Antibodies to intermediate filament proteins in the immunohistochemical identification of human tumours: an overview

Intermediate-sized filament proteins (IFP) are tissue specific in that antibodies to keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and the neurofilament proteins can distinguish between cells of epithelial and mesenchymal origin as well as of myogenic and neural origin respectively. Malignant cells retain their tissue-specific IFP, which makes it possible to use these antibodies in tumour diagnosis. Carcinomas are exclusively detected by antibodies to keratin. Monoclonal antibodies to keratin have allowed the differentiation between subgroups of epithelial tumours until now between adenocarcinomas and squamous cell carcinomas. Lymphomas, melanomas and several soft tissue tumours are distinctly recognized by antibodies to vimentin. On the other hand, rhabdomyosarcomas and leiomyosarcomas are positive for desmin, while astrocytomas give a strong reaction with GFAP antibodies. Thus, antibodies to IFP are useful tools for differential diagnosis in surgical pathology.     

Origin of cells in human oral mucosa heterotransplants in nude mice

Species identification of cells was performed in a model to study epithelial-mesenchymal interactions using combinations of human and murine tissue. The study comprised 34 successfully recovered transplants of human palatal mucosa with 15 human epithelial outgrowths formed on uncertainly species-identified connective tissue. Formalin-fixed and paraffin-embedded 5-micron sections were stained with bisbenzimide (0.8 microgram/ml) and examined in an epifluorescence microscope. The nuclear-staining pattern revealed limited peripheral invasion of the transplanted connective tissue by murine fibroblasts. In areas of epithelial outgrowth a small number of fibroblasts could not be identified but most subepithelial fibroblasts were murine. The study supports previous findings which indicate that the altered differentiation of the human epithelial outgrowths was caused by the murine connective tissue underlying the human epithelium.     

Skin structure and vascularization in the Antarctic notothenioid fish Gymnodraco acuticeps

The scaleless notothenioid Gymnodraco acuticeps is a bottom dweller beneath the sea ice of McMurdo Sound, Antarctica. Gymnodraco experience unusual environmental conditions, including highly oxygenated subzero water. Skin morphology is evaluated with reference to its potential as a barrier to ice propagation and as a surface for cutaneous respiration. Light and electron microscopy and histochemistry reveal skin structure that is generally similar to that of other teleosts. In the epidermis, epithelial cells are arranged in nine to fifteen layers, and two types of mucous cells are also present. Large mucous cells are most common on external epidermal surfaces, whereas small cells are more frequent on internal epithelial surfaces. Epithelial cell junctions have extensive areas of desmosomes as well as interdigitations of the cell membranes, especially in the basal and midepidermis. The dermis consists of an exceptionally dense stratum compactum. The skin is thicker than that of Bovichtus, a scaleless temperate notothenioid from New Zealand. Mean skin thicknesses at sites on the trunk are 371–711 μm. With the exception of fins that contact the substrate, epidermal thickness between rays of most fins is 70–118 μm. The epithelial surfaces of the oral and branchial cavities are 27–50 μm thick. An unusual type of connective tissue is present beneath the epidermis of the pelvic fin. It contains abundant ground substance and is similar to mucous connective tissue of the mammalian umbilical cord. Perfusions of a microvascular filling agent reveal a moderately developed cutaneous vasculature. These vessels have the dimensions of capillaries (mean external diameter 11 μm). They are confined to the dermis and are more prominent on the head than on the trunk. The skin is secondary to the gills as a respiratory surface in Gymnodraco.     

Restoration of the epidermal phenotype by follicular outer root sheath cells in recombinant culture with dermal fibroblasts

In order to better understand how outer root sheath (ORS) cells are able to reepithelialize superficial skin wounds, the level of epidermal differentiation achieved by isolated ORS cells in vitro was determined. Using postmitotic human dermal fibroblasts (HDF) as efficient feeder cells, large numbers of ORS cells from individual follicles were generated. Passaged ORS cells were grown exposed to air on HDF-populated collagen gels in the CRD device (Noser and Limat, In vitro 23, 541-545, 1987) which allows histiotypic tissue organization. In such recombinant organotypic cultures, ORS cells developed distinct epidermal strata comparable to interfollicular keratinocytes (NEK). Ultrastructurally, desmosomes and intermediate filaments increased in number toward the epithelial surface and small keratohyalin (KH) granules (but no large irregular KH granules as in NEK) were abundant, adjacent to an electrondense stratum corneum. Also, synthesis of epidermal suprabasal keratins (K1 and 10;2D gels) was lower in ORS cultures, but clearly visible suprabasally by immunofluorescence along with other epidermal markers (involucrin, filaggrin, surface glycoprotein gp80, pemphigus vulgaris antigen). Basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) were detectable in both ORS and NEK in these assays. Thus, phenotypic expression was largely comparable, but, whereas terminal differentiation (keratinization) was progressing in NEK cultures limiting their lifespan, this seemed to be better controlled in ORS cultures and viable cell layers persisted resulting in longer survival time.     

