Identification of bladder tumor-derived hyaluronidase: Its similarity to HYAL1

The glycosaminoglycan hyaluronic acid (HA) and its degrading enzyme, hyalur onidase, are intricately associated with tumor metastasis and angiogenesis. HA promotes tumor cell adhesion and migration, whereas its small fragments stimulate angiogenesis. Such small HA fragments are generated from the deg radation of HA by hyaluronidase. We have previously shown (V. B. Lokeshwar et al., Cancer Res., 57: 773-777, 1997) that the HA levels are elevated in the urine and tumor tissues of bladder cancer patients regardless of the tu mor grade (G). The hyaluronidase levels were found to be elevated in the ur ine and tumor tissues of G2 and G3 bladder cancer patients. Furthermore, an giogenic HA fragments mere isolated from the urine of G2/G3 bladder cancer patients, which stimulated endothelial cell proliferation, a key event in a ngiogenesis. In this study, me characterized the bladder tumor-derived hyal uronidase. Analysis of hyaluronidase activity in the culture-conditioned me dia (CM) of 11 bladder cancer cell lines, using an ELISA-like assay and a s ubstrate (HA)-gel technique, showed that the invasive bladder cancer cell l ines secrete elevated levels of a M-r similar to 60,000 hyaluronidase. Reve rse transcription-polymerase chain reaction, cloning, and sequence analyses revealed the expression of an HYAL1 transcript in bladder cancer lines. HY AL1 encodes for a hyaluronidase that is present in serum. Immunoblot analys is using an anti-HYAL1 peptide IgG confirmed the presence of a M-r similar to 60,000 HYAL1-related protein in the CM of bladder cancer cell lines, in the urine specimens from G2 and G3 bladder cancer patients, and in the part ially purified preparations of bladder tumor-derived hyaluronidase. No HYAL 1-related protein was detected in urine specimens from normal individuals, G1 bladder cancer patients, and patients with a history of bladder cancer b ut no disease at the time of testing. The bladder tumor-derived hyaluronida se present in CM and partially purified preparations was found to have maxi mum activity at a pH range of 4.1-4.3. The identification of bladder tumor- derived hyaluronidase should help in elucidating its role in bladder tumor progression.