In Vitro Cell. Dev. Biol. 27A:562-568, July 1991 © 1991 Tissue Culture Association 0883-8364/91 $01.50+0.00
GROWTH REGULATION OF SERUM-FREE CULTURES OF EPITHELIAL CELLS FROM NORMAL HUMAN BUCCAL MUCOSA KRISTINA SUNDQVIST, YUN LIU, KRISTINA ARVIDSON, KARl ORMSTAD, LENNART NILSSON, RUNE TOFTGARD, ~I~ ROLAND C. GRAFSTROMl Department of Toxicology (K. S., Y. L., R. C. G.), Department of Prosthetic Dentistry (K. A.), and Department of Forensic Medicine (K. 0., L. N.), Karolinska lnstitutet, Box 60400, S-I04 01 Stockholm, Sweden; and Center for Biotechnology (R. T.), Karolinska lnstitutet, Box 60400, Novum, S-141 52 Huddinge, Sweden (Received 9 January 1991; accepted 27 February 1991)
SUMMARY
Human buecal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins 5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings per day on flbronectin/collagen-eoated dishes at clonal density. Studies of markers of proliferation and differentiation in buecal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factor/~-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylpborbol-13-aeetate or an elevated Ca2+ concentration (from 0.l to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area, involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reprodueibly derived from explant cultures of human buecal mueosa specimens and the cells transferred under serum-free conditions. Buccal epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably respond to several agents shown to modulate growth of cells that originate from other types of epithelia. Key words: human buecal mucosa; epithelial cells; serum-free culture; growth and differentiation markers. epithelial growth and differentiation are complex and interdependently regulated (6,7,18). Human buccal mucosa in vivo exhibits a stratified and parakeratinized epithelium that is structurally different from epidermis and bronchial epithelium, as well as adjacent gingiva and palate, indicating that epithelial growth and differentiation may be inherently different in buccal tissue. Therefore, the aims of the present study were to develop procedures for propagation of human buccal epithelial cells under serum-free conditions, to verify the epithelial identity of these cell cultures, and to investigate their growth and differentiation. Herein we describe for cultured buccal epithelial cells, phenotypic characteristics (i.e. morphology and keratin expression), cloning ability, longevity in culture, growth rate, migration, surface area and their ability to express involucrin and to form cross-linked envelopes after exposure to several agents that modulate growth and differentiation, including EGF, cholera toxin, retinoic acid, Caz+, TGF-~, TPA, fetal bovine serum, and extracts of human or bovine pituitaries.
INTRODUCTION Cultures of human oral epithelial cells have been commonly obtained using methods applied to other epithelia, e.g., epidermis, including the use of feeder cells and fibroblast-conditioned and serum-supplemented media (10). For several types of human epi° thelial cells, serum-free cell growth based on the MCDB 153 medium formulation (2) have resulted in the identification of various mitogenie factors, including epidermal growth factor (EGF), cholera toxin (an agent that elevates the intraeellular content of adenosine 3',5'-monophosphate), and retinoids (6). Also, factors that induce growth arrest or terminal squamous differentiation or both, including serum fractions, Ca2+, transforming growth factor-~l, (TGF-~), and the tumor-promoting agent 12-O-tetradeeanoylphorbol-13-acetate (TPA) have been identified (6). Induction of squamous differentiation commonly involves changes in many cellular functions including a decrease in cell migratory activity, an increase of the cell surface area, and synthesis of involucrin and a cross-linked envelope (6, ] ]). Overall, comparisons of serum-free cultures of epithelial cells from various human tissues have indicated a certain degree of tissue specificity; further, the cellular programs that modulate
MATERIALSANDMETHODS Culture conditions. Human buccal tissue was obtained from donors without clinical evidence of pathology at autopsy or surgery.
1 To whom correspondence should be addressed. 562
CULTURED HUMAN BUCCAL EPITHELIAL CELLS
A
t::
Fro. l. Characteristics of primary cultures of normal human buccal epithelial cells. A, culture dish showing outgrowth of cells and satellite colony (after removal of explant). X 1. B, phase contrast micrograph of outgrowth. >90. C, ceils showing rnierovilIi (SEM ) 9 8 % were epithelial cells as determined by immunostaining for keratin) were generally obtained, although during the third or later transfers of explants, some fibroblastic cells were occasionally seen (not shown). Mostly small, polygonal cells typical of epithelial cells were observed (Fig. 1 B). Scanning and transmission elec-
tron microscopic analysis of primary epithelial cell cultures demonstrated typical microvilli and microprojections (Fig. 1 C-E) with interdigitations between adjacent cells (Fig. 1 E). Epithelial cells from the outgrowths showed few tonofilaments and desmosomes (Fig. 1 E).
