Dorothea L. Schiller

The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells

Roland Mall, Werner W. Franke and Dorothea L. Schiller Division of Membrane Biology and Biochemistry institute of Cell and Tumor Biology German Cancer Research Center D-6900 Heidelberg, Federal Republic of Germany Benjamin Geiger Department of Chemical Immunology The Weizmann Institute of Science Rehovot, Israel Reinhard Krepler Department of Pathology University of Vienna School of Medicine A-l 090 Vienna, Austria Introduction A large proportion of the cytoplasm of vertebrate cells, normal or transformed, is represented by components of the cytoskeleton, including actin-containing micro- filaments, tubulin-containing microtubules and fila- ments of intermediate size, with diameters of 7-l 1 nm. Although such structures have a widespread oc- currence in diverse cell types, examples have been reported in which they are formed in different cell types from different proteins of a multigene family of proteins, or from different subunit polypeptides of a class of related proteins. For example, differentiation specificity of expression of different actins has been described in different cell types of mammals (Vande- kerckhove and Weber, 1979). By far the most striking differentiation specificity of composition has been ob- served for the intermediate-sized filaments. Although all filaments of this category are morphologically iden- tical in different cell types, are insoluble in solutions of a broad range of low or high salt concentrations and non-ionic detergents and seem to share some common assembly properties (Steinert et al., 1981 b) and antigenic determinants (Pruss et al., 1981) im- munological and biochemical criteria allow us to dis- tinguish at least five different types of intermediate filaments (Bennett et al., 1978; Franke et al., 1978a, 1981f; Hynes and Destree, 1978; Lazarides, 1980; Anderton, 1981 ; Holtzer et al., 1981; Osborn et al., 1981). First, filaments containing keratin-like proteins (“cytokeratins”) are characteristic of epithelial cells. Second, vimentin filaments occur in mesenchymally derived cells, in astrocytes, in Sertoli cells, in vascular smooth muscle cells and in many cultured cell lines. Third, desmin filaments are typical of most types of myogenic cells. Fourth, neurofilaments are typical of neuronal cells. Fifth, glial filaments are typical of as- trocytes. During cell transformation and tumor devel- opment this cell type specificity of intermediate fila- ments is largely conserved’ (Franke et al., 1978a, 1978b, 1979a; Hynes and Destree, 1978; Sun and Green, 1978a; Sun et al., 1979; Bannasch et al., 1980; Battifora et al., 1980; Schlegel et al., 1980a; Altmannsberger et al., 1981; Gabbiani et al., 1981; Denk et al., 1982) and classification of tumors by their specific type of intermediate filaments has re- cently become very valuable in clinical histodiagnosis (see, for example, Schlegel et al., 1980a; Gabbiani et al., 1981; Ramaekers et al., 1981). The intermediate filaments of the vimentin, desmin or glial types all consist usually of only one type of subunit protein (desmin and vimentin can occur in the same filament in BHK cells and vascular smooth mus- cle cells; Steinert et al., 1981 a; Quinlan and Franke, 1982). In contrast with these, the cytokeratin fila- ments, which are composed of proteins related to, but not identical with, epidermal (Y keratins, are a complex family of many different polypeptides. These cytoker- atins, which show biochemical and immunological re- lationships of various degrees, are expressed, in dif- ferent epithelia, in different combinations polypep- tides ranging in their isoelectric pH values from 5 to 8 and in their apparent molecular weights from 40,000 to 68,000 (Doran et al., 1980; Winter et al., 1980; Fuchs and Green, 1980, 1981; Franke et al., 1981 a, 1981 b, 1981 c; Milstone and McGuire, 1981; Wu and Rheinwald, 1981). A given epithelium or epithelial cell can therefore be characterized by the specific pattern of its cytokeratin components. Human Cytokeratin Polypeptides and Their Tissue Distribution Cytoskeletal preparations from epithelial tissues ex- tracted in high salt buffer and Triton X-l 00 are highly enriched in intermediate-sized filaments containing proteins that react specifically with antibodies to au- thentic epidermal [Y keratin (see, for example, Sun and Green, 1977; Fuchs and 1978, 1980, 1981; Franke et al., 1978b, 1980, 1981a, 1981 b, 1981~; Wu and Rheinwald, 1981) and that are recovered in filaments reconstituted in vitro from denatured mono- mers (Tezuka and Freedberg, 1972; Lee and Baden, 1976; Steinert et al., 1976, 1981 a; Sun and Green, 1978b; Gipson and Anderson, 1980; Milstone, 1981; Franke et al., 1981 b, 1981~; Renner et al., 1981). When such preparations are made from different hu- man tissues and examined by two-dimensional gel electrophoresis, with the aid of isoelectric focusing as well as nonequilibrium pH gradient electrophoresis for better resolution of basic polypeptides, complex pat- terns of cytokeratin polypeptides are found. The dis- tinct cytokeratin polypeptides that we have so far identified in various human tissues are schematically summarized and arranged according to their specific coordinates on two-dimensional gel electrophoresis in Figure 1, and the corresponding tissue distribution is shown in Table 1 A. Typically, the cytokeratin polypep- tides appear in series of isoelectric variants; all but the most basic spot usually represent phosphorylated

