Expression of α2,6-sialic acid-containing and Lewis-active glycolipids in several types of human ovarian carcinomas

Abstract

To identify glycolipid antigens associated with histologically defined types of ovarian carcinomas, we determined the amounts of α2,6-sialyl and Lewis-active glycolipids, the specific activities of the α2,3- and α2,6-sialyltransferases, and the gene expression of sugar transferases in mucinous and serous cystadenocarcinoma, clear cell adenocarcinoma and endometrioid carcinoma tissues and cell lines derived from them. α2,6-sialyl glycolipid IV(6)NeuAcα-nLc(4)Cer detected with a newly developed monoclonal antibody, Y916, was present in 5/7 serous cystadenocarcinoma cases in relatively higher amounts than those in the other carcinoma tissues. On the other hand, the amounts of Lewis-active glycolipids in serous cystadenocarcinoma tissues were lower than those in the other carcinoma tissues. No correlation was observed between the structures of Lewis glycolipids and the histological classification. The gene expression of α2,3- and α2,6-sialyltransferases and α1,3/4-fucosyltransferase for the synthesis of Lewis-active glycolipids was not positively correlated with the amounts of the respective glycolipids, probably due to the epigenetic regulation of transferases in the overall metabolic pathways for lacto-series glycolipids. However, the amounts of GM3 and GD3 with short carbohydrate chains correlated with the relative intensities of GM3 and GD3 synthase gene expression, respectively. Among ovarian carcinoma-derived cell lines, the serous cystadenocarcinoma-derived ones exhibited a lower frequency of Lewis-active glycolipid expression than the other carcinoma-derived ones, which was similar to that in the respective tissues. Thus, malignancy-related Lewis-active glycolipids were shown to be regulated in different modes in ovarian serous cystadenocarcinomas and the other carcinomas.

Figure 1

Thin layer chromatography (TLC) and TLC-immunostaining of glycolipids with monoclonal antibodies Y916 and 5h6. (1) GM3, (2) IV³NeuAcα-nLc₄Cer, (3) GD3 and (4) IV⁶NeuAcα-nLc₄Cer were developed on plastic-coated TLC plates with chloroform/methanol/0.5% CaCl₂ in water (55:45:10, by volume). Spots were visualized with orcinol-H₂SO₄ reagent and monoclonal antibodies Y916 and 5h6.

Figure 2

TLC-immunostaining of glycolipids from human ovarian carcinoma tissues. Glycolipids, corresponding to 0.2 mg dry weight, were developed with chloroform/methanol/0.5% CaCl₂ in water (55:45:10, by volume). Spots were visualized by TLC-immunostaining with anti-Le^b, anti-Le^Y, anti-Le^X, anti-sialyl Le^a, and Y916 and 5h6 antibodies. The specimen numbers are the same as those shown in Table I. Standard glycolipids for Y916 was a mixture of GD3 and IV⁶NeuAcα-nLc₄Cer.

Figure 3

TLC-immunostaining of glycolipids from human ovarian mucinous and serous cystadenocarcinoma tissues. Glycolipids were obtained from an additional five specimens, respectively, i.e., other than those in Fig. 2 and Table I. The glycolipids, corresponding to 0.1 mg dry weight, were used for TLC-immunostaining with monoclonal Y916 and anti-Le^b antibodies as described in Materials and methods. Standard glycolipids for Y916 was a mixture of GD3 and IV⁶NeuAcα-nLc₄Cer.

Figure 4

TLC-immunostaining of glycolipids from ovarian carcinoma-derived cells. Glycolipids, corresponding to 0.2 mg dry weight, were used for TLC-immunostaining with anti-Le^b and anti-Le^Y antibodies. Lanes: 1, RMG-1; 2, HAC-2; 3, HMKOA; 4, HNOA; 5, 2008 and 6, KF28.

Figure 5

TLC-immunostaining of products following reactions of α2,3- and α2,6-sialyltransferases (SAT). The products following the reactions with 50 μg of enzyme proteins were developed with chloroform/methanol/0.5% CaCl₂ in water (55:45:10, by volume), and detected by TLC-immunostaining with monoclonal antibodies 5h6 for IV³NeuAcα-nLc₄Cer (α2,3SAT) and Y916 for IV⁶NeuAcα-nLc₄Cer (α2,6SAT), respectively. Standard glycolipids IV³NeuAcα-nLc₄Cer for α2,3SAT and IV⁶NeuAcα-nLc₄Cer for α2,6SAT, respectively. The specimen numbers are the same as those in Fig. 2 and Table I.

Figure 6

RT-PCR analysis of the glycosyltransferase genes in human ovarian carcinoma tissues. A, GAPDH; B, GM3 synthase; C, GD3 synthase; D, α2,3-sialyltransferase; E, α2,6-sialyltransferase and F, α1,3/4-fucosyltransferase 3. The specimen numbers are the same as those in Fig. 2 and Table I.

Figure 7

Metabolic pathways for the syntheses of IV⁶NeuAcα-nLc₄Cer and Lewis-active glycolipids. SAT, sialyltransferase; FUT2, α1,2-fucosyltrans-ferase; FUT3, α1,3/4-fucosyltransferase; H, blood group H antigen.