Fie. 5. Ultrastruetures in buccal epithelial cells exposed to 1 mM Ca2+. Section showing cells with numerous tonofilament bundles (F), desmosomal junctions between cells (D), and microvilli (M). Transmission electron mierograph, ×33 000.
567
CULTURED HUMAN BUCCAL EPITHELIAL CELLS Immunoblot analyses of keratin extracts from confluent cultures demonstrated expression of five major keratin subunits (Fig. 2) Based on relative mobility and reactivity with the AE1 and AE3 antibodies these were tentatively identified as keratins K5, K6, K14, K16/17, and K19. The expression of K6 was confirmed by immunoblot analyses using a subunit specific antibody (16). Over 100 buccal specimens were established in explant culture and greater than 90% produced outgrowths. Usually, good outgrowth of epithelial cells was obtained from autopsy specimens harvested within 6 h after death, although ceil growth was occasionally obtained from tissue received as late as 72 h after death. The initial three explant outgrowths contained mitotically active ceils exhibiting up to 6% cloning efficiency when subcultured on fibronectin/ collagen-coated dishes. Ceils at higher density (>5 X 103 cells/ cm2) could be subcultured 3 to 5 times in BEG-2 medium with 4 to 6 population doublings per passage occurring during a period of about 2 mo. Using buccal epithelial cells derived in chemically defined BEG1 medium, extracts of either human or bovine pituitaries were mitogenic at concentrations between 10 and 30 ttg/ml (Fig. 3), indicating no species difference between these extracts. Determinations of growth rate, planar ceil surface area, and migration of buccal epithelial cells derived in BEG-2 medium and grown at clonal density in the presence of various mitogens are shown in Table 1. Subcultured cells replicated at about 0.8 population doublings per day during an 8-day clonal growth assay without significant differences among the three different BEG-media. The presence of EGF markedly enhanced migration and mean planar surface area; these effects were partially counteracted by PEX. Cholera toxin significantly enhanced growth in all three BEG media. In addition, the ceil surface area and migration were decreased; these effects were more pronounced in the absence of EGF (with or without the simultaneous presence of PEX). Retinoic acid stimulated growth, decreased cell surface area, and enhanced migration of cells. These responses were not affected by EGF with or without PEX. The effects of fetal bovine serum, Ca2+, TGF-/~, and TPA on colony forming efficiency growth rate, ceil surface area, migration, involucrin expression, and Ca ionophore-induced formation of cross-linked envelopes of buccal epithelial cells are shown in Table 2 and Fig. 4. Each of these agents was growth-inhibitory and decreased both the colony forming efficiency and the clonal growth rate. Fetal bovine serum also markedly increased surface area, decreased migration, and increased involucrin expression and the ability of ceils to form cross-linked envelopes (Table 2). Moreover, ceils exhibited multiple nuclei and vacuoles, typical of cells undergoing squamous differentiation (not shown). The concentration of 100 ttM Ca2+ in BEG-1 medium clearly resulted in the most rapid growth, whereas higher concentrations in a dose-response fashion decreased the growth rate (Fig. 4). A concentration of 1 mM Ca2+ inhibited migration and caused the formation of cohesive cell aggregates without significantly affecting the mean surface area (Table 2). Moreover, this elevated Ca2+ concentration enhanced severalfold the ability of cells to express involucrin and to form crosslinked envelopes. Transmission electron microscopic analysis showed that Ca2+-exposed cells were stratified and contained numerous desmosomal junctions and an extensive network of tonofilaments (Fig. 5). TGF-/~ significantly inhibited growth of buccal epithehal cells at 0.1 pM; 50% growth inhibition occurred at 0.6 pM
(Fig. 4). Cells exposed to TGF-/~ exhibited an increased surface area, whereas migration, involucrin expression, and the ability of cells to form cross-linked envelopes was similar to that of unexposed controls (Table 2). When the effect of 1 nM TPA was assayed, involucrin expression was increased, whereas migration, surface area, and cross-linked envelope formation were insignificantly affected (Table 2). Although the mean surface area was not altered, TPA-exposed cells exhibited various phenotypical changes, including squamous morphology and formation of long cytoplasmic extensions from and between ceils (not shown). No clonal growth was obtained at 10 nM TPA whereas this concentration caused a 10fold increase in the ability of cells to form