Mechanisms Involved in Retinoic Acid-Induced Inhibition of Metastatic Melanoma Cell Lung Colonization

Gold, Jean Anderson Gordon (1993) Mechanisms Involved in Retinoic Acid-Induced Inhibition of Metastatic Melanoma Cell Lung Colonization. PhD thesis, University of Glasgow.

Abstract

This project was initiated to study some of the mechanisms by which retinoic acid inhibits the experimental lung colonization of mice by murine melanoma cells. Using the metastatic cell line B16F1M2, this was investigated in three ways. When B16F1M2 cells treated with 10-6M retinoic acid are injected via the tail vein into syngeneic mice, the number of lung colonies produced is reduced when compared to those established from control cells. In vitro, retinoic acid has an inhibitory effect on B16F1M2 cell growth and the morphology of the cells is affected with a shifting to a more "normal" form. The first stage of the project was to examine the effect of retinoic acid on the adhesion of these cells to basement membrane components, intact extracellular matrix and to other cells (both homotypic and heterotypic). It was found that retinoic acid pretreatment inhibited the adhesion of the cells to laminin/nidogen and type IV collagen in a time-dependent and concentration-dependent manner, while adhesion to fibronectin remained unaffected. The adhesion of the cells to intact subendothelial extracellular matrix was also inhibited. The ability of the cells to spread on the substrates was also affected by pretreatment with retinoic acid, with cells remaining rounded on laminin/nidogen and type rv collagen. The density of the cultures also affected cell adhesion, with low-density cultures exhibiting reduced adhesion when compared to almost confluent cultures. Reports have suggested that some of the effects of retinoic acid are reversible once the molecule is removed from the system. This was found to be the case for adhesion to laminin/nidogen and type IV collagen, although removal of the retinoid did not allow cells to return to control levels within the two days of the experiment. Addition of the retinoid after two days did reduce the degree of adhesion. Cell-cell adhesion was also investigated. B16F1M2 homotypic adhesion was inhibited by retinoic acid treatment. If both the monolayer and the seeding cell suspension were exposed to retinoic acid, an inhibition in adhesion was also observed. Cell aggregate formation was similarly affected with cells treated with 10-6M retinoic acid forming aggregates which were more readily dispersed and, indeed, these were also smaller than those formed by control cells. Heterotypic adhesion to endothelial cells was inhibited and this was accompanied by a change in cell morphology with adherent retinoic acid-treated cells remaining rounded on the endothelial monolayer. The second part of the project was to investigate the ability of the cells to degrade the extracellular matrix. Subendothelial extracellular matrix was prepared and labelled with either [3H] proline which labelled collagen or newly synthesised collagens present or 35SO4 which labelled the sulphated glycosaminoglycans. Retinoic acid was found to have an inhibitory effect on the release of products labelled with 35S, but a variable effect on the release of 3H-labelled products. This is in disagreement with many other reports. One specific enzyme, plasminogen activator, was investigated more closely. B16F1M2 cells grown in the presence of 10-6M retinoic acid were found to exhibit increased activity of this enzyme, both intracellularly and extracellularly. The third part was to examine the changes, both qualitative and quantitative, in cell surface glycosaminoglycans induced by retinoic acid. In the initial separation the glycosaminoglycans from retinoic acid-treated cells eluted as two peaks when the first separation was carried out as compared to one for the control cells. Enzymatic digestion and further chromatography - both DEAE cellulose and gel filtration - revealed little or no difference in the profiles obtained from both sets of cells. Quantitatively, there was a decrease in sulphated glycosaminoglycan production in cells grown in the presence of retinoic acid. Both the heparan sulphates and the chondroitin sulphates were demonstrated to have a reduced degree of sulphation following exposure to retinoic acid.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: R M MacKie
• Keywords: Cellular biology
• Date of Award: 1993
• Depositing User: Enlighten Team
• Unique ID: glathesis:1993-74991
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 14:44
• Last Modified: 27 Sep 2019 14:44
• URI: https://theses.gla.ac.uk/id/eprint/74991