A new colchicine binding assay for tubulin

A simple and sensitive procedure is described for assaying tubulin, the subunit protein of microtubules. Tubulin is equilibrated with [³H]colchicine after which the remaining free colchicine is removed by adsorption to activated charcoal. Values for the amount of tubulin in various rat tissues have been determined by both the charcoal method and by gel filtration on Sephadex G-100, and in all cases the values determined by the two methods correspond closely.     

Localization of betav tubulin in the cochlea and cultured cells with a novel monoclonal antibody

Tubulin, the dimeric structural protein of microtubules, is a heterodimer of alpha and beta subunits; both alpha and beta exist as numerous isotypes encoded by different genes. In vertebrates the sequence differences among the beta(I), beta(II), beta(III), beta(IV) and beta(V) isotypes are highly conserved in evolution, implying that the isotypes may have functional significance. Isotype-specific monoclonal antibodies have been useful in determining the cellular and sub-cellular distributions and possible functions of the beta(I), beta(II), beta(III), and beta(IV) isotypes; however, little is known about the beta(V) isotype. We here report the creation and purification of a monoclonal antibody (SHM.12G11) specific for beta(V). The antibody was designed to be specific for the C-terminal sequence EEEINE, which is unique to rodent and chicken beta(V). The antibody was found to bind specifically to the C-terminal peptide EEEINE, and does not cross-react with the carboxy-termini of either alpha-tubulin or the other beta-tubulin isotypes. However, the antibody also binds to the peptide EEEVNE, but not to the peptide EEEIDG, corresponding respectively to the C-terminal peptides of bovine and human beta(V). Immunofluorescence analysis indicates that beta(V) is found in microtubules of both the interphase network and the mitotic spindle. In gerbils, beta(V) also occurs in the cochlea where it is found largely in the specialized cells that are unique in containing bundled microtubules with 15 protofilaments.     

A monoclonal antibody that recognizes Golgi-associated protein of cultured fibroblast cells

We have obtained a hybridoma clone, JLJ5a, which secretes monospecific antibody directed against a 110-kdalton protein of gerbil fibroma cells, Rat-1 fibroblasts, and L6 myoblasts. It appears to be localized in the Golgi apparatus by the following criteria: (a) In double- staining experiments the localization of the 110-kdalton protein by the JLJ5a monoclonal antibody was coincident with the reaction products of thiamine pyrophosphatase (one of the enzyme markers of the Golgi apparatus; Novikoff and Goldfischer, 1961, Proc. Natl. Acad. Sci. U.S.A. 47:802-810) in the same cells. (b) The staining pattern of the JLJ5a monoclonal antibody became fragmented and dispersed into vacuoles after pretreatment of the cells with Colcemid or monensin.     

Localization of human sarcoma with radiolabeled monoclonal antibody

Summary We performed a randomized controlled study of postoperative adjuvant immunochemotherapy with Nocardia rubra cell wall skeleton (N-CWS) and Tegafur for gastric carcinoma between September 1979 and March 1983. A total of 309 patients were entered into this trial. Of the 309 patients, there were 98 evaluable patients in the chemotherapy group and 115 evaluable patients in the immunochemotherapy group. In both groups, Tegafur was given as chemotherapy at a daily dose of 400 to 800 mg, starting at 24–29 days after gastrectomy. In the immunochemotherapy group, 400 g of N-CWS was injected i. d. within the 2nd postoperative week. It was given weekly during the first month and subsequently monthly for as long as practicable. The patients were surveyed for length of survival in March 1985. The postoperative survival rate was analyzed for all cases, and for patients with various histopathological stages of carcinoma for comparison between the two treatment groups. No statistical difference was detected between the two groups in terms of age, sex, surgical curabilities, or stage of carcinoma. The overall survival rate for all patients was significantly higher in the immunochemotherapy group than in the chemotherapy group (p<0.05). With stage III plus IV disease, 53 patients from the chemotherapy group and 61 patients from the immunochemotherapy group were included for the analysis. As a consequence, a highly significant survival rate was observed in patients with stage III plus IV carcinoma in the immunochemotherapy group (p<0.005) as compared to the chemotherapy group. The overall 5-year (1800 days) survival rate after surgical treatment was 60.2% for the chemotherapy group and 73.2% for the immunochemotherapy group. In patients with stage III plus IV disease, the 5-year survival rates of the two treatment groups were 28.8% and 52.4%, respectively. Accordingly, the 50% survival period of patients with stage III plus IV cancer was 1800 days or more in the immunochemotherapy group, whereas it was only 722 days in the chemotherapy group. These results emphasize the effectiveness of N-CWS as an adjuvant immunotherapeutic agent in postoperative gastric cancer patients.The main side effects of N-CWS were skin lesions in the injected sites and fever, but these were temporary and not serious.     

