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The role of heparanase, an endo-β β β β-glucuronidase specifically degrading heparan sulfate (HS) glycosaminoglycans, in the mechanism of cancer cell invasion was investigated. Three human oral squamous cell carcinoma (SCC) cell lines (i.e., HSC-2, HSC-3 and LMF4), exhibiting various degrees of invasiveness to their surrounding tissues, were xenografted to the tongue of SCID mice in order to establish experimental cancer foci. Cancer cells and their surrounding tissues were examined for the expression of heparanase mRNA by an in situ hybridization technique, and for various basement membrane (BM)-associated molecules (i.e., perlecan, laminins and type IV collagen) by immunohistochemical procedures. BM structures surrounding cancer tissues were also examined by electron microscopy. Increasing levels of heparanase mRNA expression were observed with the progression of cancer invasiveness, as manifested by the destruction of BM structures. rocesses of cancer invasion and metastasis involve distinct biological steps such as destruction of basement membrane (BM), invasion into stroma, intravasation, attachment to endothelia at distant sites and extravasation.1, 2) Among them, the destruction of BM is one of the initial processes characterizing malignant behavior of cancer cells. It is well known that the loss of major BM components such as type IV collagen or the degradation of BM by degradative enzymes such as matrix metalloproteinases (MMPs) plays an important role 3-5) in cancer invasion.Heparan sulfate proteoglycans (HSPG) are distributed widely in higher animals especially in the extracellular matrix (ECM) of BM and as a basic cell surface constituent.6, 7) Their carbohydrate component, heparan sulfate (HS), belongs to a class of highly sulfated polysaccharides called glycosaminoglycans, and has unique characteristics, exhibiting specific molecular interactions with a variety of biologically active molecules, including growth factors, ECM molecules, cell-attachment proteins, enzymes and enzyme inhibitors. [7][8][9][10][11][12] Thus, HSPGs have been demonstrated to participate in the regulation of biological activities involving these molecules. Perlecan, a HSPG, is one of the major BM components and forms a highly organized molecular complex with type IV collagen and laminins, providing a primary scaffold structure to the BM. [13][14][15] Through its HS component, perlecan has also been demonstrated to bind and sequester various growth factors and MMPs in inactive forms in the BM. 16,17) This reservoir function of perlecan in the BM, and controlled release of biologically active molecules when necessary, may be utilized to regulate signal transduction mechanisms between epithelial cells and BM.Heparanase, an endo-β-glucuronidase specifically degrading HS, has been molecularly cloned recently, and proposed to have a role in cancer invasion. [18][19][20][21] It has been shown that heparanase is involved in the process of metastasis in many malignant tumors, such as melanoma, malignant lymphoma, breast cancer, fibro...

Cell surface heparan sulfate proteoglycans undergo unique intracellular degradation pathways after they are endocytosed from the cell surface. Heparanase, an endo-␤-glucuronidase capable of cleaving heparan sulfate, has been demonstrated to contribute to the physiological degradation of heparan sulfate proteoglycans and therefore regulation of their biological functions. A rat parathyroid cell line was found to produce heparanase with an optimal activity at neutral and slightly acidic conditions suggesting that the enzyme participates in heparan sulfate proteoglycan metabolism in extralysosomal compartments. To elucidate the detailed properties of the purified enzyme, the substrate specificity against naturally occurring heparan sulfates and chemically modified heparins was studied. Cleavage sites of rat heparanase were present in heparan sulfate chains obtained from a variety of animal organs, but their occurrence was infrequent (average, 1-2 sites per chain) requiring recognition of both undersulfated and sulfated regions of heparan sulfate. On the other hand intact and chemically modified heparins were not cleaved by heparanase. The carbohydrate structure of the newly generated reducing end region of heparan sulfate cleaved by the enzyme was determined, and it represented relatively undersulfated structures. O-Sulfation of heparan sulfate chains also played important roles in substrate recognition, implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of heparan sulfate proteoglycans. Further elucidation of the roles of heparanase in normal physiological processes would provide an important tool for analyzing the regulation of heparan sulfate-dependent cell functions.

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