texas-red and Colonic-Neoplasms

Pentosan polysulphate (PPS), a highly negatively charged polysaccharide, is a significant inhibitor of an isoenzymic form of a cell surface protease referred to as guanidinobenzoatase GB, associated with colonic carcinoma tissues in frozen sections and free GB in solution, in a concentration-dependent manner. However PPS failed to recognise and bind to the isoenzymic form of GB associated with normal colon epithelial cell surfaces. Texas red labelled PPS (TR-PPS) binds to the tumour cell surfaces of colonic carcinoma and colonic polyps and these cells fluoresce red, whilst the normal colon cell surfaces failed to bind the TR-PPS, and hence lacked red fluorescence. Polysulphonated suramin also selectively recognised and inhibited the colonic carcinoma GB isoenzyme. The kinetic data indicated that this inhibition was not caused by a mere polyanionic effect, since highly sulphated heparin failed to show a significant inhibition of colonic carcinoma GB, however trypan blue did show 50% inhibition. Kinetic studies have also shown that PPS is a non-competitive, reversible inhibitor of colonic carcinoma GB, with an apparent Km 6.8 x 10(-7) M. Gel analysis has shown that PPS binds to another site, distinct from the active centre, and after binding PPS changed the conformation of GB. These studies suggest that TR-PPS is a potent inhibitor of colonic carcinoma GB, and can be used as a novel fluorescent probe for the location of tumour cells in frozen sections of human colon tissues. PSS could also have potential as a vehicle for the transport of cytotoxic compounds to carcinoma cells of the colon.

Topics: Carboxylic Ester Hydrolases; Cell Membrane; Chromatography, Affinity; Colonic Neoplasms; Dansyl Compounds; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Fluorescent Dyes; Heparin; Humans; Hymecromone; Isoenzymes; Kinetics; Microscopy, Fluorescence; Pentosan Sulfuric Polyester; Protease Inhibitors; Protein Conformation; Suramin; Trypan Blue; Xanthenes

A novel method was developed to measure the effective permeability of microvessels in three-dimensional tumors. Two unique features characterized our approach: (i) Texas Red (with peak excitation and peak emission wavelengths of 596 and 615 nm, respectively) was used for macromolecular labeling, to minimize the absorption of fluorescence light by hemoglobin in blood. Thus the tumor tissue could be treated approximately as a uniform medium with respect to light absorption. (ii) The light absorption and scattering in tumor tissues were accounted for in relating the fluorescence intensity to the amount of Texas Red-labeled macromolecules extravasated. The vascular permeability of Texas Red-labeled bovine serum albumin in human tumor xenograft LS174T implanted in dorsal skin-fold chamber in severe combined immunodeficient mice was measured using this method. The average permeability-surface area product per unit volume (PS/V, x 10(-4) sec-1) and the average effective permeability (P, x 10(-7) cm/sec) were found to be 1.26 +/- 0.72 and 6.06 +/- 4.30, respectively; the fractional volume of tumor vessels (Vves/V, %) was found to be 9.2 +/- 2.9, and the total surface area of vessels per unit volume (S/V, cm2/cm3) was found to be 239 +/- 82. The errors in the estimation of these parameters are discussed. The method described here is general and can be adapted to study the microvascular permeability of superficial tumors in various organs in patients or animals.

Topics: Adenocarcinoma; Animals; Capillary Permeability; Colonic Neoplasms; Fluorescent Dyes; Mice; Mice, SCID; Microcirculation; Microscopy, Fluorescence; Neoplasm Transplantation; Nephelometry and Turbidimetry; Prostheses and Implants; Serum Albumin, Bovine; Skin; Specific Pathogen-Free Organisms; Transplantation, Heterologous; Videotape Recording; Xanthenes

Adenocarcinoma cells often form intracellular lumens and intercellular cysts. In order to study the structural relationships between these lumens and the apical domain of normal enterocytes, we have applied electron microscopy and confocal microscopy to a cloned cell line derived from the human colon adenocarcinoma cell line LoVo which express a high number of intracellular lumens and intercellular cysts. Microvilli reminiscent of those detected in the brush border of small intestinal cells are formed in the two types of compartments. By immunofluorescence, we found that a 135 kDa membrane glycoprotein characterized by a monoclonal Ab and normally associated with the brush-border of enterocytes is expressed at the surface of the intracellular lumens and intercellular cysts present in the adenocarcinoma cells. Comparison of fluorescence and reflection contrast micrographs obtained by confocal microscopy demonstrate the presence of spherical intracellular lumens in the juxtanuclear region of single cells, and of more complex shaped intercellular cysts located within clusters of cells. The later cells form junctional complexes limiting an apical plasma membrane domain in contact with the intercellular cyst. It is suggested that the intracellular lumens may represent the abortive form of an apical plasma membrane due to the lack of components required to establish epithelial cell contacts. As opposed to conventional fluorescence microscopy, confocal microscopy allows rapid inspection of the tridimensional organization of intracellular lumens and intercellular cysts even when they are located in cell multilayers.

Topics: Adenocarcinoma; Cell Compartmentation; Cell Membrane; Colonic Neoplasms; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Microscopy, Electron; Microscopy, Fluorescence; Microvilli; Tumor Cells, Cultured; Xanthenes