialyl Le(x) antigen on CD4+CD45+ cells, which is an ATL cell-rich population, was higher in patients with skin involvement (50.1 +/- 23.1% were positive) than in patients without skin involvement (18.1 +/- 12.5%) (P < 0.01). The other surface markers including classical sialyl Le(x) antigen defined by SNH3 or FH6 and LFA-1, VLA-4, CD4, CD25, ICAM-1, Leu8, and HLA-DR did not show a significant difference regardless of skin involvement. In the skin lesion of four patients that we could examine, infiltrating leukemia cells strongly expressed 2F3-defined sialyl Le(x) antigen. In one patient, we could also examine the expression of classical sialyl Le(x) antigen defined by SNH-3 and CSLEX-1, but this was almost negligible. Both skin and lymph node biopsy specimens were examined in two patients. Leukemia cells in the skin strongly expressed 2F3-defined sialyl Le(x) antigen, while its expression was almost negligible on the leukemia cells in the lymph node. These findings suggest that the expression of 2F3-defined sialyl Le(x) antigen on ATL cells is associated with skin involvement of ATL.

First establishment of a human monoclonal antibody directed to sulfated glycosphingolipids SM4s-Gal and SM4g, from a patient with lung cancer.

Several human monoclonal antibodies directed to tumor-associated glycolipid antigens have been established, but more than one-half of them react with gangliosides and the others react with neutral glycolipids. We report here the first establishment of a human IgM monoclonal antibody directed to the sulfated glycolipid. This monoclonal antibody, M14-376, did not react with SM3 and SB1a which have a terminal HSO3----3Gal beta 1----R1, but with the simple sulfolipids SM4s-Gal and SM4g which contain a terminal HSO3----3Gal beta 1----O----CH2----R2; however, lyso-SM4s-Gal and lyso-SM4g did not bind M14-376. These results suggest that terminal HSO3----3Gal and part of the hydrophobic region of the glycolipid are recognized by M14-376. Directly biotinylated M14-376 was used for immunohistochemical staining of 140 formalin-fixed, paraffin-embedded lung cancer tissue sections to study the distribution of the antigen. A high incidence of positive staining was found in adenocarcinoma (39.5%, 17 of 43), followed by large cell carcinoma (20.0%, 5 of 25), while this antigen was rarely detected in small cell carcinoma (4.7%, 1 of 21) and squamous cell carcinoma (3.9%, 2 of 51). Thin layer chromatography immunostaining of glycolipids extracted from lung cancer tissues showed the presence of only SM4s-Gal in adenocarcinoma, but SM4g was not found in any subtype of lung cancer. Immunohistochemical staining revealed that this antigen was expressed in normal kidney, testis, and brain, but erythrocytes, granulocytes, and lymphocytes were negative in cytofluorometric analysis.

Stage-specific expression of SSEA-1-related antigens in the developing lung of human embryos and its relation to the distribution of these antigens in lung cancers.

