Jurij Rozhin

Cathepsin B and its endogenous inhibitors: the role in tumor malignancy

SummarySeveral lysosomal proteinases including the cysteine proteinase cathepsin B have been implicated in malignant progression of tumors. Many investigators have demonstrated correlations between increased activity of cathepsin B and increased metastatic capability of animal tumors or malignancy of human tumors. Such increases in cathepsin B activity in malignant tumors may reflect alterations in synthesis, in activation and processing, and/or in intracellular trafficking and delivery as well as in the endogenous inhibitors of cathepsin B. Increases in mRNA transcripts for cathepsin B have been observed in both murine and human tumors and multiple transcripts for cathepsin B have been identified, but an association of multiple transcripts with malignancy has not been confirmed. Cathepsin B precursors found in human malignant ascites fluid do not possess mannose-rich carbohydrates suggesting that a defect in the post translational processing of carbohydrate moieties on tumor cathepsin B may be responsible for the release of cathepsin B observed in many tumor systems. However, the intracellular trafficking of cathepsin B responsible for its association with plasma membrane/endosomal systems and for its release willrequire further study as both latent, precursor forms of cathepsin B and native forms of cathepsin B are involved. We speculate that malignant tumor cells adherent to basement membrane are capable of forming a digestive microenvironment in which lysosomal proteinases such as cathepsin B function optimally, a microenvironment similar to that formed between adherent osteoclasts and bone. One of the endogenous cysteine proteinase inhibitors, stefin A, also is affected by malignancy. Reduced expression (mRNA and protein) of stefin A is found as well as a reduction in its inhibitory capacity against cysteine proteinases. The data to date at both the molecular and protein levels supporting a functional role(s) for cathepsin B and its endogenous inhibitors in cancer progression are only correlative. Experimental approaches utilizing well-defined model systems in conjunction with genetic manipulation of cathepsin B and its endogenous inhibitors are needed to provide convincing evidence that cathepsin B has an important role in cancer.

Plasma membrane-associated cysteine proteinases in human and animal tumors

The ability of tumor cells to invade into and through normal tissue during the metastatic cascade has been attributed to tumor-associated degradative enzymes including proteinases of the metallo, serine and cysteine classes. Work from several laboratories has established that the cysteine proteinases cathepsins L and B are released from tumor cells, primarily as latent precursor forms. In addition, a cathepsin B-like cysteine proteinase has been shown to be associated with the plasma membrane fraction of several animal and human tumors. This form of the enzyme retains activity under physiologic (or pathologic) conditions including at neutral pH and in the presence of low Mr inhibitors. Since we have established that cathepsin B can degrade the basement membrane attachment glycoprotein laminin, we speculate that plasma membrane-associated cathepsin B may participate in focal dissolution of the basement membrane during tumor cell extravasation.

Enhanced levels of cathepsin B mRNA in murine tumors

Relative amounts of mRNA for cathepsin B were measured in normal murine liver and three murine tumors, an invasive liver tumor (hepatoma, Hepa cl 9) and two melanoma variants (B16‐F1 and B16 amelanotic melanoma, B16a). Using a human cDNA to the cathepsin B coding region as a hybridization probe, we detected two species of cathepsin B specific RNA transcripts (2.2 and 4.1 kb) in total RNA preparation of all four tissues. The concentrations of the 2.2 and 4.1 kb species were 3.6 and 2.7‐fold greater in the highly metastatic B16a melanoma than in normal liver. The concentration of the 2.2 kb species in the invasive hepatoma wa 1.7‐fold greater than in normal liver. The increased levels of the 2.2 kb message were reflected in increases in activity of cathepsin B in both Hepa cl 9 and B16a.

A possible role for cysteine proteinase and its inhibitors in motility of malignant melanoma and other tumour cells.

The metastasis of malignant tumour cells depends on their rapid replication, and their ability to adhere to the matrix of a biological barrier such as basement membrane, to degrade the matrix, and to migrate through this more permeable barrier. Secreted enzymes, including the cysteine proteinases cathepsins B and L, are known to degrade basement membrane components. Using a barrier-free substratum we studied the possible role of cysteine proteinases in influencing the motility per se of metastatic cells. We found that stefins, the natural inhibitors of cysteine proteinases, markedly decreased the stimulated motility of both human melanoma cells and W256 carcinosarcoma cells at low concentrations (0.5 μM). A stefin also inhibited melanoma cell adherence, but to a lesser extent than motility. Additionally, synthetic inhibitors (E-64, diazomethyl ketones) of cysteine proteinases were found to depress stimulated motility of W256 cells. These results suggest that cysteine proteinases and their inhibitors may have a direct role in the development of a migratory response per se in tumour cells.

