Expression of Epithelial Antigens Exo-1 and EPM-1 in Human Epidermal Keratinocyte Maturation and Benign and Malignant Neoplasia1

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[CANCER RESEARCH 50. 7668-7676. December I, 19901 Expression of Epithelial Antigens Exo-1 and EPM-1 in Human Epidermal Keratinocyte Maturation and Benign and Malignant Neoplasia1

Reinhard Klingel,2 Petra Boukamp, Roland Moll, Wolfgang Tilgen, Norbert E. Fusenig, Karl-Hermann Meyer zum BÃ¼schenfelde, and Wolfgang G. Dippold First Department of Internal Medicine, University of Mainz, D-6500 Main: [R. K., K-H. M. :. B., W. tÃ.I).I: Division of Differentiatinn and Carcinogenesis in Vitro, Institute of Biochemistry, Deutsches Krebsforschungs:entrum, D-6900 Heidelberg Â¡P.B., N. E. F.J; Department of Pathology, University of Main:, D-6500 Mainz [R. M.I; and Department of Dermatology, University of Heidelberg, D-6900 Heidelberg Â¡W.T.], Federal Republic ofdermany

ABSTRACT lated but still tissue restricted (5-7). In human skin, proliferation and differentiation of keratino Exo-1, a polar neutral glycolipid, and EPM-1, a high molecular weight cytes and the development of benign and malignant neoplasia glycoprotein, are developmental antigens of human epithelial cells, ini are reflected by characteristic changes in tissue architecture. In tially described as components both on the cell surface and in secretions of gastrointestinal epithelia and respective tumors. In order to assess the turn, modifications in cell shape can act as a signal for terminal biological significance of both antigens for epithelial cell differentiation differentiation and inhibition of proliferation (8). Maturation and neoplastic transformation, their expression during human skin de and differentiation in human epidermis can be evaluated by velopment and benign and malignant neoplasia was analyzed in fresh determination of synthesis and modification of structural pro frozen tissue specimens of skin biopsies and of human epidermal keratin- teins and of alterations in the expression of cell surface antigens, ocytes growing in experimental model systems. Antigen expression was receptors, and epidermal lipids. Differentiation-related struc assessed immunohistochemically with specific monoclonal antibodies. tural proteins that are best defined include the keratins (9-12), During fetal development Exo-1 was temporarily expressed in interme filaggrin (13), and the cornified envelope proteins, e.g., involu- diate cells but was absent in normal adult human skin. Exo-1 expression crin (14), which are expressed and modified in a specific se reemerged in neoplasias, both benign and malignant, but was restricted to spinous-like differentiated cells. Similarly, Exo-1 was not expressed quence which closely parallels the morphological changes in in transplants of normal keratinocytes mimicking the normal epidermis human epidermis (11, 12, 15, 16). Similarly, changes in cell but was clearly visible in differentiated areas of transplants of malignantly surface carbohydrates occur during maturation of the epidermis transformed keratinocytes. EPM-1 appeared first in basal epidermal and can, for example, be visualized by different blood group cells in the second half of gestation and remained detectable in the antigens, which allow the distinction of different epidermal cell stratum basale of adult skin. While squamous cell carcinomas continued compartments (17-21). We recently identified and character to express EPM-1, it was not detectable in basal cell epitheliomas and ized two new antigens of normal and neoplastic human epithelia in normal epidermis after invasion by neuroectodermal tumor cells. In by monoclonal antibodies. These antigens were primarily ob experimental models, EPM-1 was present in the basal layers of normal served in normal and neoplastic gastrointestinal epithelia. Exo- human keratinocytes and of transformed keratinocytes with benign 1, a polar neutral glycolipid (22), and EPM-1, a high molecular growth characteristics whenever a well stratified and keratinized epider mis-like epithelium had formed in transplants. In transformed keratino weight glycoprotein (23), were present both on cell surfaces and cytes with malignant growth behavior, EPM-1 was expressed irregularly, in secretions. Other recently described differentiation-, devel as in squamous cell carcinomas in xita. Thus, expression of Exo-1 is a opment-, or tumor-associated markers of human epithelia, in marker for an early embryonic differentiation pathway of human keratin cluding the epithelial membrane antigen EMA, the high molec ocytes and in adult tissue reveals abnormal differentiation associated ular weight glycoproteins CA 19-9 and DU-PAN-2, and the H, with certain stages of hyperproliferation. EPM-1 expression is part of Le X, and Le Y blood group antigens, are different from Exo- developmental programs and is influenced by microenvironmental inter 1 and EPM-1 (18, 19, 24-31). The receptors for transferrin and actions and alterations of tissue homeostasis. epidermal growth factor, both detectable in epidermal basal cells, do not show characteristic changes during differentiation INTRODUCTION and transformation events in human skin (32, 33). In this paper, we report on the expression of Exo-1 and Glycolipids and carbohydrate structures of cell membrane EPM-1 in different stages of development and carcinogenesis glycoproteins undergo significant changes in their expression of human skin keratinocytes. Our particular interest was to during embryonic and fetal development and malignant trans analyze whether Exo-1 and EPM-1 expression patterns would formation of human cells (1-3). Glycosylation plays an impor indicate membrane changes associated with fetal maturation tant role in cytokine-mediated processes of normal tissue re and proliferation, as well as differentiation in the adult tissue. modeling and the maintenance of tissue homeostasis (4). The Disease entities of human skin as well as benign and malignant phenotype of cancer cells is a result of many genetic and tumors were studied to evaluate the influence of alterations in epigenetic changes, as well as altered microenvironmental in growth and differentiation occurring during hyperproliferation teractions. When cells are at ectopie sites, as is the case for and different stages of neoplastic transformation. In addition, metastatic tumors or tissue culture models, their phenotype normal keratinocytes and cell lines were analyzed in culture reflects the expression of genetic programs which are deregu- and transplantation models. These systems mimic in a tissue- specific way the in vivo situation of different phases of epithelial Received 4/20/90; accepted 7/13/90. regeneration and early tumor development. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. MATERIALS AND METHODS 1This work was supported by grants from the Bundesministerium fÃ¼rFor schung und Technologie (DI 01 GA 054/6) and Deutsche Forschungsgemein Tissue Samples. Tissue samples of normal skin, various neoplastic schaft (Kl 578/2-1). This work was also partly supported by a Fellowship from lesions, and experimental models were snap-frozen and stored in liquid the DKFZ (Stiftung SondervermÃ¶gen) to P. B. 2To whom requests for reprints should be addressed, at First Department of nitrogen. Cryostat sections (4-6 Â¿im)wereobtained according to stand Internal Medicine, University of Mainz. Langenbeckstr. I. D-6500 Mainz, FRG. ard procedures. 7668

