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.     

Evidence for inhibitors of the cell surface protease guanidinobenzoatase

Guanidinobenzoatase is a protease present on the surface of tumour cells. The present study describes the isolation of a protein inhibitor of guanidinobenzoatase obtained from extracts of liver and pancreas and purified by affinity techniques. Pancreatic acinar cells have been shown to possess a latent form of guanidinobenzoatase and this latency is due to complex formation with the inhibitor. A fluorescent marker has been employed to demonstrate the presence or absence of the inhibitor on sections of pancreatic tissue. The inhibitor has been shown to be exchangeable with liver and pancreatic inhibitors obtained from different species. It is postulated that these inhibitors may play a role in enzyme control.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Temporary competitive inhibition of a tumour cell surface protease as a protective mechanism in the preparation of the membrane bound native enzyme in the presence of excess cytoplasmic inhibitors.

Tumour cells possess a cell surface protease which is recognised and inhibited by a cytoplasmic protein extractable from frozen sections of tumour cells. In order to prepare sections with tumour cells carrying cell surface-bound native protease in the absence of this internal inhibitor we have used a reversible competitive inhibition step as a temporary measure to protect the active centre of GB whilst the cytoplasmic inhibitor is extracted from the frozen sections. These sections are described as protected in the sense that the enzyme is native and fully functional now that potential inhibitors have been extracted. The protected cell surface protease immobilised in the cell surface of squamous cell carcinoma cells has been used as the target for inhibition studies and displacement studies. The ability to follow these inhibition and exchange reactions concerning the cell surface protease has been made possible by virtue of the fluorescent probe, 9-amino acridine, which locates the active centre of the protease. Cells with active protease bind 9-amino acridine and fluoresce yellow; cells lacking this protease or having inhibited protease fail to bind 9-amino acridine and do not fluoresce.     

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.     

Inhibition of a cell surface protease after cisplatin chemotherapy.

The epithelial cells in squamous carcinoma and leukoplakia of the oral cavity possess the cell surface protease, guanidinobenzoatase (GB), in an active form. GB is closely similar to plasminogen activator, a protease associated with both transformed cells and tumour cells. The active centre of GB binds the fluorescent probe 9-aminoacridine (9-AA) enabling cells containing active GB to be visualised by fluorescent microscopy. It was observed that chemotherapy with cisplatin resulted in a marked decrease in cell surface GB activity and this decrease was due to the formation of an enzyme-inhibitor complex. One of the results of chemotherapy was shown to be the suppression of a cell surface protease which is known to be associated with migration and malignancy of cells in vivo.     

Molecular forms of chicken embryo acetylcholinesterase in vitro and in vivo. Isolation and characterization

The four molecular forms of chick embryo leg muscle acetylcholinesterase have been isolated by velocity sedimentation; their apparent sedimentation coefficients are 19.5 S, 11.5 S, 7.1 S, and 5.4 S. All four forms are glycoproteins, exhibit the same Km for acetylcholine, and are inhibited to the same extent by specific inhibitors of acetyl- and buryrylcholinesterase. Treatment of the 19.5 S form of acetylcholinesterase with trypsin generates an array of molecular forms, several of which have sedimentation coefficients identical with the naturally occurring forms. Collagenase treatment of the 19.5 S acetylcholinesterase results in a somewhat different pattern of acetylcholinesterase forms including a novel 20.6 S form. Only the 19.5 S acetylcholinesterase is sensitive to collagenase treatment. Our results indicate that the several acetylcholinesterase forms share a common catalytic subunit, and suggest that the molecular forms of acetylcholinesterase in the chick represent different ensembles of a common monomer. In culture, the muscle cells contain only the 11.5 and 7.1 S acetylcholinesterase forms; however, they also secrete substantial amounts of enzyme into the medium. These secreted acetylcholinesterases have sedimentation coefficients of 9 S and 15 S. The relative abundance of the different acetylcholinesterase molecular forms changes during muscle development, both in vivo and in vitro, suggesting that the assembly and distribution of this family of membrane glycoproteins is developmentally regulated.     

