Medical Applications of Hyaluronan and its Derivatives

Human-embryo fibroblasts were synchronized by means of colchicine and cytochalasin, and the production of hyaluronate was determined by [3H]glucosamine incorporation and ion-exchange chromatography. Cells arrested by colchicine synthesized small amounts of hyaluronate, whereas cells blocked by cytochalasin were stimulated in hyaluronate production. When the colchicine block was released, there was an increased synthesis of hyaluronate, which appeared first in the cellular fraction and was then shed into the culture medium. After release of the cytochalasin block, the hyaluronate production declined to that found with unsynchronized cells. A comparable increase of hyaluronate synthase activity was observed during mitosis. When hyaluronate synthesis was blocked by periodate-oxidized UDP-glucuronic acid, the cells were arrested in mitosis before rounding of cells. These results suggest that hyaluronate synthesis is required for detachment and rounding of cells during mitosis.

The hybrid cell B6 line, which synthesizes large amounts of hyaluronate as the predominant glycosaminoglycan, was grown in the presence of [3H]glucosamine. The [3H]hyaluronate has a high molecular weight and was excluded by Sephacryl S-1000. After disruption of the cells the [3H]hyaluronate could further be elongated by incubation with UDP-GlcNAc and UDP-[14C]GlcA, yielding a hybrid molecule of hyaluronate labelled with [3H]GlcNAc and [14C]GlcA. Treatment of the cells with hyaluronidase before disruption eliminated the large [3H]hyaluronate and elongation of nascent chains in vitro commenced from low-molecular-weight chains. Thus nascent hyaluronate chains were degraded extracellularly by hyaluronidase and were therefore synthesized at the inner side of plasma membranes and extruded to the cell surface.