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1990 American Association for Cancer Research. EPITHELIAL ANTIGENS IN KERATINOCYTE MATURATION AND NEOPLASIA Table 1 Exo-l and EPM-I expression in normal adult skin, fetal skin, and skin Monoclonal Antibodies. Mouse monoclonal antibody Pa-25 (IgM) appendages recognizes the high molecular weight glycoprotein EPM-1, and mono Reactivity for clonal antibody Pa-G-14 (IgM) the polar neutral glycolipid Exo-1 (22, Number 23). Both monoclonal antibodies were raised against the human pan Tissue tested Exo-l EPM-I creatic cancer cell line Capan-1, and pools of (5x) concentrated hybrid- Adult skin 25 oma culture supernatant (immunoglobulin concentration, 200 /Â¿g/ml) Basal cell layer + were used throughout the whole study. Spinous cell layer Immunocytochemical Staining. Fresh frozen, nonfixed tissue sections Skin appendages (4-6 Â¿im)werestained with Pa-25 and Pa-G-14 by an indirect immu- Hair follicle noperoxidase method, essentially as described previously (34). The Outer root sheath + immunochemical reaction was developed with the red dye 3-amino-9- Inner root sheath + ethyl-carbazole and counterstained with blue Meyer's Hemalum solu Sebaceous gland Peripheral germinative cells â€” + tion (Merck, Darmstadt, FRG).3 A monoclonal antibody to the mouse Centrally located vacuolated cells lymphocyte antigen Lyt.l.l (IgM) served as a negative control and the Eccrine sweat gland + + anti-HLA-ABC monoclonal antibody W6/32 as a positive control for Fetal skin 17 the immunostaining (35). Additionally, the indirect immunofluores- Periderm + cence method was used as described previously (36). As second anti Basalcells body, we used fluorescein isothiocyanate-labeled anti-mouse 7-globulin Before 17-week EGA 17-24-week EGA +/-" (Fab fragment; Dianova). After 24-week EGA + Cell Lines. The spontaneously immortalized human skin keratino- Intermediate cells/upper suprabasal layers cyte cell line designated HaCaT, described in detail previously (36), Before 14/15-week EGA exhibited a transformed phenotype in vitro but was nontumorigenic in 14/15-24-week EGA +/- After 24-week EGA - -_ vivo and had maintained a high degree of epidermal differentiation properties. Transfection of the HaCaT cells with the cellular Harvey- " +/â€”,presence of both positive and negative cells. mi oncogene gave rise to tumorigenic clones. After s.c. injection into nude mice, these clones either formed benign cystic tumors or formed invasively growing squamous cell carcinomas (37). Human skin kera- (Table 1). One-layered epidermis, i.e., up to 4-week EGA, was tinocytes transfected with origin-defective SV-40 DNA gave rise to an not available. In the early stratified epidermis (from 14- up to immortalized cell line designated HaSV (38). These initially nontu 24-week EGA), Exo-l was expressed in the intermediate cells morigenic HaSV cells progressed to tumorigenicity with passaging and (Fig. \A). With further stratification, the expression of Exo-l formed differentiating squamous cell carcinomas (beyond passage 70) after s.c. injection into nude mice. became restricted to the uppermost cell layers and after week Experimental in Vivo Model Systems. The procedures to establish 32 the antigen could no longer be detected. The epidermis of and cultivate normal human keratinocytes from different body sites in adult skin was completely negative for Exo-l, except for cells vitro, originally developed by Eisinger et al. (39), are described in detail of the inner root sheath of the hair follicle (not shown). There elsewhere.4 For evaluation of in vivo growth, normal and transformed was no staining in the sebaceous gland. human keratinocytes were transplanted onto and s.c. injected into nude EPM-1 showed a completely different distribution. The an mice, respectively. All cells were grown on plastic dishes (Falcon) in tigen was first detected in the periderm of the two-layered modified minimum essential medium (40) containing 10% fetal calf serum, at 37Â°Cin a humidified atmosphere of 5% CO2 in air. For epidermis of a 9-week-old fetus and persisted there until the periderm was lost between weeks 20 and 24 (Fig. IB). This passaging, cells were detached by preincubating the cultures with 0.2% EDTA for approximately 10 min, followed by incubation in a 0.1% result confirmed the similarity of this primary lining epithelium EDTA/trypsin solution (final concentration), and were disaggregated of the early embryo with simple epithelia of mucous membranes by pipetting. (15, 23). With 18-week EGA, the cells of the basal layer became In transplantation assays, IO5 cells were seeded onto a collagen EPM-1 positive, and this coincided with the development of substratum (forming organotypical cultures), as described in detail rete ridges. Up to week 24, the expression was confined to the elsewhere (41, 42). Medium was drained after 24 h and the tissue basal cells on top of the dermal papillae, while those delineating culture chambers were covered with a (silicon) transplantation chamber the deeper rete ridges remained negative or showed only weak (Renner, Dannstadt, FRG). The whole unit was then transplanted onto staining (Fig. 1C). In specimens from 36-week-old fetuses, the the dorsal muscle fascia of nude mice, as described (42). The transplants were dissected en bloc and frozen in liquid nitrogen-cooled isopentane. epidermis showed a pattern similar to that in adult skin, i.e., Exo-l was absent and EPM-1 was present in the cytoplasm of Tumor formation by human keratinocyte cell lines was assessed after s.c. injections of up to 5 x IO6cells, in 100-^1 culture medium, into the all basal cells. interscapular region of 4-6-week-old nude mice (BALB/c nu/nu back- The pattern of adult skin was observed irrespective of the crosses) and was monitored for an observation period of up to 6 months. topology (arm, leg, abdomen, or sole) (Fig. 2). Moreover, the Tumors were excised, frozen, and processed as described above. positive staining of basal cells for EPM-1 continued in the skin appendages in the basal layers of the outer root sheath of the RESULTS hair follicle and in the peripheral germinative cells of the sebaceous gland (not shown). Luminal and apical cytoplasmic Exo-l/EPM-1 Expression in Fetal and Normal Adult Human staining for EPM-1 and Exo-l was seen in the eccrine sweat Skin. Fetal human skin specimens of the sole of the foot of different gestational ages (9- to 36-week EGA5) were tested gland, in accordance with earlier observations in other secretory epithelial cells such as salivary gland, breast, and pancreas (22, 23). 1Red positive reactions and the blue counterstain gave similar contrast in the Exo-l/EPM-1 Expression in Epidermal Hyperplasia and Be noncolored figures, which is explained in the legends. 4 P. Boukamp, D. Breitkreutz, H-J. Stark, and N. E. Fusenig. Mesenchyme- nign Neoplasia. Tissue specimens of four different disease en mediated and endogenous regulation of growth and differentiation of human skin tities were analyzed concerning their Exo-l/EPM-1 expression keratinocytes derived from different body sites. Submitted for publication. 5The abbreviations used are: EGA, estimated gestational age; SCC, squamous and each revealed an individual antigen pattern. The tissue cell carcinoma. samples were derived from basal cell papillomas, verruca vul- 7669

