Mucin 21 in esophageal squamous epithelia and carcinomas: analysis with glycoform-specific monoclonal antibodies

Monoclonal antibodies (mAbs) against mucin 21 (MUC21), a human counterpart of mouse epiglycanin/Muc21, were prepared using human embryonic kidney 293 cells transfected with MUC21 as the immunogen. The specificity of these mAbs was examined by flow cytometry, immunoprecipitation and western blotting focusing on the differential glycosylation of MUC21 expressed in variant Chinese hamster ovary (CHO) cells (ldlD cells and Lec2 cells) and CHO-K1 cells. One of these mAbs, heM21D, bound to both the unmodified core polypeptide of MUC21 and MUC21 attached with N-acetylgalactosamine (Tn-MUC21). Six antibodies, including mAb heM21C, bound to MUC21 with Tn, T or sialyl-T epitopes but not the unmodified core polypeptide of MUC21. Esophageal squamous carcinomas and adjacent squamous epithelia were immunohistochemically examined for the binding of these mAbs. MUC21 was expressed in esophageal squamous epithelial cells, and its O-glycan extended forms were observed in the luminal portions of squamous epithelia. As revealed by the binding of mAb heM21D and the absence of reactivity with mAb heM21C, esophageal squamous carcinoma cells produce MUC21 without the attachment of O-glycans. This is the first report to show that there is a change in the glycoform of MUC21 that can be used to differentiate between squamous epithelia and squamous carcinoma of the esophagus. Thus, these antibodies represent a useful tool to characterize squamous epithelial differentiation and carcinogenesis.     

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.     

Keratin polypeptide distribution in benign and malignant breast tumors: subdivision of ductal carcinomas using monoclonal antibodies

Monoclonal antibodies which recognize one or only a few keratin polypeptides have been used to study the distribution of different keratins in benign and malignant breast lesions by immunocytochemical methods. Seven monoclonal antibodies which recognized either different keratin polypeptides by immunoblotting techniques, or identified different epithelial cell types in complex tissues were used. In two mastopathies and three fibroadenomas the antibody lu5 stained luminal cells as well as myoepithelial cells. In contrast the antibodies CK7, Troma 1, CK2 and KA4 labeled only luminal cells, whereas antibody CKB1 decorated only myoepithelial cells. All 15 ductal carcinomas showed a uniform staining of tumor cells with the antibodies Troma 1, CK2, KA4 and lu5. The antibody CK7 also stained all ductal carcinomas, but in two specimens the staining was heterogeneous. The antibody CKB1 decorated only the pre-existing myoepithelial cells in 11 of 12 ductal carcinomas but in the remaining specimen the tumor cells were also strongly positive. Tumor cells in lobular carcinomas were labeled by antibodies CK7, Troma 1, CK2, KA4, bu not by CKB1. The antibody CKS1 showed no staining of any of the benign and malignant breast lesions.     

Taurine levels and localisation in the stratified squamous epithelia

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.     

Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family

Glycosylation of proteins is an essential process in all eukaryotes and a great diversity in types of protein glycosylation exists in animals, plants and microorganisms. Mucin-type O-glycosylation, consisting of glycans attached via O-linked N-acetylgalactosamine (GalNAc) to serine and threonine residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The GalNAc-T family is the largest glycosyltransferase enzyme family covering a single known glycosidic linkage and it is highly conserved throughout animal evolution, although absent in bacteria, yeast and plants. Emerging studies have shown that the large number of genes (GALNTs) in the GalNAc-T family do not provide full functional redundancy and single GalNAc-T genes have been shown to be important in both animals and human. Here, we present an overview of the GalNAc-T gene family in animals and propose a classification of the genes into subfamilies, which appear to be conserved in evolution structurally as well as functionally.     

Initiation of assembly of the cell envelope barrier structure of stratified squamous epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.     

Tight junction-related structures in the absence of a lumen: occludin, claudins and tight junction plaque proteins in densely packed cell formations of stratified epithelia and squamous cell carcinomas