Effects of 12-Hydroxyeicosatetraenoic Acid on Release of Cathepsin B and Cysteine Proteinase Inhibitors from Malignant Melanoma Cells

The cysteine proteinase cathepsin B as well as the endogenous inhibitors of this enzyme have been implicated in the progression of tumors from a premalignant to a malignant state (for review see 1,2). Activity of cathepsin B has been shown to be elevated in parallel with malignancy or metastatic potential of both human and rodent tumors. These increases in cathepsin B activity correspond in part to increases in mRNA for cathepsin B and in part to reduced regulation by the endogenous low Mr cysteine proteinase inhibitors (CPIs). The inhibition constants for the interaction between stefin A purified from human tumors and cysteine proteinases are an order of magnitude greater than those for stefin A purified from human liver. Most properties of tumor cathepsin B appear to be similar to those of cathepsin B from normal tissues. However, the subcellular distribution of cathepsin B and CPIs is altered in tumors, resulting in association of cathepsin B and CPIs with plasma membrane fractions or in release of CPIs and of high Mr forms of cathepsin B (native and latent) into the extracellular mileau.

Enzyme assay in cultures of Escherichia coli by a continuous flow method based on lysis from without by a phage ghost

Abstract Lysis of Escherichia coli from without by excess of phage ghost has been shown to give excellent yield of several enzymes. The application of lysis from without to a continuous determination of enzymes in growing cultures of E. coli is illustrated with β-galactosidase. This application can be used in studies on changes of a large number of enzymes during metabolic perturbation (induction, repression, etc.) of growing cultures of E. coli .

Cathepsin B-Like Cysteine Proteinases and Metastasis

During the process of metastasis tumor cells traverse several extracellular matrix barriers in order to gain entry to the vascular space at the site of the primary tumor and to the perivascular space at the sites of metastatic tumors. A number of in vitro model systems have been designed to study tumor cell invasion through extracellular matrices. Several of these model systems including systems to study the concomitant digestion of these matrices by tumor cells are discussed elsewhere in this volume (see Chapters 18–21). A focus of much recent research is the basement membrane which underlies endothelial cells. The physical location of the basement membrane suggests that it must be traversed for tumor cells to form hematogenous metastases. Since the basement membrane in contrast to other extracellular matrix barriers contains type IV collagen a number of investigators have hypothesized that tumor cells must be able to degrade type IV collagen in order to invade through the basement membrane (see also Chapter 19). Metastatic variants of the B16 melanoma have been shown to secrete a type IV collagenase (1). However, type IV collagenase is secreted by tumor cells in a latent form which requires activation (2) suggesting that type IV collagenase is not by itself sufficient for basement membrane invasion by tumor cells and that additional proteinases or a proteolytic cascade may participate in basement membrane invasion.

Studies on bovine adrenal estrogen sulfotransferase. V. Synthesis and assay of analogs of 3′-phosphoadenosine 5′-phosphosulfate as cosubstrates for estrogen sulfurylation

Abstract Analogs of adenosine 3−phosphate 5′-phosphosulfate (3′PAdo5′PS ∗ ) have been prepared by an extension of a method for the synthesis of 3′PAdo5′PS, itself. Reaction of the ribonucleoside 2′,3′-cyclic phosphate 5′-phosphate with triethylamine sulfonic acid leads to a ribonucleoside 2′,3′-cyclic phosphate 5′-phosphosulfate. The latter, on treatment with ribonuclease-T 2 , provides the 3′-phosphate analog. Spleen phosphodiesterase, on the other hand, promotes opening of the 2′,3′-cyclic phosphate residue to a 2′-phosphate analog. In this manner, the 2′,3′-cyclic phosphates of 8-bromoadenosine-, nebularine-, inosine-, tubercidin-, and formycin-, 5′-phosphosulfates have been converted to corresponding 2′-, and 3′-phosphate analogs. V for the utilization of 3′PAdo5′PS analogs in the sulfurylation of estrone, as mediated by bovine adrenal estrogen sulfotransferase (3′-phosphoadenylylsulfate : estrone 3-sulfotransferase, EC 2.8.2.4), is decreased more than 50% by the replacement of the 7-(ring)nitrogen atom by carbon or of the 8-H atom by bromine in the adenine moiety. However, binding of these analogs at the active site of the enzyme is still very efficient (small K m and K i values; competitive inhibition). The replacement of the 9-nitrogen atom of adenine by carbon lowers V even more, and the replacement of the 6-amino group by H, i.e., nebularine, or by 0 (inosine) results in 90 and 100% loss of enzyme activity, respectively. Shifting of the 3′-phosphate to the 2′ position in the ribose of adenosine is also detrimental and results in noncompetitive inhibition of 3′PAdo5′PS utilization.