Diseases in North Sea fishes

Prior to the studies reviewed here, only lymphocystis and skeletal deformities of a variety of fish species and certain diseases of eel were known to occur in the German Bight (North Sea). From 1977 until now, 9 externally visible lesions on North Sea fishes were observed; in addition to those mentioned before, they comprise: fin rot, ulcerations, epidermal papilloma, hyperplasia, pseudobranchial tumour, eye diseases and gill swellings. With the exception of information on changes in frequencies of vertebral deformities of herring from the 1950's to the 1970's, there are no long-term data characterizing changes in frequencies of the diseases under study. For pseudobranchial tumours of cod and epidermal papilloma of dab, information is provided on occurrence and abundance. The distribution pattern of cod afflicted with pseudobranchial tumours is strongly influenced by the migratory behaviour of the fish. Epidermal papillomas of dab were more frequently found at stations within the inner German Bight than in neighbouring areas. The Bight is used for dumping of wastes from titaniumdioxide production. Further disease hot spots are areas off the Humber estuary and the British coast. Analysis of chromium in dab from the German Bight revealed elevated concentrations in epidermal tissues of specimens from the dumping area compared with that found in dab from neighbouring localities. Particulate iron was demonstrated to occur in mucous cells of dab from the dumping area. From increased levels of heavy metals with cancerogenic potential in sensitive target tissues and from increased prevalences of diseased fish in the dumping area it is concluded that these phenomena are possibly causally linked. In the vicinity of the Humber estuary high disease rates were encountered and areas with high prevalences of dab afflicted with epidermal papilloma extended over regions shown to be transport routes for persistent pollutants such as radioactive materials. It is therefore suggested that the long-range distribution of fish diseases in the southern North Sea might reflect the long-range transport of persistent pollutants.     

Actin typing of rhabdomyosarcomas shows the presence of the fetal and adult forms of sarcomeric muscle actin

We analyzed actin expression in two human rhabdomyosarcomas as well as in three rhabdomyosarcomas induced in rats by the injection of nickel sulfide. All five tumors exhibited appreciable amounts of the sarcomeric alpha-actin types, in line with their myogenic differentiation. The level of these actins was particularly high in the rat tumors, which according to morphological criteria, all showed a higher degree of differentiation than the human tumors. Interestingly, in both human tumors and in two of the three rat tumors, the level of the cardiac alpha-actin type was significantly higher than that of adult skeletal muscle alpha-actin. Taken together with the results of recent reports indicating that the cardiac alpha-actin type is a marker of embryonic and fetal skeletal muscle, our findings indicate that rhabdomyosarcomas express the embryonic sarcomeric actin isoform.     

Progressive stages of "transdifferentiation" from epidermal to mesenchymal phenotype induced by MyoD1 transfection, 5-aza-2''- deoxycytidine treatment, and selection for reduced cell attachment in the human keratinocyte line HaCaT

The ability of the myogenic determination gene (MyoD1) to convert differentiating human keratinocytes (HaCaT cell-line) to the myogenic pathway and the effect of MyoD1 on the epidermal phenotype was studied in culture and in surface transplants on nude mice. MyoD1 transfection induced the synthesis of myosin, desmin, and vimentin without substantially altering the epidermal differentiation properties (morphology, keratin profile) in vitro nor epidermal morphogenesis (formation of a complex stratified squamous epithelium) in surface transplants, demonstrating the stability of the keratinocyte phenotype. 5-Aza-CdR treatment of these MyoD1-transfected cells had little effect on the cultured cells but a morphologically unstructured epithelium was formed with no indications of typical cell layers including cornification. Since prevention of epidermal strata in transplants was not accompanied by blocked epidermal differentiation markers (keratins K1 and K10, involucrin, and filaggrin), the dissociation of morphogenesis and expression of these markers argues for independently controlled processes. A subpopulation of less adhesive cells, isolated from the 5-aza-CdR treated MyoD1-transfectants, had lost most epithelial characteristics in culture (epidermal keratins, desmosomal proteins, and surface-glycoprotein Gp90) and had shifted to a mesenchymal/myogenic phenotype (fibroblastic morphology, transactivation of Myf3 and myogenin, expression of myosin, desmin, vimentin, and Gp130). Moreover, the cells had lost the ability to stratify and remained as a monolayer of flat elongated cells in transplants. These subsequent changes from a fully differentiated keratinocyte to a mesenchymal/myogenic phenotype strongly argue for a complex "transdifferentiation" process which occurred in the original monoclonal human epidermal HaCaT cells.     

