Macrophage polarization as a novel weapon in conditioning tumor microenvironment for bladder cancer: can we turn demons into gods?

Macrophages are major components of the immune infiltration in cancer where they can affect tumor behavior. In the bladder, they play important roles during the resolution of infectious processes and they have been associated with a worse clinical prognosis in bladder cancer. The present review focused on the characteristics of these important immune cells, not only eliciting an innate immune surveillance, but also on their importance during the cancer immunoediting process. We further discuss the potential of targeting macrophages for anticancer therapy, the current strategies and the state of the art as well as the foreseen role on combined therapies on the near future. This review shows how a comprehensive understanding of macrophages within the tumor should translate to better clinical outcome and new therapeutic strategies focusing especially on bladder cancer.

Effect of proteasome inhibitors on the release into the cytosol of free polymannose oligosaccharides from glycoproteins.

Prompted by previous observations which suggested that the release of polymannose oligosaccharides shortly after the cotranslational N-glycosylation of proteins is a function of the ER-associated quality control system (Moore and Spiro (1994) J. Biol. Chem., 269, 12715-12721), we evaluated the effect which proteasome inhibitors have on the appearance of these free saccharide components. Employing as a model system castanospermine-treated BW5147 mouse T-lymphoma cells in which accelerated degradation of the T-cell receptor (TCR) alpha subunit takes place (Kearse et al. (1994) EMBO J., 13, 3678-3686), we noted that both lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal, but not leupeptin, brought about a rapid and substantial reduction in the release of free polymannose oligosaccharides into the cytosol during pulse-chase studies, while the oligosaccharides in the intravesicular compartment remained unchanged, as measured by streptolysin O permeabilization. This inhibition was furthermore selective in that it affected solely the components terminating in a single N-acetylglucosamine residue (OS-GlcNAc(1)) and not the oligosaccharides terminating in a di-N-acetylchitobiose sequence (OS-GlcNAc(2)), which reside primarily in the intravesicular compartment. Despite the quantitative effect of the proteasome inhibitors on the cytosolic oligosaccharides, the molar distribution of the triglucosyl OS-GlcNAc(1) species was unaffected. The decrease in cytosolic oligosaccharides brought about by proteasome inhibition was reflected in a pronounced increase in the stability of the TCRalpha subunit. Our findings suggest that the N-deglycosylation and proteasome mediated degradation are coupled events. On the basis of our data and those of others we propose that the quality control mechanism involves proteasomes associated with the cytosolic side of the endoplasmic reticulum acting in concert with a membrane situated N-glycanase. Such a complex by removing the carbohydrate units could facilitate the retrograde ER to cytosol translocation of glycoproteins.

Characterization of the epitope on murine T-cell receptor (TCR) alpha proteins recognized by H28-710 monoclonal antibody.

Antigen recognition by alphabeta T lymphocytes is mediated via the multisubunit T-cell receptor (TCR) complex consisting of invariant CD3-gamma,delta,epsilon, and zeta chains associated with clonotypic TCRalpha,beta molecules. In the current report, we evaluated the molecular basis for recognition of murine TCRalpha proteins by H28-710 monoclonal antibody (MAb), specific for the constant region of murine TCRalpha chains. H28-710 is widely used in the study of the TCR complex as it is the only reagent currently available that recognizes all murine TCRalpha proteins, regardless of their clonotype. These data show that H28-710 is useful for the immunoprecipitation of TCRalpha proteins not associated with CD3 subunits, and that H28-710 effectively recognizes denatured TCRalpha proteins synthesized in several different cell types. Most importantly, these results demonstrate that H28 binding involves a serine/threonine-rich region between amino acids 150-177 on murine TCRalpha polypeptides.

Processing of viral envelope glycoprotein by the endomannosidase pathway: evaluation of host cell specificity.