The localization of three carbohydrate antigens, Lex, Ley, and sialylated Lex-i, which are closely related to stage-specific embryonic antigen 1, in the lung of developing human embryos was investigated using specific monoclonal antibodies. In the 38-day-old embryo, when the primitive lung bud has appeared and developed into two lung sacs, only Ley antigen was specifically positive in the proliferating cells in the terminal portion of lung bud. In the 50-53-day-old embryos, the future bronchi were actively developing from the bronchial buds. At this stage, the Ley antigen was maximally expressed and the Lex antigen appeared in the bud cells. In the lung of the 12-week-old embryo, buds for the future bronchioles were lined by simple cuboidal epithelial cells, which were strongly positive for Lex antigen, weakly positive for Ley antigen, and still completely negative for sialylated Lex-i antigen. Sialylated Lex-i antigen finally appeared in 18-week-old embryos, in the cells of the terminal buds for the future alveoli. At this stage, the Lex and Ley antigens were already beginning to disappear and were only weakly positive in cells of terminal buds. At 20 weeks, only sialylated Lex-i antigen was weakly detected in the cells in the terminal buds; after 8 months, all three antigens were essentially not detected in the respiratory cells in most of the embryos examined in this study. Formation of bronchial glands was detected at 18 weeks, where the developing gland cells were specifically positive for sialylated Lex-i antigen. Ciliation of the bronchial epithelial cells started at 12 weeks and propagated thereafter. The ciliation was accompanied by the reappearance of Ley and Lex antigen in the epithelial cells. These findings collectively indicated that the three antigens all have a physiological significance as stage-specific developmental antigens of the human lung; those antigens were specifically present in the bud cells at each important step of the morphogenesis of the human lung, such as cells in the lung buds, bronchial buds, and terminal buds for the formation of the alveolus, and cells differentiating into bronchial gland cells. The three antigens gradually disappear in the later stage of development along with the maturation process of the lung. Stage-specific embryonic antigen 1 and related antigens are known to be associated with various human cancers, including lung cancers. We suggest that the expression of these antigens in the lung cancer cells is the result of the retrodifferentiation of the cancer cells to the stages of immature embryonic lung cells.

Accumulation of highly acidic sulfated glycosphingolipids in human hepatocellular carcinoma defined by a series of monoclonal antibodies.

An accumulation of sulfated and very complex, highly acidic glycolipids was observed in cultured human hepatocellular carcinoma cells. Among the cells tested, PLC/PRF/5 cells contained a significant amount of very complex sulfated acidic glycolipids, and HepG2 cells were characterized as having a large amount of relatively simple sulfated glycolipids. Several monoclonal antibodies (all IgM) directed to these sulfated and highly acidic glycolipids were established. Among them, 49-D6 and 7-E10 were both directed to SM3 (LacCer-II3-sulfate), a relatively simple sulfated glycolipid, and 34-A4 was directed to SD1a (GgOse4Cer II3,IV3-disulfate) and more complex sulfated glycolipids. The other four antibodies, 26-A10, 34-B9, 79-C8, and 16-E10, reacted with unknown highly acidic glycolipids, which were eluted in 0.9-2.7 M ammonium acetate in DEAE chromatography, indicating that these antigenic glycolipids were far more acidic than the usual glycolipids described until now. Analysis of the glycolipids extracted from the hepatocellular carcinoma tissues and cirrhotic livers of patients and from a normal liver with these monoclonal antibodies revealed that sulfated glycolipids having simple carbohydrate structures such as SM3 accumulated significantly in the cirrhotic liver (2 of 4 cases) as well as hepatocellular carcinoma tissue (15 of 17 cases, 88%), and more complex sulfated glycolipids and highly acidic glycolipids were much more specific to hepatocellular carcinoma tissues (10 of 17 cases, 59%) compared to the cirrhotic liver (0 of 4 cases).

Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium.

The carbohydrate antigen, sialyl Lex, is known to be a ligand for the cell adhesion molecule called ELAM-1 (E-selectin, endothelial cell leukocyte adhesion molecule-1), which is present on cytokine-activated human endothelial cells. Recently, we reported that another carbohydrate antigen, sialyl Lea, can also serve as a ligand for ELAM-1 (A. Takada, K. Ohmori, N. Takahashi, K. Tsuyuoka, K. Yago, K. Zenita, A. Hasegawa, and R. Kannagi, Biochem. Biophys. Res. Commun., 179: 713-719, 1991). Both sialyl Lex and sialyl Lea are expressed in many human malignant cells. In order to assess the contribution of these carbohydrate antigens to the adhesion of human malignant cells to vascular endothelium, we selected a panel of 12 cultured human epithelial cancer cell lines and a panel of 12 human leukemia cell lines which express sialyl Lex and/or sialyl Lea antigens. All 12 epithelial cancer cell lines exhibited a clearly ELAM-1-dependent adhesion to cytokine-activated human umbilical vein endothelial cells, while only 3 of the 12 leukemia cell lines exhibited significant participation of ELAM-1 in the adhesion. With regard to epithelial cancer cells, the adhesion of 6 cancer cell lines, mostly of colon and pancreas origin, was dependent almost exclusively on sialyl Lea. A significant contribution of the sialyl Lex antigen was noted in the adhesion of the other 6 cell lines, including cancers of lung and liver origin. These results imply that the sialyl Lea/ELAM-1 adhesion system, as well as the sialyl Lex/ELAM-1 adhesion system, plays an important role in the adhesion of human cancer cells to human umbilical vein endothelial cells. With regard to leukemia cells, on the other hand, adhesion of the 3 leukemia cell lines that showed ELAM-1-dependent adhesion was mediated by the sialyl Lex antigen, and none of these leukemia cell lines expressed sialyl Lea or exhibited sialyl Lea-dependent adhesion.

