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Analysis of CD44/hyaluronan binding in lymphoid and fibroblast cell lines

This thesis set out to determine how regulatory factors, such as possible cytoskeletal associations or cytoplasmic Proteins, could affect how CD44 expressing cells bind hyaluronan. Cell lines expressing CD44 were used as a tool to determine if associations between the cytoskeleton and CD44 could account for the variable hyaluronan binding in cell lines with different binding abilities towards hyaluronan. Analysis of CD44-cytoskeletal associations in lymphoid and fibroblast cell lines suggested no direct link between CD44 and actin in lymphoid cells nor between CD44 and actin, vimentin or keratin in NIH 3T3 fibroblast cells that could regulate hyaluronan binding. It was shown that in one cell line, the T-cell lymphoma WH(4).l in which hyaluronan binding was induced with the phorbol ester, Phorbol 12-Myristate 13- Acetate (PMA), that the increase of hyaluronan binding was prevented by an inhibitor of Protein synthesis and this inhibition was shown to be reversible. L-cells expressing CD44 but lacking the glycosaminoglycans (GAG's) heparan sulphate or chondroitin sulphate showed no differences in their ability to bind hyaluronan or in their distribution of CD44 before or after Triton X-100 solubilization. However, L-cells lacking chondroitin sulphate were unable to be activated to bind hyaluronan with a CD44 monoclonal antibody that normally induces CD44 dependent hyaluronan binding. The distribution of CD44 following Triton X-100 solubilization of NIH 3T3 cells displayed some similarities to that of Thy-1, a glycophosphoinositol Lipid Linked Protein. In addition, both these Proteins were insoluble in Triton X-100 in these cells but were solubilized by a different non-ionic detergent octyl glucoside.