Keratin and carcinoembryonic antigen (CEA) in human melanoma cells.

Human melanomas are known to contain vimentin intermediate filaments but there has been some dispute about their expression of cytokeratins. The cytoplasm of human M21 melanoma cells maintained in culture reacted with a rabbit anti-keratin antibody and two monoclonal anti-keratin antibodies AE1 and AE2. Cells derived directly from subcutaneous xenografts of M21 melanoma in nude mice, however, failed to express cytokeratins. The presence of keratin filaments in cultured M21 cells was confirmed by electronmicroscopic and immuno-electronmicroscopic examinations of cell extracts. Polyacrylamide gel electrophoresis (PAGE), revealed 46 KD keratin proteins in cultured M21 cells. Small amounts of these low molecular weight keratins were detected by PAGE in M21 melanoma xenografts even though immunofluorescence and immunoperoxidase assays failed to demonstrate keratin at the light microscopic level. Immunofluorescence revealed keratin and carcinoembryonic antigen (hitherto undetected in human melanomas) first on the 9th day of culture of xenograft-derived M21 cells. The appearance of keratin and CEA in M21 melanoma cells in vitro was not affected by inhibition of cellular proliferation or as a result of exposure to methotrexate or adriamycin. However, adriamycin altered the cytoplasmic distribution of keratin.     

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.     

Cytoskeleton of human embryonal carcinoma cells

Monoclonal antibodies to cytoskeletal proteins were used to study the intermediate filament proteins of human embryonal carcinoma (EC) cell lines, tumors produced in nude mice from these cell lines, and surgically removed testicular germ cell tumors. It was found that all cells of tumor lines 2102Ep, 1156 and Tera 1 react with antibodies to low molecular weight keratin proteins. By immunoblotting of SDS gels it was found that these lines expressed three keratin polypeptides (40K, 45K and 52K). Clonal line NTera-2 derived from Tera-2 differed from the above listed cell lines in that only 10% of the cells expressed the 40K keratin polypeptide. Upon treatment with retinoic acid 70% of NTera-2 cells became reactive with the antibody to the 40K keratin polypeptide. All cell lines contained a small population of vimentin-positive cells. The number of vimentin-positive cells could be increased by retinoic acid treatment of NTera-2 cells or by seeding the 2102Ep cells at low cell density. Neurofilament-positive cells could be induced in the cell line NTera-2 by retinoic acid treatment. Tumors produced from NTera-2 cells injected into nude mice contained cells reacting with antibodies to keratin, vimentin, neurofilament proteins and desmin. Keratin polypeptides were immunohistochemically demonstrated in embryonal carcinoma, yolk sac carcinoma and trophoblastic components of solid human germ cell tumors. Atypical intratubular cells ('carcinoma in situ') also reacted with antibodies to keratin.     

Defining the cellular target(s) of porcine reproductive and respiratory syndrome virus blocking monoclonal antibody 7G10

We produced a monoclonal antibody (MAb) (7G10) that has blocking activity against porcine reproductive and respiratory syndrome virus (PRRSV). In this study, we identified the components of the 7G10 MAb-bound complex as cytoskeletal filaments: vimentin, cytokeratin 8, cytokeratin 18, actin, and hair type II basic keratin. Vimentin bound to PRRSV nucleocapsid protein and anti-vimentin antibodies showed PRRSV-blocking activity. Vimentin was expressed on the surface of MARC-145, a PRRSV-susceptible cell line. Simian vimentin rendered BHK-21 and CRFK, nonsusceptible cell lines, susceptible to PRRSV infection. These results suggest that vimentin is part of the PRRSV receptor complex and that it plays an important role in PRRSV binding with the other cytoskeletal filaments that mediate transportation of the virus in the cytosol.     

Mutational analysis of the human p53 gene in malignant melanoma

Nine metastatic melanoma cell lines and two melanocyte cell lines were analyzed for point mutations in highly conserved regions of the p53 gene. No mutations were detected in the two melanocytic cell lines and in eight melanoma cell lines. However, a C----T transition at codon 248, resulting in a substitution of tryptophan for arginine, was found in one melanoma cell line. On immunohistochemical staining, only this cell line showed reactivity for mouse monoclonal antibody 1801, which is immunoreactive with human p53 protein. The original paraffin-embedded specimen from which this mutant cell line was established was obtained, and sequence analysis detected the identical mutation in the p53 gene as that seen in the derived cell line. This is the first report indicating point mutations in the p53 gene in malignant melanocytic tissues.     

