Cancer cell biology / edited by Takeo Nagayo, Wataru Mori.

Date:
[1980]
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Cancer cell biology / edited by Takeo Nagayo, Wataru Mori. Source: Wellcome Collection.

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    induced not only aggregation of tumor cells but also adhesiveness of the cells. In order to act on the tumor cells, this adhesive factor must bind to their surfaces, presumably through specific surface receptors. The purified cell surface-associated adhesive factor was labeled with 125 I. The labeled product was homogeneous and its biological activity remained unchanged after purification by affinity chromatography; its binding to AH109A cells noted above was specifically inhibited by D -mannose. The binding to the cells exhibited a simple saturation pattern, reaching its peak at a concentration of 50 /<g/ml (Fig. 1A). By plotting the binding data according to the Steck and Wallach (43) equations, the total numbers of binding sites per cell were calculated to be 6x 10 5 and the affinity constant (A a ) was 1.1 xlO 6 (Fig. IB) {14,21). The binding of this adhesive factor to the cells was also demonstrated by the indirect immunofluorescent technique using rabbit antibody against the purified adhesive factor; this bound ad hesive factor was found localized on the cell surface. Synthesis of Cell Surface-associated Adhesive Factor 1. Synthesis of the factor by cancer cells and its localization As mentioned above, the cell surface-associated adhesive factor functions as a trigger factor to develop junctional complexes in tumor cells, provided it was exoge- neously applied to the cells. In order to form cell islands characterized by junctional complexes without any addition of this component, tumor cells, e.g., AH136B and AH7974 cells themselves, must synthesize this adhesive component. It seems possible that if these tumor cells could be maintained under physiological conditions but pre vented from aggregating at a reasonably low cell density, the cells may continue to synthesize this component. The tumor cells, which were mechanically dissociated in Rabinowitz' balanced salt solution containing EDTA (1.2 mM), were suspended at a density of 2xl0 4 cells/ml for cultivation. The synthesis (or regeneration) of this ad hesive component was examined using 125 I-labeled rabbit antibody (IgG fraction) against the purified adhesive factor; 125 I-labeled rabbit IgG served as a control. The cultured cells were harvested after different cultivation times (0 to 24 hr). The synthesis (as shown in the form of increased radioactivity specifically bound to the cells) began to rise rapidly; it increased 8-fold in 12 hr of cultivation, reaching its peak, i.e., a 10-fold increase, in 24 hr of cultivation (14, 21) (Fig. 2). Since the adhesive component, which previously existed in the cells, was revealed during cell dissociation, it was scarcely detected in the inhibition of cultivation. This finding indicated that the release of this component from tumor cells occurs more easily than that of the cognin (12) from embryonic chick neural retina cells, because the adhesiveness of the tumor cells was less strong than that of the neural retina cells. The regeneration of the factor was strongly inhibited by the addition of actinomycin D or puromycin, indicating that the regeneration required protein synthesis (Fig. 2). In contrast, this type of adhesive component was not detectable in AH109A and YS cells during 24 hr of cultivation (Fig. 2), although there remained some questions that this component might exist in an inaccessible state (hidden within the surface mem brane layer or situated at the submembrane layer), or in a smaller quantity not demon strable by this technique. However, biochemical evidence that this component could not be separated from those tumor cells by the same procedures as noted above (Table