Tight junctions (TJs), hallmark structures of one-layered epithelia and of endothelia, are of central biological importance as intramembranous "fences" and as hydrophobic "barriers" between lumina represented by liquid- or gas-filled spaces on the one hand and the mesenchymal space on the other. They have long been thought to be absent from stratified epithelia. Recently, however, constitutive TJ proteins and TJ-related structures have also been identified in squamous stratified epithelia, including the epidermis, where they occur in special positions, most prominently in the uppermost living epidermal cell layer, the stratum granulosum. Much to our surprise, however, we have now also discovered several major TJ proteins (claudins 1 and 4, occludin, cingulin, symplekin, protein ZO-1) and TJ-related structures in specific positions of formations of epithelium-derived tissues that lack any lumen and do not border on luminal or body surfaces. Using immunohistochemistry and electron microscopy we have localized TJ proteins and structures in peripheral cells of the Hassall's corpuscles of human and bovine thymi as well as in specific central formations of tumor nests in squamous cell carcinomas, including the so-called "horn pearls". Such structures have even been found in carcinoma metastases. In carcinomas, they often seem to separate certain tumor regions from others or from stroma. The structural significance and the possible functional relevance of the locally restricted synthesis of TJ proteins and of the formations of TJ-related structures are discussed. It is proposed to include the determination of the presence or absence of such proteins and structures in the diagnostic program of tumor pathology.     

Genomic organization and chromosomal localization of three members of the UDP-N-acetylgalactosamine: polypeptide N- acetylgalactosaminyltransferase family

A homologous family of UDP- N -acetylgalactosamine: polypeptide N - acetylgalactosaminyltransferases (GalNAc-transferases) initiate O- glycosylation. These transferases share overall amino acid sequence similarities of approximately 45-50%, but segments with higher similarities of approximately 80% are found in the putative catalytic domain. Here we have characterized the genomic organization of the coding regions of three GalNAc-transferase genes and determined their chromosomal localization. The coding regions of GALNT1 , -T2 , and -T3 were found to span 11, 16, and 10 exons, respectively. Several intron/exon boundaries were conserved within the three genes. One conserved boundary was shared in a homologous C. elegans GalNAc- transferase gene. Fluorescence in situ hybridization showed that GALNT1 , -T2 , and -T3 are localized at chromosomes 18q12-q21, 1q41-q42, and 2q24-q31, respectively. These results suggest that the members of the polypeptide GalNAc-transferase family diverged early in evolution from a common ancestral gene through gene duplication.      

The development of a standardised protocol to measure squamous differentiation in stratified epithelia, by using the fluorescein cadaverine incorporation technique

Fluorescein cadaverine (FC) incorporation into cornified envelopes during squamous differentiation in stratified epithelia acts as a fluorescent substitute for endogenous transglutaminase substrates that can be visualised and quantified. The FC incorporation technique has been used to evaluate squamous differentiation in keratinocytes cultured in a medium that stimulates differentiation and in response to modulation by chemicals. A Standard Operating Procedure for the measurement of squamous differentiation is required as part of the prevalidation procedure for in vitro assays. In the present study, keratinocytes were isolated from the epidermis of 34 human donors. Cellular metabolic activity (resorufin production), total protein (kenacid blue uptake) and transglutaminase activity (FC incorporation) were measured in 87 and 21 independent runs at 6 and 12 days, respectively. Analysis of the control data showed that the cultures had a mean resorufin production that decreased over 12 days. This was inversely related to FC incorporation, which increased over 12 days. Mean protein concentration was reduced over the 12 days, but not in analyses that used donors for whom both 6-day and 12-day data were available. This information was used to define the normal limits within which the data should fall (mean +/- 1 SD). Data sets falling outside the normal limits performed statistically no differently from the normal responders, in experiments conducted to investigate the effects of chemicals on the modulation of squamous differentiation in keratinocytes. This was demonstrated by using compounds that modify transglutaminase expression and/or activity. All-trans retinoic acid significantly inhibited FC incorporation, but stimulated resorufin production at 1 x 10(-7)M and above. Nicotine significantly up-regulated both FC incorporation and resorufin production at 125 microg/ml. Hence, it was concluded that this robust assay approach, in which keratinocytes from a range of donors respond predictably to the test chemicals employed, did not justify the limitations that would be imposed by setting criteria that eliminated all data lying outside the normal range.     

Heterogeneity in the immunolocalization of cytokeratin-specific monoclonal antibodies in the rat lung: evaluation of three different alveolar epithelial cell types

The distribution of individual cytokeratin polypeptides in the adult rat lung parenchyma was investigated by immunohistochemistry with 44 monoclonal and 2 polyclonal antibodies. Simple epithelial cytokeratins 7, 8, 18 and 19 were found to be expressed differently in alveolar and bronchial epithelial cells. Three distinct types of alveolar cells were detected according to their pattern of immunoreactivity: type II cells strongly expressing cytokeratins 8 and 18 and weakly expressing cytokeratins 7 and 19 in the cell periphery; type I cells predominantly positive for cytokeratins 7 and 19 and weakly for cytokeratin 8; and a newly defined third cell type III (alveolar brush cell) with cytokeratin 18 abundantly expressed but organized in an unusual intracellular (globular) structure. The latter cell type failed to bind the type II specific Maclura pomifera lectin, and contained no surfactant proteins. Bronchial epithelial cells exhibited a more or less uniform staining pattern for cytokeratins 8, 18 and 19 and focally for cytokeratins 4 and 7.This work was supported by Bundesminister für Forschung und Technologie (07NBL03) and Dakopatts (Glostrup, Denmark)     