Microinjection of IFA antibody induces intermediate filament aggregates in epithelial cell lines but perinuclear coils in fibroblast-like lines.

The murine monoclonal IFA antibody recognizes a conserved sequence present in almost all intermediate filament (IF) proteins. When IFA antibody was injected into 13 different primary or established cell lines, striking differences were detected between epithelial and fibroblastic cell lines. In epithelial cells keratin IFs were broken down within 4 h into numerous spheroid aggregates scattered throughout the cytoplasm. Keratin aggregates were first detected in the cytoplasmic periphery. In contrast, in fibroblastic cells, injection of IFA antibody led to the formation of perinuclear coils of vimentin. IFA antibody at a concentration of greater than 1 mg/ml had to be injected to initiate these transitions. When HeLa cells, which contain separate networks of vimentin and keratin filaments, were injected with IFA antibody, vimentin did not form perinuclear coils but was instead found together with keratin in aggregates. Electron micrographs of HeLa cells injected with IFA antibody showed that the aggregates have diameters between 0.5 and 2.6 microns and resembled the keratin aggregates observed in certain mitotic epithelial cells. Although the ultrastructural studies support an association of some aggregates with desmosomes, aggregates were, however, also induced by injection of IFA antibody into human keratinocytes in low calcium medium under conditions where desmosomes were not present.     

SARS-CoV单克隆抗体的制备及抗原表位的初步鉴定

参照已发表的SARS冠状病毒BJ01株基因序列 ,利用计算机软件预测并选取该病毒S、M、N三种主要结构蛋白部分抗原性优势区域 ,以编码Gly-Pro-Gly序列相连接合成两段嵌合基因A和B。并分别克隆于pGEX -6p- 1载体上用IPTG进行诱导表达 ,以纯化的嵌合蛋白A和B为抗原 ,分别免疫BALB c小鼠制备单克隆抗体。利用单克隆抗体亚型检测试剂盒和SARS CoV商品化ELISA检测试剂盒对其进行亚型和特异性鉴定。结果表明融合表达两段嵌合基因产物 ,其大小分别为 34kD和35kD ,Westernblot分析证实两种表达产物都能被SARS病人康复期血清所识别。获得了 6株能稳定分泌特异性抗体的阳性细胞克隆株。亚型鉴定结果除D3C5为IgG2a外其他单抗均为IgG1,而且所有单抗的轻链均为κ链。特异性鉴定发现除D3D1外 ,其余的 5株单抗均能与SARS CoV商品化ELISA检测试剂盒发生特异性反应。将D3D1与灭活后经超声波裂解的SARS CoV进行Westernblot分析 ,发现它能特异性识别 180kD的蛋白带。分别融合表达了 6个S蛋白的寡肽 (S1- S6 ) ,并对筛选出的单克隆…     

Cell density and cell shape-related regulation of vimentin and cytokeratin synthesis : Inhibition of vimentin synthesis and appearance of a new 45 kD cytokeratin in dense epithelial cell cultures

The pattern of the intermediate type filament protein synthesis was examined in cultured bovine mammary gland epithelial (BMGE) cells under conditions of varied cell shape and cell-cell contact. In dense monolayer and suspension cultures BMGE cells expressed a new cytokeratin of 45 kD identified as a member of the acidic subfamily of cytokeratins. This polypeptide has a phosphorylated component and is dissociated from the cytokeratins complex in the presence of 6.5 M urea. The mRNA of the new cytokeratin accumulated in dense cell cultures, as revealed by in vitro translation in a cell-free system. In BMGE-H cells that express also vimentin, the synthesis of vimentin decreased dramatically in dense cell cultures, while the synthesis of the 45 kD cytokeratin was maximal under these conditions. The results suggest that the expression of certain cytokeratins and that of vimentin can be coordinately regulated by factors in the cellular environment that effect cell shape and cell surface contacts.     

