ROLE OF URINARY HYALURONATE ASSAYS IN THE DETECTION OF BLADDER CARCINOMA

I. Production of Hyaluronate by Established Cell Lines from Human Bladder Carcinoma In Vitro

Abstract

The purpose of the present study is to examine the hypothesis that human urothlial carcinoma produces hyaluronate, which is thought to be one of migration factors of malignant cells in the event of invasion into the connective tissue. In order to clarify whether hyaluronate is produced in a significant amount by urothelial carcinoma in vitro, the hyaluronate concentration was measured in incubation medium of cell lines derived from human bladder carcinomas (NBT-2, MGH-U1, T-24, KU-1 and KU-7) and these results were compared with those of cell lines derived from human fibroblast, uterus carcinoma (Hela) and human renal cell carcinoma (KU-2). Furthermore, histological study was performed to evaluated the distribution of acidic glycosaminoglycans (GAG) in the cytoplasm of these cells.
The original tumors of NBT-2, MGH-U1 and T-24 were high grade, non-papillary and invasive bladder urothelial tumors, while KU-1 and KU-7 were derived from papillary, non-invasive urothelial carcinoma.
For the determination of GAG as a total amount, the incubation medium was collected for 10 days from culture bottles and passed through 8.0μ-pore filter to exclude cell debris. Aliquats were used for the measurement of hyaluronate by a hyaluronate-specific radioassay. The amount of total GAG was measured by the carbazole reaction reported by Bitter & Muir in the incubation medium which had been digested in pronase-E solution, and the results were expressed as the amount of uronic acid per mg protein of cells used for the assay.
The amount of total GAG in the incubation medium was highest in fibroblast (181±15μg/mg protein; mean±S.E.M.) and this was followed, in a decreasing order, by T-24, MGH-U1, KU-7, NBT-2 and KU-1. The hyaluronate production in the medium was also highest in fibroblast. In the 5 cell lines originated from human bladder carcinomas, those from high grade, invasive tumors (MGH-U1, T-24) seemed to have higher hyaluronate production rates than those from non-invasive tumors. Moreover, the histochemical study with colloidal iron stain revealed the presence of abundant ferrisol particles in the perinuclear region (perinuclear granules) predominantly in those cell lines with higher HA production. The amount of uronic acid as well as hyaluronate in the incubation medium of Hela and KU-2 was not measurable, as these data were subtracted with blank values in control medium.
The distribution of abundant GAG in the cytoplasm and significant hyaluronate production in the cell lines from highly malignant bladder carcinomas suggest that hyaluronate and other related GAG might be involved in the characterization of the aggressiveness of the original tumors.