Characterization of monoclonal antibodies specific for rabbit renal brush-border hydrolases: application to immunohistological localization

By use of immunodepletion studies, we characterized four monoclonal antibodies reactive with rabbit brush-border (BB) as specific for aminopeptidase N (AP), dipeptidylpeptidase IV (DPPIV), neutral endopeptidase (EP), and angiotensin-converting enzyme (ACE), and we used these antibodies for immunohistochemical detection of these four hydrolases. Expression within the kidney was studied by light and electron microscopy. All four hydrolases are expressed on the various segments of the proximal tubule. In addition, EP and DPPIV are detectable on visceral epithelial cells of the glomerulus and AP on the cells of Bowman's capsule. Outside the kidney, the four hydrolases are expressed within the digestive and genital tracts, where AP, EP, and DPPIV predominate on epithelial structures, whereas ACE is essentially located in vascular structures. The latter localization is also characteristic of ACE in the other organs studied, where clear-cut systematic distribution of the other hydrolases was often difficult to demonstrate. In addition, AP, DPPIV, and EP were detected on lymphoid cells. As compared to reports of data obtained essentially by enzymatic or immunoradiometric assays, these observations suggest considerable interspecies variations of extrarenal expression of the major BB hydrolases. This should be taken into account in attempting to define a general physiological role for a given enzyme.     

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies

The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes. These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes.     

The highly modified membrane of cornified cells in stratified squamous epithelia: A comparison of heterogenous deposits in keratinized and nonkeratinized epithelia

Summary The chemical nature of the thickened plasma membrane of cornified cells in stratified squamous epithelium was investigated in comparison with that in noncornified epithelium. Localizations of transglutaminase, molecular weight 92000 daltons, and detection of epidermal cysteine proteinase inhibitor were effected with a monoclonal antibody and a monospecific rabbit anti-inhibitor immunoglobulin, respectively, directed to the antigens. N-(7-dimethylamino-4-methylcoumarinyl) maleimide was used to demonstrate S-S cross-linking. In all keratinizing epithelia, the enzyme and inhibitor were deposited on membranes of granular cells. S-S bonds were formed in cornification with the appearance of electron-dense material by the inner leaflet. Both enzyme and inhibitors occurred on the corneal epithelium, but S-S linkage and the thickened plasma membrane did not form even at the last stage of maturation. On the other hand, the internal vaginal epithelium in the proestrous stage without keratinization contained the enzyme, but neither inhibitor nor S-S linkage. Both antigens and S-S bonds were detected when keratinization proceeded during estrus. The staining patterns in the epithelium near the vaginal introitus were identical to those in the skin. Cuboidal and simple epithelia exhibited none of those constituents. The findings indicated that heterogenous components contribute to modification of the plasma membrane of cornified cells, but S-S cross-linkages are associated exclusively with formation of the ultrastructurally unique membrane structure. In addition, findings suggested hormonal regulation in the chemical modification of the membrane in estrogen-sensitive internal vaginal epithelium.     

Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana

Calcium-dependent protein kinases (CDPKs) belong to a unique family of enzymes containing a single polypeptide chain with a kinase domain at the amino terminus and a putative calcium-binding EF hands structure at the carboxyl terminus. From Arabidopsis thaliana, we have cloned three distinct cDNA sequences encoding CDPKs, which were designated as atcdpk6, atcdpk9 and atcdpk19. The full-length cDNA sequences for atcdpk6, atcdpk9 and atcdpk19 encode proteins with a molecular weight of 59343, 55376 and 59947, respectively. Recombinant atCDPK6 and atCDPK9 proteins were fully active as kinases whose activities were induced by Ca²⁺. Biochemical studies suggested the presence of an autoinhibitory domain in the junction between the kinase domain and the EF hands structure. Serial deletion of the four EF hands of atCDPK6 demonstrated that the integrity of the four EF hands was crucial to the Ca²⁺ response. All the three atcdpk genes were ubiquitously expressed in the plant as demonstrated by RNA gel blot experiments. Comparison of the genomic sequences suggested that the three cdpk genes have evolved differently. Using antibodies against atCDPK6 and atCDPK9 for immunohistochemical experiments, CDPKs were found to be expressed in specific cell types in a temporally and developmentally regulated manner.     

