Functional differences between two morphologically distinct cell subpopulations within a human colorectal carcinoma cell line

The LISP-I human colorectal adenocarcinoma cell line was isolated from a hepatic metastasis at the Ludwig Institute, Säo Paulo, SP, Brazil. The objective of the present study was to isolate morphologically different subpopulations within the LISP-I cell line, and characterize some of their behavioral aspects such as adhesion to and migration towards extracellular matrix components, expression of intercellular adhesion molecules and tumorigenicity in vitro. Once isolated, the subpopulations were submitted to adhesion and migration assays on laminin and fibronectin (crucial proteins to invasion and metastasis), as well as to anchorage-independent growth. Two morphologically different subpopulations were isolated: LISP-A10 and LISP-E11. LISP-A10 presents a differentiated epithelial pattern, and LISP-E11 is fibroblastoid, suggesting a poorly differentiated pattern. LISP-A10 expressed the two intercellular adhesion molecules tested, carcinoembryonic antigen (CEA) and desmoglein, while LISP-E11 expressed only low amounts of CEA. On the other hand, adhesion to laminin and fibronectin as well as migration towards these extracellular matrix proteins were higher in LISP-E11, as expected from its poorly differentiated phenotype. Both subpopulations showed anchorage-independent growth on a semi-solid substrate. These results raise the possibility that the heterogeneity found in the LISP-I cell line, which might have contributed to its ability to metastasize, was due to at least two different subpopulations herein identified

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis

Evaluation of an anti-carcinoembryonic monoclonal antibody suitable for immunoscintigraphy

An anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb 6D1.1) was evaluated in vitro and in vivo to determine its suitability as a tracer for immunoscintigraphy of colorectal carcinomas. Determination of mAb affinity for CEA showed a constant of association of 0.63 + or - 0.11 x 109 M-1. Binding of technetium-99m (99mTc)-6D1.1, labeled by a direct method, to human cultured lineages was highly specific. Binding to only CEA-positive LS-174T cells resulted in a saturable curve inhibited by pre-incubation with unlabeled mAb. No binding at all was observed for the human lineages MeWo (melanoma) or ZR75-30 (breast carcinoma), neither of them expressing CEA cells. Intravenous injection of 99mTc-6D1.1 into nude mice xenografted with human LS-174T tumors resulted in planar images of excellent quality. Localization of an irrelevant mAb labeled with either 99mTc or iodine-125 was never observed in tumor masses. Biodistribution studies on excised tumoral tissue showed retention of 28.48 percent of the injected dose per gram of LS-174T tumor. The tumor-to-blood ratio was 3.46. The same analysis performed on the other three human xenografted tumors studied demonstrated that only the CEA-producing HT-29 (colorectal adenocarcinoma) retained 99mTc-6D1.1 while the other two (ZR75-30 and MeWo) did not. These data demonstrate that this mAb is an adequate tool for targeting CEA-expressing tumors in experimental models

Monoclonal antibody-based immunoassay for carcinoembryonic antigen (CEA) measurement in human serum

Clinical oncology requires methods to detect tumor markers in patients sera and tissues Presently, monoclonal antibodies (MAbs based enzyme immunoassays are amongst the most advantageous techniques. Here we present a sensitive and specific double-antibody enzyme immunoassay for serum me surement of carcinoembryonic antigen (CEA). It has been developed with MAbs which recognize nonoverlapping peptide epitopes on the antigen molecule. The capture MAb 6C7 is GOLD 1 highly specific antibody while the tracer MAb 5.D11, labeled with biotin, is GOLD 4 that cross-reacts with nonspecific cross-reacting antigen (NCA) expressed on granulocytes. In addition the biotinylate MAb is shown to be also useful to detect CEA by immunohistochemistry.

Participation of glycosylation sites in the binding of Staphylococcus aureus to laminin

1. Microbial pathogenicity is in many instances associated with the ability to adhere to host surfaces or to extracellular matrix components. 2. Laminin is a major glycoprotein of basement membranes which can promote specific bacterial adhesion. Staphylococcus aureus is a pathogenic bacterium which presents a laminin receptor of about 50-kDa molecular mass (Lopes JD, Reis M per cent Brentani RR (1985). Science, 229: 275-277). 3. Adhesion inhibition assays of [125iodine]-labeled bacteria to laminin demonstrate that the receptor binding site is contained in the pepsin-derived (P1) laminin fragment. 4. Cell adhesion to laminin is unaffected by periodate oxidation of sugars on the surface of bacteria or by removal of divalent cations by ethylenediaminetetraacetic acid (EDTA). In contrast, bacterial adhesion is reduced when laminin is deglycosylated with N-glycosidase F or when bacteria are submitted to controlled trypsin digestion. 5. Laminin binding to the S. aureus 50-kDa band in immunoblotting assays has confirmed all of these results obtained in cell adhesion experiments

The immunodominant surface antigen of Plasmodium gallinaceum is present in both the salivary gland and oocyst sporozoites

1. Monoclonal antibodies (MAbs) against surface antigens of Plasmodium gallinaceum sporozoites, an avian malaria parasite, were produced using spleen cells from mice immunized with sporozoites from mosquito salivary glands (SGS) or from midadgusts containing oocysts (OoS). 2. All of the 15 MAbs teted (11 anti-SGS and 4 anti-OoS) reacted with SGS and OoS by indirect immunofluorescence and circumsporozoiter precipitation reactions. Fourteen of these MAbs (11 anti-SGS and 3 anti-OoS) produced a Western blot (WB) patten identical to that produced with serum from mice lyperimmunized with viable intacts sporozoites. 3. All MAbs and the immune sera recognized only two polypeptide bands of approximate molecutlar weight 78 and 64KDa. 4. No difference in the WB pattern was observed when-or 12-day SGS or OoS extracts were used as antigens in WB. This antigenic similary was confirmed when the total protein extracts were visualized on silver-stained SDS-PAGE gel

Evolutionary conservation of laminin-binding proteins

The virulence of pathogens and metastatic capacity of cancer cells seems to correlate with the ability to adhere to cells and/or to basement components. A key feature of this mechanism in the expression of specific receptors for the basement membrane protein laminin. There different receptors have been already described in cells phylogenetically very distant, such as human white blood cells, Trichomonas vaginalis and Stapgylococcus aureus, all recognizing laminin with the same range of affinity. We have shown that laminin, which is also found in the circulation, enchances phagocytosis of S. aureus by macrophages in a species-specific fashion. Also, monoclonal antibodies (MAb) raised against the bacterial receptor inhibit the phagocytic enhancement mediated by laminin and recognize laminin-binding proteins in unicellular parasites and mammalian cells. The same Mab 1.H12 elutes a 52-kDa protein from bacterial extracts and a 67-kDa band from cancer cells extracts. Since the MAb is a monospecific reagent, results with 1.H12 strongly suggest and evolutionary conservation of the biding site of phylogenetically different laminin receptors