Monoclonal antibodies directed to a disulfated glycosphingolipid, SB1a (GgOse4Cer-II3IV3-bis-sulfate), associated with human hepatocellular carcinoma.

Two murine monoclonal antibodies, 2H6G5 (IgM) and 4A9E10 (IgG3), were obtained by using either cultured human hepatocellular carcinoma cells (PLC/PRF/5) or the acidic glycolipid mixture prepared from the same cells as immunogens. The antigen in PLC/PRF/5 cell membranes recognized by both antibodies was identified as a disulfated acidic glycolipid, GgOse4Cer-II3IV3-bis-sulfate (SB1a). Both antibodies reacted specifically with SB1a, and no significant reactivity was noted with other sulfated glycolipids or gangliosides except that the antibody 2H6G5 showed a weak cross-reactivity with LacCer-II3-sulfate (SM3), another sulfated glycolipid which partly shares the same carbohydrate structure as SB1a. The SB1a antigen is a relatively minor glycolipid in PLC/PRF/5 cells, but it was strongly expressed at the surface of PLC/PRF/5 cells as ascertained by cytofluorometry using both antibodies. A significant amount of SB1a antigen was present in 3 of the acidic glycolipid fractions isolated from 15 human hepatocellular carcinoma tissues as well as in the acidic glycolipid fraction prepared from PLC/PRF/5 cells, while all the acidic glycolipid fractions prepared from cirrhotic livers and a normal liver were essentially negative for SB1a, as ascertained by both solid phase enzyme immunoassay and the thin-layer chromatography-immunostaining method. These results strongly suggest that the SB1a antigen as defined by these new monoclonal antibodies is associated with human hepatocellular carcinoma.

Expression of alpha-(1,3)-fucosyltransferases which synthesize sialyl Le(x) and sialyl Le(a), the carbohydrate ligands for E- and P-selectins,in human malignant cell lines.

