Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-07-10
2001-02-20
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S201000
Reexamination Certificate
active
06190875
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to novel qualitative and quantitative glycosidases catalytic activity assays. More particularly, the present invention relates to a method of screening for potential anti-metastatic and anti-inflammatory agents and, most particularly, to a method of screening for potential anti-metastatic and anti-inflammatory agents using mammalian heparanase, preferably purified human recombinant heparanase, as a probe.
heparan sulfate proteoglycans (HSPGs): HSPGs are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (
1
-
5
). The basic HSPG structure consists of a protein core to which several linear heparan sulfate chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups (
1
-
5
). Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPGs in embryonic morphogenesis, angiogenesis, metastasis, neurite outgrowth and tissue repair (
1
-
5
). The heparan sulfate (HS) chains, which are unique in their ability to bind a multitude of proteins, ensure that a wide variety of effector molecules cling to the cell surface (
4
-
6
). HSPGs are also prominent components of blood vessels (
3
). In large vessels they are concentrated mostly in the intima and inner media, whereas in capillaries they are found mainly in the subendothelial basement membrane where they support proliferating and migrating endothelial cells and stabilize the structure of the capillary wall. The ability of HSPGs to interact with ECM macromolecules such as collagen, laminin and fibronectin, and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion. Cleavage of HS may therefore result in disassembly of the subendothelial ECM and hence may play a decisive role in extravasation of normal and malignant blood-borne cells (
7
-
9
). HS catabolism is observed in inflammation, wound repair, diabetes, and cancer metastasis, suggesting that enzymes which degrade HS play important roles in pathologic processes.
Involvement of heparanase in tumor cell invasion and metastasis: Circulating tumor cells arrested in the capillary beds of different organs must invade the endothelial cell lining and degrade its underlying basement membrane (BM) in order to escape into the extravascular tissue(s) where they establish metastasis (
10
). Several cellular enzymes (e.g., collagenase IV, plasminogen activator, cathepsin B, elastase) are thought to be involved in degradation of the BM (
10
). Among these enzymes is an endo-&bgr;-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites (
7
,
9
,
11
-
12
). Expression of a HS degrading heparanase was found to correlate with the metastatic potential at mouse lymphoma (
11
), fibrosarcoma and melanoma (
9
) cells. The same is true for human breast, bladder and prostate carcinoma cells (see U.S. pat application Ser. No. 09/109,386, which is incorporated by reference as if fully set forth herein). Moreover, elevated levels of heparanase were detected in sera (
9
) and urine (U.S. patent application Ser. No. 09/109,386,) of metastatic tumor bearing animals and cancer patients and in tumor biopsies (
12
). Treatment of experimental animals with heparanase alternative substrates and inhibitor (e.g., non-anticoagulant species of low MW heparin, laminarin sulfate) markedly reduced (>90%) the incidence of lung metastases induced by B16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells (
8
,
9
,
13
), indicating that heparanase inhibitors may be applied to inhibit tumor cell invasion and metastasis.
Our studies on the control of tumor progression by its local environment, focus on the interaction of cells with the extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells (EC) (
14
,
15
). This ECM closely resembles the subendothelium in vivo in its morphological appearance and molecular composition. It contains collagens (mostly type III and IV, with smaller amounts of types I and V), proteoglycans (mostly heparan sulfate- and dermatan sulfate-proteoglycans, with smaller amounts of chondroitin sulfate proteoglycans), laminin, fibronectin, entactin and elastin (
13
,
14
). The ability of cells to degrade HS in the ECM was studied by allowing cells to interact with a metabolically sulfate labeled ECM, followed by gel filtration (Sepharose 6B) analysis of degradation products released into the culture medium (
11
). While intact HSPG are eluted next to the void volume of the column (Kav<0.2, Mr of about 0.5×10
6
), labeled degradation fragments of HS side chains are eluted more toward the Vt of the column (0.5<kav<0.8, Mr of about 5-7×10
3
) (
11
). Compounds which efficiently inhibit the ability of heparanase to degrade the above-described naturally produced basement membrane-like substrate, were also found to inhibit experimental metastasis in mice and rats (
8
,
9
,
13
,
33
). A reliable in vitro screening system for heparanase inhibiting compounds may hence be applied to identify and develop potent anti-metastatic drugs.
Possible involvement of heparanase in tumor angiogenesis: We have previously demonstrated that heparanase may not only function in cell migration and invasion, but may also elicit an indirect neovascular response (
15
). Our results suggest that the ECM HSPGs provide a natural storage depot for PFGF and possibly other heparin-binding growth promoting factors. Heparanase mediated release of active &bgr;FGF from its storage within ECM may therefore provide a novel mechanism for induction of neovascularization in normal and pathological situations (
6
,
18
).
Expression of heparanase by cells of the immune system: Heparanase catalytic activity correlates with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses. Interaction of platelets, granulocytes, T and B lymphocytes, macrophages and mast cells with the subendothelial ECM is associated with degradation of heparan sulfate (HS) by heparanase catalytic activity (
7
). The enzyme is released from intracellular compartments (e.g., lysosomes, specific granules) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens, mitogens), suggesting its regulated involvement and presence in inflammatory sites and autoimmune lesions. Heparan sulfate degrading enzymes released by platelets and macrophages are likely to be present in atherosclerotic lesions (
16
). Treatment of experimental animals with heparanase alternative substrates (e.g., non-anticoagulant species of low molecular weight heparin) markedly reduced the incidence of experimental autoimmune encephalomyelitis (EAE), adjuvant arthritis and graft rejection (
7
,
17
) in experimental animals, indicating that heparanase inhibitors may be applied to inhibit autoimmune and inflammatory diseases (
7
,
17
). A reliable in vitro screening system for heparanase inhibiting compounds may hence be applied to identify and develop non-toxic anti-inflammatory drugs for the treatment of multiple sclerosis and other inflammatory diseases.
Cloning and expression of the heparanase gene: A purified fraction of heparanase isolated from human hepatoma cells was subjected to tryptic digestion. Peptides were separated by high pressure liquid chromatography and micro sequenced. The sequence of one of the peptides was used to screen data bases for homology to the corresponding back translated DNA sequence. This procedure led to the identification of a clone containing an insert of 1020 base pairs (bp) which included an open reading frame of 963 bp foll
Ayal-Hershkovitz Maty
Ben-Artzi Hanna
Miron Daphna
Pecker Iris
Peleg Yoav
Friedman Mark M.
Insight Strategy & Marketing Ltd.
Weber Jon P.
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