Inhibition of guanidinobenzoatase: evidence for multiple forms of this protease on different tumour cells

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. 9-Aminoacridine is a competitive inhibitor of this enzyme and has been used to locate cells possessing this enzyme in wax embedded sections by means of fluorescent microscopy. Naturally occurring inhibitors of guanidinobenzoatase can be extracted from different tissues. These inhibitors show selectivity in their ability to inhibit the binding of 9-aminoacridine to different types of tumour cells which have invaded human liver tissue. Inhibition is non-competitive and reversible. The results indicate that guanidinobenzoatase exists in a number of different forms on the surface of different tumour cells. These different forms of the enzyme were recognised by inhibitors obtained from different organs. It is suggested that these inhibitors may have a regulatory role in tumour cell migration.     

The role of inhibitors in the fluorescent staining of benign naevus and malignant melanoma cells with 9-amino acridine and acridine orange

Guanidinobenzoatase is a trypsin-like protease capable of degrading fibronectin. An inactive form of guanidinobenzoatase is present on the surface of benign naevus cells and these cells stain very weakly with 9-aminoacridine, a known competitive inhibitor of guanidinobenzoatase. Malignant melanoma and metastatic malignant melanoma cells exhibit strong surface staining with 9-aminoacridine and also exhibit strong staining of cytoplasmic RNA with acridine orange. These simple fluorescent techniques have been used to distinguish benign naevus cells from malignant melanoma cells in human skin sections. This difference in cell surface staining with 9-aminoacridine has been demonstrated to be caused by the presence or absence of an inhibitor. The inhibitor can be displaced from the cell surface enzyme and then replaced by an affinity purified inhibitor obtained from fresh liver homogenates. It is proposed that the inhibition or control of cell surface guanidinobenzoatase may be one of the regulatory mechanisms by which benign naevus cells are prevented from developing into malignant melanoma cells.     

The Role of Inhibitors in the Fluorescent Staining of Benign Naevus and Malignant Melanoma Cells with 9-Amino Acridine and Acridine Orange

Abstract

Guanidinobenzoatase is a trypsin-like protease capable of degrading fibronectin. An inactive form of guanidinobenzoatase is present on the surface of benign naevus cells and these cells stain very weakly with 9-aminoacridine, a known competitive inhibitor of guanidinobenzoatase. Malignant melanoma and metastatic malignant melanoma cells exhibit strong surface staining with 9-aminoacridine and also exhibit strong staining of cytoplasmic RNA with acridine orange. These simple fluorescent techniques have been used to distinguish benign naevus cells from malignant melanoma cells in human skin sections. This difference in cell surface staining with 9-aminoacridine has been demonstrated to be caused by the presence or absence of an inhibitor. The inhibitor can be displaced from the cell surface enzyme and then replaced by an affinity purified inhibitor obtained from fresh liver homogenates. It is proposed that the inhibition or control of cell surface guanidinobenzoatase may be one of the regulatory mechanisms by which benign naevus cells are prevented from developing into malignant melanoma cells.     

Fluorescent inhibitors of a cell surface protease used to locate leukaemia cells in kidney sections

Guanidinobenzoatase is a trypsin-like protease on the surface of cells capable of migration, for example leukaemia cells. We have used a number of fluorescent probes that are competitive inhibitors of guanidinobenzoatase to locate leukaemia cells in resin sections of kidney tissue obtained from leukaemic rats. We have demonstrated how this competitive inhibition system can be used to direct desired molecules (such as cytotoxic drugs) to these cells and to monitor the arrival of such compounds at the active site of guanidinobenzoatase. The principles developed in this study could equally well be applied to other enzymes on other cells provided suitable competitive inhibitors were designed. The presence of an enzyme on the surface of a cell can be used to direct molecules to that cell provided that these molecules contain a functional group that acts as an inhibitor for the chosen enzyme.     

Inhibition of a protease associated with tumour cells and the probable mechanism of regulation of this interaction in vivo

The regulation of activity of a protease on the surface of human colonic tumour cells implanted in nude mice has been studied. A synthetic fluorescent probe was used to locate cells possessing active guanidinobenzoatase in frozen sections of tumour bearing tissues. These studies demonstrated the presence of intracellular protein inhibitors of guanidinobenzoatase which could be extracted in isotonic saline and transferred to the outer surface of the tumour cells. This study showed that the inhibition of guanidinobenzoatase was pH dependent and that the tumour cell may regulate the activity of its surface guanidinobenzoatase by exporting lactic acid.     