Fig. 2. Regularly stratified adult human skin. I. no staining for Exo-1. /<. EPM-1-positive basal cell layer; dark cells in the granular layers are caused by the blue counterstain.

Table 2 Exo-1 and EPM-1 expression in hyperprolifertive skin lesions and benign neoplasms of the skin Reactivity for Number Tissue tested Exo-1 EPM-1 Hyperproliferative and neoplastie keratinocytes Basal cell papilloma Basaloid cells Cells delineating horn pearls

Verruca vulgaris Fig. 1. Fetal human skin, f Exo-1-positive intermediate cells at 18-week Basal cell layer EGA; B. EPM-1-positive periderm at 13-week EGA; C, EPM-1-positive basal Spinous cell layer cells restricted to the top of the dermal papillae at the end of 22-week EGA. Psoriasis Basal cell layer garis, psoriasis, and reactive acanthosis (Table 2). Spinous cell layer Basal cell papillomas are characterized by branches of prolif Reactive acanthosis Basal cell layer erating benign basaloid cells with horn pearl formation. Typical Spinous cell layer suprabasal strata are missing. These papillomas expressed EPM-1 in all basal cells. Weak Exo-1 staining was present in Hyperproliferative nonepilhelial cells located intracpidermally spinous-like cells directly surrounding horn pearls. Nevus cell nevus 15 Verruca vulgaris, a virally induced epidermal papillomatosis, " (+), weak but significant staining. is characterized by hyperplasia and hyperkeratosis. These tis sues expressed both antigens. Exo-1 was clearly restricted to liferation with hyperplasia and disturbed differentiation (43). the suprabasal cells as a membrane-accentuated staining (Fig. In skin specimens of this disease, both antibodies reacted pos 3A). EPM-1, in addition to the basal layer, was also present in itively but differently when compared to verruca vulgaris. While the lower suprabasal layers. EPM-1 was restricted to the basal layer, Exo-1 was, in addition Psoriasis is described as a parakeratotic epidermal hyperpro- to the spinous layers, also present in the basal cells (Fig. 3B). 7670

Fig. 3. Exo-1 staining in the spinocellular layer of a verruca vulgaris, with negative basal cells l.li. and in the basal and spinocellular layer of psoriatic epidermis (B).

In reactive acanthosis, i.e., in areas of epidermis with ex panded spinous layers surrounding or adjacent to nevus cell nevi or malignant melanomas, the fourth characteristic Exo-1/ EPM-1 expression pattern was found. Exo-1 was present exclu sively in the hyperproliferative spinous cell compartment. Basal cells underneath such Exo-1-positive areas of reactive acan thosis did not express EPM-1, in contrast to basal layers of nromal epidermis, verruca vulgaris, and psoriasis. Exo-l/EPM-1 Expression in Malignant Skin Tumors. Epider mal malignancies were analyzed in biopsies from basal cell epitheliomas, tumors composed of basaloid malignant cells; M. Bowen, i.e., carcinoma in situ of the skin located intraepidermally above an intact basement membrane but lacking a regular stratification; and SCCs (Table 3). In contrast to normal skin and the cells of basal cell papi Ilomas, EPM-1 was not detectable in the basal cell epithelioma. In the surrounding epidermis, the normal distribution of EPM-1 in basal cells was maintained (Fig. 4A). A clear demarcation was made between EPM-1 - positive normal basal cells and EPM-1-negative malignant cells forming nodular clusters or lacelike strands. Exo-1 could not be detected in these tumors. In precancerous and malignant lesions of the squamous cell lineage, such as M. Bowen and SCCs, Exo-1 and EPM-1 were generally coexpressed. Compared to the benign hyperplastic diseases, the distribution was less organized. Approximately 50% of the tumor cells were Exo-1 positive; the staining was

Table 3 Exo-1 and EPM-1 expression in malignant lesions ana tumors of the skin forExo-1 TissueMalignant tested10 EPM-1+ keratinocytes Fig. 4. A, nest of EPM-1-negative tumor cells of a basal cell epithelioma, with Basal cell epithelioma EPM-1-positive adjacent basal cell layers. B, Exo-1 expression in a keratinizing Precancerous lesions (M. Bowen) 4 + area of a moderately differentiated SCC. Squamous cell carcinomaNumber 6Reactivity

