Roland Moll

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.

Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases

Abstract We report the human DNA and protein sequence of adipophilin and its association with the surface of lipid droplets. The amino acid sequence of human adipophilin has been determined by using cDNA clones from several tissues and confirmed by the reverse transcription/polymerase chain reaction method and Edman sequencing. The open reading frame of adipophilin encodes a polypeptide with a calculated molecular weight of 48.1 kDa and an isoelectric point of 6.72. By immunofluorescence and electron-microscopic localization with newly raised specific poly- and monoclonal antibodies, we show that this protein is not restricted to adipocytes as previously indicated by studies of the mouse homologous protein, adipose-differentiation-related protein. Adipophilin occurs in a wide range of cultured cell lines, including fibroblasts and endothelial and epithelial cells. In tissues, however, expression of adipophilin is restricted to certain cell types, such as lactating mammary epithelial cells, adrenal cortex cells, Sertoli and Leydig cells of the male reproductive system, and steatosis or fatty change hepatocytes in alcoholic liver cirrhosis. Our results reveal adipophilin as a possible new marker for the identification of specialized differentiated cells containing lipid droplets and for diseases associated with fat-accumulating 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.

Biological and prognostic significance of stratified epithelial cytokeratins in infiltrating ductal breast carcinomas.

Abstract The biological significance of the differential expression of cytokeratin (CK) polypeptides in breast carcinomas is unclear. We examined the CK profiles of 101 primary infiltrating ductal breast carcinomas using monoclonal antibodies directed against 11 different CKs and against vimentin. Two major CK phenotypes were distinguished: first, a phenotype expressing only the simple-epithelial CKs 7 (variably), 8, 18 and 19, and secondly, a bimodal phenotype co-expressing significant amounts of one or more of the stratified-epithelial CKs 4, 14 and 17. The vast majority of G1 and G2 carcinomas had the simple-epithelium phenotype, as did a subgroup of G3 carcinomas. Interestingly, the majority (62%) of G3 carcinomas exhibited the bimodal phenotype, with the expression of CKs 4, 14 and 17 being statistically correlated with poor histological differentiation and absence of steroid hormone receptors. The distribution of vimentin only partially overlapped with that of these stratified-epithelial CKs. Prognostic analyses suggested that the presence of CKs 4, 14 and/or 17 was associated with short overall and disease-free survival in subgroups comprising G3, oestrogen-receptor-negative and vimentin-negative tumours. In node-positive tumours the correlation between these CKs and a shorter disease-free interval attained statistical significance (log rank, 0.0096). Thus, abnormal CK profiles in ductal breast carcinomas appear to reflect disturbed regulation of differentiation-related gene expression programmes and may prove to be of clinical value.

Cytokeratins in normal lung and lung carcinomas. I. Adenocarcinomas, squamous cell carcinomas and cultured cell lines.