Transcripts of alpha-(1,3)-fucosyltransferases in human epithelial cancer and leukemia cell lines were analyzed by Northern blotting and reverse transcriptase mediated-polymerase chain reaction using specific probes and primers which can discriminate between the transcripts derived from the four alpha-(1,3)-fucosyltransferase genes Fuc-TIII, IV, V, and VI. Flow cytometric analysis of the sialyl Le(x) and sialyl Le(a) antigens was also performed on the same cell lines. The sialyl Le(x) antigen was expressed on 14 of 15 epithelial cancer cell lines, and the sialyl Le(a) antigen was detected on 8 of them. The message of Fuc-TIII was detected in most of the epithelial cancer cell lines (14 of 15), which correlated with the surface expression of these carbohydrate determinants. In addition, the messages of Fuc-TIV and Fuc-TVI were detected in most epithelial cancer cell lines, while the message of Fuc-TV was undetectable in most of them. On the other hand, all leukemia cell lines were positively stained for sialyl Le(x), but none of them was stained for sialyl Le(a) in flow cytometry. The messages of Fuc-TIV++ were detected in all leukemia cell lines tested. Small quantities of Fuc-TIII, V, and/or VI messages were also detected in some leukemia cell lines in reverse transcriptase mediated-polymerase chain reaction analysis. These studies indicate that alpha-(1,3)-fucosyltransferase activities in epithelial cancer and leukemia cell lines are mixtures of multiple molecular species of alpha-(1,3)-fucosyltransferases. It is natural that epithelial cancer cells contain a significant amount of Fuc-TIII mRNA and leukemia cell lines contain Fuc-TIV mRNA, since their normal counterparts, normal epithelial cells and leukocytes, respectively, are known to contain these fucosyltransferases. The unexpectedly frequent occurrence of Fuc-TIV mRNA in epithelial cancer cell lines may be related to their retro-differentiation associated with tumorigenesis. Another unexpected finding was a weak but significant expression of the alpha-(1,3)-fucosyltransferases Fuc-TIII, V, and/or VI in leukemia cell lines detected by reverse transcriptase mediated-polymerase chain reaction analysis. Since these enzymes are known to be capable of synthesizing the sialyl Le(x) determinant, this finding implies a possibility that some of them may be involved in the synthesis of sialyl Le(x) in leukemia cells.

Stage-specific expression of cancer-associated type 1 and type 2 chain polylactosamine antigens in the developing pancreas of human embryos.

Expression of type 1 and type 2 chain carbohydrate antigens during the course of morphogenesis of human embryonic pancreas was investigated using specific monoclonal antibodies and compared with the carbohydrate antigen profiles of human pancreatic cancers. The type 2 chain antigens, such as stage-specific embryonic antigen 1 (Le(x)) and I-antigens, appeared much earlier than the type 1 chain antigens; the epithelial cells of primitive foregut were Le(x)+I-antigen- in the embryos at Carnegie stages 16-23, while the pancreatic primordial cells, which had differentiated from the Le(x)+ gut epithelial cells, were Le(x)-I-antigen+ at Carnegie stages 22-23. The type 1 chain antigens, such as Le(a), Le(b), Le(c), and their sialylated derivatives, were not expressed in any cells at these stages and appeared much later in the pancreas of the 10-12-week embryos, when the primitive pancreatic ductal cells in the primordia exhibited an extensive budding of the daughter cells. At this stage, Le(a) appeared and was expressed strongly in the epithelial cells of primitive pancreatic ducts as well as in the daughter cells that were destined to differentiate into future centroacinar cells; Le(b) was localized in the daughter cells which were to become future acinar cells; and Le(c) was specifically expressed in the daughter cells which were to form future Langerhans islets. With regard to the sialylated derivatives of Le(a), expression of the 2-3 sialyl Le(a) antigen was limited to the epithelial cells of the primitive pancreatic ducts, while the 2-6 sialyl Le(a) antigen was strongly expressed in the future centroacinar cells, which had differentiated from the corresponding daughter cells. Among these antigens, the Le(a) and 2-3 sialyl Le(a) antigens showed the highest incidence in human pancreatic cancer tissues. These results indicate that the expression of these carbohydrate antigens in embryonic pancreas is differentiation dependent and cell lineage specific and that most human pancreatic cancer cells mimic the carbohydrate antigen profile of the epithelial cells of the primitive pancreatic ducts in human embryos.

Factors affecting expression of glycolipid tumor antigens: influence of ceramide composition and coexisting glycolipid on the antigenicity of gangliotriaosylceramide in murine lymphoma cells.