Direct evidence for the cell surface location of a protease-inhibitor complex on intact leukaemia cells

The interaction of a protease with two fluorescent inhibitors has been studied using intact fixed leukaemia cells as the source of the membrane bound enzyme. Fresh rat leukaemia cells were disrupted and the cytosol collected; this extract was known to contain a protein inhibitor of guanidinobenzoatase (GB) associated with leukaemia cells. All the cytosolic proteins were derivatised with Texas red acid chloride. Leukaemia cells with latent GB failed to bind the Texas red inhibitor protein but did so after activation of GB. Competition experiments with 9-amino acridine (a fluorescent marker for the active site of GB) demonstrated that the Texas red-inhibitor protein could only bind to intact leukaemia cells when the active centre of GB was not already occupied by 9-amino acridine. This competition between these two fluorescent inhibitors demonstrated their specificity for GB. The use of intact leukaemia cells and the high molecular weight of the inhibitor protein precludes the possibility of any interaction between GB and inhibitor within the cells. It is concluded that GB and the GB-inhibitor complex of latent GB are located on the external surface of intact leukaemia cells.     

The inhibitor reacting with a tumour cell surface protease can be exchanged with plasminogen activator inhibitor (PAI-1)

Tumour cells possess the cell surface protease guanidinobenzoatase (GB) which can be located by the fluorescent probe 9-amino acridine (9-AA). Frozen sections and formaldehyde fixed sections of tumour tissue were used to demonstrate the interactions between GB, 9-AA and two protein inhibitors of GB. A cytoplasmic extract from the tumour tissue, and a purified inhibitor of plasminogen activator (PAI-1) were shown to be exchangeable components of the enzyme-inhibitor complex on the fixed tumour cell surfaces. The evidence suggests that GB is functionally very similar to plasminogen activator and that this enzyme can be regulated by protein inhibitors in vivo and also by changes in the redox potential at the cell surface.     

Fluorescent location of human colonic tumour cells by means of an enzyme-inhibitor complex

We studied the enzymic status of the tumour cell surface protease, guanidinobenzoatase (GB) in frozen sections of a human colonic tumour grown in nude mice and also in human colons. Active enzyme was demonstrated by the binding of a synthetic fluorescent probe for the active centre of guanidinobenzoatase (GB). It was observed that tissue derived inhibitors of GB blocked the binding of this fluorescent probe and that enzyme inhibitor complex formation could be controlled by lowering the pH of the medium with lactic acid. The presence of an inhibitor of GB in the mouse tumour extract was taken advantage of by making two fluorescent derivatives of this inhibitor; both of which located GB on colonic tumour cells in frozen sections of human colon.     

The protective role of a natural inhibitor in the fluorescent location of cells possessing a latent form of cell surface protease.

Leukaemia cells possess a latent form of a cell surface protease referred to as guanidinobenzoatase. Latency is due to complex formation between an inhibitor protein and the cell surface enzyme which is stable under acid conditions but is dissociated with formaldehyde treatment. The latent form of the cell surface protease has been used as a protecting mechanism during a preliminary step to stain all the nuclei of cells with haematoxylin. The enzyme-inhibitor complex was then dissociated and a combination of 9-amino acridine and propidium iodide employed to enable the fluorescent location of cells possessing active guanidinobenzoatase. We were thus able to visualise the nuclei by conventional light microscopy and simultaneously visualise the cell surface of leukaemia cells by fluorescent microscopy. This simple model system has provided technology applicable to the more complex analysis of neoplastic cells in cervical smears.     

Fluorescent Location of Human Colonic Tumour Cells by Means of an Enzyme-Inhibitor Complex

Abstract

We studied the enzymic status of the tumour cell surface protease, guanidinobenzoatase (GB) in frozen sections of a human colonic tumour grown in nude mice and also in human colons. Active enzyme was demonstrated by the binding of a synthetic fluorescent probe for the active centre of guanidinobenzoatase (GB). It was observed that tissue derived inhibitors of GB blocked the binding of this fluorescent probe and that enzyme inhibitor complex formation could be controlled by lowering the pH of the medium with lactic acid. The presence of an inhibitor of GB in the mouse tumour extract was taken advantage of by making two fluorescent derivatives of this inhibitor; both of which located GB on colonic tumour cells in frozen sections of human colon.     

The design of fluorescent probes which bind to the active centre of guanidinobenzoatase. Application to the location of cells possessing this enzyme

Cells possessing a known enzymic activity may be located by fluorescent probes designed to act as competitive inhibitors of this enzyme. We have prepared a series of dansyl N-substituted guanidino derivatives which bind to the active centre of guanidinobenzoatase. 9-Aminoacridine also acts as a competitive inhibitor and behaves similarly to these guanidino derivatives. These fluorescent probes have been used to locate tumour cells possessing this enzyme in thin sections of fixed tissue by employing fluorescent microscopy.     

