Heparan sulphate alterations in tumour cells.

Heparan sulphate proteoglycan is widely distributed, being found inserted into plasma membranes of most cells (Kjellenetal., 1981 ; Norlingetal., 1981)and asa significant component of basement membranes (Kanwar et al., 1980; Hassel et al., 1980). As a strong polyanion, heparan sulphate interacts with many components under physiological conditions, the specificity of these interactions depending on the distribution of charged groups along the chain. Such interactions are thought to be involved in cell adhesion (Culp et al., 1979), organization and function of basement membranes (Kanwar et al., 1980; Vlodavsky et al., 1980) and regulation of enzyme activity [e.g. lipoprotein lipase (Shimada et al., 1981; Cheng et al., 1981) and serine proteases (Colburn & Buonassisi, 1982)l. These functions could underlie the role that heparan sulphate appears to play in growth control. Heparan sulphate obtained from cells transformed by viruses has consistently been found to have a lower degree of sulphation than the glycosaminoglycan obtained from control cells (Underhill & Keller, 1975; Winterbourne & Mora, 1978; Fransson et al., 1981). A similar defect has been observed when heparan sulphate from both rat and human hepatomas has been compared with normal hepatocytes (Nakamura et al., 1978; Nakamura & Kojima, 1981). In our studies we have used established mouse cell lines which have overcome the normal restrictions on indefinite growth in vitro. Such immortalized cells have progressed along the multi-step route to carcinogenesis to a stage where single-hit events may be capable of inducing tumourigenesis (Land et al., 1983). Such events can occur during normal tissue culture, and highly tumourigenic variants which arise in culture of cloned cells can be selected by inoculation of a sufficiently large number of cells into syngeneic animals. Analysis of the heparan sulphate produced by two such variants, isolated from separate clones, demonstrated that in both cases the glycosaminoglycan was reduced in sulphation (Winterbourne & Mora, 1981). More recently we have repeated these studies with eight tumour cell lines isolated separately from one of the clones. In each case the heparan sulphate bound less strongly to DEAE-cellulose than the heparan sulphate from the parent clone. A typical result for cell surface glycoconjugates is shown in Fig. 1. The same alteration was observed in heparan sulphate secreted into the medium and material isolated from trypsinized cells. These results show that reduced sulphation of heparan sulphate is closely associated with the progression to highly tumourigenic cells. At least five enzymes are involved in the maturation of the glycosaminoglycan side chains of heparan sulphate. The concerted action of these enzymes result in the introduction of Nand 6-0-sulphate groups on glucosamine residues and 2-0-sulphate groups on iduronic residues (which arise by epimerization of glucuronic acid residues within the polymer) (Feingold et al., 1981). The structural complexity of heparan sulphate is increased because these reactions do not go to completion. Sulphate and iduronic acid residues are not distributed regularly along the chain, but occur in highly substituted stretches separated by varying lengths of unmodified polysaccharide chain. Structural analysis of the heparan sulphate from the cell surface of control and tumour cells demonstrated that the reduced affinity for DEAE-cellulose of tumour cell heparan sulphate arose from a reduction in the degree of O-sulphation with unchanged or even increased N-sulphation (Winterbourne & Mora, 1981). The reduction occurred in 60-sulphate groups located in regions of alternating Nsulphated and N-acetylated glucosamine residues. Excision of these regions by nitrous acid, which cleaves the polysaccharide at N-sulphated glucosamine residues, yields a variety of tetrasaccharides. It is within the group of monosulphated tetrasaccharides that the defect was located (Winterbourne & Mora, 1981). These results were confirmed in the analysis of the heparan sulphate from the eight recently isolated cell lines (not shown). Such subtle changes in the structure of heparan sulphate have a marked affect on the properties of the glycosaminoglycan as judged by the inability of these molecules to selfassociate (Fransson et al., 1981) and their reduced affinity for cell-surface receptors (Winterbourne, 1982). These alterations might be involved in defective cellkell or cellmatrix interactions, possibly resulting in the reduced capacity of transformed cells to organize an extracellular matrix at their surface (Norling et al., 1981 ; Alitalo et al., 1982). Where altered sulphation of proteoglycans has been reported previously, the increased (Conrad & Woo, 1980) or decreased (Sugahara & Schwartz, 1979) sulphation has been assigned to alterations in levels of enzymes involved in sulphate activation but not to altered sulphotransferases. Using similar techniques we found increased levels of the sulphate activation enzymes when assayed in vitro, suggesting that defective sulphation in tumour cells occurs by a different mechanism from that previously reported. In agreement with this, the specific activities of both Nand 0-sulphotransferases in the tumour(2 19CT) and virustransformed (21 SCSC) cells were reduced by 30% compared with control cells (210C, Table 1).