SummaryThe various epithelial cells of the lower respiratory tract and the carcinomas derived from them differ markedly in their differentiation characteristics. Using immunofluorescence microscopy and two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissues we have considered whether cytokeratin polypeptides can serve as markers of cell differentiation in epithelia from various parts of the human and bovine lower respiratory tract. In addition, we have compared these protein patterns with those found in the two commonest types of human lung carcinoma and in several cultured lung carcinoma cell lines. By immunofluorescence microscopy, broad spectrum antibodies to cytokeratins stain all epithelial cells of the respiratory tract, including basal, ciliated, goblet, and alveolar cells as well as all tumor cells of adenocarcinomas and squamous cell carcinomas. However, in contrast, selective cytokeratin antibodies reveal cell type-related differences. Basal cells of the bronchial epithelium react with antibodies raised against a specific epidermal keratin polypeptide but not with antibodies derived from cytokeratins characteristic of simple epithelia. When examined by two-dimensional gel electrophoresis, the alveolar cells of human lung show cytokeratin polypeptides typical of simple epithelia (nos. 7, 8, 18 and 19) whereas the bronchial epithelium expresses, in addition, basic cytokeratins (no. 5, small amounts of no. 6) as well as the acidic polypeptides nos. 15 and 17. Bovine alveolar cells also differ from cells of the tracheal epithelium by the absence of a basic cytokeratin polypeptide. All adenocarcinomas of the lung reveal a “simple-epithelium-type” cytokeratin pattern (nos. 7, 8, 18 and 19). In contrast, squamous cell carcinomas of the lung contain an unusual complexity of cytokeratins. We have consistently found polypeptides nos. 5, 6, 8, 13, 17, 18 and 19 and, in some cases, variable amounts of cytokeratins nos. 4, 14 and 15.Several established cell lines derived from human lung carcinomas (SK-LU-1, Calu-1, SK-MES-1 and A-549) show a uniform pattern of cytokeratin polypeptides (nos. 7, 8, 18 and 19), similar to that found in adenocarcinomas. In addition, vimentin filaments are produced in all the cell lines examined, except for SK-LU-1.From these analyses we conclude, in contrast to some previous reports, (1) that all epithelial cells from the trachea and lung contain cytokeratins, (2) that epithelial cells from different parts of the respiratory tract express different cytokeratins, and (3) that the bronchial epithelium is characterized by the occurrence of basic (nos. 5 and 6) and acidic (nos. 15 and 17) polypeptides which are absent from alveoli. A marked, probably related difference in cytokeratin expression is also seen between squamous cell carcinomas and adenocarcinomas.The possible value of analyses of cytokeratin polypeptide patterns in lung carcinomas and metastases is discussed in relation to the diagnosis and histogenesis of lung neoplasms. We propose to use monoclonal antibodies specific for individual cytokeratin polypeptides as selective diagnostic tools for the differentiation and classification of lung carcinomas.

Differences of expression of cytokeratin polypeptides in various epithelial skin tumors

SummaryIn normal skin, cytokeratin polypeptides are expressed in different cell-type-specific patterns, in the keratinocytes of the different epidermal cell strata as well as in different lateral epithelial domains. Using light microscopically controlled microdissection of defined regions from frozen sections of biopsies, we have prepared cytoskeletons of various benign and malignant keratinocyte-derived tumors of human skin and analyzed their cytokeratin polypeptide patterns by two-dimensional gel electrophoresis. Premalignant fibro-epitheliomas and basal cell epitheliomas display a relatively simple cytokeratin pattern (cytokeratins nos. 5, 14, 15, and 17). Pseudocarcinomatous hyperplasia, some squamous cell carcinomas, and a certain subtype of condylomata acuminata present a hair-follicle-like pattern (nos. 5, 6, 14, 16, 17). In addition to these components, variable, mostly low amounts of cytokeratins nos. 1 (Mr 68,000), and 11 are detected in most squamous cell carcinomas, in keratoacanthomas, verruca vulgaris, and another type of condylomata acuminata. In molluscum contagiosum, verruca plana, solar keratosis, and seborrheic keratosis, the cytokeratin expression is shifted more towards the normal epidermal pattern (polypeptides nos. 1, 2, 5, 10, 11, 14, 15 and traces of nos. 6 and 16 in the latter two tumors). No tumor-specific cytokeratins have been found. We conclude that keratinocyte-derived skin tumors contain various combinations of cytokeratins of the subset typical for normal keratinocytes of skin, but no cytokeratins typical for internal, simple epithelia. Different groups of tumors can be distinguished by their specific cytokeratin patterns. Possible applications of cytokeratin typing in clinical diagnosis are discussed.

Tumor dedifferentiation: An important step in tumor invasion

Tumor invasionin vivo was studied by light and electron microscopy as well as by immunofluorescence microscopy. Special regard was paid to the grade of tumor differentiation. Dimethylhydrazine-induced murine colonic carcinomas comprising a differentiated and an undifferentiated tumor type with low and high invasiveness respectively, were used. At the invasion front of both tumor types a striking dissociation of the organized tumor cell complexes into isolated tumor cells was found together with a loss of most of the cytological features of differentiation. It is supposed that this process mobilizes the tumor cells from the main tumor bulk enabling them to invade the host tissue by active locomotion. This view is strongly supported by the demonstration of morphological equivalents of active cell movement such as pseudopodia-like cytoplasmic extrusions, adaptive changes of the cell shape and microfilament bundles. Although the proposed mechanism of tumor invasion is essentially the same in both tumor types, the grade of differentiation is nevertheless critical, as in the undifferentiated carcinomas only subtle dedifferentiation steps (loss of basement membrane and cell junctions) are necessary to acquire an invasive status. This fact may explain the comparatively high invasiveness and poor prognosis of undifferentiated carcinomas.

