Characterization of N-glycolylneuraminic acid-containing gangliosides as tumor-associated Hanganutziu-Deicher antigen in human colon cancer

Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid (NeuGc)-containing gangliosides were isolated and characterized from human colon cancer tissues. The antigenic gangliosides were detected by thin-layer chromatography by our newly developed method (H. Higashi, Y. Fukui, S. Ueda, S. Kato, Y. Hirabayashi, M. Matsumoto, and M. Naiki. J. Biochem., 95: 1517-1520, 1984) of enzyme immunostaining using affinity-purified chicken antibody against hematoside containing NeuGc (II3NeuGc-LacCer) and horseradish peroxidase-conjugated rabbit anti-chicken lgG. One to six species of the antigenic gangliosides were isolated from seven of 16 cases of colon cancer, whereas no antigenic compound was detected in all apparently normal colorectal tissues from 17 individuals without colorectal cancer. Tissues from different patients showed different patterns of molecular species of the antigenic gangliosides. Densitometric determination indicated that HD antigenic sialic acid, NeuGc, accounted for about 1% or less of the total lipid-bound sialic acids. Four species of antigenic gangliosides were identified as hematoside and hematoside-containing O-acyl ester (II3NeuGc-LacCer and II3 4- or 7-O-acyl-NeuGc-LacCer), GM2-containing NeuGc (II3NeuGc-GgOse3Cer), and sialylparagloboside (IV3NeuGc-nLcOse4Cer) by their behaviors on 2-dimensional thin-layer chromatography, and the effects of mild alkaline treatment, sialidase treatment, periodate oxidation, and endo-beta-galactosidase treatment.     

Specific expression of unusual GM2 ganglioside with Hanganutziu-Deicher antigen activity on human colon cancers

This paper reports the presence of GM2 ganglioside containing N-glycolylneuraminic acid (NeuGc) in human colon cancer tissues. GM2(NeuGc) was detected by two-dimensional thin layer chromatography (2d-TLC)/enzyme-immunostaining using affinity-purified chicken antibody against GM3(NeuGc) and horseradish peroxidase-conjugated rabbit anti-chicken IgG antibody. Like usual GM2 ganglioside containing N-acetylneuraminic acid (NeuAc) isolated from Tay-Sachs brain, GM2(NeuGc) in colon cancer could be converted into GM3(NeuGc) by human kidney beta-N-acetylhexosaminidase A in the presence of a GM2-specific activator protein isolated from guinea pig kidney. Three of 7 specimens of Hanganutziu-Deicher (HD) antigen-positive human colon cancer tissues so far examined expressed this unique ganglioside. In order to detect and determine specifically GM2(NeuGc) on human colon cancers, specific antibody against GM2 (NeuGc) has been prepared by immunizing chickens. By a sensitive TLC/immunostaining method using the antibody, the amounts of the antigen were determined to be 0.3-3% of total lipid-bound sialic acid. NeuGc-containing gangliosides were also detected in meconium and fetal intestinal tissues. Three species of antigenic gangliosides in pooled meconium were tentatively identified as GM3(NeuGc), sialylparagloboside and sialylhexaosylceramide on the basis of their migration positions on 2d-TLC and the results of endo-beta-galactosidase treatment. GM3(NeuGc) was the sole HD-active ganglioside in fetal intestinal tissue from one of 3 individuals tested; the other two showed no HD-active ganglioside at all. GM2(NeuGc), however, could not be detected in either meconium or fetal tissues so far examined, suggesting that this unique ganglioside is a tumor-specific antigen, at least for human intestinal tissues.     

Hypoxic culture induces expression of sialin, a sialic acid transporter, and cancer-associated gangliosides containing non-human sialic acid on human cancer cells

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.     

Detection of Gangliosides as N-Glycolylneuraminic Acid-specific Tumor-associated Hanganutziu-Deicher Antigen in Human Retinoblastoma Cells

Gangliosides were shown to bear the tumor-associated N -glycolylneuraminic acid (NeuGc)-specific Hanganutziu-Deicher (HD) antigen expressed in human retinoblastoma cells. HD antigenie gangliosides were detected by thin-layer chromatography/enzyme-immunostaining using affinity-purified chicken antibody against GM3 containing NeuGc and horseradish peroxidase-conjugated anti-chicken IgG. One to four species of the antigenic gangliosides were detected from all of 4 cell lines, Y79, WERI-Rb1, TOTL1, and YK, as well as freshly cultured retinoblastoma cells and isolated tumor tissue. All cases contained GMS(NeuGc) as an HD antigen. No HD antigenic ganglioside was detected in normal retinal tissues by the same procedure.     