Nonepithelial malignant tumor cells predominantly on the cell membrane but was also observed in located intraepidermally the cytoplasm (not shown). In certain areas of the M. Bowen Malignant melanoma 15 specimens where the stratification was not completely irregular, 7671

Exo-1 was confined to lower cell layers. EPM-1 was detected into nude mice) were still able to form structured epithelia in in 30-50% of the cells, in single cells or clusters of cells in all transplants (37). In early stages (1 to 2 weeks after transplan layers of the precancerous lesions. The staining intensity was tation), when the multilayered epithelium was less organized, often stronger in basally located cells. Exo-1 was expressed suprabasally (Fig. 5B) while EPM-1 was In squamous cell carcinomas, the ratio between Exo-1- and negative. At later stages (3-4 weeks after transplantation), when EPM-1-positive cells largely depended on the degree of mor the epithelium showed all characteristic strata (Fig. 5C), Exo- phological differentiation, i.e., keratinization and horn pearl 1 could no longer be detected, but EPM-1 was expressed (Fig. formation. In well differentiated SCC, where keratinization of 5D). The distribution of EPM-1 discriminated between trans individual cells and formation of keratin pearls are common, plants of benign and malignant HaCaT-ras cells. The benign the spinous-like cells expressed Exo-1 mainly localized at the cells, which were more controlled in their growth behavior, cell membrane (Fig. 4B). EPM-1 was present in about 50% of stayed as superficial epithelia (Fig. 5C). Here EPM-1 was the tumor cells, in the cytoplasm. In less differentiated SCCs, restricted to the basal cells, similar to normal keratinocytes the number of Exo-1-positive cells decreased to 20-30%, with (Fig. 5D). In the malignant cells infiltrating the mouse mesen- cytoplasmic staining, while the percentage of EPM-1-positive chyme (Fig. 5E), in addition to the positive basal cells a patch- tumor cells increased to 80%. wise suprabasal staining of EPM-1 could be seen, as well as Exo-l/EPM-1 Expression in Nonepithelial Skin Tumors. Be clusters of positive cells within the tumor tissue (Fig. 5F). nign nevus cells and tumor cells of malignant melanomas (both When the HaCaT-ros clones were injected s.c., tumors were of neuroectodermal origin) expressed neither Exo-1 nor EPM- formed which were either large epidermal cysts (benign tumors) 1 (Tables 2 and 3). In areas where these nonepithelial tumor or highly differentiated squamous cell carcinomas (37). Similar cells had built up neoplastic structures in the epidermis, leading to normal skin, the lining epithelium of the benign cysts still to reactive acanthosis, basal keratinocytes became negative for showed an ordered stratification and cornification, leading to EPM-1, while spinous cells still expressed Exo-1 (see above). an epidermis-like epithelium. The basal layer of the epithelium Exo-l/EPM-1 Expression in Tissues Derived from Normal was positive for EPM-1 (Fig. 6A) and Exo-1 was not expressed. and Transformed Keratinocytes in Experimental Systems. In In the sqamous cell carcinomas, both antigens were present, addition to normal human skin and benign and malignant comparable to tumor biopsies as described above (Fig. 6B). The neoplastic skin disease entities, we tested normal and in vitro same was found for moderately to well differentiated sqamous transformed human skin keratinocytes after transplantation cell carcinomas obtained after s.c. injection of tumorigenic and s.c. injection, respectively (Table 4). Normal human kera HaSV cells, which were immortalized with SV-40 DNA and tinocytes, when transplanted onto the back of nude mice as spontaneously transformed (38). intact cultures (i.e., grown on a collagen matrix), form a well stratified and differentiated epidermis within 1 week.4 When DISCUSSION these transplants were tested for the two antigens, we found a pattern identical to that of normal skin; EPM-1 was expressed Exo-1, a polar neutral glycolipid, and EPM-1, a high molec in the basal cells while Exo-1 was absent (not shown). At the ular weight glycoprotein, represent two antigens of human edges of the transplants where stratification and differentiation epithelial cells initially detected on the cell surface and in was less pronounced, EPM-1 was not detected. This suggests secretions of gastrointestinal epithelial tissues and tumors (22, that a strict correlation exists between EPM-1 antigen expres 23). In order to obtain further insight into the biological signif sion and regular tissue architecture. icance of these epithelial cell markers, human keratinocytes Spontaneously immortalized but nontumorigenic HaCaT were studied. The epidermis was selected, because different cells, functionally similar to normal keratinocytes, form a well stages such as development, differentiation, and transformation organized epidermis-like epithelium following transplantation are well characterized morphologically and by the expression (36). Within 1 week, the basal layer of the epithelium was of structural proteins. The expression of Exo-1 was found to be positive for EPM-1 (Fig. 5A), while Exo-1 could not be detected. related to an early embryonic differentiation pathway, which The Harvey-ras oncogene-transfected HaCaT-ras clones (which can reemerge abnormally in the adult, associated with hyper- formed benign and malignant tumors following s.c. injection proliferation. EPM-1 expression is part of developmental pro-

Table 4 Expression of Exo-1 and EPM-1 in experimental model systems Reactivity for Cells Growth condition Exo-1 EPM-1 Normal keratinocytes Transplant (1 week)

Immortalized nontumorigenic cells HaCaT Transplant (1 week)

Tumorigenic cells with benign growth behavior HaCaT-ras (benign clone) Transplant (1-2 weeks) Transplant (3-4 weeks) s.c. injection/cyst

Tumorigenic cells with malignant growth capacity HaCaT-rai (malignant clone) Transplant (1-2 weeks) Transplant (3-4 weeks) s.c. injection/solid tumor HaSV s.c. injection/solid tumor Â°+/â€”,presence of both positive and negative cells. '' In addition to basal, also suprabasal staining. 7672

