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7.1 Introduction

Many studies have focused on the role of the four main families of adhesion molecules in metastasis; these being the cadherins (Takeichi, 1993), integrins (Lauri et al., 1991a), the immunoglobulin superfamily (Rice and Bevilacqua, 1989) and the selectins (Iwai at a!., 1993). However, it is possible that glycosaminoglycans may also have a role to play in metastasis. As described in Chapter 1 glycosaminoglycans (GAGs) are linear, polymeric, carbohydrate molecules which are generally highly negatively charged due to frequent sites of sulphation and carboxylation. In nature, all GAGs except HA, appear as part of a proteoglycan molecule. Proteoglycans are expressed on the surface of many cell types including endothelial cells (Oohira at a/., 1983). Indeed, most of the negative charge on the endothelial cell surface is due to HS and DS proteoglycans (Klein

at a/., 1992). Endothelial GAGs exhibit a range of different functions including providing an antithrombogenic surface (Marcum and Rosenberg, 1984), and the binding of growth factors such as FGF (Tanaka at a/., 1993b).

GAGs can directly mediate cell-cell adhesion by acting as the ligand of other adhesion molecules. For instance, CD31/PECAM-1 (Ig-superfamily) which is constitutively expressed by endothelial cells, mediates the calcium-dependant, heterophilic aggregation of transfected mouse L-cells by binding HN and OS on the opposing cell surface (de Lisser at a/., 1993). NOAM, another member of the Ig- superfamily, also has a GAG binding consensus sequence and has been shown to bind HS (Cole at a/., 1986). In this instance, the binding of HS to NCAM may enhance hemophilic binding (Reyes at a/., 1990). GAGs may act in association with other adhesion molecules to mediate cell adhesion. For example, melanoma cell surface CSPGs (NG2) appear to collaborate with the a4pi integrin (VLA-4) during cell spreading and focal contact formation (lida at a!., 1995). Ligand binding by NG2 leads to communication with the integrins by inside-out signalling, with the result that the cells spread and form focal contacts on fibronectin. Without collaboration from NG2 the integrins only mediate adhesion. Members of the selectin family of adhesion molecules also adhere to GAGs. L-selectin interacts

with endothelial HN/HS chains (Norgard-Sumnicht et al., 1993). Similarly, endothelial P-selectin may interact with HN/HS based ligands expressed by neutrophils (Skinner et a!., 1991). GAGs are also involved in embryo implantation as a HS-like GAG mediates the initial adhesion of a human trophoblastic cell line to epithelial cells (Rhode and Carson, 1993), although the epithelial ligand has not been identified. In addition to interacting with specific cell surface and matrix components, GAGs can also non-specifically modulate the behaviour of other adhesion molecules. They probably do so by stearic hindrance due to their extended, anionically charged structures. For instance, large PGs on the surface of invasive epithelial cell lines inhibit the function of E-cadherin (VIeminckx et a/., 1994).

Proteoglycans, and in particular their GAG components, have been linked to various aspects of the spread of malignant cells during metastasis. Malignant transformation of cells leads to altered production of PG, both by tumour cells and surrounding normal tissue (lozzo, 1985). However, no consistent differences in proteoglycan synthesis have been identified. In some tumours there may be an increase in the expression of CSPG, concurrent with a decrease in HSPG. The HSPG that are produced in these cases tend to be poorly sulphated (Winterbourne and Mora, 1981; Robinson et a!., 1984). As CSPGs are capable of altering the adhesiveness of tumour cells to ECM (Brennan et a!., 1983) such changes have been postulated to lead to reduced adhesion to the ECM (Liotta et a/., 1986). A CSPG, which was originally identified on malignant melanoma cells, is expressed by malignant astroglial cells and proliferating brain endothelial cells. This molecule may be a marker for the onset of angiogenesis within glioblastomas (Schrappe et

a/., 1991).

Other investigators have linked increased expression of HS and HA, and decreased expression of CS, with the malignant transformation of glial-derived cells (Steck et a/., 1989). Interestingly, tumour formation by CHO cells in nude mice is dependant upon the production of HSPG, but not CSPG, possibly because the HSPGs protect tumour cells from attack by host B-cells (Esko et a/., 1988). Perlecan, an ECM HSPG, is deposited in larger amounts by invasive melanoma cells than by their non-invasive counterparts. It has been suggested that disruption