Gangliotriaosylceramide (Gg3Cer) was previously described as a tumor-associated antigen in murine L5178Y lymphoma [Young, W. W., Jr., and Hakomori, S., Science (Wash. D.C.), 211: 487-489, 1981]. This paper describes the major factors affecting the expression of Gg3Cer at the surface of various clones of L5178Y lymphoma. Of 26 sublines that were recloned, six cell lines showing different degrees of Gg3Cer expression at the cell surface were used for analysis of the glycolipid composition as related to its cell surface antigenicity. Three remarkable correlations between glycolipid composition and the antigenicity of Gg3Cer have been found: (a) high-expressor sublines were characterized by a large proportion of a unique molecular species of Gg3Cer having alpha-hydroxypalmitic acid in its ceramide moiety in striking contrast to low expressors which did not contain this molecular species; (b) low expressors contained a large quantity of ganglio-N-tetraosylceramide (Gg4Cer) and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1 Cer (GM1b) gangliosides, whereas these glycolipids were almost absent in high-expressor clones; and (c) nonexpressors, which were converted from the high expressors in vivo through immunotherapy with the monoclonal antibodies to Gg3Cer, contained a large quantity of ganglio-N-tetraosylceramide and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1Cer. The nonexpressors should have an induced enzyme system to metabolize Gg3Cer to ganglio-N-tetraosylceramide and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal beta 1 leads to 4Glc beta 1 leads to 1Cer. Three factors, i.e., ceramide composition, coexisting glycolipids, and an antibody-dependent glycolipid change, are therefore important in determination of glycolipid antigenicity and antigen modulation by antibodies. The ceramide composition may affect glycolipid organization in membranes, and the coexisting glycolipid having a longer carbohydrate chain may mask the accessibility of antibody to the antigenic glycolipid. The antigenic modulation by the action of the antibody in vivo may be based on activation of a new glycosyltransferase.

Location and distribution of difucoganglioside (VI3NeuAcV3III3Fuc2nLc6) in normal and tumor tissues defined by its monoclonal antibody FH6.

The distribution of a novel difucoganglioside (6B ganglioside, NeuAc alpha 2----3Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4Glc beta 1----1Cer) in various normal adult and fetal tissues, as well as in cancer tissues, has been studied by immunoperoxidase staining with a specific monoclonal antibody, FH6, directed to this antigen. A large variety of embryonic and fetal tissues (stomach, colon, small intestine, pancreas, esophagus, lung, and heart) showed a diffuse, weakly positive staining, particularly in the epithelial layer, up to the 70th to 80th day of gestation. However, no staining was observed in various normal adult tissues, including gastrointestinal and glandular epithelial tissues which were stained positively by antibody N-19-9 (directed to sialyl-Lea) or CSLEXI (directed to sialyl-Lex). FH6-positive loci were limited to the proximal convoluted tubuli in kidney and granulocytes. In contrast, 44 of 76 cases of cancer tissue tested, including gastric, colonic, lung, breast, and renal cancers, showed clearly positive staining. The intensity of staining in gastric and colonic cancer tissues by FH6 antibody was weaker and less frequent, although the incidence of positive staining for lung (50%) and breast cancer (86%) was significantly higher than that of the antigen stained by monoclonal antibody FH4 (Y. Fukushi, S. Hakomori, and T. Shepard, J. Exp. Med., 159: 506-520, 1984), which is directed to the asialo core of the FH6 antigen. The antigen levels in the serum of patients with various cancers, inflammatory diseases, and normal subjects were determined by radioimmunoassay. The antigen level was found to be significantly higher in the serum of some patients with cancer, particularly lung, liver, and pancreatic cancers, as compared with the serum levels in other types of cancer, noncancerous diseases, and normal subjects.

Increased expression of UDP-galactose transporter messenger RNA in human colon cancer tissues and its implication in synthesis of Thomsen-Friedenreich antigen and sialyl Lewis A/X determinants.