Fig. 5. Exo-1 and EPM-I expression in epithelium of the experimental cell systems transplanted onto the backs of nude mice. I. EPM-1 -positive basal cells in well stratified epithelium 1 week after transplantation of immortalized nontumorigenic HaCaT cells. Immunofluorescence. B, Exo-l expression in the upper and sometimes intermediate cell layers of a hyperplastic but still poorly differentiated epithelium 2 weeks after transplantation of a benign HaCaT-nu clone. Immunofluorescence. ( . well differentiated epithelium of a benign HaCaT-ras clone 4 weeks after transplantation. H & E staining. /'. same epithelium with positive EPM-I immunofluorescence in the basal layer. Â£,well differentiated epithelium of a malignant HaCaT-ras clone invading the host mesenchyme 4 weeks after transplantation. H & E staining. /'. same epithelium stained with monoclonal antibody against EPM-I. 7673

Fig. 6. EPM-I expression in tissue of lu- morigenic HaCaT-ra.v clones injected s.c. A, positive cells in the basal compartment of a cystic tumor of a benign HaOaT-ras clone; dark cells in the granular layer are caused by the blue countcrstain. B, positive tumor cells of a M < hiÂ«'growing malignant Ha('aT-nu clone.

grams and indicated a steady state of proliferation and differ injection but are undistinguishable from nontumorigenic cells entiation in the completely developed epidermis. These conclu in our short term transplantation assay. In malignant squamous sions were supported by results obtained from biopsies as well cell tumors either derived from patients or induced experimen as tissues developed in experimental model systems. tally (after s.c. injection of malignant HaCaT-ros or HaSV Exo-1 was expressed suprabasally in the fetus (weeks 15 to cells), the expression of Exo-1 largely correlated with the pres 24) when the epidermis developed from a one- to a multilayered ence of differentiated structures. Keratinizing areas of the tested tissue. Exo-1 could not be detected in the mature epidermis, SCCs showed typical Exo-1 staining. In the tested cases of less where proliferation is reduced to a low level. The horny layer well differentiated tumors, the percentage of positive cells de and the sebaceous gland were negative; thus, Exo-1 should not creased. be considered one of the skin lipids involved in the epidermal The regulation of EPM-1 expression was substantially differ barrier function (44, 45). ent and independent of Exo-1. The complete basal layer of Hyperproliferation in the adult is found in the disease entities adult human epidermis in a state of tissue homeostasis is I I'M of psoriasis, verruca vulgaris, and reactive acanthosis of skin, 1 positive. No difference was detected between the two types of for example adjacent to nonepithelial tumors. Under such human adult basal cells: the nonserrated basal cells confined to conditions, where hyperproliferation is characterized by the the tips of the deep rete ridges and the serrated cells along the presence of keratins K6 and K.16 (12), Exo-1 was detectable thinner, more flattened portions of the epidermis (48, 49). and accentuated at cell membranes. While in verruca vulgaris EPM-1 expression in basal cells started at about 17-week EGA, and reactive acanthosis the typical Exo-1 staining pattern was when rete ridges were formed. At this stage it was confined to' present only in suprabasal spinous layers, in psoriasis Exo-1 the basal cells on top of the dermal papillae, while those of the started to occur already in basal cells. This observation confirms developing rete ridges remained negative. When the process of that psoriatic keratinocytes follow a different maturation path ridge formation was complete, all basal cells were EPM-1 way with respect to kinetics and sequence of the differentiation positive. Therefore, EPM-1 is not a marker for proliferation process (43, 46, 47). itself but is related to the proliferative compartment at the Other evidence for a positive correlation between hyperpro steady state level between differentiation and proliferation, i.e., liferation and expression of Exo-1 was drawn from the reformed tissue homeostasis. Such a pattern is unique and so far not seen epidermal structures obtained in our experimental models. Exo- with other developmental markers such as keratins or the H, 1 was present suprabasally when tumorigenic HaCaT-ras cells LeX, and LeY blood group antigens (17-19). formed a hyperproliferative epithelium with incomplete differ In the hyperproliferative situations of psoriasis, verruca vul entiation, up to about 2 weeks after transplantation. Exo-1 was garis, and reactive acanthosis, EPM-1 expression indicated no longer detectable after 3 to 4 weeks, when the epidermis differences between their proliferative compartments. A single appeared thinner and well differentiated. Transplants of normal layer of basal cells was EPM-1 positive in psoriasis. This finding as well as immortalized nontumorigenic keratinocytes, which confirms studies by Leigh et al. (50). Morphologically, the form a well differentiated epidermis already after 1 to 2 weeks, proliferative compartment in psoriatic lesions appears to be did not express Exo-1. Therefore, in this special experimental enlarged to three cell layers. Analyzing the stratum basale in condition, the expression of Exo-1 distinguished between non psoriatic lesions by its characteristic composition of keratin tumorigenic and tumorigenic cells. This is of particular impor filaments, only one basal layer could be detected, as in normal tance in the case of cells which form benign tumors after s.c. skin (50). In contrast, the suprabasal expression of EPM-1 in 7674

