Carbohydrate moieties on the cell surface play an important role in cellular recognition.
Increased sialylation of cell surface glycoproteins has been reported in malignant tumours and has been correlated with the invasive and metastatic potential of the cells.
The aim of this work was to investigate the expression of cell membrane glycoproteins on four lung cancer cell lines and to identify potential changes in their sialylation
employing the tools of proteomics and functional genomics.
Three highly metastatic cell lines: PC 14- human lung adenocarcinoma, PLC- primary liver carcinoma and T24- human bladder carcinoma were chosen for the preliminary
studies. Methods optimized on the above cell lines were then employed to isolate and purify membrane proteins from four human lung cancer cell lines, including two
adenocarcinoma cell lines (PC 14 and SK-Lu 1), squamous cell carcinoma (SK-Mes 1) and small cell carcinoma (NCI-H82).
Two methods were used to assess the state of glycosylation of the proteins. Firstly the samples were treated with endoglycosidases (Peptide N-glycosidase and O-glycanase)
and changes in electrophoretic mobility between enzyme-treated and non-treated controls were used to reveal removal of glycans. In the second method proteins were
transferred from gels by Western blotting and the blots probed with lectins. Four biotinylated lectins were used: concanavalin A (Con A), wheat germ agglutinin (WGA),
Sambucus nigra agglutinin (SNA) and Maackia amurensis (MAA II). Significant differences in the binding patterns for the lectins were observed. Binding of MAA II and SNA confirmed the presence of a2,3-linked and cc2,6-linked sialic acids respectively. Binding of Con A suggested extensive expression of N-linked glycans with a high content of mannose. It was suspected that results obtained by Western blotting were affected by shedding of sialic acid residues during protein purification.
Hence, in a second approach to the study of glycoprotein sialylation the expression of two sialyltransferase genes at the mRNA level was investigated. The enzyme mainly
responsible for the addition of a2,6-linked sialic acids is p-galactoside cc2,6- sialyltransferase (ST6Gal I) and mRNA for this enzyme was detected in all four lung tumour cell lines examined. This was not the case with the p-galactoside a2,3- sialyltransferase III (ST3Gal III). A very weak response was detected in PC 14 cell line only, but not in the other three cell lines. It was concluded that oc2,3 sialylation detected by binding of MAA II lectin was due to the action of other a2,3-sialyltransferases.
A final part of the work concentrated on a specific glycoprotein, mainly CD44.
CD44 is a major hyaluronan-binding protein and is implicated in cell-cell adhesion, cell-matrix interaction and metastasis. The presence of the CD44 antigen on cell
membranes and expression of the CD44 gene were nvestigated. The CD44 gene contains over 20 exons and 12 of them are alternatively spliced. It was observed that
different histologic types of lung cancer expressed different forms of the CD44 antigen.
Adenocarcinoma of lymphoblastoid origin (PC 14 cell line) showed the expression of several isoforms including the standard form CD44S, epithelial form CD44R and variant form CD44v. Small cell carcinoma (NCI-H82 cell line) did not express CD44 at either the protein or mRNA level. Two other cell lines examined (SK-Lu 1 adenocarcinoma and SK-Mes 1 squamous cell carcinoma) expressed only the CD44S form. In summary the results obtained suggest that the expression of the CD44 molecule on lung cancer cell lines differs between histological types of tumour and could be related to the metastatic potential of the cells.
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