Extinction of the HPV18 upstream regulatory region in cervical carcinoma cells after fusion with non-tumorigenic human keratinocytes under non-selective conditions.

'Universal fuser' clones of a human papillomavirus type 16 positive cervical carcinoma cell line (SiHa) were established to study the effect of a non-tumorigenic fusion partner on the regulation of a stably integrated chloramphenicol acetyltransferase (CAT) gene controlled by the HPV18 upstream regulatory region under non-selective conditions. The CAT expressing cells were fused with both non-tumorigenic, spontaneously immortalized human keratinocytes (HaCaT) and non-modified SiHa cells. The resulting hybrids were characterized by restriction enzyme fragment length polymorphism analysis and flow cytometry. While the non-selectable, HPV18-driven indicator gene is constitutively expressed in SiHa cells, the CAT activity is extinguished in SiHa x HaCaT cells, but still present in SiHa x SiHa hybrids. Examination of the cytokeratin expression pattern reveals that the keratinocyte phenotype seems not only to be dominant in terms of the extinction of the HPV18 regulatory region but also by the conservation of most of the differentiation markers of the non-tumorigenic fusion partner. Cycloheximide treatment and intracellular competition experiments using the transient COS7 fusion-amplification technique are accompanied by the reactivation of the marker gene in previously CAT- SiHa x HaCaT hybrids. These data strongly suggest that trans-acting negative regulatory factors derived from the non-malignant human keratinocytes are responsible for the extinction phenomenon.

An epithelium-type cytoskeleton in a glial cell: astrocytes of amphibian optic nerves contain cytokeratin filaments and are connected by desmosomes.

In higher vertebrates the cytoskeleton of glial cells, notably astrocytes, is characterized (a) by masses of intermediate filaments (IFs) that contain the hallmark protein of glial differentiation, the glial filament protein (GFP); and (b) by the absence of cytokeratin IFs and IF-anchoring membrane domains of the desmosome type. Here we report that in certain amphibian species (Xenopus laevis, Rana ridibunda, and Pleurodeles waltlii) the astrocytes of the optic nerve contain a completely different type of cytoskeleton. In immunofluorescence microscopy using antibodies specific for different IF and desmosomal proteins, the astrocytes of this nerve are positive for cytokeratins and desmoplakins; by electron microscopy these reactions could be correlated to IF bundles and desmosomes. By gel electrophoresis of cytoskeletal proteins, combined with immunoblotting, we demonstrate the cytokeratinous nature of the major IF proteins of these astroglial cells, comprising at least three major cytokeratins. In this tissue we have not detected a major IF protein that could correspond to GFP. In contrast, cytokeratin IFs and desmosomes have not been detected in the glial cells of brain and spinal cord or in certain peripheral nerves, such as the sciatic nerve. These results provide an example of the formation of a cytokeratin cytoskeleton in the context of a nonepithelial differentiation program. They further show that glial differentiation and functions, commonly correlated with the formation of GFP filaments, are not necessarily dependent on GFP but can also be achieved with structures typical of epithelial differentiation; i.e., cytokeratin IFs and desmosomes. We discuss the cytoskeletal differences of glial cells in different kinds of nerves in the same animal, with special emphasis on the optic nerve of lower vertebrates as a widely studied model system of glial development and nerve regeneration.

Cytokeratin filaments and desmosomes in the epithelioid cells of the perineurial and arachnoidal sheaths of some vertebrate species.