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1990 American Association for Cancer Research. EPITHELIAL ANTIGENS IN KERATINOCYTE MATURATION AND NEOPLASIA verruca vulgaris implied that functional characteristics of the Feizi, T. Demonstration by monoclonal antibodies that carbohydrate struc tures of glycoproteins and glycolipids are oncodevelopmental antigens. Na basal cell compartment expanded in this lesion. These data ture (Lond.), 314: 53-57, 1985. relating psoriasis and verruca vulgaris indicate that the hyper- 3. Dabelsteen, E., and Clausen, H. Tumor-associated carbohydrate antigens. J. Oral. Pathol., 16: 196-198, 1987. proliferation in these two disease entities does not prevent the 4. Vlassara, H., Brownlee, M., Manogue, K. R., Dinarello, C. A., and Pasagian, establishment of a state of tissue homeostasis. A. Cachectin/TNF and IL-1 induced by glucose-modified proteins: role in Essentially different were the results in the third hyperproli- normal tissue remodeling. Science (Washington DC), 240:1546-1548, 1988. ferative situation of reactive acanthosis; EPM-1 was missing in 5. Marks, P. F., ShefTery, M., and Rifkind, R. A. Induction of transformed cells to terminal differentiation and the modulation of gene expression. Cancer the basal layer. Probably due to the permanent irritating inter Res., 4 7: 659-666, 1987. action with the growth of neuroectodermal cells (nevus cells 6. Nicolson, G. L. Tumor cell instability, diversification, and progression to the metastatic phenotype: from oncogene to oncofetal expression. Cancer Res., and melanoma cells), tissue homeostasis is constantly disturbed. 47: 1473-1487, 1987. Further away from the involved area, EPM-1 was seen again. 7. Sutherland, R. M. Cell and environment interactions in tumor microregions: the multiceli spheroid model. Science (Washington DC), 240:177-184, 1988. A recent report by Stoler et al. (51) describes as a similar 8. Walt, F. M., Jordan, P. W., and O'Neill, C. H. Cell shape controls terminal phenomenon the unusual keratin pattern in epidermis overlying differentiation of human epidermal keratinocytes. Proc. Nati. Acad. Sci. dermofibromas. In addition to the expression of K6 and K16 USA. 85: 5576-5580, 1988. 9. Lane, E. I!, Bartek, J., Purkis, P. E., and Leigh, I. M. Keratin antigens in in suprabasal cells, indicating hyperproliferation, the keratin differentiative skin. Ann. NY Acad. Sci., 455: 241-258, 1985. K14 (typical of basal cells) was altered in its expression. 10. Woodcock-Mitchell, J., Eichner, R., Nelson, W. G., and Sun, T-T. Immu- Whether this interaction is due to pure mechanical irritation or nolocalisation of keratin polypeptides in human epidermis using monoclonal antibodies. J. Cell Biol., 95: 580-588, 1982. to tumor-derived factors should be studied. When dealing with 11. Fuchs, E., and Green, H. Changes in keratin gene expression during terminal malignant melanomas, the reappearance of EMP-1 might serve differentiation of the keratinocyte. Cell, 19: 1033-1042, 1980. as an additional marker to determine the area of the epidermis 12. Stoler, A., Kopan, R., Duvic, M., and Fuchs, E. Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during influenced by the tumor growth. normal and abnormal epidermal differentiation. J. Cell Biol., 107: 427-446, The characteristic regulation of EPM-1 was also demon 1988. 13. Dale, B. A., Resing, K. A., and Lonsdale-Eccles, J. D. Filaggrin: a keratin strated in experimental conditions. In transplants of normal as filament associated protein. Ann. NY Acad. Sci., 455: 330-342, 1985. well as immortalized nontumorigenic and tumorigenic cells, 14. Watt, F. M., and Phil, D. Involucrin and other markers of keratinocyte EPM-1 was expressed in the basal layers only when a well terminal differentiation. J. Invest. Dermalol., 81: 100S-103S, 1983. 15. Moll, R., Moll, I., and Wiest, W. Changes in the pattern of cytokeratin differentiated epithelium had formed. In neoplastic basal cells, polypeptides in epidermis and hair follicles during skin development in EPM-1 expression clearly correlated with the benign (basal cell human fetuses. Differentiation, 23: 170-178, 1982. papi 111una; EPM-1 positive) and the malignant phenotype 16. Bowden, P. E., Stark, H. J., Breitkreutz, D., and Fusenig, N. E. Expression and modification of keratins during terminal differentiation of mammalian (basal cell epithelioma; EPM-1 negative). Monoclonal antibod epidermis. Curr. Top. Dev. Biol., 22: 35-68, 1987. ies against cytokeratin determinants or recognizing other basal 17. Dale, B. A., Holbrock, K. A., Kimhall. J. R., Hoff, M., and Sun, T-T. cell antigens, including the epidermal growth factor and trans- Expression of epidermal keratins and filaggrin during human fetal skin development. J. Cell Biol., 101: 1257-1269, 1985. ferrin receptors, do not distinguish between these two types of 18. Fukushi, V.. Hakomori, S-I., and Shepard, T. Localization and alteration of neoplasia (52-60). Disregulated expression of EPM-1 was seen mono-, di- and trifucosyl 1-3 type 2 chain structures during human embry- in squamous cell carcinomas either derived from patients or ogenesis and in human cancer. J. Exp. Med., 159: 506-520, 1984. 