Further inhibition studies on guanidinobenzoatase, a trypsin-like enzyme associated with tumour cells

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasminogen activator, plasmin, thrombin and a newly described tumour associated enzyme specific for guanidino phenylalanine residues. These conclusions have been derived from inhibition studies employing 4-methyl-p-guanidinobenzoate as substrate. Three active site titrants for trypsin have been shown to be good substrates for guanidinobenzoatase. A new active site titrant for trypsin, rhodamine bisguanidinobenzoate, can also be used to assay guanidinobenzoatase in a stoichiometric manner. This active site titrant can be employed to label guanidinobenzoate on the surface of leukaemia cells.     

The estrogen-regulated destabilization of Xenopus albumin mRNA is independent of translation

Protein synthesis inhibitors have been shown to increase the stability of a number of labile mRNAs. In Xenopus laevis serum albumin mRNA is destabilized in the liver cell cytoplasm following estrogen administration. The present study examined the effect of translation inhibitors on this process. The initiation inhibitor 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide causes accumulation of albumin mRNA in 20-80S mRNP particles whereas the elongation inhibitor cycloheximide causes albumin mRNA to accumulate in polysomes. Neither inhibitor blocked the disappearance of albumin mRNA from liver cell cytoplasm when added with estradiol to the medium of liver explant cultures. We conclude that unlike a number of labile mRNAs the instability of Xenopus albumin mRNA following estradiol is independent of translation.     

Hepatic proliferation inhibitor

Summary Although a long held tenet of biology has been that endogenous inhibitors can modulate cell proliferation, little progress was made in purifying any such inhibitor. This was largely due to the rarity of non-malignant cell cultures in which regulation of cell division was still operative, and to problems in separating cytotoxic and cytostatic effects in the complex biological extracts which were being studied. During the last decade, hepatic proliferation inhibitors of varying degrees of purity have been isolated using regenerating rat liver or hepatoma cell cultures as test systems. In these early studies, a number of inhibitors with differing molecular weights, physicochemical properties and biological responses were purified from liver cytosol and/or serum. Some of them could inhibit DNA synthesis or mitosis and thus were considered to be G1 or G2 inhibitors. However, experiments which could give precise answers about mechanisms of action could not be done until an inhibitor purified to homogeneity was available.Using well-characterized rat liver diploid epithelial cell cultures, which maintain a number of liver properties and which do not possess any transformation markers or malignant properties, we recently purified an hepatic proliferation inhibitor to a homogenous protein. It has a molecular weight of 26 000 daltons and an isoelectric point of 4.65. It specifically inhibits cell division and DNA synthesis in a number of non-malignant rat liver epithelial cell types, and has no effect on transformed liver cells, or hepatoma cells, in culture. Its effect is not mediated through destruction or sequestration of essential nutrients or calcium ions. Nor have preliminary experiments shown the hepatic proliferation inhibitor to interfere with the binding of epidermal growth factor to its receptors. The majority of the cells treated with the inhibitor are blocked in the G1 phase. Further experiments to study its mechanism of action and the inter-relationship, if any, between the cell cycle block induced by serum or nutrient deprivation, and the inhibitor-induced cycle block are in progress.     

Depression of macrophage function by a factor produced by neoplasms: a merchanism for abrogation of immune surveillance.

The possibility that macrophages mediate surveillance against the development of neoplasms has been reciving increasing support. The acquisition, by neoplastic cells, of the capacity to subvert macrophage function may be an important mechanism by which they escape destruction by the host and become established tumors. Indeed, animals implanted with syngeneic neoplasms developed depressed macrophage migratory ability in vivo and chemotactic responsiveness in virto. It therefore seemed plausible that neoplasms might be capable of producing inhibitors of macrophage function. The present report describes the identification of such a low molecular weight (6,000 to 10,000), heat-stable inhibitor of murine macrophage accumulation in vivo and chemotaxis in vitro. The inhibitor of macrophages was present in four different murine neoplasms, but not present in normal liver, spleen, or inflammatory exudate cells and did not affect PMN chemotaxis in vitro. When given with low numbers of neoplastic cells, the inhibitor increased both the frequency of tumor development and rate of tumor growth. By producing inhibitors of macrophage function, neoplasms may escape initial host surveillance mechanisms.     

