IMPORTANCIA DE LAS MACRORREGLAS:

To demonstrate that the transfected ay formed a functional receptor with B^ adhesion assays were carried out on vitronectin, the ligand for the integrin ayBg in kératinocytes (Adams and Watt 1991).

«V Transfection

3.6.1 Preparation of Vitronectin

H357 cells have been shown to adhere to commercially available vitronectin

(Sugiyama et al. 1993), despite having no ay receptor for vitronectin. This may be due

to commercial vitronectin containing other proteins, such as fibronectin, that the cells are able to adhere to. Vitronectin was therefore purified as part of this project.

Vitronectin was purified from human plasma according to the method of Yatogho

et al. (1988). The final step involves elution of vitronectin from a heparin-Sepharose

column with a buffer containing a high salt concentration. 1 0 aliquots of lOmls were

collected, dialyzed and then concentrated. Samples 1-10 were run on a 10% SDS-

polyacylamide gel to determine which of the aliquots contained vitronectin. Figure 3.8a

shows the SDS-PAGE gel: two bands of around 6 8 kDa are present in samples 1, 2 and

5. Yatohgo et al. (1988) reported a doublet of 75kDa and 65kDa bands collected following elution of the column which corresponds to the molecular weight of vitronectin. Vitronectin

is composed of a mixture of 65kDa and 75kDa polypeptides, the former is an

endogenously proteolytically nicked product of the latter, lacking a carboxyl terminal fragment (Suzuki et al. 1984). Samples 1 and 2 were combined and along with sample 5 their protein concentrations were determined using a BioRad protein assay. 5mg of vitronectin was produced from 200ml of plasma, yields of between 4-6mg vitronectin/200ml of plasma are expected (D. Cheresh-personnal communication).

Samples 1 + 2 combined and sample 5 were run on another SDS-PAGE gel after equal volumes of protein had been loaded, (Figure 3.8b). Sample 1 + 2 were used for the adhesion assays, as sample 5 appeared to contain other contaminating proteins.

3.6.2 Adhesion to Vitronectin

Initial adhesion assays were carried out to determine optimal conditions for adhesion of the H357 cells. Non-specific adhesion was blocked by coating the wells with heat-denatured bovine serum albumin (BSA) for 1 hour at 37®C.

Initially cells were allowed to adhere for 1 hour to a range of concentrations of

vitronectin (0.5/xg/ml, 0.75^g/ml, lug/ml, 5^g/ml and 10/ig/ml). Using this regime it was

not possible to demonstrate any increase in adhesion to vitronectin in the «y-positive

«V Transfection

clones. Adhesion of VI and 1A4 is shown in Figure 3.9.

Adhesion assays were then carried out on higher concentrations of vitronectin (5|xg/ml, 10/xg/ml 20/xg/ml and 50/ig/ml) and the cells were allowed to attach for 3 hours. This resulted in an increase in adhesion of the «y-positive clones to vitronectin compared to the parental cells. However, there was a high level (around 30%) of adhesion of the parental cells and the «y-positive clones on BSA alone. This is demonstrated in Figure 3.10, with clones V4 and V5 and the parental cells. The cells may have been secreting their own extracellular matrix during the time course of the assay enabling them to adhere

to a greater extent than they could after only 1 hour. To prevent this happening

cycloheximide, which inhibits de novo synthesis of proteins, was added to the cells before

they were used in the adhesion assays.

Adhesion to high concentrations of vitronectin in the presence of cycloheximide for

1 and 3 hours is shown in Figure 3.11a and b. After 1 hour there was an increase in

adhesion in «y-positive clones compared with the parental cells at all concentrations of vitronectin used. After 3 hours much greater numbers of cells adhered and there was a marked difference in adhesion between the «y-positive clones at all concentrations of vitronectin compared to the parental cell line. Further adhesion assays carried out for 3

hours in the presence of cycloheximide confirmed that 2 0/xg/ml vitronectin supported

maximal adhesion of the «y-positive transfectants.

3.6.3 Blocking Adhesion with Antibodies

To show that the adhesion to vitronectin was mediated through the «y integrin receptor attempts were made to block the adhesion with antibodies to «y (13C2) and «yllg (P1F6). The concentration of vitronectin was decreased as previous work carried out in the laboratory had demonstrated that blocking antibodies were more effective against lower

concentrations of extracellular matrix proteins (personal communication). A range of

concentrations of 13C2 were used on 5^g/ml vitronectin and a slight inhibition was noticed

on 5/ig/ml vitronectin with 80/;ig/ml 13C2. These conditions were used for further

adhesion assays.

a y Transfection

adhesion on 5/ig/ml vitronectin in the presence of 1:100 concentration of P1F6. Differences in adhesion were seen between the different clones, but all showed reduced

adhesion to vitronectin in the presence of P1F6, confirming that the transfected ay subunit

was a functional subunit. Anti-ay^s antibodies produced a more profound effect on adhesion compared to that achieved with the anti-ay antibody alone, possibly reflecting differences in the binding sites of the antibodies. The ability of anti-ayBg antibodies to

inhibit adhesion of the positive clones confirmed that the B5 subunit was paired with the uy

subunit.

3.7 Growth of ay Transfectants