Cytokeratin polypeptide patterns of different epithelia of the human male urogenital tract: immunofluorescence and gel electrophoretic studies.

Intermediate filament proteins of normal epithelia of the human and the bovine male urogenital tract and of certain human renal and bladder carcinomas have been studied by immunofluorescence microscopy and by two-dimensional gel electrophoresis of cytoskeletal fractions from microdissected tissue samples. The patterns of expression of cytokeratin polypeptides differ in the various epithelia. Filaments of a cytokeratin nature have been identified in all true epithelial cells of the male urogenital tract, including renal tubules and rete testis. Simple epithelia of renal tubules and collecting ducts of kidney, as well as rete testis, express only cytokeratin polypeptides nos. 7, 8, 18, and 19. In contrast, the transitional epithelia of renal pelvis, ureter, bladder, and proximal urethra contain, in addition to those polypeptides, cytokeratin no. 13 and small amounts of nos. 4 and 5. Most epithelia lining the human male reproductive tract, including those in the epididymis, ductus deferens, prostate gland, and seminal vesicle, synthesize cytokeratin no. 5 in addition to cytokeratins nos. 7, 8, 18, and 19 (cytokeratin no. 7 had not been detected in the prostate gland). Cytokeratin no. 17 has also been identified, but in very low amounts, in seminal vesicle and epididymis. The cytokeratin patterns of the urethra correspond to the gradual transition of the pseudostratified epithelium of the pars spongiosa (cytokeratins nos. 4, 5, 6, 13, 14, 15, and 19) to the stratified squamous epithelium of the fossa navicularis (cytokeratins nos. 5, 6, 10/11, 13, 15, and 19, and minor amounts of nos. 1 and 14). The noncornified stratified squamous epithelium of the glans penis synthesizes cytokeratin nos. 1, 5, 6, 10/11, 13, 14, 15, and 19. In immunofluorescence microscopy, selective cytokeratin antibodies reveal differential staining of different groups or layers of cells in several epithelia that may relate to the specific expression of cytokeratin polypeptides. Human renal cell carcinomas show a simple cytokeratin pattern consisting of cytokeratins nos. 8, 18, and 19, whereas transitional cell carcinomas of the bladder reveal additional cytokeratins such as nos. 5, 7, 13, and 17 in various proportions. The results shows that the wide spectrum of histological differentiation of the diverse epithelia present in the male urogenital tract is accompanied by pronounced changes in the expression of cytokeratin polypeptides and suggest that tumors from different regions of the urogenital tract may be distinguished by their cytokeratin complements.

Prognostic value of E‐cadherin expression in 413 gastric carcinomas

E‐cadherin is Ca2+‐dependent intercellular adhesion molecule known to exert an invasion‐suppressor function. In the present study, E‐cadherin expression was immunohistochemically investigated in a retrospective series of 413 RO‐resected gastric carcinomas using the monoclonal antibody (MAb) 5H9. Of these cases, 108 tumors revealed a preserved E‐cadherin expression similar to that of normal gastric mucosa. In 95 tumors, E‐cadherin expression was moderately reduced and in 86 tumors highly reduced. In 124 tumors, no or only a very weak dotted expression could be detected. There was a significant correlation between the degree of E‐cadherin expression and the grade of tumor differentiation, as well as with histological type according to the Laurén and the WHO classifications. In contrast, no correlation could be demonstrated between E‐cadherin expression and the prognostic parameters depth of invasion, lymph node involvement and vascular invasion. As shown by univariate Cox regression analysis, patients with E‐cadherin‐positive tumors had significantly better 3‐ and 5‐year survival rates than patients with E‐cadherin‐negative tumors. This prognostic impact remained present in a multivariate Cox regression analysis, including the prognostic parameters pT category, pN category and vascular invasion.