Sialosyl Le(a)-carrying gangliosides present on the surface of colon carcinoma cells are not directly involved in adhesion to E-selectin.

We have shown previously that human colon cancer CX-1 cells contain lipid- and protein-bound sialosyl Lewis(a) structures that support the adhesion of these cells to E-selectin. Treatment of cancer cells with O-sialoglycoprotease did not decrease either the binding of anti-sialosyl Le(a) antibodies or binding to E-selectin-expressing CHO cells. This suggested that cleavage of sialomucins uncovered cryptic sialosyl Le(a) gangliosides that support such interactions. In the present study, inhibitors of glycolipid and O-glycan biosynthesis, d,l-threo-PPPP and GalNAc-alpha-O-benzyl, respectively, were used to study whether the binding of anti-sialosyl Le(a) antibody and adhesion of CX-1 cells to E-selectin can be mediated by sialosyl Le(a) gangliosides. Treatment of cancer cells with each of the inhibitors decreased the expression of the respective glycoconjugates as shown by TLC-binding assay and immunoblotting with anti-sialosyl Le(a) antibody. However, only slight differences in binding of antisialosyl Le(a) antibody to the surfaces of control and inhibitor-treated CX-1 cells were found by flow cytometry, as well no differences were observed in binding of control and inhibitor-treated CX-1 cells to E-selectin-expressing CHO cells, supporting the earlier hypothesis on the involvement of gangliosides in binding of anti-sialosyl Lewis(a) in the partial absence of mucin O-glycans. This hypothesis was further proven by electron microscopy data. Both native CX-1 and d,l-threo-PPPP-treated cells were labelled with anti-sialosyl Lewis(a) antibody mostly at a distance 70-90 nm from cell surface, suggesting interaction with protein-bound carbohydrate structures only. In contrast, the cancer cells treated with GalNAc-alpha-O-benzyl showed most of the staining around 20 nm distance from the plasmalemma, implying that the antibody interacts with lipid-bound sialosyl Lewis(a) instead. The electron microscopy data in conjunction with other results described in this report strongly support the hypothesis that sialosyl Lea gangliosides are not involved in the adhesion of CX-1 cells to E-selectin when mucins are present on the cell surface, but they may be involved in binding to E-selectin in their absence.

Characterization of RNABP, an RNA binding protein that associates with RNase L.

The 2',5'-oligoadenylate (2-5A)/RNase L pathway is one of several enzymatic pathways induced by interferons (IFN). RNase L is a latent endoribonuclease that is activated on its binding by 2-5A and inhibited by the ribonuclease L inhibitor (RLI). We have shown previously by coimmunoprecipitation that RNase L may be associated with a 90-kDa RNA binding protein (RNABP), identified with a monoclonal antibody (mAb) raised against an RNase L complex purified under native conditions on 2-5A-sepharose. Here we confirm, by gel-filtration and pull-down analysis, the association of RNase L and RNABP, and we demonstrate that this association is significantly increased in the presence of 2-5A. Moreover, we found that RNABP protein levels decrease during terminal differentiation in various cell lines but do not vary during vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) infection or following IFN-alpha/beta treatment. In this latter case, although total cellular RNABP levels do not vary, the amount of RNABP found in the cytoplasm increases in comparison to that found in the nucleus, indicating a cytoplasmic localization of RNABP after IFN-alpha/beta treatment. Finally, we demonstrate the interaction between RNase L and RNABP in intact cells. Microinjection of an mAb against RNABP into HeLa cells inhibits RNase L antiviral activity and partially inhibits the IFN-alpha/beta-induced antiviral activity.

Metastatic potential of human uroepithelial cancer cells is not dependent on their adhesion to E-selectin.