Endo-alpha-D-mannosidase is an enzyme involved in N-linked oligosaccharide processing which through its capacity to cleave the internal linkage between the glucose-substituted mannose and the remainder of the polymannose carbohydrate unit can provide an alternate pathway for achieving deglucosylation and thereby make possible the continued formation of complex oligosaccharides during a glucosidase blockade. In view of the important role which has been attributed to glucose on nascent glycoproteins as a regulator of a number of biological events, we chose to further define the in vivo action of endomannosidase by focusing on the well characterized VSV envelope glycoprotein (G protein) which can be formed by the large array of cell lines susceptible to infection by this pathogen. Through an assessment of the extent to which the G protein was converted to an endo-beta-N-acetylglucosaminidase (endo H)-resistant form during a castanospermine imposed glucosidase blockade, we found that utilization of the endomannosidase-mediated deglucosylation route was clearly host cell specific, ranging from greater than 90% in HepG2 and PtK1 cells to complete absence in CHO, MDCK, and MDBK cells, with intermediate values in BHK, BW5147.3, LLC-PK1, BRL, and NRK cell lines. In some of the latter group the electrophoretic pattern after endo H treatment suggested that only one of the two N-linked oligosaccharides of the G protein was processed by endomannosidase. In the presence of the specific endomannosidase inhibitor, Glcalpha1-->3(1-deoxy)mannojirimycin, the conversion of the G protein into an endo H-resistant form was completely arrested. While the lack of G protein processing by CHO cells was consistent with the absence of in vitro measured endomannosidase activity in this cell line, the failure of MDBK and MDCK cells to convert the G protein into an endo H-resistant form was surprising since these cell lines have substantial levels of the enzyme. Similarly, we observed that influenza virus hemagglutinin was not processed in castanospermine-treated MDCK cells. Our findings suggest that studies which rely on glucosidase inhibition to explore the function of glucose in controlling such critical biological phenomena as intracellular movement or quality control should be carried out in cell lines in which the glycoprotein under study is not a substrate for endomannosidase action.

Sulphation of N-linked oligosaccharides of vesicular stomatitis and influenza virus envelope glycoproteins: host cell specificity, subcellular localization and identification of substituted saccharides.

The presence of sulphate groups on various saccharide residues of N-linked carbohydrate units has now been observed in a number of glycoproteins. To explore the cell specificity of this post-translational modification, we evaluated sulphate incorporation into virus envelope glycoproteins by a variety of cells, since it is believed that assembly of their N-linked oligosaccharides is to a large extent dependent on the enzymic machinery of the host. Employing the vesicular stomatitis virus (VSV) envelope glycoprotein (G protein) as a model, we noted that the addition of [35S]sulphate substituents into its complex carbohydrate units occurred in Madin-Darby canine kidney (MDCK), Madin-Darby bovine kidney, LLC-PK1 and BHK-21 cell lines but was not detectable in BRL 3A, BW5147.3, Chinese hamster ovary, HepG2, NRK-49F, IEC-18, PtK1 or 3T3 cells. The sulphate groups were exclusively located on C-3 of galactose [Gal(3-SO4)] and/or C-6 of N-acetylglucosamine [GlcNAc(6-SO4)] residues in the N-acetyllactosamine sequence of the branch chains. Moreover, we observed that the pronounced host-cell-dependence of the terminal galactose sulphation was reflected by the 3'-phosphoadenosine 5'-phosphosulphate:Gal-3-O-sulphotransferase activity assayed in vitro. Comparative studies carried out on the haemagglutinin of the influenza virus envelope formed by MDCK and LLC-PK1 cells indicated that sulphate in this glycoprotein was confined to its complex N-linked oligosaccharides where it occurred as Gal(3-SO4) and GlcNAc(6-SO4) on peripheral chains as well as on the mannose-substituted N-acetylglucosamine of the core. Since sulphation in both internal and peripheral locations of the virus glycoproteins was found to be arrested by the alpha1-->2 mannosidase inhibitor, kifunensine, as well as by the intracellular migration block imposed by brefeldin A, it was concluded that this modification is a late biosynthetic event which most likely takes place in the trans-Golgi network.