Various keratin antibodies produce immunohistochemical staining of human myocardium and myometrium

Various polyclonal and monoclonal antibodies to keratins were used to stain different human muscle tissues by paired immunofluorescence and the unlabelled antibody peroxidase-anti-peroxidase method. In the myocardium, distinct coloration of the intercalated discs was produced by two polyclonal reagents to human epidermal keratins but not by two monoclonal antibodies to cytokeratins from pig renal tubular cells. In the myometrium--mainly in the middle layer of the uterine wall--cytoplasmic coloration of a varying fraction of the smooth muscle bundles was produced, especially by one of the polyclonal and by both monoclonal reagents. The staining was often confined to the perinuclear region. The keratin-positive myometrial cells usually coexpressed vimentin and actin in various proportions. These findings indicated that intermediate filaments of the keratin type, or antigenically similar elements, are not restricted to cells of epithelial origin. Other types of muscle cells did not react with keratin antibodies, but keratin-positive macrophages were occasionally found in tongue musculature and in inflamed epicardium. Altogether, our observations emphasize that keratin reactivity cannot be considered specific for epithelial (or mesothelial) cells without reservation.     

Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.     

Detection of phenotypic differences on human malignant melanoma lines and their variant sublines with monoclonal antibodies

The hybridoma technique was used to generate monoclonal antibodies against a wide spectrum of melanoma-associated surface antigens. Mice were immunized against the human melanoma lines Mel A-375, SK Mel-25, and Mel S-5 (subclone of SK Mel-25), which differ with respect to a number of biological and biochemical properties. Spleen cells were fused with P3 X 63-AG8.653 myeloma cells. Twenty hybridomas producing antibodies that were negative on platelets, leukocytes, and monocytes but positive on melanoma cells were isolated and recloned. The specificity of antibodies was investigated on 30 human melanoma and nonmelanoma lines. Five groups of antibodies could be distinguished by their reactivity (1) with few melanoma lines and embryonic fibroblasts; (2) with melanoma, neuroblastoma, and teratoma; (3) with melanoma, neuroblastoma, glioblastoma, teratoma, and carcinoma; (4) with melanoma, teratoma, and carcinoma; and (5) with melanoma, neuroblastoma, teratoma, glioblastoma, carcinoma, embryonic fibroblasts, and B-lymphoblastoid cells. The antigen expression was qualitatively and quantitatively different from cell line to cell line. No evidence for melanoma-specific antigens was found. Eight antibodies were isolated detecting phenotypic differences on sublines of SK Mel-25.     

Early expression of vimenti in human mammary cultures

Summary The intermediate filaments of most epithelial cells in vivo consist solely of cytokeratins. Using monoclonal antibodies to vimentin or keratin, we have examined the expression of vimentin in homologous specimens of frozen tissue sections and primary cultures of normal human mammary epithelium. In frozen sections, only epithelial cells reacted with the antikeratin antibody, whereas antivimentin reactivity was associated with stromal cells. All epithelial cultures were positive for cytokeratin and in addition coexpressed vimentin as strongly as cultured fibroblasts and as early as the 4th d after initiation of the culture. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of cytoskeletal preparations of secondary cultures of normal mammary epithelium have also demonstrated the appearance of a moiety identical to the vimentin found in cultured fibroblasts. Our observations are consistent with the hypothesis that vimentin expression is induced, possibly as a result of changes in cell shape or growth rate, when cells are freed from three-dimensional restirctions imposed by the tissue of origin     

Identification and characterization of a novel mammalian intermediate filament-associated protein