Lack of correlation between differentiation status and response to radiation of three murine squamous cell carcinomas

Summary The gross growth rate, histology, cellular kinetics, andin situ radiobiological response have been measured for three murine, keratinising squamous cell carcinomas that differed in their degree of differentiation. Growth rate was fastest in the least-differentiated tumour, slowest in the best-differentiated. However, the kinetics of the compartment of undifferentiated cells that are likely to be radiotherapeutically important, were the same for the three lines. There was no correlation between degree of differentiation and intrinsic or apparent radiosensitivity as measured by the growth delay assay. The radiobiologically best-oxygenated tumour was that which had the largest stromal component and this was not the best-differentiated tumour.     

Generation of monoclonal antibodies detecting specific epitopes in olfactory and respiratory epithelia

Summary Two panels of monoclonal antibodies have been generated, each panel having a distinct specificity for antigens located in the ciliary zone of either the olfactory or respiratory epithelium of rats. Tissue specificity was confirmed in enzyme-linked immunosorbent assays on membrane fractions from various tissues. During ontogeny, the expression of olfactory-specific antigens preceeds that of respiratory-specific antigens; this observation correlates with differences in the genesis of the respective cilia type and confirms that different molecular entities are recognized. A spatial segregation of immunoreactivity in the chemosensory epithelium was observed for one of the olfactory-specific monoclonal antibodies; negative zones were located in the dorsal recess of the nasal cavity and on the tips of the turbinates. Olfactory-specific antibodies reacted with distinct polypeptide bands on Western blots from olfactory ciliary preparations.     

A histological method for the visualization of the intercellular permeability barrier in mammalian stratified squamous epithelia

Summary Mammalian epidermis and oral epithelia possess an intercellular permeability barrier which is located in the superficial region of the tissue. This study reports a staining reaction which appears to demonstrate a histological correlate of this functional property. Specimens of ear skin, palate, buccal and oesophageal mucosa and of cornea and bladder were obtained from adult rabbits and rats, bisected and either incubatedin vitro with 2.5% horseradish peroxidase as a tracer or fixed and processed for light microscopy and stained with a modification of Hart's elastin stain. Examination of specimens prepared by each procedure showed a complementary staining pattern in the intercellular spaces of the stratum corneum or in the superficial region of the non-keratinized tissue. In the epidermis and oral and oesophageal epithelia, the region which excluded the tracer stained with the modified elastin stain. In contrast, the corneal and bladder epithelia neither excluded the tracer nor showed intercellular staining. This relationship between staining of the intercellular space and the exclusion of tracer suggests that the intercellular material in the superficial region of epithelia may be chemically altered to form a barrier substance, possibly as the result of the discharge of the contents of the membrane-coating granules which are present in all the epithelia examined except the cornea and bladder.     

Evidence for a role of the monoclonal antibody E48 defined antigen in cell-cell adhesion in squamous epithelia and head and neck squamous cell carcinoma

Monoclonal antibody (MAb) E48 recognizes a 20- to 22-kDa antigen expressed by human squamous and transitional epithelia and their neoplastic counterparts. Histochemical examination of these tissues revealed distinct surface labeling with MAb E48. To investigate the subcellular localization of the E48 antigen we have performed electron microscopical analysis. In cells of normal oral mucosa, the E48 antigen was expressed on the plasmalemma, particularly associated with desmosomes, suggesting involvement of the E48 antigen in intercellular adhesion. Furthermore, the level of expression of the E48 antigen appeared to be influenced by the cellular organization. In squamous cell carcinoma (SCC) cell lines grown in vitro as subconfluent monolayer cultures, the E48 antigen expression was low. However, E48 antigen expression increased when SCC cells were grown to confluency. E48 antigen expression was similarly high when SCC cell lines were cultured under conditions promoting three-dimensional growth either as colonies within floating collagen gels or as xenograft in tumor-bearing nude mice. Further evidence for the involvement of the E48 antigen in cell-cell adhesion was found when SCC cells were grown within collagen gels in the presence of MAb E48: no spherical colonies were formed, but cells grew out to colonies composed of single cells. Moreover, in this culture system the percentage of SCC cells growing out to colonies was decreased by the presence of MAb E48. These findings indicate that the E48 antigen is involved in the structural organization of squamous tissue and might have a role in intercellular adhesion.