A series of human nucleotide sugar transporters of the Golgi apparatus was recently cloned, including the transporters for UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc) and CMP-sialic acid (CMP-SA). We have examined the mRNA expression of these three transporters in human colon cancer tissues by reverse transcription-PCR analysis and compared it with that in nonmalignant colonic mucosa prepared from the same patients. The amount of mRNA for UDP-Gal transporter was significantly increased in colon cancer tissues compared with nonmalignant mucosa tissues (P = 0.035; n = 20). The increase was more prominent in patients with advanced colorectal cancer of Dukes' stages C and D, in which the amount of UDP-Gal transporter mRNA in cancer tissues showed on average about a 3.6-fold increase over the paired nonmalignant mucosa (statistically significant at P = 0.004; n = 14). The mRNA content of the other two transporters showed no significant difference between the paired cancer and normal tissues. When UDP-Gal transporter cDNA was stably transfected to cultured human colon cancer cells, the expression of Thomsen-Friedenreich (TF) antigen and of sialyl Lewis A (NeuAcalpha2-->3Galbeta1-->3[Fucalpha1-->4]GlcNAcbeta1-->R) and sialyl Lewis X (NeuAcalpha2-->3Galbeta1-->4[Fucalpha1-->3]GlcNAcbeta1-->R) determinants was significantly induced on transfectant cells, which resulted in markedly enhanced cell adhesion to vascular E-selectin. These findings suggest that the increase of UDP-Gal transporter mRNA is involved in the enhanced expression of cancer-associated carbohydrate determinants such as TF and sialyl Lewis A/X antigens in colon cancers.

Generation of two murine monoclonal antibodies that can discriminate N-glycolyl and N-acetyl neuraminic acid residues of GM2 gangliosides.

Since the preliminary analyses of the glycolipids of small cell carcinomas of the lung showed an increase of GM2 ganglioside, we generated new murine monoclonal antibodies directed to GM2 to identify the molecular species of the glycolipid. The monoclonal antibodies MK2-34 and MK1-16 (both IgM), which specifically detect N-glycolyl GM2 and N-acetyl GM2, respectively, were generated by immunizing mice with liposomes containing monophosphoryl lipid A, trehalose dimycolate, and the antigenic ganglioside. Among the glycolipid preparations extracted from the cancer tissues of 39 patients with lung cancer, a significant amount of N-acetyl GM2 was detected with MK1-16 antibody in 70% of the squamous cell carcinoma cases, 50% of the lung adenocarcinoma cases, 33% of the large cell carcinoma cases, and 100% of the cases of small cell carcinoma of the lung. On the other hand, N-glycolyl GM2 which was defined by the monoclonal MK2-34 was not found in any of the glycolipid fractions prepared from the lung cancer tissues examined in this study. Immunohistochemical studies of the lung cancer tissues with the MK1-16 antibody showed that the N-acetyl GM2 was present not only in small cell carcinoma tissues as one of the antigens related to tumors of neuroectodermal origin, but also in the squamous cell carcinoma and adenocarcinoma of the lung with a comparable frequency. The appearance of the N-acetyl GM2 antigen correlated well with the degree of differentiation of the cancer cells in patients with squamous cell carcinoma and adenocarcinoma of the lung.

Quantitative and qualitative characterization of human cancer-associated serum glycoprotein antigens expressing epitopes consisting of sialyl or sialyl-fucosyl type 1 chain.