Transfection of the EJ rasHa gene into keratinocytes derived from carcinogen-induced mouse papillomas causes malignant progression.

The development of malignant tumors in carcinogen-treated mouse skin appears to involve several genetic changes. Genetic changes which initiate the process are believed to induce alterations in the normal pattern of epidermal differentiation, resulting in the formation of benign tumors, i.e., epidermal papillomas. Subsequent changes appear to be required for the malignant conversion of papillomas to epidermal, squamous-cell carcinomas. Activation of the rasHa gene occurs frequently in chemically induced benign skin papillomas as well as squamous cell carcinomas and thus may represent one mechanism to achieve the initiation step. In the present study, we analyzed several cell lines derived from chemically induced mouse skin papillomas for the presence of transforming oncogenes by transfection of their DNA into NIH 3T3 cells. These papilloma cell lines exhibit an altered differentiation program, i.e., the ability to proliferate under culture conditions favoring terminal differentiation. When DNA from six separate cell lines was tested in the NIH 3T3 transfection assay, active transforming activity was not detected. However, when the EJ rasHa gene was introduced into three of the papilloma cell lines by DNA transfection, transfectants showed an enhanced capacity to proliferate under differentiating culture conditions and formed rapidly growing, anaplastic carcinomas in nude mice. Our findings suggest that in some papilloma cells, a genetic change distinct from rasHa activation may produce an altered differentiation program associated with the initiation step, and this genetic alteration may act in a cooperating fashion with an activated ras gene to result in malignant progression.     

Abnormal deposition of basement membrane and connective tissue components in dimethylbenzanthracene-induced rat mammary tumours: an immunocytochemical and ultrastructural study

Summary Dimethylbenzanthracene-induced rat mammary tumours consist of lobules of tumours cells surrounded by connective tissue. The interstitial connective tissue proteins, collagen types I, III and V, fibronectin and elastin are largely restricted to the interlobular connective tissue. The tumour lobules are surrounded by a basement membrane that stains with antiserum to laminin. Electron microscopy reveals a greatly thickened basement membrane to which striated interstitial collagen fibres are closely juxtaposed. The lumina within the tumour lobules are of two types. In the first type, the luminal surface is characterized by the presence of microvilli and tight junctions are reacts with antiserum to rat milk fat globule membrane. In the second type, the luminal surface is flattened and lined by a thickened basement membrane that stains with antiserum to laminin and type IV collagen. These abnormal patterns of growth and differentiation may be partly a consequence of the disorganization of extracellular matrix components at the interface between the tumour epithelial cells and the surrounding stroma.     

Coexistence of different tissue tumourigenesis in an N-methyl-N-nitrosourea-induced mammary carcinoma model: a histopathological report in Sprague-Dawley rats

N-methyl-N-nitrosourea (MNU), a highly potent carginogen, is widely used to generate mammary tumours in murine species. In a model of MNU-induced mammary carcinogenesis using immature female Sprague-Dawley rats, large mammary tumours (largest dimension > or =0.5 cm) were obtained within a very short period of time. In addition, in the rats bearing MNU-induced mammary carcinomas, there were a number of tumours whose origins were not from mammary tissue but from several different tissues and from mammary non-epithelial tissue. The tumours were of mesenchymal or epithelial origin and they were located in the inguinal region. These tumours were diagnosed as fibroadenoma, combined tubular adenoma and fibroadenoma, hyperkeratotic papilloma, keratinous cyst and malignant peripheral nerve sheath tumour (MPNST) with smooth muscle differentiation. The occurrence of these other tumours in addition to the development of the mammary carcinomas may be attributed to a direct local effect of the intraperitoneal administration of MNU during the sexual development of the immature rats. In the MNU-induced mammary tumour model, coexistence of tumourigenesis in various non-mammary tissues should be considered an important factor that may interfere with experimental procedures and results and also the quality of life of the tumour-bearing animals.     