A tubulin identified by a monoclonal antibody is not present in mitotic spindles

A monoclonal antibody, G8, which recognizes a form of tubulin (G8-tubulin) with a novel distribution in Rat-1 cells and Potorous tridactylis kidney (Ptk-2) cells was isolated. G8 labeled the interphase cytoskeleton of Rat-1 fibroblasts but not mitotic spindles or midbodies. G8 also stained a fiber network in some but not all Ptk-2 interphase cells but did not label mitotic spindles or midbodies in these cells. G8-tubulin is the only identified tubulin known to be absent from these structures. This distribution may indicate that G8-tubulin possesses functional specificity.     

A monoclonal antibody recognizing cytoskeletal keratins of stratified epithelia and bladder carcinomas

Monoclonal antibodies were generated by immunizing rats with mouse bladder carcinomas, fusing their spleen cells with NS-1 myeloma cells and selecting for antibodies that bound to mouse bladder carcinomas. One of the antibodies, IG5, recognizes an antigenic determinant which is present in mouse bladder carcinoma cytoskeletons and is not detectable in the normal bladder epithelium. Indirect immunofluorescence studies revealed that the antigen is expressed intracellularly and is organized in the form of filamentous arrays. The antigen was detected by peroxidase--antiperoxidase immunocytochemistry in stratified epithelia and glands derived from these, but has not been observed in any tissues of mesenchymal or neuronal origin. Various normal and neoplastic human tissues were subsequently tested for reactivity with antibody IG5. Antigen expression in normal tissues was similar to that in the mouse. Most carcinomas of the bladder and lung were stained, while all of eleven colon carcinomas were negative. Antibody IG5 immunoprecipitated radio-iodinated peptides of 58, 56, 52 and 43 kD molecular weight from mouse bladder carcinomas. Western blotting experiments with antibody IG5 demonstrated bands of 56 and 50 kD in a keratin-enriched fraction of the bladder carcinoma cytoskeleton. Antibody IG5 reacted with molecules which have several properties typical of cytoskeletal keratin peptides. Our findings are discussed in the context of previously described keratin peptides and relevant monoclonal antibodies.     

Use of radiolabeled monoclonal antibody to enhance vaccine-mediated antitumor effects

Radiolabeled monoclonal antibodies (mAb) have demonstrated measurable antitumor effects in hematologic malignancies. This outcome has been more difficult to achieve for solid tumors due, for the most part, to difficulties in delivering sufficient quantities of mAb to the tumor mass. Previous studies have shown that nonlytic levels of external beam radiation can render tumor cells more susceptible to T cell-mediated killing. The goal of these studies was to determine if the selective delivery of a radiolabeled mAb to tumors would modulate tumor cell phenotype so as to enhance vaccine-mediated T-cell killing. Here, mice transgenic for human carcinoembryonic antigen (CEA) were transplanted with a CEA expressing murine carcinoma cell line. Radioimmunotherapy consisted of yttrium-90 (Y-90)-labeled anti-CEA mAb, used either alone or in combination with vaccine therapy. A single dose of Y-90-labeled anti-CEA mAb, in combination with vaccine therapy, resulted in a statistically significant increase in survival in tumor-bearing mice over vaccine or mAb alone; this was shown to be mediated by engagement of the Fas/Fas ligand pathway. Mice receiving the combination therapy also showed a significant increase in the percentage of viable tumor-infiltrating CEA-specific CD8(+) T cells compared to vaccine alone. Mice cured of tumors demonstrated an antigen cascade resulting in CD4(+) and CD8(+) T-cell responses not only for CEA, but for p53 and gp70. These results show that systemic radiotherapy in the form of radiolabeled mAb, in combination with vaccine, promotes effective antitumor response, which may have implications in the design of future clinical trials.     

Quantitative assay for binding of Bradyrhizobium japonicum to cultured soybean cells.