Characterization and purification of fucosyltransferases from the cytosol of rat colon.

The occurrence and baseline characteristics of fucosyltransferases (alpha-1,2, alpha-1,3 and alpha-1,4) in the cytosol (soluble) and pellet (membrane-bound) of rat colon have been studied since the fucosylation process is known to alter in colon pathology. All enzymes studied in the colon pellet had higher activity when compared to the cytosol. The colon pellet alpha-1,3 fucosyltransferase preferred desialylated alpha 1-acid glycoprotein as acceptor substrate. Both soluble and membrane-bound enzymes, alpha-1,2 and alpha-1,3 fucosyltransferases, required Mn2+, Mg2+ and Ca2+ for maximum activity but were inactivated by Cu2+ ions. Both soluble alpha-1,2 and alpha-1,3 fucosyltransferases showed optimal activity at pH 6.0, whereas the optimum for their membrane-bound activities were at pH 5.8 and 6.2, respectively. Furthermore, a soluble alpha-1,3 fucosyltransferase from rat colon was purified and during purification the co-presence of alpha-1,3/4 fucosyltransferase was detected. The acceptor of preference for the purified soluble alpha-1,3 fucosyltransferase was desialylated glycoprotein while low molecular weight substrates were poor acceptors. Both the purified fucosyltransferases were inhibited by N-ethylmaleimide. The M(r) values determined by SDS-PAGE electrophoresis of alpha-1,3/4 fucosyltransferase and of alpha-1,3 fucosyltransferase were 68,780 and 40,680 respectively. In conclusion, based on their properties, the purified soluble colon alpha-1,3 fucosyltransferase appeared to be of plasma-type (or FT-I) while the soluble alpha-1,3/4 fucosyltransferase corresponded to Lewis-type or FT-III.

Activity and characterization of sialyltransferase from serum of normal rats, of rats bearing Zajdela ascitic hepatoma, in normal host liver and in Zajdela hepatoma cells.

Comparative studies on the content of sialic acid and on the sialyltransferase activity in normal serum and in serum of rats with Zajdela ascitic hepatoma in different phases of tumor development have been conducted. Unlike the serum from animals with tumors, in which the sialic acid quantity increases in dependence of the stage of tumor development, the activity of serum sialyltransferase statistically augmented only in serum of rats at the final stage of tumor progression. The sialyltransferase activity towards asialofetuin as an acceptor in normal liver and in Zajdela hepatoma cells, was measured and a decrease in this activity in tumor cells as well as in host liver was found. When lactose was used as acceptor, again lower enzyme activity in the tumor cells in comparison with that in liver was established, but in liver and in hepatoma cells the predominant 14C-labelled product of the sialyltransferase assay was alpha (2-6) sialyllactose isomer. The results contribute to the biochemical characterization of rat Zajdela hepatoma.

Comparative studies on the uridine-5'-diphosphate-galactose: glycoprotein galactosyltransferase activity in rat liver and Zajdela ascitic hepatoma.

We report here data on the galactosyltransferase activity in serum, liver and Zajdela ascitic hempatoma cells and about the rate of galactose attachment to appropriate acceptors by beta(1-4, beta(1-3) and alpha(1-3) linkages in liver and hepatoma cell homogenates. It was established that in serum of Zajdela bearing rats, in host liver and in Zajdela ascitic cells, galactosyltransferase activity towards ovomucoid was elevated in comparison with control serum and liver. The activity towards low molecular acceptor (N-acetylglucosamine) in liver and hepatoma was nearly the same. The predominant isomer synthesized in both tissues was beta 1-4-galactose-N-acetylglucosamine. When asialofetuin was used as acceptor the activity of alpha(1-3) galactosyltransferase was measured. In Zajdela ascitic hepatoma cells alpha(1-3) galactosyltransferase activity was 3 times higher than that in liver. The elevated ability of the tumor cells to attach galactose residues with alpha(1-3) linkage to Gal beta 1-4GlcNAc-R sequence should be considered as a characteristic feature of these malignant cells.