Detection of gangliosides as N-glycolylneuraminic acid-specific tumor-associated Hanganutziu-Deicher antigen in human retinoblastoma cells

Gangliosides were shown to bear the tumor-associated N-glycolylneuraminic acid (NeuGc)-specific Hanganutziu-Deicher (HD) antigen expressed in human retinoblastoma cells. HD antigenic gangliosides were detected by thin-layer chromatography/enzyme-immunostaining using affinity-purified chicken antibody against GM3 containing NeuGc and horseradish peroxidase-conjugated anti-chicken IgG. One to four species of the antigenic gangliosides were detected from all of 4 cell lines, Y79, WERI-Rb1, TOTL1, and YK, as well as freshly cultured retinoblastoma cells and isolated tumor tissue. All cases contained GM3(NeuGc) as an HD antigen. No HD antigenic ganglioside was detected in normal retinal tissues by the same procedure.     

Exogenous incorporation of neugc-rich mucin augments n-glycolyl sialic acid content and promotes malignant phenotype in mouse tumor cell lines

Background

Carbohydrates embedded in the plasma membrane are one of the main actors involved in the communication of cells with the microenvironment. Neuraminic sialic acids are glycocalyx sugars that play important roles in the modulation of malignant cell behaviour. N-glycolylneuraminic acid (NeuGc) is synthesized by the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), an enzyme expressed in all mammals except humans. In mice, this sugar is synthesized in several somatic tissues.

Methods

We used the B16 melanoma and F3II mammary carcinoma mouse tumor cell lines. By CMAH directed RT-PCR and NeuGc detection with the specific anti-NeuGc-GM3 antibody 14F7 we evaluated enzyme and ganglioside expression in tumor cells, respectively. Expression of NeuGc-GM3 ganglioside was reached by in vitro incubation with NeuGc-rich bovine submaxillary mucin and evaluated by slot-blot and immunohistochemistry assays using the 14F7 antibody. Tumor cells treated with mucin or purified NeuGc were injected s.c. and i.v. in syngeneic mice to evaluate tumor and metastatic growth.

Results

In the present work we demonstrated the absence of expression of CMAH enzyme in B16 melanoma and F3II mammary carcinoma cells. In vitro incubation of these NeuGc-negative cells with NeuGc-rich mucin increased the presence of NeuGc in cell membranes for at least 48-72 h, as a component of the GM3 ganglioside. Preincubation with NeuGc-rich mucin reduced tumor latency and increased the metastatic potential of tumor cells in syngeneic animals. Similar results were obtained when cells were incubated with purified NeuGc alone.

Conclusion

Our results indicate that B16 and F3II mouse tumor cell lines do not express NeuGc in cell membranes but they are able to incorporate NeuGc from an exogenous source, contributing to the malignant phenotype of melanoma and mammary carcinoma cells.     

Monoclonal antibody to chicken fetal antigen on Marek's disease lymphoblastoid cell line MDCC-MSB1

A monoclonal antibody (2H3) to chicken fetal antigen (CFA) expressed on the cell surface of the Marek's disease lymphoblastoid cell line MDCC-MSB1 was generated. 2H3 reacted specifically with various cells from chicken embryos, especially with fetal red blood cells, fetal bursa-derived lymphocytes, fetal liver cells, and embryo fibroblasts but not with fetal peripheral blood lymphocytes. 2H3 reacted also with all lymphoblastoid cell line cells tested: Marek's disease, avian leukosis, and reticuloendotheliosis line cells. On the basis of 2H3 specificity, CFA detected by 2H3 seemed to be similar to chicken fetal red blood cell antigen previously reported, but not to chicken alpha-fetoprotein. A transient increase of reactivities with 2H3 was observed in lymphocytes prepared from thymus, spleen, and bursa of chickens inoculated with Marek's disease virus or herpesvirus of turkeys from 7 to 21 days postinoculation. The average percentage of CFA-positive cells in lymphoid cells from Marek's disease virus-infected chickens at the peaks was higher than that of the cells in Marek's disease tumor cells from 146- and 156-day-old chickens in the field. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis revealed that the monoclonal 2H3 is recognized a Mr 50,000 oncodevelopmental cell surface antigen present on MDCC-MSB1 cells. The possible differences between CFA detected by 2H3 and chicken fetal red blood cell antigen expressed on normal chicken fetal erythrocytes are discussed.     