Incubation of Bradyrhizobium japonicum with the cultured soybean cell line SB-1 resulted in the adhesion of the bacteria to the plant cells. An antiserum was raised against B. japonicum, and the 125I-labeled immunoglobulin fraction was used to quantitate the number of bacteria bound to the soybean cells. The measurement of 125I-labeled antibody binding correlated well with parallel assays by microscopic observation. Using this quantitation, we have optimized the parameters of the assay in terms of time course, ratio of B. japonicum to SB-1 cells, and pH. We then explored the effects of saccharides, NaCl, EDTA, and culture age of the bacteria and SB-1 cells on B. japonicum binding under these optimal assay conditions. The results showed good correlation between conditions that govern B. japonicum binding to SB-1 cells in culture and those that regulate B. japonicum-induced nodulation in legume roots. Together, they suggest that this binding event may be important in controlling host specificity.     

A monoclonal antibody with binding and inhibiting activity towards human pancreatic carcinoma cells

Summary The murine monoclonal antibody (MAb) BW 494 was characterized in relation to its tissue specificity, the epitope recognized, in vitro and in vivo radiolocalization and its potential to mediate antibody dependent cellular cytotoxicity (ADCC) and complement mediated cytolysis (CMC). The MAb defined carbohydrate epitope located on a >200 k daltons glycoprotein was mainly expressed on the majority of well differentiated adenocarcinomas of the pancreas. Furthermore, the epitope is accessible to MAb BW 494 in vivo, allowing an enrichment of radioactive antibody at the tumor site in nude mice. Additionally, MAb BW 494 is able to use human peripheral blood lymphocytes as effector cells for ADCC reactions against appropriate tumor target cells in vitro. In contrast, the antibody does not mediate human or rabbit CMC.     

A monoclonal antibody to alpha tubulin recognizes host cell and Trypanosoma cruzi tubulins

A mouse monoclonal anti-alpha-tubulin antibody was used to investigate the disposition of the cytoskeletal microtubules of three tissue culture cell lines--J774 macrophages, BSC-1, and Vero cells--infected with the Brazil strain of Trypanosoma cruzi. Indirect immunofluorescence light microscopy was used to demonstrate the antigenic response in host cells and parasites, simultaneously. In all morphotypes of T. cruzi, the monoclonal antibody reacted with all subpopulations of microtubules, inclusively, the subpellicular, flagellar, cytopharyngeal, and mitotic. The host cell cytoskeletal microtubule framework was revealed and the redistribution and destruction of the microtubular lattice in response to parasite infection over a 120 h period recorded. Our results show that after the initial inoculation of tissue cultures with trypomastigotes, the parasites penetrate the cells and locate in the perinuclear region of the cell where they multiply. The number and distribution of host cell microtubules were altered during the infection. The normal radial distribution of microtubules extending from the center of the cell to the periphery was destroyed. The remaining microtubules were observed at the periphery encircling, but well removed from the proliferating parasites. The complete transformation of the parasites was monitored throughout the infection with the end result being the liberation of parasites and the near complete destruction of the microtubular framework of the host cell. A residual population of dividing spheromastigotes was observed in cells liberating trypomastigotes. Colloidal gold labeling of thin sections as seen in the electron microscope affirmed the specificity of our monoclonal antibody to all subpopulations of microtubules in T. cruzi.     

A radioligand binding assay for antitubulin activity in tumor cells

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.     

Hydrogen peroxide-induced cytoskeletal rearrangement in cultured pulmonary endothelial cells

Although the signaling pathways leading to hydrogen peroxide (H₂O₂)-induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. We have recently demonstrated that thrombin-induced actin reorganization in bovine pulmonary artery endothelial cells (BPAEC) requires activation of both myosin light chain kinase (MLCK) and protein kinase C (PKC). Therefore, the present study was designed to investigate the effects of H₂O₂ on actin reorganization in BPAEC. H₂O₂ initiated sustained recruitment of actin to the cytoskeleton and transient myosin recruitment in a time- and concentration-dependent manner. The H₂O₂-induced actin recruitment was significantly inhibited by the calmodulin antagonists, W7 and TFP, but not by the MLCK inhibitor, KT5926, nor the PKC inhibitors, H7 and calphostin C. H₂O₂ also caused actin filament rearrangement in BPAEC with disruption of the dense peripheral bands and formation of stress fibers. These alterations occurred prior to actin translocation to the cytoskeleton and are prevented by inhibition of either MLCK or PKC. High concentrations of H₂O₂ transiently attenuated PKC activity but slightly increased the phosphorylation of the prominent PKC substrate and actin-binding protein, myristoylated alanine-rich C kinase substrate (MARCKS), by 5 min. However, MARCKS phosphorylation was reduced to below basal levels by 30 min. On the other hand, H₂O₂ induced a time- and dose-dependent phosphorylation of myosin light chains which was eliminated by both MLCK and PKC inhibitors. These data suggest that MLCK contributes to H₂O₂-induced myosin light chain phosphorylation and actin rearrangement and that PKC may play a permissive role. Neither of these enzymes appears to be involved in the H₂O₂-induced recruitment of actin to the cytoskeleton. J. Cell. Physiol. 174:370–379, 1998. © 1998 Wiley-Liss, Inc.     