CONTROL OF GRANULOCYTE PRODUCTION: SEPARATION AND CHEMICAL IDENTIFICATION OF A SPECIFIC INHIBITOR (CHALONE)

Abstract. It has previously been shown by others that blood serum contains inhibitors of blood cell production acting on the proliferation of granulocy tic and erythrocytic precursor cells in the bone marrow. It is now shown that the active extract from calf blood serum can be further subfractionated into six different components, all of them exhibiting inhibitory effects on the proliferation of rat bone marrow cells in vitro. Ascitic fluid from rats treated intraperitoneally with polyvinylpyrrolidone contains inhibitors which apparently are the same as those found in calf serum.
It was further possible to demonstrate that only one of these inhibitors is contained in mature granulocytes where it is actively synthesized from amino acids and subsequently released into the surrounding medium. By chromatography on Sephadex G-25 of this conditioned medium the inhibiting substance could be obtained in relatively pure form being contaminated only by low amounts of two ninyhdrin-positive substances. the experiments allow the granulocytic inhibitor to be identified as a polypeptide with a molecular weight below 5000. the results suggest that this substance is the granulocytic chalone.     

Catalytic properties of monooxygenases from isolated immunocompetent cells

The kinetic parameters of two monoxygenase (N-demethylase and benzo(a)pyrene hydroxylase) reactions occurring in murine immunocompetent cells were determined. It was shown that the pH optimum for macrophage enzymes lies at 7.4, whereas that for thymocytes and spleen cells at 8.5. The effects of cytochrome P-450 inhibitors (methyrapone, SKF-525A, tilorone) on monooxygenase systems of immunocytes were investigated. Methyrapone and SKF-525A were shown to be effective inhibitors of N-demethylase. The mode of inhibition changes from non-competitive to competitive, depending on the inhibitor concentration. Tilorone, an immunostimulator and inhibitor of liver cytochrome P-450-dependent enzymes, competitively inhibits N-demethylase and benzo(a)pyrene hydroxylase of immunocytes.     

Inhibition of guanidinobenzoatase by a substrate for trypsin-like enzymes

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasmin and thrombin, the latter enzymes can be assayed with bis(carbobenzyloxycarbonyl-L-argininamido)-Rhodamine or BZAR. Guanidinobenzoatase is inhibited by BZAR when the enzyme is assayed in free solution and when the enzyme is cell-bound in frozen sections of tumour containing tissues. It is proposed that BZAR and its analogues may be of value in inhibiting tumour cell invasion in vivo and also that the selectivity of BZAR may be used to direct cytotoxic drugs to tumour cells possessing active guanidinobenzoatase.     

A theoretical consideration of fundamental biological phenomena on cell-specific mitosis-inhibiting protein excretion hypothesis

A new hypothesis on mitotic control mechanism in eukaryotic cells has been recently proposed. The hypothesis is based on a concept obtained from studies on mitotic control mechanism in the regenerating rat liver. The concept is that there exists a double layered pattern of humoral factors; a primary mitotic inhibitor and secondary mitotic inhibitors or stimulators. The primary mitotic inhibitor is a protein and has been termed cell-specific mitosis-inhibiting protein.According to the concept several aspects of mitosis of the hepatocyte and the related phenomena are discussed. The putative form of the cell-specific mitosis-inhibiting protein is searched for in some fundamental biological phenomena such as immune response (antibody or T-cell factor), fibrogenesis (procollagen) and myogenesis (myosin). The origin of the polypeptide hormone or the organizer is tentatively inferred on the same line of consideration. Some biological aspects of senescence are also discussed.The hypothesis, cell-specific mitosis-inhibiting protein excretion hypothesis, may offer a new theoretical framework to our modern biology.     

A letrozole-based dual aromatase-sulphatase inhibitor with in vivo activity

The role of aromatase inhibitors in the treatment of hormone-dependent breast cancer is well established. However, it is now recognised that steroid sulphatase (STS) inhibitors represent a new form of endocrine therapy. To explore the potential advantage of dual inhibition by a single agent, we recently developed a series of dual aromatase-sulphatase inhibitors (DASIs) based on the aromatase inhibitor YM511. We report here a new structural class of DASI obtained by obtained introducing the pharmacophore for STS inhibition, i.e. a phenol sulphamate ester into another established aromatase inhibitor letrozole. Hence, the bis-sulphamate 9 was synthesised which exhibited IC(50) values of 3044 nM for aromatase and >10 microM for STS in JEG-3 cells. However, at a single oral dose of 10mg/kg, 9 inhibited aromatase and rat liver STS by 60% and 88%, respectively, 24h after administration. A proposed metabolite of 9, carbinol 10, was synthesised. Despite also showing weak STS inhibition in JEG-3 cells, 10 inhibited rat liver STS activity to the same extent as 9 at a single oral dose of 10mg/kg. Thus, the concept of a letrozole-based DASI has been validated and could be further developed and modified for therapeutic exploitation.