Morphological and functional differentiation in epithelial cultures obtained from human skin explants

The present study was undertaken to characterize primary epithelial cultures obtained from human skin explants as experimental systems for studies of the differentiation process. When human skin explants were incubated at 34-35 degrees C, fibroblastic growth was strongly inhibited, whereas the epithelial growth proceeded unchanged. The lateral growth of the epithelial cells could be divided into two phases - a migratory and a proliferative one. Only cultures incubated at 35 degrees C or below completed the morphological differentiation process before sloughing, whereas no qualitative difference in protein synthesis was observed between cultures incubated at temperatures from 33-37 degrees C. Cultured epidermal cells were labelled with 3H-thymidine and analysed by flow cytometry and cell sorting. Cells sorted from the S- and G2-phase populations were further analysed by autoradiography and a considerable heterogeneity as to the nuclear labelling was disclosed. A large fraction of S-phase cells were found to be totally unlabelled. The grain count distributions revealed similar cell cycle subpopulations as have been shown to occur in vivo. The relationship of these subpopulations to the differentiation process is discussed.     

Regulation of connective tissue collagenase production: stimulators from adult and fetal epidermal cells

We have examined the ability of primary adult rabbit skin cells to regulate collagenase production in vitro. Dermal cells constitutively produce collagenase in culture, and enzyme production by these cells can be influenced by epithelial cells. Co-culture with skin epidermal cells resulted in more enzyme production by dermal cells, whereas co- culture with corneal epithelial cells yielded less enzyme activity. Connective tissue cells from a different source, cornea, also produced collagenase when co-cultured with skin epidermal cells, although the stromal cells alone made no enzyme. The drug cytochalasin B had very little influence on collagenase production by dermal cells, either alone or in co-culture with epidermal cells, but did significantly potentiate enzyme production by corneal stromal cells responding to epidermal effector molecules. Epidermal-cell-conditioned medium from both fetal and adult rabbit skin was a potent source of stimulators (apparent mol wt 20,500 and 55,000) of connective-tissue-cell collagenase production. Stimulator production by epidermal cultures was cell density dependent. Optimal production of stimulators occurred in adult cultures containing 10(6) epidermal cells/ml of medium, and in fetal cultures containing 10(5) cells/ml. Inhibitors of connective tissue cell enzyme production were not detected in conditioned medium from either adult or fetal epidermal cells.     

Growth of sebaceous cells in monolayer culture

Summary Rat preputial cells were grown in an epithelial cell primary monolayer culture system identical to that used for culturing epidermal cells, which were studied for comparison. Despite similar appearance when observed by phase contrast microscopy, other features identified the preputial cells as a unique epithelial cell population. Preputial cells grew as a relatively small number of large colonies, formed domes before confluence, and expressed a specific acinar keratin, K4, which had previously been found in human sebaceous glands. In addition, preputial cells formed fewer cornified envelopes than epidermal cells, too few to discern the reduction of envelope formation by retinoic acid treatment in vitro which was found in epidermal cells. Rat preputial cells in monolayer culture, therefore, are a promising model for studying the effects of hormones on sebaceous cell growth and differentiation.     

In vitro differentiation of chicken embryo skin cells transformed by Rous sarcoma virus

The epidermal cells isolated from 14-day chicken embryo shank skin epidermis were infected in vitro with Rous sarcoma virus (RSV). Within a few weeks, rapidly growing colonies of epithelial cells appeared among the sea of transformed fibroblastic cells. When isolated and subcultured, these cells were found to possess typical markers of skin epidermis. The presence of major keratin and typical epithelial cell type morphology strongly suggested that these cells were transformed epidermal cells retaining their differentiated characteristics but having the capacity to propagate in cell culture. If RSV tsNY68, an RSV mutant having a temperature lesion in the src gene, was used, similar transformed epidermal cells were obtained at 36 degrees C (permissive temperature). At the nonpermissive temperature (41 degrees C) the growth rate of these cells decreased and additional keratin species appeared. At 41 degrees C the cells were flattened and lost the refractivity in their peripheries. All the keratins which are synthesized at the nonpermissive temperature were present in normal differentiated shank skin of 19-day old chick embryo. These cells also had "cornified envelop," indicating extensive differentiation. Viral production was as efficient as transformed fibroblasts during the rapid growth phase, while it declined significantly after the cells reached confluency, exhibiting the differentiated characteristics. Since no normal epidermal cells could be cultured under our experimental conditions, these results represent examples in which the src gene is essential for propagation of differentiated cells in cell culture while it abolishes only a part of differentiated characteristics.     