19. Dabelsteen, E., Holbrook, K., Clausen, H., and Hakomori, SI. Cell surface induced experimentally. The percentage of antigen-positive carbohydrate changes during embryonic and fetal skin development. J. Invest. cells in the tissue specimens studied correlated inversely with Dermatol., 87: 81-85, 1986. Klein, C. E., Cordon-Cardo, C., Soehnchen, R., Cote, R. J., Oettgen, H. F., the degree of differentiation. 20. Eisinger, M.. and Lloyd, M. Changes in cell surface glycoprotein expression In conclusion, we could demonstrate in human epidermal during differentiation of human keratinocytes. J. Invest. Dermatol., 89: 500- keratinocytes that the epithelial antigens Exo-1 and EPM-1 506, 1987. 21. Dabelsteen, E., Buschard, K., Hakomori, S-I., and Young, W. W. Pattern of represent valuable markers to distinguish normal, hyperplastic, distribution of blood group antigens on human epidermal cells during mat and neoplastic stages in the epidermis. Recently described hu uration. J. Invest. Dermatol., 82: 13-17, 1984. man epithelial and tumor-associated differentiation markers, 22. Dippold, W. G., Klingel, R., Bernhard, H., Dienes, H. P., Knuth, A., and Meyer zum BÃ¼schenfelde,K-H. Secretory epithelial cell marker on gastroin including the epithelial membrane antigen EMA and the high testinal tumors and in human secretions defined by a monoclonal antibody. molecular weight glycoproteins CA 19-9 and DU-PAN-2, to Cancer Res., 47: 2092-2097, 1987. our knowledge do not show comparable disease-specific 23. Dippold, W. G., Bernhard, H., Klingel, R., Dienes, H. P., KrÃ¶n,G., Schnei der, R.. Knuth, A., and Meyer zum BÃ¼schenfelde,K-H. A common epithelial changes in their expression patterns (18, 19, 24-31). Further cell surface antigen (EPM-1) on gastrointestinal tumors and in human sera. studies regarding the expression of Exo-1 and EPM-1 should Cancer Res., 47: 3873-3879, 1987. 24. Gioanni, J., Samson, M., Zanghellini, E., Mazeau, C., Ettore, F., Domarci, include regenerating epidermis from wounded areas, because F., Chauvel, P., Duplay, H., Schneider, M., Laurent, J-C, and Lalanne, C. there hyperproliferation and reorganization of tissue architec M. Characterization of a new surface epitope specific for human epithelial ture simultaneously occur and are accompanied by aberrant cells defined by a monoclonal antibody and application to tumor diagnosis. keratin expression (61-63), as well as epithelial tissues from Cancer Res., 47: 4417-4424, 1987. 25. Hamburger, A. W., Reid, Y. A., Pelle, B., Milo, G. E., Noyes, L, Krakauer, other organs. Exo-1 and EPM-I are comprehensive markers to H., and Fuhrer, J. P. Isolation and characterization of a monoclonal antibody study both directions of epithelial cell differentiation: simple specific for epithelial cells. Cancer Res., 45: 783-790, 1985. 26. Knight, J. C., Davis, P. A., and Gusterson, B. A. A new marker of terminal and stratified epithelia, which are fundamentally different in differentiation in keratinizing epithelia. J. Pathol., 148: 19-27. 1986. terms of keratin expression (9, 64). 27. Momburg, F., Moldenhauer, G., HÃ¤mmerling,G. J., and MÃ¶ller,P. Inumi nohistochemical study of the expression of a M, 34,000 human epithelium- specific surface glycoprotein in normal and malignant tissues. Cancer Res., ACKNOWLEDGMENTS 4 7: 2883-2891, 1987. We would like to especially thank R. KÃ¼hnl-Bontsoland co-workers 28. Overbeck, J., von Statili, C., Gudat, F., Carmann, H., LautenschlÃ¤ger, C., DÃ¼rmÃ¼ller,U.,Takacs, B., Miggiano, V., Staehlein, T., and Heitz, P. U. for excellent photo work. Immunohistochemical characterization of an antiepithelial monoclonal an tibody (mAb lu-5). Virchow Arch. Pathol. Anal., 407: 1-12, 1985. REFERENCES 29. Cordell, J., Richardson, T. C., Pulford, K. A. F., Ghosh, A. K., Gatter, K. C., Heyderman, E., and Mason, D. Y. Production of monoclonal antibodies 1. Hakomori, S-I. Aberrant glycosylation in cancer cell membranes as focussed against human epithelial membrane antigen for use in diagnostic immuno- on glycolipids: overview and perspectives. Cancer Res., 45:2405-2414,1985. cytochemistry. Br. J. Cancer, 52: 347-354, 1985. 7675