Using electron microscopy and immunohistochemistry with a large panel of antibodies to various cytoskeletal proteins we have noted that the single- or multi-layered sheaths of epithelioid cells ("neurothelia") surrounding peripheral nerves (perineurial cells) or structures of the central nervous system, including the optic nerve (arachnoid cells), show remarkable interspecies differences in their cytoskeletal complements. In two anuran amphibia examined (Xenopus laevis, Rana ridibunda), the cells of both forms of neurothelia, i.e., perineurial and arachnoid, are interconnected by true desmosomes and are rich intermediate-sized filaments (IFs) of the cytokeratin type. Among higher vertebrates, a similar situation is found in the bovine and chicken nervous systems, in which the arachnoid cells of the meninges contain desmosomes and IFs of both the cytokeratin (apparently with restricted epitope accessibilities in the chicken) and the vimentin type, whereas the perineurial cells of many nerves contain cytokeratin IFs, often together with vimentin, but no desmosomes. In contrast, in rat arachnoidal and perineurial cells significant reactions have been observed neither for cytokeratins nor for desmosomes. In the human nervous system, cytokeratins and desmosomes have also not been seen in the various perineuria studied whereas desmosomes are frequent in arachnoidal cell layers which are dominated by vimentin IFs and only in certain small regions of the brain contain some additional cytokeratins. The occurrence of cytokeratins in the tissues found positive by immunohistochemistry has been confirmed by gel electrophoresis of cytoskeletal proteins, followed by immunoblotting. Our results emphasize both similarities and differences between the neurothelia on the one hand and epithelia or endothelia on the other, justifying classification as a separate kind of tissue, i.e., neurothelium. The observations of interspecies differences lead to the challenging conclusion that neither desmosomes nor cytokeratins are essential for the basic functions of neurothelial sheaths nor does the specific type of IF protein expressed in these cells appear to matter in this respect. The results are also discussed in relation to the cytoskeletal characteristics of other epithelioid tissues and of human neurothelium-derived tumors.

Cytokeratins in normal and malignant transitional epithelium. Maintenance of expression of urothelial differentiation features in transitional cell carcinomas and bladder carcinoma cell culture lines.

The pattern of cytokeratins expressed in normal urothelium has been compared with that of various forms of transitional cell carcinomas (TCCs; 21 cases) and cultured bladder carcinoma cell lines, using immunolocalization and gel electrophoretic techniques. In normal urothelium, all simple-epithelium-type cytokeratins (polypeptides 7, 8, 18, 19) were detected in all cell layers, whereas antibodies to cytokeratins typical for stratified epithelia reacted with certain basal cells only or, in the case of cytokeratin 13, with cells of the basal and intermediate layers. This pattern was essentially maintained in low-grade (G1, G1/2) TCCs but was remarkably modified in G2 TCCs. In G3 TCCs simple-epithelial cytokeratins were predominant whereas the amounts of component 13 were greatly reduced. Squamous metaplasia was accompanied generally by increased or new expression of some stratified-epithelial cytokeratins. The cytokeratin patterns of cell culture lines RT-112 and RT-4 resembled those of G1 and G2 TCCs, whereas cell line T-24 was comparable to G3 carcinomas. The cell line EJ showed a markedly different pattern. The results indicate that, in the cell layers of the urothelium, the synthesis of stratification-related cytokeratins such as component 13 is inversely oriented compared with that in other stratified epithelia where these proteins are suprabasally expressed, that TCCs retain certain intrinsic cytoskeletal features of urothelium, and that different TCCs can be distinguished by their cytokeratin patterns. The potential value of these observations in histopathologic and cytologic diagnoses is discussed.

Expression of simple epithelial type cytokeratins in stratified epithelia as detected by immunolocalization and hybridization in situ.

Multi-layered ("stratified") epithelia differ from one-layered ("simple") polar epithelia by various architectural and functional properties as well as by their cytoskeletal complements, notably a set of cytokeratins characteristic of stratified tissue. The simple epithelial cytokeratins 8 and 18 have so far not been detected in any stratified epithelium. Using specific monoclonal antibodies we have noted, in several but not all samples of stratified epithelia, including esophagus, tongue, exocervix, and vagina, positive immunocytochemical reactions for cytokeratins 8, 18, and 19 which in some regions were selective for the basal cell layer(s) but extended into suprabasal layers in others. In situ hybridization with different probes (riboprobes, synthetic oligonucleotides) for mRNAs of cytokeratin 8 on esophageal epithelium has shown, in extended regions, relatively strong reactivity for cytokeratin 8 mRNA in the basal cell layer. In contrast, probes to cytokeratin 18 have shown much weaker hybridization which, however, was rather evenly spread over basal and suprabasal strata. These results, which emphasize the importance of in situ hybridization in studies of gene expression in complex tissues, show that the genes encoding simple epithelial cytokeratins can be expressed in stratified epithelia. This suggests that continual expression of genes coding for simple epithelial cytokeratins is compatible with the formation of squamous stratified tissues and can occur, at least in basal cell layers, simultaneously with the synthesis of certain stratification-related cytokeratins. We also emphasize differences of expression and immunoreactivity of these cytokeratins between different samples and in different regions of the same stratified epithelium and discuss the results in relation to changes of cytokeratin expression during fetal development of stratified epithelia, in response to environmental factors and during the formation of squamous cell carcinomas.