Diversity of cytokeratins: Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues☆

Abstract Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [32P]phosphate labeling in vivo. It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.

Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one‐ or two‐dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the ‘basic cytokeratin subfamily’ defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis‐specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad‐range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.

A subfamily of relatively large and basic cytokeratin polypeptides as defined by peptide mapping is represented by one or several polypeptides in epithelial cells.

Epithelial cells contain a class of intermediate‐sized filaments formed by proteins related to epidermal alpha‐keratins (‘cytokeratins’). Different epithelia can express different combinations of cytokeratin polypeptides widely varying in apparent mol. wt. (40 000‐68 000) and isoelectric pH (5.0‐8.5). We have separated, by two‐dimensional gel electrophoresis, cytokeratin polypeptides from various tissues and cultured cells of man, cow, and rodents and examined their relatedness by tryptic peptide mapping. By this method, a subfamily of closely related cytokeratin polypeptides has been identified which comprises the relatively large (greater than or equal to mol. wt. 52 500 in human cells) and basic (pH greater than or equal to 6.0) polypeptides but not the smaller and acidic cytokeratins. In all species examined, the smallest polypeptide of this subfamily is cytokeratin A, which is widespread in many simple epithelia and is the first cytokeratin expressed during embryogenesis. This cytokeratin polypeptide subfamily is represented by at least one member in all epithelial and carcinoma cells examined, indicating that polypeptides of this subfamily serve an important role as tonofilament constitutents . Diverse stratified epithelia and tumours derived therefrom contain two or more polypeptides of this subfamily, and the patterns of expression in different cell types suggest that some polypeptides of this subfamily are specific for certain routes of epithelial differentiation.

An epithelial cell line with elongated myoid morphology derived from bovine mammary gland. Expression of cytokeratins and desmosomal plaque proteins in unusual arrays

Cells of a clonal line (BMGE + HM) selected from bovine mammary gland epithelial cell cultures are described which, after reaching confluence, do not assume typical epithelioid morphology, but form elongated cells with long slender processes extending over the surfaces of other cells. However, cells of this line which display non-epithelioid morphology and are exceptionally rich in actin microfilaments are identified as epithelial cells by their synthesis of cytokeratins and desmosomal plaque proteins, as demonstrated by immunofluorescence and immunoelectron microscopy and by gel electrophoresis of cytoskeletal proteins. The cells do not produce vimentin and desmin filaments. The specific cytokeratin polypeptides of these myoid cells are identical to those present in normal epithelioid BMGE + H cells but are arranged in unusual arrays of meshworks of finely dispersed, non-fasciated filaments and granular structures. Desmosomal plaque proteins, notably desmoplakins, are abundant, but the electron microscopic appearance of the desmosomes is abnormal in that most of them are associated with a second accessory plaque formed at a distance of 0.1-0.15 micron from the normal desmosomal plaque. Both cytokeratin filaments and desmosomal structures are found throughout the whole cytoplasm, including the extended cell processes. The existence of an epithelial cell line with such an unusual morphology demonstrates the importance of non-morphological criteria in identifying epithelium-derived cells. Our findings also indicate that dramatic differences of cell shape and organization of epithelial cells need not necessarily be associated with changes in the expression of specific cytoskeletal proteins. The possible origin of this cell line from myoepithelial cells is discussed.