Modulation of the epidermal growth factor receptor by the human papillomavirus type 16 E5 protein in raft cultures of human keratinocytes

It has been shown that the E5 protein of the human papillomavirus type 16 modulates epidermal growth factor receptor downregulation in monolayer cultures of human keratinocytes and mouse fibroblasts. We have now analysed the effect of this protein on the expression, the distribution and the activation of EGF receptors in raft cultures derived from an E5-transfected human keratinocyte cell line. The epithelia generated in these cultures were stratified and exhibited suprabasal expression of cytokeratins 1 and 10, which are known markers of early epidermal differentiation. In situ hybridization with an antisense riboprobe to the human papilloma virus type 16 E5 protein revealed a homogeneous gene expression within the entire epithelium of E5-transfected but not empty vector-transfected control cultures. Treatment of serum-starved rafts with EGF for 48 hours led to a strong decrease of suprabasal EGF receptors in control cultures, but not in rafts of E5-expressing cells. Under these conditions, no activated receptors were observed in control cultures, but activated receptors were still present in E5-raft cultures. Our results indicate that human papilloma virus type 16 E5-mediated modulation of EGF receptor expression occurs in a time- and structure-dependent manner in epithelial equivalents of human keratinocytes.     

Programmed cell death and heterolysis of larval epithelial cells by macrophage-like cells in the anuran small intestine in vivo and in vitro.

The degenerative processes in the larval small intestine of Xenopus laevis tadpoles during spontaneous metamorphosis and during thyroid hormone-induced metamorphosis in vitro were examined by electron microscopy. Around the beginning of spontaneous metamorphic climax (stages 59-61), both apoptotic bodies derived from larval epithelial cells and intraepithelial macrophage-like cells suddenly increase in number. The macrophage-like cells become rounded and enlarged because of numerous vacuoles containing the apoptotic bodies. Mitotic profiles of the macrophage-like cells, however, are localized in the connective tissue where different developmental stages of macrophage-like cells are present. After stage 62, the intraepithelial macrophage-like cells decrease in number, while large macrophage-like cells which include the apoptotic bodies and retain intact cell membranes and nuclei appear in the lumen. Degenerative changes similar to those during spontaneous metamorphosis described above could be reproduced in vitro. In tissue fragments isolated from the small intestine of stage 57 tadpoles and cultured in the presence of thyroid hormone, the number of intraepithelial macrophage-like cells reaches its maximum around the 3rd day of cultivation when the larval epithelial cells most rapidly decrease in number. These results suggest that the rapid degeneration of larval epithelial cells occurs not only because of apoptosis of the epithelial cells themselves but also from heterolysis by macrophages. The macrophages probably originate in the connective tissue, actively proliferate, migrate into the larval epithelium around the beginning of metamorphic climax, and are finally extruded into the lumen.     

Monoclonal antibodies against group A streptococcal polysaccharide reacting with basal-cell antigen of tumors histogenetically related to epidermal tissues

Immunofluorescence of tumour tissues histogenetically related to tissues containing cross-reacting squamous epithelial basal-cell antigen (BCAg) was performed using monoclonal antibodies (McAb) A6/I-D to squamous epithelial basal-cell antigen cross-reacting with group A staphylococcal polysaccharide. BCAg was found in tumour cells arising from surface tissues of the epidermal type (basal-cell cancer, squamous-cell cancer of the skin, esophagus, cervix uteri and other organs) and was not found in glandular tumours (adenocarcinoma of the stomach, intestine, mammary glands). The results obtained indicate that McAb A6/I-D against cross-reacting BCAg may be used while characterizing the majority of normal epidermal tissues and tumours arising from tissues containing cross-reacting BCAg.     

Synthesis and evaluation of new 5-fluorouracil antitumor cell differentiating derivatives

Three new antitumour drugs containing two 5-fluorouracil moieties at both ends of the structure and a two amide bond linker were synthesized. Appropriated bis-acetal were reacted with two equivalents of 5-FU to afford the desired compounds. These drugs were evaluated for their ability to induce myogenic maturation in vitro on human rhabdomyosarcoma cells in an experimental model. Compounds 5 and 6 induced morphological and phenotypical differentiation in rhabdomyosarcoma cells at 4.5 and 3.5 microM, respectively. These new cell differentiating agents could be used as an alternative to selective destruction of undifferentiated cells. A potential role of the differentiation therapy as an alternative approach to the treatment of rhabdomyosarcomas is suggested.