Monoclonal cytokeratin antibody recognizing a heterotypic complex: immunological probing of conformational states of cytoskeletal proteins in filaments and in solution.

A novel type of monoclonal murine antibody (Ks18.18) directed against an epitope depending on human cytokeratin (CK) 18, a member of the acidic (type I) CK subfamily, is described. We show by SDS-PAGE immunoblots and dot-blot assays that this antibody is unreactive with both the denatured and the renatured individual polypeptides but binds strongly to heterotypic coiled-coil complexes of CK 18 with several members of the complementary basic (type II) CK subfamily, notably with CK 8; i.e., its most frequent natural partner. We also show that specific interactions between complementary CK polypeptides take place during the incubation steps of immunoblotting procedures as polypeptides, or fragments thereof, that detach from the substrate can bind to complementary polypeptides attached to the substratum, which may result in false assignments of antibody reactivities. The conformation-specific, CK 18-dependent epitope of Ks18.18 was detected in intermediate filaments (IFs) of cultured cells, simple epithelia, and many carcinomas and, surprisingly, also in the basal cells of some stratified epithelia. Ks18.18 also reacts with altered CK configurations as present in the spheroidal bodies of mitotic cells and in the Mallory bodies of hepatocytes intoxicated with certain drugs, thus indicating that the heterotypic CK complexes are maintained in these structures. We have also used antibody Ks18.18 to demonstrate the existence of heterotypic CK 8 and 18 complexes in a distinct soluble form among supernatant proteins from cell homogenates which is indistinguishable from the heterotypic tetramer obtained after experimental disintegration of IFs. The potential value of such IF conformation-specific antibodies in cell biological research and pathology is discussed.

Immunohistochemical investigation of different cytokeratins and vimentin in the prostate from the fetal period up to adulthood and in prostate carcinoma.

From the fetal period up to puberty the immature epithelium of the prostate glands, the prostatic ducts, the ejaculatory ducts and the seminal vesicles as well as the urothelium of the prostatic urethra are extensively positive for different keratin antibodies (antibody against keratins from human stratum corneum, broadly reacting antibody "AE1 and AE" and antibodies against the keratins 7, 8, 18 and 19) immunohistochemically. The epithelium of the ejaculatory ducts and seminal vesicles in addition regularly exprimates vimentin which is found in the epithelium of the prostate glands focally. During puberty, the immature epithelium of the prostate glands differentiates into the two cell types basal cell and secretory epithelium which differ immunohistochemically: Keratins from human stratum corneum are exclusively demonstrable in the basal cells, the keratins 8 and 18 only in the secretory epithelium. For keratin 7, 19 and the antibody "AE1 and AE3" both cell types are positive. Keratin 7 is demonstrable only focally. The secretory epithelium partly co-exprimates keratins and vimentin. Prostatic carcinomas of different grades virtually contain no keratins from stratum corneum. All other keratins are found in variable extension in the vast majority of the tumors independent of the differentiation. Vimentin is positive mostly focally in about 50% of the tumors. Prostatic carcinoma and the secretory epithelium of the prostate glands share identical immunohistochemical features and differ from the basal cell by several markers. This indicates that prostatic carcinoma rather derives directly from the secretory epithelium than from the basal cell.

Co-expression of cytokeratins and neurofilament proteins in a permanent cell line: cultured rat PC12 cells combine neuronal and epithelial features.