Pattern of sialoglycoproteins obtained by chromatofocusing of chicken liver and hepatoma Mc-29 microsomal preparations labelled in vivo with 3H-leucine and N-acetyl-14C-mannosamine.

Chromatofocusing has been used for separation of chicken liver and virus-induced hepatoma Mc-29 microsomal glycoproteins double labelled in vivo with 3H-leucine and N-acetyl-14C-mannosamine. The sialoglycoprotein profile was obtained by plotting the pH-values, as well as the values of the calculated specific activity (SA-cpm/mg protein) in each fraction, in the graphs. Different patterns for liver and hepatoma sialoglycoproteins were detected. Unlike liver microsomes in which the highest labelled compounds were registered in the alkaline zone of the pH-gradient, special feature for the hepatoma sialoglycoprotein pattern was the presence of highly labelled fraction eluted in the acidic zone of the pH-gradient. A term named "sialylation rate" of a separated sialoglycoproteins was involved. It has been found that liver sialoglycoproteins are more or less uniformly sialylated, independently of the pI-values, while those from hepatoma with acidic pI were sialylated at a higher extent in comparison to the fractions with alkaline pI.

Partial characterization of microsomal sialyltransferase from chicken liver and hepatoma Mc-29: I. Effect of nucleotides and metal ions.

CMP-N-acetylneuraminic acid: glycoprotein sialyltransferase activities were assayed in microsomal fractions from chicken liver and hepatoma, induced by the leukosis virus strain Mc-29, using asialofetuin as the substrate acceptor of N-acetylneuraminic acid. The effect of some nucleotides and metal ions on the enzyme activity was investigated. Kinetic studies revealed that the Km values toward asialofetuin at a saturation concentrations of CMP-N-acetylneuraminic acid for both liver and hepatoma enzymes are very closed, while V value was lower for the tumor enzyme. The liver and hepatoma enzymes have no exogenous Mn cations requirement and are inhibited by CTP, CMP and ATP. CMP was shown to act as a competitive inhibitor with an apparent Ki of 0.24 mM for the liver and 0.16 mM for hepatoma enzyme, respectively.

Partial characterization of microsomal sialyltransferase from chicken liver and hepatoma Mc-29: II. Measurement of enzyme activities utilizing microsomal glycoproteins as exogenous acceptors.

Microsomal sialyltransferase was assayed in chicken liver and hepatoma Mc-29 utilizing liver and hepatoma microsomal glycoprotein fractions, treated with Triton X-100, as exogenous acceptors. In a homologous assay system containing enzyme and acceptor from one and the same tissue no quantitative dependence of enzyme activity was revealed with increasing amount of the acceptor. In mixed experiments in which liver enzyme activity was tested towards hepatoma acceptor glycoproteins, a gradual drop in sialyltransferase activity occurred with increasing quantities of the acceptor. This effect seems to be a consequence of the presence of some inhibitor in the microsomal fractions from the hepatoma cells.

Physico-chemical analysis of systems with a main component acetylsalicylic acid in ethanol-water mixtures. Part 3: Systems containing calcium acetylsalicylate.

The systems C9H8O4-(C9H7O4)2Ca-25% ethanol-water mixture (I), C9H8O4-(C9H7O4)2Ca-50% ethanol-water mixture (II), C9H8O4-(C9H7O4)2Ca-95% ethanol-water mixture (III) have been investigated at 15 degrees C by physico-chemical analytical methods. It was established that under the conditions of the no experiments no double or triple compounds of acetylsalicylic acid and calcium acetylsalicylate have been formed. The fields of the crystallization of the pure components are outlined. In systems I and II there exist crystallization fields of (C9H7O4)2Ca - 2H2O and in system III field of (C9H7O4)2Ca. The anhydrous salt and the crystalline hydrate were isolated and were investigated by chemical analysis and X-ray diffraction analysis.