A rapid quantitative assay for the detection of mammalian heparanase activity.
Heparan sulphate (HS) is an important component of the extracellular matrix and the vasculature basal laminar which functions as a barrier to the extravasation of metastatic and inflammatory cells. Cleavage of HS by endoglycosidase or heparanase activity produced by invading cells may assist in the disassembly of the extracellular matrix and basal laminar, and thereby facilitate cell migration. Heparanase activity has previously been shown to be related to the metastatic potential of murine and human melanoma cell lines [Nakajima, Irimura and Nicolson (1988) J. Cell. Biochem. 36, 157-167]. To determine heparanase activity, porcine mucosal HS was partially de-N-acetylated and re-N-acetylated with [3H]acetic anhydride to yield a radiolabelled substrate. This procedure prevented the masking of, or possible formation of, new heparanase-sensitive cleavage sites as has been observed with previous methods of radiolabelling. Heparanase activity in a variety of tissues and cell homogenates including human platelets, colonic carcinoma cells, umbilical vein endothelial cells and rat mammary adenocarcinoma cells (both metastatic and non-metastatic variants) and liver homogenates all degraded the substrate in a stepwise fashion from 18.5 to approximately 13, 8 and finally to 4.5 kDa fragments, as assessed by gel-filtration analysis, confirming the substrate as suitable for the detection of heparanase activity present in a variety of cells and tissues. A rapid quantitative assay was developed with the HS substrate using a novel method for separating degradation products from the substrate by taking advantage of the decreased affinity of the heparanase-cleaved products for the HS-binding plasma protein chicken histidine-rich glycoprotein (cHRG). Incubation mixtures were applied to cHRG-Sepharose columns, with unbound material corresponding to heparanase-degradation products. Heparanase activity was determined for a variety of human, rat and murine cell and tissue homogenates. The highly metastatic rat mammary adenocarcinoma and murine lung carcinoma cell lines had four to ten times the heparanase activity of non-metastatic variants, confirming the correlation of heparanase activity with metastatic potential. Human cancer patients had twice the serum heparanase levels of normal healthy adults. The assay will be valuable for the determination of heparanase activity from a variety of tissue and cell sources, as a diagnostic tool for the determination of heparanase potential, and for the development of specific inhibitors of heparanase activity and metastasis.
Freeman
C
C
Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT2601, Australia.
Parish
C R
CR
D000328:Adult; D000818:Animals; D002460:Cell Line; D002645:Chickens; D005260:Female; D005966:Glucuronidase; D006026:Glycoside Hydrolases / Q000032:analysis / Q000097:blood / Q000378:metabolism*; D006497:Heparan Sulfate / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa; D007700:Kinetics; D051379:Mice; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000097:blood / Q000201:enzymology*; D011506:Proteins; D051381:Rats; D012016:Reference Values; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D000099053:Histidine-Rich Glycoprotein; D000099175:Heparanase
D005966:Glucuronidase; D006026:Glycoside Hydrolases; D006497:Heparan Sulfate; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; D000099175:Heparanase