Interaction of the antitumor drug 9-aminoacridine with guanidinobenzoatase studied by spectroscopic methods: a possible tumor marker probe based on the fluorescence exciplex emission

Fluorescence spectroscopy, surface-enhanced Raman spectroscopy (SERS), and analytical centrifugation are applied in this work to study the interaction of the antitumor drug 9-aminoacridine (9AA) with a trypsin-like protease, guanidinobenzoatase (GB), extracted from an Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission can be detected at a certain drug and enzyme concentration. The 9AA exciplex emission was also studied for 9AA interacting with others serin proteases: alpha-chymotrypsin, trypsin, and penicillin G-acylase (PGA), as well as with bovine serum albumin (BSA) in order to obtain information about the active center of GB. We have found that the exciplex 9AA emission may be induced by a ring-stacking interaction between the monomeric drug, under the amino form, and an aromatic residue placed in the catalytic site of the protein. The results derived from Raman spectroscopy corroborate this interaction mechanism, as demonstrated by the existence of typical protonated amino 9AA marker bands as well as an important modification of the ring vibrations, thus indicating the existence of an interaction through ring stacking. The analytical centrifugation technique was applied to study the GB association in aqueous solution, demonstrating that the 9AA/GB interaction depends on the enzyme quaternary structure. An interaction of 9AA with an associate form of GB, which may be the actual enzyme active form, is suggested.     

Surface-enhanced Raman and steady fluorescence study of interaction between antitumoral drug 9-aminoacridine and trypsin-like protease related to metastasis processes, guanidinobenzoatase

Fluorescence spectroscopy and surface-enhanced Raman spectroscopy (SERS) were applied to study the interaction of the antitumoral drug 9-aminoacridine (9AA) with a trypsin-like protease guanidinobenzoatase (GB) extracted from a mouse Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission was detected in the emission fluorescence spectra at certain drug and enzyme concentrations. A SERS study was accomplished on silver colloids at several excitation wavelengths in order to obtain more information about the interaction mechanism. The results derived from Raman spectroscopy indicated that 9AA in the amino monomeric form may interact with the enzyme by means of two different bonds: an ionic bond with a negatively charged amino acid and a ring stacking interaction with an aromatic residue placed in the catalytic site of GB. This interaction mechanism was responsible for a strong exciplex emission detected at a longer wavelength than the expected value of the normal fluorescence emission. Moreover, the GB concentration dependence of the interaction suggested that the drug was sensitive to the quaternary structure of the enzyme.     

Genome wide CRISPR/Cas9 screen identifies the coagulation factor IX (F9) as a regulator of senescence

During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.Subject terms: Senescence, Tumour biomarkers     

Hyaluronidase in sera of tumour-bearing nude mice

Cancer cell lines often secrete hyaluronidase, suggesting that this enzyme could be used as a marker of growing tumours. We have measured hyaluronidase in the sera of non-grafted mice and mice grafted with human tumour-derived hyaluronidase-secreting H460M and SA87 cells or non-secreting CB 193 cells. Mouse serum hyaluronidase was measured at pH 3.8 using the enzyme-linked sorbent assay (ELSA) technique by reference to human serum whose activity at pH 3.8 was determined by the Reissig technique. The serum hyaluronidase in non-grafted mice ranged from 310-520 mU l^?1 (mean±SD 432±70 mU l^?1, median 440 mU l^?1). Hyaluronidase increased in the sera of tumour-bearing mice grafted with H460M cells or with SA87 cells, but not in the sera of mice grafted with CB 193 cells. Serum hyaluronidase activity in H460M or SA87 tumour-bearing mice correlated with the tumour mass, increased with time, and decreased after tumour removal. Zymography detected two different hyaluronidase forms in the sera of non-grafted mice: type 1 had only one hyaluronidase band and type 2 had five different bands. In both types, enzyme augmentation in tumour-bearing mice correlated with the presence of an additional enzyme band that was not seen in normal sera and that migrated as the cancer cell enzyme did; there was no augmentation of the normal isoform(s). These results show that serum hyaluronidase can be used to follow the development of tumours in mice grafted with hyaluronidase-secreting cells.