In our previous studies we have found that human uroepithelial cell lines differ in their expression of sialosyl LewisA antigen. We have also shown that among the studied cell lines, only Hu 1703He cells with the highest expression of this tetrasaccharide bind to E-selectin-expressing cells. In the present study we put forward the question, of whether sialosyl LewisA-mediated adhesion of uroepithelial cells to E-selectin is important in the formation of metastases. The HCV 29T and Hu 1703He cells, representing two uroepithelial cell lines, were transplanted into NCr nu/nu mice. Hu 1703He cells express on their surface a high level of sialosyl LeA antigen, while HCV 29T cells are sialosyl LewisA-negative. We have shown that human uroepithelial cancer cells, in addition to their tumorigenic and invasive properties, are highly metastatic when inoculated into athymic nu/nu mice. The ability to form secondary tumour foci in lung and liver seems to be independent of the expression of sialosyl LewisA antigen.

CEA-related proteins on human urothelial cell lines of different transformation grades.

CEA family proteins from human urothelial cell lines of different transformation grades were characterized by flow cytometry and Western blotting using monoclonal antibodies: 26/3/13, D14HD11, 9A6 and 4/3/17. The following observations were made: (i) the urothelial cell lines, representing transformation grade III (TGr III, tumorigenic, invasive cells), were characterized by the presence of a component with molecular mass 110-135 kDa, most probably representing biliary glycoprotein (BGP); (ii) BGP was absent in non-tumorigenic and non-invasive TGr II urothelial cell lines; (iii) a protein band with apparent molecular mass 180 kDa, and migrating as a CEA standard was detected in only one of seven urothelial cell lines analyzed; (iv) a broad band of apparent molecular mass migrating at 65-90 kDa, probably representing NCA-50/90, was found in two tumorigenic and invasive cell lines, HCV 29T and Hu 1703He.

Aldehyde dehydrogenase isoenzymes in tumours--assay with possible prognostic value for oxazaphosphorine chemotherapy.

A novel fluorimetric assay, allowing independent measurement of the activities of two principal cytosolic forms of human aldehyde dehydrogenase, ALDH-1 and ALDH-3 (known as a tumour-associated ALDH) was applied to estimate the activities of these isoenzymes in human liver and thyroid tumours. The assay is based on two artificial substrates, 6-methoxy-2-naphthaldehyde (MONAL-62) and 7-methoxy-1-naphthaldehyde (MONAL-71), exhibiting excellent substrate properties toward various forms of human ALDH (see Wierzchowski et al., 1997, Anal. Biochem. 245, 69-78). We have found significant differences in ALDH activities between malignant and non-malignant tissue fragments, particularly in cancerous livers. Out of 16 tumours examined, only 4 exhibited ALDH-1 activities comparable to that found in the tumour-free tissue (0.5-2.5 U/g), while in the remaining 12 this activity was at least 10-fold lower. The ALDH-3 activity was detectable in about 40% of both tumour and tumour-free liver samples (maximum value 1.5 U/g). Comparison of 13 pathological thyroid fragments revealed ALDH activities in the range of 0.02 to 0.35 U/g, with two malignant samples showing activities of 0.27 and 0.18 U/g. Both substrate specificity and kinetic behaviour of the thyroid ALDH (Km values for the fluorogenic naphthaldehydes as well as propanal inhibition profile) were similar to those of the purified ALDH-1. In 5 thyroid samples traces of ALDH-3 activity was detected, using MONAL-62 and NADP+ as substrates (maximum value 0.04 U/g). Possible prognostic value of the foregoing measurements for cyclophosphamide chemotherapy is discussed.

Role of sialosyl Lewis(a) in adhesion of colon cancer cells--the antisense RNA approach.