The levels of carbohydrate antigens having epitopes consisting of type 1 chain (R----Gal beta 1----GlcNAc beta 1----3Gal beta 1----R) in the sera of patients with various malignant and nonmalignant disorders have been investigated with the use of three monoclonal antibodies, N-19-9, FH-7, and FH-9. Serum levels of 2----3 sialylated Lea antigen and 2----6 sialylated Lea antigen, defined respectively by antibodies N-19-9 and FH-7, were found to be frequently high in patients with cancer of the digestive system, particularly pancreatic cancer. High levels of 2----3,2----6 disialylated Lc4 antigen, defined by antibody FH-9, were less frequent in cancer patients when compared with the other two antigens. In patients with nonmalignant disorders, especially renal and autoimmune diseases, serum levels of the two type 1 chain antigens defined by FH-7 and FH-9 were more frequently high than that defined by N-19-9. Molecular weights and other general biochemical characteristics of serum mucin carrying the type 1 chain determinants were not significantly different in cancer patients as compared with patients with nonmalignant disorders. However, the degree of glycosylation of the antigen, as assessed by its solubility in perchloric acid, showed significant differences; i.e., the mucin antigen carrying 2----6 sialylated Lea determinant in the sera of patients with nonmalignant disorders had the highest carbohydrate/protein ratio, followed by the mucin carrying the same determinant in the sera of cancer patients. Mucin antigen carrying 2----3 sialylated Lea antigen or 2----3, 2----6 disialylated Lc4 antigen in cancer patients had the lowest carbohydrate/protein ratio among the four groups tested. Thus, the carbohydrate/protein ratio in the type 1 chain mucin antigens in sera of normal subjects is higher than that in sera of cancer patients (P less than 0.05). This finding is in contrast to previous findings on the mucin antigens carrying the type 2 chain determinant (R. Kannagi et al., Cancer Res., 46: 2619-2626, 1986), in which the mucin antigen in cancer patients was found to have a much higher carbohydrate/protein ratio than that carrying the same antigenic determinants in patients with nonmalignant disorders.

Gangliosides and sialoglycoproteins carrying a rare blood group antigen determinant, Cad, associated with human cancers as detected by specific monoclonal antibodies.

Two murine monoclonal antibodies, 2A3D2 and 2D11E2 (both IgM), which are directed to the gangliosides and sialoglycoproteins related to a rare blood group antigen, Cad, were obtained by using a ganglioside mixture prepared from human hepatocellular carcinoma cells (PLC/PRF/5) as the immunogen. These two monoclonal antibodies detected multiple ganglioside antigens present in the PLC/PRF/5 cells, and the major antigenic ganglioside was characterized as IV4GalNAc beta-GD1a, which has the carbohydrate structure GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3GalNAc beta 1---- 4(NeuAc alpha 2----3)Gal beta 1----Cer. The two antibodies also reacted with GM2 (GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer) and a Cad-active lactoseries ganglioside (IV4GalNAc beta-sialosylparagloboside, GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4GlcNAc beta 1---- 3Gal beta 1----4Glc beta 1----Cer), which have carbohydrate structures related to IV4GalNAc beta-GD1a. Beside gangliosides, both antibodies recognized the carbohydrate determinant carried by glycophorin A on very rare Cad-positive human RBC; the structure of which is GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3(NeuAc alpha 2---- 6)GalNAc alpha 1----Ser/Thr. From these findings, it is clear that monoclonal antibodies 2A3D2 and 2D11E2 both recognize the nonreduced carbohydrate terminus composed of three sugar residues, GalNac beta 1----4(NeuAc alpha 2----3)Gal beta 1----R, and are useful for detecting the Cad-related antigen in cells and tissues. By using these monoclonal antibodies, it was revealed that many cultured human hepatocellular carcinoma cell lines and cancer tissues taken from patients with hepatocellular carcinoma contain both Cad-active glycoprotein antigens and related gangliosides, while normal liver tissues contain no appreciable amount of either species of antigen. The Cad-active glycoprotein antigens in cultured human hepatocellular carcinoma cells appeared as triplet bands having molecular weights of 92,000, 75,000, and 61,000, under either reducing or nonreducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Essentially the same triplet proteins were observed in as many as 4 of 9 cases (44%) of cancer tissue from patients with hepatocellular carcinoma, but not in neighboring cirrhotic tissues or normal livers tissues. These results suggest that the rare blood group antigen Cad is associated with human cancers, especially hepatocellular carcinoma.

Quantitative and qualitative characterization of human cancer-associated serum glycoprotein antigens expressing fucosyl or sialyl-fucosyl type 2 chain polylactosamine.