Pathogenesis of Marek's disease: early appearance of Marek's disease tumor-associated surface antigen in infected chickens.

Marek's disease tumor-associated surface antigen (MATSA) appeared in different lymphoid tissues of P-line chickens soon after they were infected with BC-1 strain of Marek's disease virus. MATSA-bearing cells first appeared in spleens by 5 days post infection (PI) and were observed through a 21-day experimental period at mean levels varying from 3.8 to 21.9% of the total cells examined. Lower percentages of MATSA-bearing cells were observed in the thymus, in the bursa of Fabricius, among peripheral blood lymphocytes, and among bone marrow cells beginning 7 days PI. The antigen was not detected on normal lymphocytes from chickens or chicken embryos, nor was it detected on cultured chicken embryo fibroblasts infected or transformed by avian RNA tumor viruses.     

Isolation from a transmissible lymphoid tumour (TLT) lymphoblastoid cell line of a herpesvirus similar to Marek's disease virus.

A chicken lymphoblastoid cell line (TLT)-6855 originally established from an avian oncornavirus-induced lymphoma (Siegfried and Olson, 1972) was studied for the presence and expression of Marek's disease virus (MDV) genome. By nucleic acid hybridization a significant amount of MDV DNA was found in this cell line. This virus DNA, however, was not expressed in either virus-specific intracellular or membrane antigens or the MD-associated tumour-specific surface antigen (MATSA). Moreover, MDV-specific antigens could not be activated in this cell line by treatment with 5-IdUrd. In several experiments, when chickens were inoculated with the cell line a herpesvirus was repeatedly isolated from the kidneys. This herpesvirus was antigenically similar to MDV but was low in oncogenicity for chickens.     

Transplantable Marek's disease lymphomas. II. Variable tumor immunity induced by different lymphoblastoid cells

The effectiveness of cells from 6 different Marek's disease (MD) lymphoblastoid cell lines to induce immunity to syngeneic transplantable MD lymphomas was investigated in 2 related inbred lines of White Leghorn chickens (lines G-B1 and G-B2) that have different major histocompatibility complex (MHC) genotypes. Cells from 2 line G-B1 lymphomas (MDCT-NYM1 and MDCT-UG1) and 1 line G-B2 lymphoma (MDCT-UG2) were used for challenge. Three of the lymphoblastoid cell lines tested were developed from these lymphomas. Growth of palpable lymphomas was lowest among G-B2 chickens immunized with syngeneic lymphoblastoid cells. Protection against the early lethal effects of the highly virulent transplantable lymphomas was greatest in both lines of chickens when the lymphoblastoid cells were syngeneic with the hosts. Lymphoblastoid cells of unknown MHC type either failed to induce immunity to the lymphomas in both lines or protected some line G-B2 chickens challenged with syngeneic MDCT-UG2 lymphoma cells.     

Detection of N‐glycolyated gangliosides in non‐small‐cell lung cancer using GMR8 monoclonal antibody

Gangliosides are glycosphingolipids found on the cell surface. They act as recognition molecules or signal modulators and regulate cell proliferation and differentiation. N‐glycolylneuraminic acid (NeuGc)‐containing gangliosides have been detected in some neoplasms in humans, although they are usually absent in normal human tissues. Our aim was to evaluate the presence of NeuGc‐containing gangliosides including GM3 (NeuGc) and assess their relationship with the prognosis of non‐small‐cell lung cancer (NSCLC). NeuGc‐containing ganglioside expression in NSCLC tissues was analyzed immunohistochemically using the mouse monoclonal antibody GMR8, which is specific for gangliosides with NeuGc alpha 2,3Gal‐terminal structures. On the basis of NeuGc‐containing ganglioside expression, we performed survival analysis. We also investigated the differences in the effects of GM3 (N‐acetylneuraminic acid [NeuAc]) and GM3 (NeuGc) on inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase in A431 cells. As a result, the presence of NeuGc‐containing gangliosides was evident in 86 of 93 (93.5%) NSCLC samples. The NSCLC patients with high NeuGc‐containing ganglioside expression had a low overall survival rate and a significantly low progression‐free survival rate. In the in vitro study, the inhibitory effect of GM3 on EGFR tyrosine kinase in A431 cells after exposure to GM3 (NeuGc) was lower than that after exposure to GM3 (NeuAc). In conclusion, NeuGc‐containing gangliosides including GM3 (NeuGc) are widely expressed in NSCLC, and NeuGc‐containing ganglioside expression is associated with patient survival. The difference in the effects of GM3 (NeuGc) and GM3 (NeuAc) on the inhibition of EGFR tyrosine kinase might contribute to improvement in the prognosis of NSCLC patients. (Cancer Sci 2013; 104: 43–47)     

Presence of natural killer cells in specific-pathogen-free chickens.