A novel monoclonal antibody, designated M1.4, recognizes the high molecular weight microtubule-associated protein MAP1A (ca. Mr 380 kD) in both bovine and rat brain. In HeLa cells, however, M1.4 binds to a 240 kD polypeptide on immunoblots and co-localizes with both vimentin and cytokeratin filaments using double-label immunofluorescence microscopy. Immunoelectron microscopy indicates that the 240 kD polypeptide localizes along bundled intermediate filaments in a periodic manner. Two-dimensional electrophoretic analysis indicates that the 240 kD polypeptide has a basic pI of 7.7. When HeLa cell intermediate filaments are isolated using standard non-ionic detergent/high-salt conditions the 240 kD polypeptide does not sediment with the intermediate filaments, unlike the established intermediate filament-associated protein plectin. Immunoblot analysis with M1.4 shows the 240 kD polypeptide is expressed in a number of mammalian cell lines. Additionally, double-label immunofluorescence shows the 240 kD polypeptide to associate with vimentin filaments in African Green Monkey kidney (CV-1) and JC neuroblastoma cells. Due to its unique biochemical and biological characteristics, the 240 kD polypeptide is clearly a novel intermediate filament-associated protein for which we have proposed the designation gyronemin (Gr. gyros: around; nemin: filament).     

Production of monoclonal antibodies against the B700 murine melanoma antigen and their antimetastatic properties.

Two unique murine melanoma antigens, termed B700 and B50, have been identified and isolated from several different murine melanoma cell lines. Both antigens can be detected on the cell surface, are actively shed in culture, and are often found in close association intracellularly. In previous studies, the antigen B700, which is related to serum albumin by biochemical and immunological criteria, was shown to function as a melanoma-specific tumor rejection antigen. We have also shown that animals sensitized to irradiated JB/RH melanoma cells produce antibodies which recognize B700 and/or B50, with B700 evoking the stronger humoral response. Animals testing positive by ELISA for antibody production to B700 or B50 were used for preparation of hybridomas and four different murine monoclonal antibodies have been produced whose specificities should facilitate epitope mapping. Clones have been used to generate ascites fluid in nude mice; the antibodies specifically recognize B700 and intact murine melanoma cells, but not B50. Two of these monoclonal antibodies have been administered systemically to C57Bl/6 mice bearing 5 day pulmonary metastases of the JB/MS melanoma, and significant inhibition of metastatic growth was observed for both antibodies.     

Microinjection of monoclonal antibodies to vimentin, desmin, and GFA in cells which contain more than one IF type

Microinjection of antibodies to vimentin into fibroblast cell lines causes intermediate filaments (IFs) to build perinuclear caps. We have extended these findings by microinjection of monoclonal antibodies specific for different IF types to non-epithelial cell lines of human origin, which co-express two different IF proteins. Thus GFA and vimentin IgGs have been microinjected in separate experiments into a glioma cell line, desmin and vimentin IgGs into RD cells, and vimentin IgGs into a cell line which co-expresses neurofilaments and vimentin. In all instances, microinjection of a single antibody causes the formation of perinuclear caps in which the two different IF proteins co-localize, suggesting that vimentin and the second IF type present in each cell line localize to the same 10-nm filaments. Immunoelectron microscopy using desmin and vimentin antibodies made in different species and appropriate second antibodies labelled with 5 and 20 nm gold particles confirm this result for RD cells. When Fab' fragments of the vimentin IgGs are microinjected into different cell types, formation of perinuclear caps is observed in immunofluorescence microscopy. In RD cells immunoelectron microscopy shows that the Fab' fragments induce caps which appear less dense than the caps seen after microinjection of IgGs.     

Biochemical analysis of a 130,000 molecular weight glycoprotein on human melanoma cells

A monomeric sialoglycoprotein of 130,000 molecular weight (gp 130) is a membrane protein detected by mouse monoclonal antibodies on human melanoma cells and in lesser amounts on a wide range of normal and malignant cell types. Eight monoclonal antibodies reacting with gp 130 detect at least four spatially distinct epitopes on the exposed surface of the gp 130 molecule. Biosynthetic studies have shown that gp 130 is synthesized through two precursor forms: a 100 kD glycosylated species and an 80 kD unglycosylated species, presumably the primary translational product of the encoding mRNA.     

