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Immunohistochemical studies on synovial sarcomas have proved the potentiality of these neoplasm for epithelial and mesenchymal differentiation and antibodies detecting epithelial cells have been found to be helpful in determining the histological types. In this study different epithelial markers directed against various cytokeratins, HMFG-2 and EMA were investigated on paraffin embedded tissues of 13 cases of synovial sarcomas, with regard to their reliability in unmasking the epithelial components demonstrable in this type of neoplasm. The results lead to three conclusions: firstly, synovial sarcomas possess the capacity for generating different epithelial cell types with uncommon compositions of intermediate filaments as well as of membrane proteins, secondly, these features may be expressed in a heterogenous pattern even within the same tumour and finally, the use of wide range anti-cytokeratin antibodies covering the spectrum of basic as well as acidic type proteins seems to be necessary for the detection of all epithelial components demonstrable in synovial sarcomas.

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The expression of microtubule-associated proteins, MAP-1 and MAP-2, was studied in human neuroblastomas at various developmental stages using the immuno-alkaline-phosphatase technique and immunofluorescence microscopy. Of 15 cases examined, including grade I, grade II, and grade III neuroblastomas (M. Hughes, H. B. Marsden, and M. K. Palmer, Cancer 34:1706, 1974), rabbit antibodies raised against individual MAP-1 and MAP-2 from mammalian brain showed strong reactions with the whole spectrum of tumor cells including the immature small neuroblasts, partially mature neuroblasts, neurofibrils, and ganglion cells. Antibodies to alpha- and beta-tubulin showed similar staining patterns. In contrast, antibodies to the Mr = 200.000 neurofilament protein were reactive only with the mature and partially mature tumor cells, as well as with neurofibrils, but not with immature "round-cell" neuroblasts. Other types of round-cell tumors examined, including several cases of Ewing's sarcoma, undifferentiated rhabdomyosarcoma, and malignant lymphoma, showed no reaction with antibodies to MAP-1 and MAP-2. These tumors were reactive, however, with antibodies to various other tumor-specific as well as nonspecific antigens. It is concluded that antibodies to neuronal MAPs provide a valuable new tool for the differential diagnosis of neuroblastomas.

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The morphological findings in three resected specimens of Barrett's ulcer in children are discussed. Nearly identical morphologic changes are found in all cases, even in adults. This pathologic condition is understood as the third or fourth stage of reflux esophagitis. Perforation or even malignant degeneration is described in these cases. Therefore mainly all therapeutic aims must be to prevent these stages.

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The occurrence of plectin in various human tissues and cell lines was investigated using immunofluorescence microscopy and antibody gel overlay/immunoblotting techniques. Plectin was identified in all tissues and cell lines tested, namely placenta, kidney, cornea, foreskin and eyelid skin, skin fibroblasts, monocytes, keratinocytes and HeLa cells. In frozen sections of cornea and skin, plectin was found to be enriched at epithelial basal cell surface membranes. Consequently, antibodies to plectin could serve as a tool in the classification of mechanobullous diseases.

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The present study was undertaken in order to elucidate the effect of liver injury exerted by the antimicrotubular drug griseofulvin on hepatic transglutaminase activity in mice. Griseofulvin treatment of mice leads to a significant increase of transglutaminase activity associated with the 105 000 X g supernatant of liver homogenate, which is readily reversible after replacement of the griseofulvin-containing diet by a normal diet. Neoplastic nodules originally induced by prolonged griseofulvin feeding also show increased transglutaminase activity in contrast to reports in the literature. The increase in hepatic transglutaminase activity in griseofulvin-fed mice could be due either to mitotic inhibition exerted by the drug or to disturbance of intracellular Ca++ homeostasis following membrane injury. The second possibility could also account for the increased enzyme activity in neoplastic nodules. Similar events have been described as occurring in aging erythrocytes and terminally differentiating keratinocytes. The pathologic consequences and the substrates of increased hepatic transglutaminase activity have still to be elucidated.