Spleen cells of normal specific-pathogen-free N, P, and 15 X 7 chickens were cytotoxic in vitro for target cells of Marek's disease lymphoma line MSB-1. The natural killer (NK) cell activity, best expressed in chickens over 7 weeks old, varied among several genetic lines of chickens tested. The NK cells were thermolabile, and incubation of the cells at 37 degrees C for 30--60 minutes resulted in substantial loss of cytotoxicity. The specificity of NK effector cells was directed against common antigen(s) on tumor cell lines of diverse origin but not on normal adult or embryonic cells.     

Establishment of Marek's disease lymphoblastoid cell lines from transplantable versus primary lymphomas

Six new Marek's disease (MD) lymphoblastoid cell lines were established in vitro by cultivation in a medium containing 2-mercaptoethanol (2-ME). Attempts using primary lymphoma cells were generally unsuccessful; only one of 28 lymphomas yielded a cell line and that one came from an experimentally immunosuppressed chicken. In contrast, two of seven low-passage, and two of two established MD transplantable lymphomas grew readily in vitro. A sixth line was obtained using buffy coat cells from a leukemic chicken. It was concluded that the use of transplantable tumor cells and a medium containing 2-ME provided a combination highly suited to the establishment of cell lines from MD.     

Characteristics of JMV Marek's disease tumor: a nonproductively infected transplantable cell lacking in rescuable Virus.

Cells of the JMV Marek's disease (MD) tumor, originally produced by rapid serial passage of MD lymphoma cells in chickens, were characterized to determine whether they were of host or donor origin and to ascertain certain virus-host cell interrelationships. Differences noted in blood group B surface alloantigens between tumor cells and host lymphocytes indicated a probable nonhost origin (i.e., transplantability) of the tumor. JMV spleen tumors contained predominantly large lymphoblasts bearing MD tumor-associated surface antigen. DNA from JMV tumor cell suspensions hybridized significantly with MD virus cRNA, which indicated that JMV cells contained at least a portion of the MD virus genome. No MD virus was rescued from JMV tumors by techniques suitable for rescue of virus from MD lymphomas. The JMV tumor cells were also devoid of MD virus-specific antigens. These properties differed markedly from those of MD lymphoma cells and make the JMV tumor cell a unique, potentially valuable, tool for further study of oncogenic herpesvirus infection and tumor immunity in the chicken.     

Inhibitory effects of macrophages against Marek's disease virus plaque formation in chicken kidney cell cultures.

Inhibition of plaque formation by Marek's disease virus (MDV) in chicken kidney cell cultures was investigated with the use of peritoneal exudate cells (PEC) from chickens. PEC from MDV-infected White Leghorn chickens inhibited the formation of MDV plaques, whereas the inhibitory effect of PEC from chickens vaccinated with herpesvirus of turkey (HVT) or PEC from normal chickens was very weak. However, PEC from either normal chickens or HVT-vaccinated chickens inhibited the MDV plaque formation in the presence of serum from MDV-infected chickens but not from normal or HVT-vaccinated chickens. The capacity of PEC to inhibit plaque formation was significantly reduced when PEC was treated with carrageenan but not with antithymus or antibursa cell serum. These results indicate that macrophages may have a role in protection against Marek's disease by reducing the number of MDV-infected cells and thereby decreasing the spread of the virus in vivo.     