Plasminogen activating enzyme in cultured glioblastoma cells. An immunofluorescence study with monoclonal antibody

A monoclonal antibody against a 52,000 dalton human plasminogen activating enzyme (HPA52) was used for immunofluorescence staining of cultured glioblastoma cells. The fluorescence was located in the cytoplasm of the cells. A pronounced variation in the staining intensity was observed between the individual cells. The specificity of the fluorescent stain was supported by the findings that 1) no staining was obtained with a monoclonal antibody of the same subclass, but with irrelevant specificity (anti-2,4,6-trinitrophenyl); 2) adsorption with HPA52 purified to homogeneity removed the ability of anti-HPA52 to mediate staining; 3) the glioblastoma cells contained HPA52, as measured by enzymatic assay, while melanoma cells that were not stained did not contain HPA52 activity; 4) dexamethasone reduced both the enzymatically determined HPA52 content and the immunofluorescence in parallel, while progesterone affected none of these parameters; 5) we have previously found that culture fluid conditioned by the glioblastoma cells apart from HPA52 does not contain detectable amounts of any protein that binds to anti-HPA52. Several advantages of immunohistochemical detection of plasminogen activators compared with enzyme histochemical methods are discussed, among these that the immunohistochemical method distinguishes between plasminogen activators of different types.     

Immunocytochemical localization of native chondroitin-sulfate in tissues and cultured cells using specific monoclonal antibody

Chondroitin-sulfate containing proteoglycan (CSPG) of the extracellular matrix (ECM) was visualized in chick tissues and cell cultures with a monoclonal antibody, CS-56. Cultured cells of various origins contained dense punctate layers of CSPG on both the substrate and the cell surface, as determined by immunofluorescent and immunogold staining. Under culture conditions the CSPG-containing matrix was usually excluded from stable cell-to-substrate focal contacts. The substrate-attached CSPG exhibited remarkable chemical stability but could be successfully removed by pronase or chondroitinases ABC and AC. Incubation of living cells with CS-56 antibodies resulted in the clustering of surface CSPG into patches, indicating that the surface-bound CSPG is free to move laterally along the plasma membrane. The unique properties of the CSPG-containing ECM revealed by CS-56 antibodies and their relationships to specific types of cell contacts are discussed.     

A fluorescent assay of proteinases in cultured mammalian cells

We have demonstrated proteinase activity in unfixed cells grown on tissue culture plates with a technique using 5-nitrosalicylaldehyde and peptide derivatives of 4-methoxy-2-naphthylamine. The 4-methoxy-2-naphthylamine liberated by proteinase activity reacts with 5-nitrosalicylaldehyde to form a fluorescent product. The substrates CBZ-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine and lysyl-alanyl-4-methoxy-2-naphthylamine, were used for the direct visual detection of two arylamidase activities in BALB/c 3T3 and C3H 10T 1/2 cells. With low magnification these enzyme activities can be detected in single clones; with higher magnification the fluorescent product can be seen within the cytoplasm of single cells.     

A cell-free binding assay maps the LSP1 cytoskeletal binding site to the COOH-terminal 30 amino acids

The leukocyte specific protein 1 or LSP1 is a multi functional protein involved in such divers biological processes as the regulation of neutrophil motility, chemotaxis, adhesion and membrane immunoglobulin M (mIgM) mediated apoptosis of B-lymphocytes. The 330-amino-acid mouse LSP1 protein contains a high-affinity F-actin binding site and in intact cells localizes to the F-actin filament containing cytoskeleton. Here we use a high-speed F-actin co sedimentation assay and transfection experiments in the LSP1- T-lymphoma cell line BW5147 to show that LSP1 interacts with F-actin and the cytoskeleton through residues downstream of amino acid residue 230. We then designed a novel cell-free cytoskeleton binding assay in which a set of GST-LSP1 fusion proteins are allowed to bind to the cytoskeleton in NP-40 soluble lysates of BW5147 cells and are recovered in the low-speed detergent insoluble pellet. Using this assay the cytoskeleton binding site of mouse LSP1 maps to the 300-330 interval. These results will allow the design of LSP1 mutants that do not bind to the cytoskeleton to determine the importance of LSP1 cytoskeleton binding for the diverse functions of LSP1.