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The occurrence and cellular localization of polypeptides related to hog brain microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2) in non-neuronal cell lines of various species and types, and in several tissues from rat was studied. When insoluble cell fractions were prepared by incubation of isotonic cell extracts with 20 microM taxol, polypeptides co-migrating with MAP-1 and MAP-2 upon gel electrophoresis were observed in virtually all cases examined. Immunoblotting of preparations from 3T6, CHO, HeLa and N2A cells, as well as pituitary, heart, testis and liver revealed immuno-reactivity with antibodies to neuronal MAP-1 for polypeptides co-migrating with MAP-1 in all cases, except for HeLa cells and liver. With similar preparations, antibodies raised to neuronal MAP-2 were barely reactive with bands of the MAP-2 size except for N2A cells and pituitary gland. In all cases of non-neuronal cells and tissues, major cross-reactive bands, however, were of mol. wt. lower than that of MAP-2, indicating, most likely, proteolytic breakdown of MAP-2 during cell fractionation. As shown by double immunofluorescence microscopy of various cultured cell lines using affinity-purified antibodies to MAPs, and monoclonal antibodies to tubulin, MAP-1-as well as MAP-2-related antigens were generally, but not exclusively, associated with typical microtubule structures of the cytoplasm, spindle, midbody and primary cilia. Antigens related to both MAPs were also localized in frozen sections of rat trachea, testis, pituitary, kidney and cardiac and skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

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Various tissues from rat were examined for the occurrence and cellular localization of plectin, a 300,000-dalton polypeptide component present in intermediate filament-enriched cytoskeletons prepared from cultured cells by treatment with nonionic detergent and high salt solution. The extraction of liver, heart, skeletal muscle, tongue, and urinary bladder with 1% Triton/0.6 M KCl yielded insoluble cell residues that contained polypeptides of Mr 300,000 in variable amounts. These high Mr polypeptide species and a few bands of slightly lower Mr (most likely proteolytic breakdown products) were shown to react with antibodies to rat glioma C6 cell plectin using immunoautoradiography and/or immunoprecipitation. By indirect immunofluorescence microscopy using frozen sections (4 micron) of stomach, kidney, small intestine, liver, uterus, urinary bladder, and heart, antigens reacting with antibodies to plectin were found in fibroblast, endothelial, smooth, skeletal, and cardiac muscle, nerve, and epithelial cells of various types. Depending on the cell type, staining was observed either throughout the cytoplasm, or primarily at the periphery of cells, or in both locations. In hepatocytes, besides granular staining at the cell periphery, conspicuous staining of junctions sealing bile canaliculi was seen. In cardiac muscle strong staining was seen at intercalated disks and, as in skeletal muscle, at Z-lines. In cross sections through smooth muscle, most strikingly of urinary bladder, antibodies to plectin specifically decorated regularly spaced, spot-like structures at the cell periphery. By immunoelectron microscopy using the peroxidase technique, antiplectin-reactive material was found along cell junctions of hepatocytes and was particularly enriched at desmosomal plaques and structures associated with their cytoplasmic surfaces. A specific immunoreaction with desmosomes was also evident in sections through tongue. In cardiac muscle, besides Z-lines, intercalated disks were reactive along almost their entire surface, suggesting that plectin was associated with the fascia adherens, desmosomes, and probably gap junctions. In smooth muscle cells, regularly spaced lateral densities probably representing myofilament attachment sites were immunoreactive with plectin antibodies. The results show that plectin is of widespread occurrence with regard to tissues and cell types. Furthermore, immunolocalization by light and electron microscopy at junctional sites of various cell types and at attachment sites of cytoplasmic filaments in epithelial and muscle cells suggests that plectin possibly plays a universal role in the formation of cell junctions and the anchorage of cytoplasmic filaments.

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The intermediate filament cytoskeleton of various types of human soft tissue tumors was analyzed by immunofluorescence microscopy with the use of specific antibodies against cytokeratins, vimentin, and desmin, as well as by one- and two-dimensional gel electrophoresis of high-salt buffer- and detergent-resistant cytoskeletal preparations. All leiomyomas as well as a leiomyosarcoma contained desmin. Leiomyomas of both gastrointestinal and uterine derivation and the retroperitoneal leiomyosarcoma showed strong reaction for desmin in the smooth muscle cells, but the latter two exhibited also vimentin staining. In embryonal rhabdomyosarcomas, desmin prevailed in the large, apparently well-differentiated rhabdomyoblasts; whereas the smaller, less differentiated tumor cells preferentially contained vimentin. Cells of malignant fibrous histiocytomas were characterized by their content of vimentin as the only intermediate filament protein present. In alveolar soft part sarcoma, a rare tumor of hitherto unknown histogenesis, vimentin and desmin co-existed within the same tumor cells, indicating, together with chemical determinations, the myogenic derivation of this neoplasm. The results show that immunologic and biochemical analysis of proteins associated with the intermediate filament cytoskeleton is a useful adjunct in the diagnosis of diverse neoplasms, particularly those with equivocal histologic features, and thus aids in the histogenetic classification of soft tissue tumors.