The cytoskeleton of the rat cultured cell line PC12, which is widely used in cell biology as a model system for neuron-like differentiation, displays an unusual combination of intermediate-sized filaments (IFs). As determined by electron microscopy, immunolocalization, and biochemical analyses, these cells contain, in addition to neurofilaments, an extended meshwork of bundles of cytokeratin IFs comprising cytokeratins A and D, equivalent to human cytokeratin polypeptides Nos. 8 and 18, irrespective of whether they are grown in the presence or absence of nerve growth factor. The two IF systems differ in their fibrillar arrays, the neurofilaments being concentrated in perinuclear aggregates similar to those found in certain neuroendocrine tumors of epithelial origin. We conclude that PC12 cells permanently co-express IFs of both the epithelial and the neuronal type and thus present an IF combination different from those of adrenal medulla cells and pheochromocytomas, i.e., the putative cells of origin of the line PC12. The IF cytoskeleton of PC12 cells resembles that of various neuroendocrine tumors derived from epithelial cells. The results show that the development of a number of typical neuronal differentiation features is compatible with the existence of an epithelial type IF cytoskeleton, i.e., cytokeratins. The implications of these findings concerning the validity of the PC12 cell line as a model for neuronal differentiation and possible explanations of the origin of cells with this type of IF co-expression are discussed.

Cell type heterogeneity of cytokeratin expression in complex epithelia and carcinomas as demonstrated by monoclonal antibodies specific for cytokeratins nos. 4 and 13.

Three monoclonal antibodies, 1C7, 2D7 and 6B10, directed against cytokeratins of human esophagus were isolated and characterized by one- and two-dimensional gel electrophoresis and by immunohistochemical staining on sections of human epithelial tissues. In immunoblot experiments, antibodies of clones 1C7 (IgG2a) and 2D7 (IgG2b) react only with cytokeratin no. 13 of the acidic (type I) subfamily of cytokeratin polypeptides (Mr 54000; pI 5.1); antibodies of clone 6B10 (IgG1) detect only cytokeratin no. 4 (Mr 59000; pI 7.3) of the basic (type II) cytokeratin subfamily and allows the detection of this protein and possible degradation products at high sensitivity. In immunohistochemical staining all three antibodies stain non-cornifying squamous epithelium (e.g., tongue, esophagus, anus) and transitional epithelium of the bladder. Antibodies of clone 6B10 also stain cells in certain ciliated pseudostratified epithelia and ductal epithelia of various exocrine glands. These monoclonal antibodies are the first examples of antibodies specific for individual cytokeratin polypeptides characteristic of certain complex epithelia. They allow the identification of distinct minor populations of cells present in certain complex and glandular epithelia and in tumors derived therefrom which hitherto have not been distinguished. The possible reasons for the occurrence of cell type heterogeneity of cytokeratin expression in complex epithelia and in some carcinomas are discussed.

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.

Patterns of expression and organization of cytokeratin intermediate filaments.

Cytokeratins are a large multigene family comprising two polypeptide types, i.e. acidic (type I) and basic (type II) ones, which are distinguished on the basis of immunological, peptide mapping, mRNA hybridization, and primary amino acid sequence data. The acidic (type I) cytokeratins can be subdivided into at least two different subtypes on the basis of their carboxy-terminal sequences. Considerable interspecies conservation of sequences exists, even extending to the 3'-non-coding mRNA regions. Different pairs of type I and II cytokeratins show different resistance to dissociation in urea. Sequence differences of the type I cytokeratins containing functional domains may be an explanation of the observed preference of co-expression with certain type II cytokeratins. The distribution of the different type I and II cytokeratins in normal epithelia and in carcinomas is differentiation related and can be used for cell typing and identification. The cell type-specific expression of cytokeratin polypeptides is recognized at both the protein and the mRNA level. The building block of cytokeratin IFs is a heterotypic tetramer, consisting of two type I and two type II polypeptides arranged in pairs of laterally aligned coiled coils. This principle of tetrameric organization is thought to be generally applicable to IFs.