Tumor cell proteins, detected using antibodies to the S- and N-terminal fragments of the fos proto-oncogene product

Antibodies to c-fos oncoprotein were produced in rabbits by immunization with synthetic peptides, corresponding to the sequences 6-15 of N-end and 371-380 of C-end of c-fos oncoprotein. C-fos expression was tested with immunoprecipitation and immunoblotting in various transformed cell lines with antibodies to N- and C-decapeptides. It was shown that antibodies to C-terminal decapeptide revealed a c-fos gene product and also some fos-related antigens FRAs 36 kD, 46 kD, 75 kD and 90 kD in rat pheochromocytoma PC-12 cells and mouse carcinoma cell lines MAC-3 and LL. In some cell lines 46 kD FRA was expressed in the absence of p62 c-fos. Besides, different clones of the same cell line cultivated in identical conditions revealed differences in the 46 kD FRA expression. Antibodies to sequence 6-15 of N-end revealed only c-fos products and no FRAs were detected. Therefore FRAs have homology with the c-fos product in the C-terminal region and differ from it in the N-terminal region.     

Monoclonal antibody-defined circulating human tumor-associated antigen with epitope shared by cytokeratins

Sera of human colonic carcinoma xenografted rnu/nu rats were used to immunize rnu/+rats in order to obtain an immune response against circulating human tumor-associated components. After fusion of rat spleen cells with mouse myeloma cells monoclonal antibody MAB 108 could be established which reacted with two 40 and 45 kD cytokeratins as well as with vimentin, with a soluble 37 kD protein apparently derived from the 45 kD protein and with a 37 kD protein released by tumor cells. The MAB 108-specific epitope was also detected in tissue polypeptide antigen (TPA), a human tumor-associated antigen originally described by Bj?rklund et al. (22).     

Expression of beta1-integrins and N-cadherin in bladder cancer and melanoma cell lines

Changes in the expression of integrins and cadherins might contribute to the progression, invasion and metastasis of transitional cell cancer of the bladder and of melanomas. The expression of alpha5 (P < 0.001), alpha2 and beta1 (P < 0.05 - P < 0.001) integrin subunits in melanoma cells from noncutaneous metastatic sites (WM9, A375) were significantly increased as compared to cutaneous primary tumor (WM35) and metastatic (WM239) cell lines. These differences might be ascribed to the invasive character of melanoma cells and their metastasis to the noncutaneous locations. The significantly heterogeneous expression of beta1 integrin subunit in two malignant bladder cancer cell lines (T24 and Hu456) and nonsignificant differences in the expression of alpha2, alpha3, and alpha5 subunits between malignant and non-malignant human bladder cell lines do not allow an unanimous conclusion on the role of these intergrin subunits in the progression of transitional cancer of bladder. The adhesion molecule, expressed in all studied melanoma and bladder cell lines, that reacted with anti-Pan cadherin monoclonal antibodies was identified as N-cadherin except in the HCV29 non-malignant ureter cell line. However, neither this nor any other bladder or melanoma cell line expressed E-cadherin. The obtained results imply that the replacement of E-cadherin by N-cadherin accompanied by a simultaneous increase in expression of alpha2, alpha3 and alpha5 integrin subunits clearly indicates an increase of invasiveness of melanoma and, to a lesser extent, of transitional cell cancer of bladder. High expression of N-cadherin and alpha5 integrin subunit seems to be associated with the most invasive melanoma phenotype.     

Coexpression of cytokeratin and vimentin in Rathke's cysts of the human pituitary gland

Summary Immunohistochemistry with monoclonal and polyclonal antibodies revealed the presence of cytokeratins in epithelial cells of Rathke's cysts in the pars intermedia of the human pituitary gland. With monoclonal antibodies specific for individual cytokeratins, the expression of CK 18, CK 8, CK 7, and CK 19 could be shown in these cells. Within the hypophysis, CK 19 and CK 7 were restricted to Rathke's cysts and a few epithelial cell clusters in the pars tuberalis, whereas other cytokeratins were also present in endocrine cells of the pars distalis. Furthermore, vimentin and, focally, glial fibrillary acidic protein (GFAP) were detected in the cystic epithelia. By double labelling, coexpression of cytokeratin and vimentin, GFAP and cytokeratin, and GFAP and vimentin could be demonstrated. Compiled data of all known cases of coexpression of cytokeratin and vimentin in normal cells reveal physiological correlations and suggest a functional significance of this rare type of coexpression of intermediate filament proteins.