The quantity of tumor-associated antigens carrying type 2 chain polylactosamines with four types of fucosyl determinants, LeX (X-hapten), poly-LeX, sialyl LeX, and LeY (Y-hapten), present in sera of patients with various malignant and non-malignant disorders, as well as the qualitative chemical properties of the carrier molecules in sera, have been investigated using four monoclonal antibodies, each of which defines one of these determinants. The following findings are of particular importance: the serum levels of LeX defined by antibody FH2 and poly-LeX defined by ACFH18 in patients with cancer were occasionally high (incidence about 10%); however, the majority of patients did not show elevated levels; the serum level of the antigen, defined by monoclonal antibody FH6 (termed sialyl LeX-i since this determinant is carried by i antigen), was significantly high in patients with cancers originating from organs from which adenocarcinomas often develop. For example, among various types of lung cancer, only adenocarcinoma but not squamous cell carcinoma, small cell carcinoma, or large cell carcinoma showed a high level of sialyl LeX-i antigen in sera. The incidence of high antigen levels in sera of patients with adenocarcinomas of lung was as high as 76% of the observed cases; the serum level of Ley (Y-hapten) was frequently high in patients with hepatoma (incidence, 34%); sialyl LeX-i antigen was separated on gel filtration as a glycoprotein with an average molecular weight greater than 10(6). It was characterized by its susceptibility to basehydrolysis, Pronase digestion, and sialidase and endo-beta-galactosidase treatment and is assumed to be a high molecular weight mucin-type glycoprotein; sialyl LeX-i antigen expressed in sera of patients with cancer was soluble in perchloric acid, while the same antigen in sera of patients with noncancerous diseases and normal subjects was mostly insoluble in perchloric acid. LeX, a poly-LeX, and essentially all LeY antigens in sera of patients with cancer were perchloric acid-insoluble.

Expression of sialyl 6-sulfo Lewis X is inversely correlated with conventional sialyl Lewis X expression in human colorectal cancer.

Sialyl 6-sulfo Lewis X determinant has been described recently as a major ligand for L-selectin on high endothelial venules of human peripheral lymph nodes. From our investigation of its distribution in human colorectal cancer tissues and cultured colon cancer cells, the sialyl 6-sulfo Lewis X determinant was preferentially expressed in the nonmalignant colonic epithelia rather than cancer cells (P < 0.001; n = 23). This was in contrast to the distribution of conventional sialyl Lewis X, which was preferentially expressed in cancer tissues rather than nonmalignant epithelia (P = 0.007; n = 23), indicating that 6-sulfation predominantly occurs in nonmalignant tissues and is suppressed upon malignant transformation. In confirmation of this, a nonsialylated determinant 6-sulfo Lewis X was also found to be preferentially localized in the nonmalignant epithelia. Significant expression of sialyl 6-sulfo Lewis X was observed in only 2 lines, whereas 8 were positive for conventional sialyl Lewis X, among 13 cultured colon cancer cell lines. Transfection of cells with fucosyltransferase (Fuc-T) VI induced expression of sialyl 6-sulfo Lewis X, whereas transfection of Fuc-T III did not, suggesting that the determinant was synthesized mainly by Fuc-T VI in colonic epithelia. Members of the sialic acid cyclase pathway, the de-N-acetyl sialyl 6-sulfo Lewis X and cyclic sialyl 6-sulfo Lewis X determinants, were also preferentially expressed in the nonmalignant epithelia rather than colonic cancer cells (P < 0.001; n = 23). Stimulation of the sialyl 6-sulfo Lewis X-positive colon cancer cell line with a calcium ionophore ionomycin markedly reduced sialyl 6-sulfo Lewis X and induced cyclic sialyl 6-sulfo Lewis X expression. These results suggested that the metabolic conversion of sialyl 6-sulfo Lewis X into cyclic sialyl 6-sulfo Lewis X by a calcium-dependent enzyme, sialic acid cyclase, as we hypothesized for human leukocytes previously (C. Mitsuoka et al., Proc. Natl. Acad. Sci. USA, 96: 1597-1602, 1999), also occurs in nonmalignant colonic epithelia.