To study whether the adhesion of colon cancer cells to E-selectin can be directly affected by changes in the expression level of sialosyl Le(a) antigen we created a specific loss-of-function phenotype. A stable subclone (CX-1.1) with high expression of sialosyl Le(a) structure, obtained from a heterogenous population of colon carcinoma CX-1 cells, was transfected with an expression vector containing a fragment of cDNA for alpha1,3/4-fucosyltransferase in antisense orientation. After transfection, the cell line was isolated which did not express sialosyl Le(a) antigen and lacked the alpha1,3/4-fucosyltransferase activity, despite an unchanged level of mRNA specific for this enzyme. It was found that the specific lack of expression of sialosyl Le(a) carbohydrate structure on the surface of colon cancer cells completely abolished their adhesion to E-selectin. To evaluate which cellular glycoconjugates carry sialosyl Le(a) antigen, glycoproteins as well as glycolipids of CX-1.1 cells were analysed for the expression of this structure. Anti-sialosyl Le(a) antibodies detected multiple glycoprotein bands with apparent molecular masses of 65-280 kDa on western blots, and an intense band representing sialosyl Le(a)-ganglioside on a thin-layer chromatogram. Using O-sialoglycoprotease from Pasteurella haemolytica and an alkaline beta-elimination procedure, it was shown that protein-linked sialosyl Le(a) structures are carried mostly by mucin-type glycoproteins. However, treatment of CX-1.1 cells with O-sialoglycoprotease did not decrease either their binding to E-selectin-expressing Chinese hamster ovary cells, or binding of anti-sialosyl Le(a) antibodies to the cell surface. These results suggested that cleavage of sialomucins uncovered cryptic sialosyl Le(a)-ganglioside, which was inaccessible for the antibody and E-selectin in untreated cells. This hypothesis was confirmed to some extent by the higher accessibility of gangliosides to galactose oxidase on the surface of O-sialoglycoprotease-treated CX-1.1 cells, comparing to untreated cells. We propose that glycoproteins as well as gangliosides carrying sialosyl Le(a) structures, when properly exposed and present in high density on surface of cancer cells, can effectively support the adhesion of cancer cells to E-selectin.

Metastatic potential of human CX-1 colon adenocarcinoma cells is dependent on the expression of sialosyl Le(a) antigen.

Several lines of evidence indicate that sialosyl Le(a), tumor-associated carbohydrate antigen present on human colon carcinoma cells, is involved in formation of metastases. To study the role of this carbohydrate structure in development of metastases, we have used the clone of human colon carcinoma CX-1 cells transfected with antisense expression vector containing fragment of cDNA for alpha1,3/4-fucosyltransferase (FT III), which is involved in synthesis of sialosyl Le(a) tetrasaccharide. It has been reported previously that, in contrast to the parental cells, the antisense-transfected CX-1.1AS5 cells do not express sialosyl Le(a) and do not adhere to E-selectin-expressing CHO cells. In the present work we have studied the formation of liver metastases by CX-1.1AS5 cells after their orthotopic or intrasplenic implantation into athymic nu/nu mice. After orthotopic implantation of sialosyl Le(a)-negative colon carcinoma CX-1.1AS5 cells, the number of mice with liver metastases was markedly lower (21% of mice) in comparison with their number after implantation of the parental CX-1.1 cells (86% of mice). However, no differences in ability to form colonies in liver were observed between parental CX-1.1 cells and antisense-transfected CX-1.1AS5 cells after intrasplenic inoculation. The liver metastases were formed in 89% and 84% of mice, respectively. Our data support the thesis on the importance of sialosyl Le(a) antigen expression in the development of liver metastases by colon cancer cells, and indicate the role of transplantation route and primary tumor localization in formation of metastases.

Mouse monoclonal IgA antibodies lack interchain disulfide bonds.

A mouse monoclonal IgA antibody (A008), anti-blood group A antigen, was shown to dissociate into heavy (H) and light (L) polypeptide chains under non-reducing conditions. The dissociation occurred in the presence of 0.2% sodium dodecyl sulphate (2 h at room temperature) or at elevated temperature (1 h at 70 degrees C). The free H and L polypeptide chains could be separated in SDS-polyacrylamide gel electrophoresis or by gel filtration in the presence of SDS. The dissociation of IgA (A008) antibody into heavy and light polypeptide chains was reversible, since the fractions containing gel filtration-isolated chains, after removing the detergent, pooling together and incubation at 4 degrees C created again the immunoglobulin molecules with activity close to the native value. Similarly, the same IgA antibody heated at 70 degrees C for 1 h recovered its antibody activity after keeping at 4 degrees C. Three other mouse monoclonal IgA antibodies showed the same ability to dissociate into heavy and light polypeptide chains after exposure to SDS or elevated temperature, which suggests that this outstanding property is a characteristic feature of the mouse monoclonal IgA antibodies and it comes out from the lack of H-H, L-L and H-L interchain disulfide bonds in these molecules.