of the ECM structure by these perlecan deposits facilitates the initial stages of melanoma invasion (Cohen et al., 1994). Timar et al. found that highly metastatic Lewis lung carcinoma lines express more HS, but less HA and CS, than the parental lines (Timar et al., 1987). Turley et al. also reported increased production of HS in highly invasive or metastatic melanoma variants (Turley and Tretiak, 1985), but in addition, these authors found that HA levels are enhanced at the onset of invasion, and are localized to the tumour periphery. As well as being concentrated around tumours, HA levels are increased in the serum of patients with disseminated neoplasms, possibly due to tumour factors which induce HA expression by normal cells (Decker et al., 1989). The role of HA in tumour metastasis has been the topic of a number of studies. Most of these studies have concentrated on the interactions of CD44, a lymphocyte homing molecule, with HA during metastasis (East and Hart, 1993; Herrlich et al., 1993). In the last chapter it was demonstrated that HA on the surface of the RPMI-7951 melanoma line mediates basal adhesion to HUVEC CD44 (Ch. 6).

GAGs and related sulphated polysaccharides have been shown to interfere with the interactions of leucocytes with blood vessel walls (Brenan and Parish, 1986; Tangelder and Arfors, 1991; Bazzoni et al., 1993); a process which is considered a paradigm for tumour cell arrest. The inhibition of inflammatory reactions by GAGs appears to be due to the binding of GAGs to active sites on molecules which mediate the adhesion of leucocytes to endothelial cells (Arfors and Ley, 1993). It is well established that soluble GAGs are capable of inhibiting haematogenous metastasis (Coombe et al., 1987; Irimura et al., 1986) as well as the extravasation of inflammatory cells. These effects are not solely due to the anti-coagulant properties that these carbohydrates may posses. Collectively these findings suggest that GAGs may be involved in the adhesion of metastasizing tumour cells to endothelial cells. The potential role of GAGs in mediating tumour cell adhesion to endothelial cells was therefore investigated in this chapter.

7.2 Specific materials and methods

Sodium Chlorate Treatment

The growth of cells in chlorate, an inhibitor of ATP sulphate adenyltransferase (ATP-sulfurylase), produces cells with under-sulphated proteins and carbohydrates (Beauerle and Huttner, 1986). This enzyme is the first required for the biosynthesis of 3-phosphoadenosine 5'-phosphosulphate (PAPS), which is the ubiquitous co­ substrate for sulphation reactions in vivo. The methods of Keller (Keller et a/., 1989) and Carew (Carew et a/., 1990) were adapted to study the importance of sulphation of cell surface components in adhesion. A stock solution of 100 mM sodium chlorate (Aldrich Chemical Company, Gillingham, UK) was made up in water and kept at 4°C. The effects of chlorate inhibition of the levels of HS sulphation were first ascertained for the tumour cell lines and HUVEC. The cells were grown in 24-well tissue culture plates until they reached confluence at which point their medium was replaced by 200 pi of low sulphate medium. The media used was Eagle's Minimum Essential Medium (MEM, Gibco BRL), 4% L- methionine (Gibco BRL), 0.5% BSA, 1% dialysed PCS and 2 mM L-glutamine. After 6 h the cells received fresh low sulphate medium containing sodium chlorate at 5, 10 or 20 mM. Controls received medium with no chlorate. After another 6 h this was replaced by medium containing chlorate plus 0.25 pCi/ml of ^®S-sulphate (Du Pont NEN, Stevenage, UK). The cells were incubated in this solution for 24 h. After radiolabelling the monolayers were washed three times with PBS/A. The cell surfaces were then subjected to digestion by heparinase III as previously described (Ch. 2). Digests were collected and pooled with two washes of 200 pl/well PBS/A. The cells were then brought into suspension with 200 pl/well trypsin/EDTA and an aliquot retained for counting. The wells were washed with 0.5 ml of PBS/A. The cells were combined with the wash and pelleted. The pellets were resuspended in 0.5 ml of 0.2% SDS and allowed to disaggregate. The amount of radioactivity in the enzyme digests of cell surface HS, and cell pellets was determined using a Beckman IS 1801 scintillation counter (Beckman Instruments Inc, High Wycombe, UK).

To determine the effects of chlorate on adhesion the cells were preincubated in DMEM (Gibco) without sulphate or cysteine, and containing 1% of the usual concentration of L-methionine. The medium contained 5% dialysed PCS for tumour

cells, or 16% for endothelial cells. The PCS was dialysed in Visking tubing (pore size 12 kD - 14 kD; Fisons Scientific Equipment, Loughborough, Leics, UK) at 4“C against 3 changes of PBS/A to remove sulphate. After 6 h the medium was replaced with Eagle's MEM containing 5% or 15 % PCS, and 10 mM sodium chlorate. The cells were incubated in this medium for 20 h and then used in adhesion assays.