This project is composed of two areas of research: (I) Cellular control of nucleoside diphosphate hexose synthesis and hyaluronan production and (II) Regulation of pyrimidine nucleotide synthesis in vivo. Hyaluronan is a glycosaminoglycan that is associated with tumor invasion and metastasis. We are using Swiss 3T3 fibroblasts as our model cell system. These cells synthesize hyaluronan as the major component of their extracellular matrix. We found that hyaluronan synthesis is stimulated by growth factors (EGF, bFGF, insulin, PDGF, TGF beta) and by media conditioned by the growth of human carcinoma cell lines. These findings support a model for tumor-associated hyaluronan synthesis in vivo whereby hyaluronan is produced by fibroblasts in response to tumor- derived hyaluronan-stimulating factors. Studies of the cellular regulation of hyaluronan synthesis are focused on four enzymes as possible regulatory control points. We found that the cellular production of hyaluronan correlates with hyaluronan synthase activity. Of the three enzymes involved in the synthesis of UDP-hexoses that are precursors for hyaluronan, activities of UDP-glucose dehydrogenase and glucosamine-6-phosphate synthetase follow a time course that is similar to hyaluronan synthase, whereas UDP-glucose synthetase does not. We explored the use of hyaluronan-derived oligosaccharides to disrupt tumor-stromal interactions. We prepared oligosaccharides of up to 16 monosaccharide units using Streptomyces hyaluronan lyase. Enzyme products were purified by gel exclusion chromatography and characterized by electrospray-ionization mass spectrometry and high-performance anion- exchange chromatography with pulsed amperometric detection. In addition to the expected even-numbered oligomers, we found minor amounts of odd- numbered oligomers that had not been reported for this enzyme. These purified oligomers are under study for possible anti-metastatic activity in an in vivo animal tumor model.