Application of tyramide signal amplification for detection of N-glycolylneuraminic acid in human hepatocellular carcinoma

Background. N-Acetylneuraminic acid and N-glycolylneuraminic acid (NeuGc) are the most common sialic acids in mammals, and NeuGc has attracted attention as a tumor-associated antigen. Methods. In frozen liver sections from patients with hepatocellular carcinoma, glycolipid-type NeuGc was detected on the surface of liver cancer cells in 9 of 17 samples (52.9%) by immunostaining, using two chicken monoclonal antibodies against NeuGc and the tyramide signal amplification method. When conventional immunostaining without amplification was used, all 17 specimens tested were negative. Results. Increased serum levels of anti-NeuGc IgG and/or IgM were observed in 13 of the17 patients with hepatocellular carcinoma (76.5%). The presence of these antibodies was mostly attributed to the expression of NeuGc on hepatocellular carcinoma cells. Of the subjects with small HCCs (diameter 3cm or less), 6 of 10 were positive for serum anti-NeuGc antibodies; however, 1 of these was negative for both serum -fetoprotein (AFP) and for prothrombin–induced vitamin K antagonist II (PIVKA-II). There was no correlation between serum AFP- or PIVKA-II, levels and the presence of NeuGc or anti-NeuGc IgG and/or IgM. Conclusion. The tyramide signal amplification method is useful for the immunohistochemical detection of low-level NeuGc expression by hepatocellular carcinoma cells. We therefore consider that measurement of serum levels of anti-NeuGc antibodies is clinically meaningful and that anti-NeuGc antibody may be a useful screening test, in combination with AFP and PIVKA-II, for the early diagnosis of hepatocellular carcinoma.     

Detection of a new antigen associated with chicken thrombocytes on Marek's disease lymphoblastoid cell line

A monoclonal antibody, 2C12, produced against MDCC-MSB1 cells, reacted with several cell lines derived from Marek's disease (MD), including MDCC-MSB1 cells, normal chicken thrombocytes, and MD tumor cells. It did not react with cell lines derived from avian leukosis or reticuloendotheliosis, cells from normal chicken thymus and bursa, chicken kidney cells and quail fibroblast cultures inoculated with MD virus, or with erythrocytes from 1-day-old and adult chickens, cow, sheep, and horse. Anti-thrombocyte serum prepared in a rabbit reacted with thrombocytes and MDCC-MSB1 cells. The specificities of antibody 2C12 and anti-thrombocyte serum against MDCC-MSB1 cells and thrombocytes were confirmed by cross-absorption, blocking, and double-membrane fluorescent antibody tests. The existence of a polypeptide with an apparent molecular weight of 103,000, common to both MDCC-MSB1 cells and thrombocytes, was demonstrated by Western blotting analyses.     

Occurrence of tumor-associated ganglioside antigens with Hanganutziu-Deicher antigenic activity on human melanomas

The specificity of antibody to NeuGc alpha 2-3Gal beta 1-4Glc-cer (GM3(NeuGc] was carefully reexamined by the method of enzyme-immunostaining on a thin layer plate. The affinity-purified antibody was found to react with NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuGc-NeuGc] and NeuGc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuGc-NeuAc], but not with NeuAc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuAc-NeuGc)) or NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuAc-NeuAc]. From this result together with the previous results, it (GD3(NeuAc-NeuAc], From this result together with the previous results, it could be concluded that the antibody recognizes the outer portion of molecular species of sialic acids in the gangliosides. By using this antibody, the expression of Hanganutziu-Deicher (HD) gangliosides could be demonstrated in human malignant melanoma. The molecular species were different among individuals examined. Among HD-antigenic gangliosides, GM3(NeuGc) was commonly found in melanoma tissues. One of the patients examined expressed GD3(NeuGc-NeuGc) and GD 3(NeuGc-NeuAc), which may be characteristic gangliosides in human melanomas, since these gangliosides could not be detected in human colon cancer or human fetal tissues.     

Differences in surface sialic acid and galactosyl residues of two autologous human melanoma cell lines

Cell surface carbohydrate differences were observed between two human cell lines initiated from primary melanoma and metastasis of the same patient. Although total sialic acid content was similar in both cell lines, neuraminidase-released sialic acid was twice as high in metastatic cells than that of primary cells. One class of Concanavalin A binding sites with similar affinity constant was found in untreated and neuraminidase-treated cells in both cell lines. Before surface sialic acid release, the primary cell line expressed two classes of Ricinus lectin binding sites with high and low affinity; the cell line of metastatic origin had only one class of Ricinus lectin binding sites with low affinity. After neuraminidase treatment, the number of Ricinus lectin binding sites with low affinity increased two- or three-fold in both cell lines, whereas the high-affinity binding sites were not observed in primary cells. The present data indicated that differences in surface sialic acid level modified the Ricinus lectin binding in two human melanoma cell lines. However, the ability of the cells to bind Concanavalin A was not changed.