Fluorescent location of malignant cells in fine needle aspirates.

Malignant cells in fine needle aspirates possess a cell surface protease which can be targeted with fluorescent affinity probes. Cells with active GB exhibit cell surface fluorescence when stained with such affinity probes. The nuclei of all cells on the slides can be counterstained with a nuclear fluorescent stain.

Evidence for the induction of a tumour associated cell surface protease on cytologically normal epithelial cells present in the sputum of patients possessing lung tumours.

Sputum obtained from healthy subjects and patients with known lung tumours has been challenged with fluorescent probes for the presence of an active cell surface protease. The mature epithelial cells from healthy patients' sputum lacked ability to bind these fluorescent probes whilst the majority of mature epithelial cells in the tumour patients' sputum bound these probes and consequently fluoresced. This demonstrable difference in the cell surface chemistry of mature epithelial cells was linked to the presence of lung tumour cells, which also possessed this cell surface protease.

Fluorescent location of lung tumour cells.

Cells were collected on glass slides by touching tumour surfaces (A) and normal regions (B) of the lung. The slides were stained with a nuclear stain and a fluorescent probe for a tumour associated cell surface protein. The (B) slides from the normal regions lacked fluorescent epithelial cells.

Sputum cells from lung tumor patients carry a cell surface marker not found in normal sputum.

Cells were collected from sites of known lung tumours and corresponding control areas of these lungs. Fluorescent staining demonstrated that the tumour cells and epithelial cells (cytologically these cells appeared normal) both possessed a receptor for these fluorescent probes. Fluorescent labelling of sputum cells from these tumour patients also resulted in fluorescent labelling of these "cyto logically normal" epithelial cells.

On the stability of a tumour cell surface protease after exposure to 6N HCl.

Well defined acinar tumour cells in frozen sections were exposed to 6NHCl for 5 h at room temperature. A technique was designed to monitor the activity of the enzyme, guanidinobenzoatase (GB), on these tumour cells; this involved cross-linking the enzyme to the cell surface and challenging the active centre with known fluorescent probes which only bind to the functional enzyme. It was demonstrated that the enzymic activity can be regained by appropriate folding of the protein.

A simple clinical method for the preparation of improved cervical smears-approximating to monolayers.

Conventionally prepared cervical smears contain multilayers of cells deposited within strands of mucin. The present study is concerned with chemical reduction of disulphide bonds in the mucin leading to depolymerisation prior to forming a smear in the conventional manner. The resultant distribution of cells on the slide is similar to that obtained by machines designed to produce monolayers of cells.

Fluorescent location of cells of cytological interest in cervical smears prestained with thionin.

Cervical cells of cytological interest were located in conventional smears, treated with thionin for quantitative DNA staining by subsequent treatment with fluorescent probes for a cell surface protease. Normal mature cervical epithelial cells failed to bind these fluorescent probes whilst metaplastic cells, glandular cells, and dyskaryotic cells were readily located. By this means, the nuclear staining of these fluorescent cells of cyotological interest enabled them to be classified by a cytologist.

Correlation of cell surface fluorescence with conventional PAP analysis of cells of cytological interest obtained from cervical scrapes.

Archival PAP stained cervical smears were destained and treated with a fluorescent probe for a cell surface enzyme (GB). Cells which exhibited cell surface fluorescence were demonstrated to be cells of cytological interest in the analysis of cervical smears. These cells could be directly related to PAP and reclassified by subsequent restaining with PAP.

Selectivity of the plasminogen activator inhibitor (PAI-1) for the iso enzyme of guanidinobenzoatase on the surface of colonic carcinoma cells.

The interaction of plasminogen activator-inhibitor (PAI-1) with a cell surface protease, guanidinobenzoatase (GB), has been studied in free solution and on the surface of colonic epithelial cells. It has been demonstrated that PAI-1 recognises and inhibits the iso enzymic form of GB associated with colonic carcinoma cells but fails to bind to the iso enzymic form of GB associated with normal donor colonic epithelial cells. This interaction is mediated by a lysyl binding site on the GB: complex formation prevents GB binding to fibrin fibrils which also involves lysyl binding sites.

Fluorescent location of malignant cells in smears obtained from sputum.

A fluorescent probe (rhodamine a-N-agmatine) has been used to locate cells possessing a surface protease in sputum smears. Malignant epithelial cells possess this protease and can be quickly located by this technique. These results have been confirmed by using a second fluorescent probe (9-animo acridine) for this same cell surface protease.

The fluorescent location of abnormal cervical cells employing the principle of differential competitive inhibition.

The screening of cervical smears is concerned with the detection of abnormal epithelial cells which may be indicative of cervical intraepithelial neoplasia (CIN). Several types of cells in cervical smears possess a cell surface protease where isoenzymic forms of this enzyme can be differentially inhibited. Using this phenomenon a simple fluorescent technique has been developed in conjunction with differential competitive inhibition which enables abnormal cervical epithelial cells to bind the fluorescent probe whilst other cells do not bind the probe.

Differential competitive inhibition of a cell surface protease on normal epithelial cells and carcinoma cells of the colon.

The cell surfaces of normal colonic epithelial cells and colonic carcinoma cells both possess a protease referred to as guanidinobenzoatase (GB). Previous studies have shown that these cells possess two distinct isoenzymic forms of GB which could be distinguished by their selective recognition of cytoplasmic protein inhibitors of GB. In the present study we have used competitive inhibitors of GB to demonstrate the differential inhibition of the GB on normal colonic epithelial cells whilst the GB on colonic carcinoma cell surfaces remains active.

Differential competitive inhibition: the theory of a concept.