30. Heyderman, E., Graham, R. M., Chapman. D. V., Richardson, T. C., and pathophysiology of psoriasis. J. Invest. Dermatol., 85: 579-583, 1985. McKee, P. H. Epithelial markers in primary skin cancer: an immunoperoxi- 47. Strefling, A. M., Knapp, A. M., and Mansbridge, J. N. Histologie distribution dase study of the distribution of epithelial membrane antigen (EMA) and of staining by a monoclonal antibody (Psi-3) in psoriasis and occurrence of carcinoembryonic antigen (CEA) in 65 primary skin carcinomas. Histopa- Psi-3 antigen in other cutaneous diseases. J. Invest. Dermatol., 84: 100-104, thology, 8: 423-434, 1984. 1985. 31. Miyachi, Y., 1mamma. S., Ohshio, G., Manabe, T., Endo, K., and Kudo, H. 48. Lavker, R. M., and Sun, T-T. Heterogeneity in epidermal basal keratinocytes: Immunohistochemical localisation of cancer associated antigens CA 19-9 morphological and functional correlations. Science (Washington DC), 2/5: and DU-PAN-2 in human skin. Acta Dermatol. (Kyoto), 83: 99-103, 1988. 1239-1241, 1982. 32. Nanney, L. B., Magid, M., Stoschek, C. M., and King, L. E. Comparison of 49. Lavker, R. M., and Sun, T-T. Epidermal stem cells. J. Invest. Dermatol., 81: epidermal growth factor binding and receptor distribution in normal human 121S-127S, 1983. epidermis and epidermal appendages. J. Invest. Dermatol., 83: 385-393. 50. Leigh, I. M., Pulford, K. A., Ramaekers, F. C. S., and Lane, E. B. Psoriasis: 1984. maintenance of an intact monolayer basal cell differentiation compartment 33. Gatter, K. C., Brown, G., Strowbridge, I., Woolston, R-E., and Mason, D. in spite of hyperproliferation. Br. J. Dermatol., 113: 53-64, 1985. Y. Transferrin receptors in human tissues: their distribution and possible 51. Stoler, A., Duvic, M., and Fuchs, E. Unusual patterns of keratin expression clinical relevance. J. Clin. Pathol., 36: 539-545, 1983. in the overlying epidermis of patients with dermatofibromas: biochemical 34. Dippold, W. G., Dienes, H. P., Knuth, A., and Meyer zum BÃ¼schenfelde,K- alterations in the epidermis as a consequence of dermal tumors. J. Invest. H. Immunohistochemical localization of ganglioside GD3 in human malig Deramtol., 93: 728-738, 1989. nant melanoma, epithelial tumors and normal tissue. Cancer Res., 45: 3699- 52. Viac, J., Staquet, M. J., Chomel, G., and Thivolet, J. Alterations in the 3706, 1985. expression of two epidermal difTerentialion antigens in human epidermal 35. Barnstable, C. J., Bodmer, W. F., Brown, G., Galfre, G., Milstein, C., disorders. Path. Res. React., ISO: 577-583, 1985. Williams, A. F., and Ziegler, A. Production of monoclonal antibodies to 53. Weiss, R. A., Guille!, G. Y. A., Freedberg, I. M., Farmer, E. R., Small, E. group A erythrocytes, HLA and other human cell surface antigens: new tools A., Weiss, M. M., and Sun, T-T. The use of monoclonal antibody to keratin for genetic analysis. Cell, 14: 9-20, 1978. in human epidermal disease: alterations in immunohistochemical staining 36. Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, pattern. J. Invest. Dermatol., 81: 224-230, 1983. A., and Fusenig, N. E. Normal keratinization in a spontaneously immortal 54. Kariniemi, A. L., HolthÃ¶fer, H., Vartio, T., and Virtanen, I. Cellular differ ized aneuploid human keratinocyte cell line. J. Cell Biol., Â¡06:761-771, entiation of basal cell carcinomas studied with fluorescent lectins and cyto- 1988. keratin antibodies. J. Cutaneous Pathol., //: 541-548, 1984. 37. Boukamp, P., Stanbridge, E. J., Foo, D. Y., Cerutti, P. A., and Fusenig, N. 55. Morhenn, V. B., Schreiber, A. B., Soriero, O., McMillan, W., and AlusiÃ³n, E. C-Ha-ros oncogene expression in immortalized human keratinocytes A. C. A monoclonal antibody against basal cells of human epidermis. J. Clin. (HaCaT) alters growth potential in vivo but lacks correlation with malig Invest., 76: 1978-1983, 1985. nancy. Cancer Res.. 50: 2840-2847, 1990. 56. Oseroff, A. R., Roth, R., Lipman, S., and Morhenn, V. B. Use of a murine 38. Fusenig, N. E., Boukamp, P., Breitkreutz, D., Karietta, S., and Petrussevska, monoclonal antibody which binds to malignant keratinocytes to detect tumor R. T. Oncogenes and malignant transformation of human keratinocytes. In: cells in microscopically controlled surgery. J. Am. Acad. Dermatol., 8: 616- P. R. Cerutti, O. F. Nygaard, and M. G. Simic (eds.), Anticarcinogenesis and 619, 1983. Radiation Protection, pp. 227-231. New York: Plenum Publishing Corp., 57. Gottlieb, A. B., Posnett, D. M., Crow, M. K., Horokoshi, T., Mayer, L., and 1987. Carter, D. M. Purification and in vitro growth of human epidermal basal 39. Eisinger, M., Lee, J. S., Hefton, J. M.. Darzynkiewicz, Z., Chiao, J. W., and keratinocytes using a monoclonal antibody. J. Invest. Deramtol., 85: 299- de Harven, E. Human epidermal cell cultures: growth and differentiation in 303, 1985. the absence of dermal components or medium supplements. Proc. Nati. 58. Patterson, J. A. K., Eisinger, D. V. M., Haynes, B. F., Berger, C. L., and Acad. Sci. USA, 76: 5340-5344, 1979. Edelson, R. L. Monoclonal antibody 4F2 reactive with basal layer keratino 40. Boukamp, P., Rupniak, H. T. R., and Fusenig, N. E. Environmental modu cytes: studies in the normal and a hyperproliferative state. J. Invest. Derma lation of the expression of differentiation and malignancy in six human tol., Â«J:210-213, 1984. squamous cell carcinoma cell lines. Cancer Res., 45: 5582-5592, 1985. 59. Bauknecht, T., Gross, G., and Hagedorn, M. Epidermal growth factor 41. Bohnert, A., Hornung, J., Mackenzie, I. C., and Fusenig, N. E. Epithelial- receptors in different skin tumors. Dermatologica (Basel), ///: 16-20, 1985. mesenchymal interactions control basement membrane production and dif 60. Gaiter, K. C., Pulford, K. A. F., Vanslapel, M. J., Roach, B., Mortimer, P., ferentiation in cultured and transplanted mouse keratinocytes. Cell Tissue Woolston, R. E., Taylor-Papadimitriou, J., Lane, E. B., and Mason, D. Y. Res., 244: 413-429, 1986. An immunohistological study of benign and malignant skin tumors: epithelial 42. Worst, P. K. M., Mackenzie, I. C., and Fusenig, N. E. Reformation of aspects. Histopathology (Oxf.), 8: 209-227, 1984. organized epidermal structure by transplantation of suspensions and cultures 61. Eisinger, M., Sadan, S., Silver, I. A., and Flick, R. B. Growth regulation of of epidermal and dermal cells. Cell Tissue Res., 225: 65-77, 1982. skin cells by epidermal cell-derived factors: implications for wound healing. 43. Bernhard, B. A., Asselineau, D., Schaffar-Deshayes, L., and Darmon, M. Y. Proc. Nati. Acad. Sci. USA, Â«5:1937-1941, 1988. Abnormal sequence of expression of differentiation markers in psoriatic 62. DÃ©marchez,M., Sengel, P., and Prunieras, M. Wound healing of human skin epidermis: inversion of two steps in the differentiation program. J. Invest. transplanted onto nude mouse. I. An immunohistochemical study of the Dermatol., 90: 801-805, 1988. reepithelialization process. Dev. Biol., 113: 90-96, 1986. 44. Elias, P. M. Epidermal lipids, barrier function and desquamation. J. Invest. 63. Mansbridge, J. N., and Knapp, A. M. Changes in keratinocyte maturation Dermatol., 80: 44S-49S, 1983. during wound healing. J. Invest. Dermatol., 89: 255-263, 1987. 45. Downing, D. T., Stewart, M. E., Wertz, P. W., CollÃ³n, S. W., Abraham, W., 64. Sun, T-T., Eichner, R., Williams, G., Nelson, B. A., Scheffer, C. G. T., and Strauss, J. S. Skin lipids: an update. J. Invest. Dermatol., 88: 2S-6S, Weiss, R. A., Jarvinen, M., and Woodcock-Mitchell, J. Keratin classes: 1987. molecular markers for different types of epithelial differentiation. J. Invest. 46. Weinstein, G. D., McCullogh, J. L., and Ross, P. A. Cell kinetic basis for Dermatol., 81: 109S-115S, 1983.

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Reinhard Klingel, Petra Boukamp, Roland Moll, et al.

Cancer Res 1990;50:7668-7676.

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