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Human epithelial cells contain, intermediate-sized filaments formed by polypeptides related to epidermal alpha-keratin ("cytokeratins") which are expressed in different combinations in different epithelia. Using cytoskeletal proteins from human biopsies and autopsies we have examined, by two-dimensional gel electrophoresis and immunoblotting experiments, the cytokeratin polypeptide patterns of diverse primary and metastatic carcinomas and have compared them with those of corresponding normal epithelial tissues and cultured cells. Five groups of carcinoma cytokeratin patterns can be discriminated. (1) Cytokeratins typical of simple epithelia (polypeptides Nos. 7, 8, 18, 19) are expressed, in various combinations, by many adenocarcinomas, for example those of gastrointestinal tract. (2) Cytokeratins typical of stratified epithelia (Nos. 1, 5, 6, 10, 11, 14-17) are found, in various combinations, in squamous cell carcinomas of skin and tongue. (3) Complex patterns showing polypeptides Nos. 7, 8, 18, 19, and one basic component (No. 5 or 6) are detected in certain carcinomas of the respiratory tract and the breast. (4) Complex patterns containing cytokeratins widespread in stratified epithelia (Nos. 4-6, 14-17) as well as components Nos. 8 and 19 occur in diverse squamous cell carcinomas derived from non-cornified stratified epithelia, with or without additional small amounts of cytokeratin No. 18. (5) Patterns of unusually high complexity can be found in some rare tumors as is shown for a cloacogenic carcinoma. No significant qualitative changes of expression of cytokeratins were found when primary tumors and metastases were compared. When compared with cytokeratin patterns of normal epithelia, carcinomas of the first type usually display a high degree of relatedness to the tissue of origin. Other carcinomas do not express some of the cytokeratins present in the tissue of their origin and, vice versa, certain components which are minor or apparently absent in normal tissue are major cytokeratins in the corresponding tumor. These differences may be explained by cell type selection during carcinogenesis, but changes of expression during tumor development cannot be categorically excluded. The possibility of cell type heterogeneity within a given tumor is also discussed. Similarly complex patterns of cytokeratin polypeptides have been noted in certain cultured human carcinoma cell lines (e.g., A-431, RPMI 2650, Detroit 562, A-549) and can also be observed in cell clones. The possible value of analyses of cytokeratin patterns, by gel electrophoresis or specific monoclonal antibodies, in distinguishing different carcinomas by non-morphologic criteria is discussed.

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Epithelial cells contain desmosomes, special intercellular junctions providing sites of membrane attachment for intermediate-sized filaments of the cytokeratin type (tonofilaments). Such sites of anchorage of tonofilaments appear as dense plaques on the cytoplasmic side of the desmosomal membrane. We have isolated desmosome-enriched fractions from bovine snout epidermis and tongue mucosa and have characterized the major protein associated with the desmosomal plaque. This protein occurs in equimolar amounts of two polypeptides of Mr 250,000 (desmoplakin I) and Mr 215,000 (desmoplakin II) which are chemically and immunologically related. Antibodies raised against desmoplakins allow the identification and localization of this protein in epithelial cells grown in tissues or in vitro and show crossreaction in species as diverse as man, mouse, and chicken. Using immunolocalization at the light and electron microscope levels, we show that these antibodies bind specifically to desmosomal plaques. Antibodies to desmoplakins have been used successfully for detection of desmosomal proteins in a broad variety of epithelium-derived human tumors, including primary carcinomas and their metastases, irrespective of the morphology of the specific tumor. Nonepithelial tumors examined have been negative. We propose to use antibodies to desmoplakins and to cytokeratins in pathological diagnosis as two independent markers for the positive immunocytochemical identification and classification of epithelium derived tumors.