The concept of differential competitive inhibition of cell surface isoenzymes is discussed. If the isoenzymes have different structures and functions it is likely that they will handle active site directed molecules at different rates. This could be tested by employing two competitive inhibitors in sequence and exploited if the second inhibitor is fluorescent.

Inhibition of a tumour protease with 3,4-dichloroisocoumarin, pentamidine-isethionate and guanidino derivatives.

Guanidinobenzoatase (GB) is a cell surface proteolytic enzyme capable of degrading fibronectin, and is associated with tumour cells and cells capable of migration. The location of active GB in sections has been demonstrated with 9-aminoacridine (9-AA), a competitive inhibitor of GB. 3,4-Dichloroisocoumarin (3,4-DCI) and pentamidine isethionate (PI) are inhibitors of trypsin-like enzymes.

Retinoic acid inhibition of a tumour protease immobilised on cell surfaces and in free solution.

Retinoids are inhibitors of tumour cell proliferation in culture and have been shown to suppress carcinogenesis and decrease the levels of proteases. The present study has demonstrated that retinoic acid is a potential non-competitive inhibitor of a protease (GB) immobilised on the surfaces of tumour cells in thin sections and free GB in solution. The enzymic status of GB on the cell surfaces in sections has been determined by challenging the retinoic acid-treated cells with a second fluorescent inhibitor (9-AA), followed by fluorescence microscopic analysis.

The interactions of protein inhibitors with tumour proteases studied in solution and immobilised on cell surfaces in frozen sections.

The cell surface protease guanidinobenzoatase (GB) has been purified from human colonic and lung carcinoma tissue by an affinity step involving the binding of the enzyme either onto fibrin fibrils or onto agmatine-sepharose. The inhibitor protein (I) was extracted from the cytoplasm of tumour cells and isolated by an affinity step involving the binding of I to GB on the surface of cultured carcinoma cells. The interaction of GB and I in solution was followed by kinetic studies employing the release of the fluorescent 4-methylumbelliferone (MU) from the synthetic substrate 4-methylumbelliferyl-p-guanidinobenzoate (MUGB).

The application of double fluorescence to the rapid selection of atypical cervical epithelial cells spread in monolayers.

Monolayer spreads of cervical cells were prepared and reacted in sequence with two fluorescent probes. The nuclei were reacted with 4,6-Diamidino-2-phenylindole (DAPI), resulting in white fluorescence of all cell nuclei. Those cells possessing active guanidinobenzoatase (GB) bound the second probe, rhodamine-alpha-N-agmatine (Rh-Agm), resulting in orange cell surface fluorescence.

Association and dissociation of a protease and its inhibitor on the surface of lung squamous cell carcinoma cells.

Squamous cell carcinoma cells possess a cell surface protease, referred to as guanidinobenzoatase (GB). GB is a plasminogen-activator-like enzyme which can be located by the fluorescent probe 9-amino acridine in frozen sections. Fluorescence microscopy has been used to study the inhibition of this GB, the displacement of inhibitor from GB, the displacement of GB from the cell surface receptor and the preparation of both active GB and inhibitor, obtained from these frozen sections of tumour tissue.

A protein inhibitor, extracted from Hela cells, which recognises a cell surface protease on preneoplastic cells obtained from cervical smears.

Hela cells originate from a clone of cervical carcinoma cells. The cytoplasm of Hela cells contains a protein which recognises and inhibits a proteolytic enzyme (guanidino-benzoatase) on the surface of Hela cells. This same protease is present on the surface of abnormal epithelial cells obtained from cervical smears, enabling a rhodamine labelled inhibitor extracted from Hela cells to locate abnormal cervical cells in monolayer spreads.

The status of the surface enzyme guanidinobenzoatase on mature epithelial cells in sputum cell monolayers.

Mature epithelial cells derived from the sputum of normal healthy adults lack GB, while the epithelial cells of sputum collected from patients with lung tumours contain a spectrum of epithelial cells with active GB, some of which are clearly tumour cells, based upon their morphology. The presence of abnormal epithelial cells was confirmed by the cytologist whilst observing the fluorescent stained cells and later by independent cytological analysis of these same cells employing conventional stains.

Affinity preparation of a protein inhibitor recognising a cell surface protease.

Epithelial cell surfaces possess a trypsin-like protease, referred to as guanidinobenzoatase (GB). The cytoplasm of these cells contains an extractable protein (I) which recognises the cell surface GB by forming an enzyme-inhibitor complex (GB-I). Rhodamine-agmatine (Rh-Agm) was designed as a red fluorescent probe, directed to the active centre of GB, which can be used to locate cells with GB, employing fluorescence microscopy.

Further evidence for different isoenzymic forms of a cell surface protease, guanidinobenzoatase, associated with tumours.

Normal colonic epithelial cells possess a cell surface protease referred to as guanidinobenzoatase (GB) and a corresponding cytoplasmic protein inhibitor of GB. Colonic carcinoma cells possess two isoenzymic forms of GB, the normal and the carcinoma specific form, each of which is recognised by the corresponding inhibitors present in the cytoplasm of colonic carcinoma cells. An affinity-purified inhibitor preparation obtained from the cytoplasm of cultured colonic carcinoma cells inhibited these two colonic carcinoma isoenzymic forms of GB but not the GB associated with other forms of tumour.

A simple fluorescent technique for screening cervical cells prior to nuclear analysis.

Monolayer spreads of cervical cells were prepared and stained with haematoxylin and rhodamine-alpha-N-agmatine, a fluorescent marker for a cell surface protease. Mature epithelial cells from normal cervices lacked this cell surface enzyme and did not fluoresce. The abnormal cells possessed the cell surface enzyme, bound the probe and were quickly detected by fluorescence microscopy.