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To study the individual location of the microtubule proteins MAP-1 and MAP-2 in neuronal tissues and cells, antisera to electrophoretically purified MAP-1 and MAP-2 components were raised in rabbits. When frozen sections through rat brain were examined by immunofluorescence microscopy the antibodies to MAP-1 strongly stained a variety of nerve cells including dendrites and myelinated axons in the cerebrum and cerebellum. Antibodies to MAP-2 showed similar staining patterns, except that myelinated axons were unstained. These results were confirmed by immunoelectron microscopy of frozen sections through cerebellum using the peroxidase technique. Thereby, the association of MAP-1 with microtubules was also clearly demonstrated. When cultured mouse neuroblastoma N2A cells were examined by immunofluorescence microscopy the antiserum to MAP-1 brightly stained filamentous structures resembling microtubules, whereas relatively weak and diffuse staining of the cytoplasm was observed with the antiserum to MAP-2. In agreement with the immunolocalization, MAP-1, but not MAP-2, was found as a prominent component of microtubules proteins polymerized in vitro by taxol from soluble N2A cell extracts. Together these results indicate that neuronal microtubules are preferentially associated with distinct high mol. wt. polypeptides. Therefore, they support the concept that different complements of associated proteins determine distinct functions of microtubules.

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Cytokeratin polypeptides of human epidermis, of epithelia microdissected from various zones of the pilosebaceous tract (outer root-sheath of hair follicle, sebaceous gland), and of eccrine sweat-glands have been separated by one- and two-dimensional gel electrophoresis and characterized by binding of cytokeratin antibodies and by peptide mapping. The epithelium of the pilosebaceous tract has three major keratin polypeptides in common with interfollicular epidermis (two basic components of mol wts 58,000 and 56,000 and one acidic polypeptide of mol wt 50,000); however, it lacks basic keratin polypeptides in the mol wt range of 64,000-68,000 and two acidic keratin-polypeptides of mol wts 56,000 and 56,500 and contains an additional characteristic acidic cytokeratin of mol wt 46,000. Another cytokeratin polypeptide of mol wt 48,000 that is prominent in hair-follicle epithelium is also found in nonfollicular epidermis of foot sole. Both epidermis and pilosebaceous tract are different from eccrine sweat-gland epithelium, which also contains two major cytokeratins of mol wts 52,500 and 54,000 (isoelectric at pH 5.8-6.1) and a more acidic cytokeratin of mol wt 40,000. A striking similarity between the cytokeratins of human basal-cell epitheliomas and those of the pilosebaceous tract has been found: all three major cytokeratins (mol wts 58,000; 50,000; 46,000) of the tumor cells are also expressed in hair-follicle epithelium. The cytokeratin of mol wt 46,000, which is the most prominent acidic cytokeratin in this tumor, is related, by immunological and peptide map criteria, to the acidic keratin-polypeptides of mol wts 48,000 and 50,000, but represents a distinct keratin that is also found in other human tumor cells such as in solid adamantinomas and in cultured HeLa cells. The results show that the various epithelia present in skin, albeit in physical and ontogenic continuity, can be distinguished by their specific cytokeratin-polypeptide patterns and that the cytoskeleton of basal-cell epitheliomas is related to that of cells of the pilosebaceous tract.

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Mallory bodies induced by long-term griseofulvin feeding in mouse liver were isolated and analyzed by one- and two-dimensional gel electrophoresis and reaction of the separated polypeptides with cytokeratin antibodies using the blotting technique. Comparison with normal intermediate filament cytoskeletons from mouse hepatocytes revealed that Mallory bodies contain two polypeptides: Component II (Mr: 55,000; apparent isoelectric point values: 6.45, 6.1, 5.9) and component III (Mr: 48,000; apparent isoelectric point values: 5.7, 5.5, 5.43, 5.38, 5.2) which appear to be similar, if not identical, to liver cytokeratins A and D, respectively. By contrast, component I of Mallory bodies (Mr: 65,000; apparent isoelectric point values: 5.4, 5.38, 5.2) was not found in appreciable amounts in normal hepatocytes. Component II was positive in immunoreaction with antibodies to murine hepatocyte keratins A and D as well as epidermal prekeratin. Component III showed reaction with the antibodies to murine hepatocyte keratins A and D but not with those raised against epidermal prekeratins. By contrast, the unusual component I reacted with antibodies to murine hepatocyte keratin D and to epidermal prekeratins. The results prove that cytokeratin polypeptides are major constituents of Mallory bodies and suggest that the pattern of liver cytokeratin polypeptides is altered during the toxic treatment and/or Mallory body formation.

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