cross-linked envelopes. DISCUSSION Growth factor-enriched, modified MCDB 153 medium efficiently cultivates epithelial ceils from explants of human buecal mueosa. Consistently, at least three consecutive outgrowths containing virtually pure populations of epithelial cells can be obtained. Transfer cultures can easily be passaged 3 or more times, and these cells exhibit up to 6% eloning ability when subcultured on fibronectin/ collagen-coated dishes. Cells exhibit typical epithelial characteristics, including cobblestone-like or squamous morphology, microvilli, desmosomes, tonofilaments, interdigitations, formation of cross-linked envelopes, and expression of involucrin and various keratin subunits. The expression of keratins K5, K6, K14, K16/ 17, and K19, as shown in this study, is common to many epithelial cells in vitro when grown as submerged cultures (4). A recent report also demonstrated that the same keratin subunits are expressed in eultures of both fetal and adult human gingival cells (12). Because keratins K6 and K16 are not expressed in buecal epithelial in vivo (3), K6 expression in cultured cells was confirmed by a subunit specific antibody (cf. Fig. 2), demonstrating that bueeal cells express a similar set of keratin subunits as cultured epidermal and gingival ceils. Investigation of growth regulation of human buccal epithelial cells now demonstrates that EGF, cholera toxin, and retinoic acid are mitogens at defined conditions, i.e. in the absence of serum and, if required, in the absence of pituitary extract (cf. Table 1). Each of the agents exhibits a distinctive mitogenic ability in that EGF significantly increases the cell yield of primary cultures, the cell migratory ability, and surface area; cholera toxin increases clonal growth and decreases both migration and surface area, whereas retinoic acid increases clonal growth and migration and decreases surface area. Except for EGF, enhanced growth by these various treatments is correlated to a decrease in cell surface area (cf. Table 1). Stimulation of migration by EGF probably relates to its mitogenic effect, as previously shown with serum and feeder-cell-dependent epidermal cells (1), although the partly divergent effects of the other mitogens on various growth-related parameters indicate that cell proliferation may be stimulated without the concomitant enhancement of migration. Fetal bovine serum inhibits growth and induces squamous differentiation of buccal epithelial cells as demonstrated by inhibition of clonal growth and migration and by an increase in surface area, involucrin expression, and cross-linked envelope formation. Such serum-dependent effects are not detected when oral epithelial cells are cultured with feeder cells in other media (10). Both growth rate and migration are also markedly decreased by elevation of the
568
SUNDQVIST ET AL.
Ca2+concentration from 0.1 to i mM, which causes cells to undergo squamous differentiation evident from increased expression of involucrin and formation of cross-hnked envelopes, stratification of cells, and the increased presence of desmosomes and tonofilaments. TGF-/3 decreases clonal growth rate and significantly increases the cellular surface area but does not affect migration, involucrin expression, or cross-linked envelope formation, indicating that this agent is primarily a growth-inhibitory agent for buccal epithelial cells, as previously shown in epidermal cells (12). Moreover, growth inhibitory effects and induction of differentiation by nanomolar concentrations of TPA in buccal cells agree with the hypothesis that certain tumor-promoting agents inhibit growth and induce differentiation of normal cells as part of the selection for growth of preneoplastic cells (13,19). In summary, selective serum-free culture conditions for maintenance of explants and for serial transfer and clonal growth of human buccal epithelial cells have now been established. Proliferating buccal epithelial cell cultures generally consist of comparatively smaller cells that exhibit a high degree of motility. In contrast, cells that undergo squamous differentiation exhibit a comparatively larger mean surface area, show less migratory activity, increased expression of involucrin, and form cross-linked envelopes. Defined physiologic factors such as EGF, retinoic acid, Ca 2+, and TGF-~ regulate the growth behavior of buccal epithelial cells without the presence of confounding serum factors. Moreover, interactive effects among various agents is clearly demonstrated by EGF-dependent inhibition of the growth-modulating effects of cholera toxin. Overall, regulation of cell proliferation and differentiation of buccal epithelial cells seems to be highly integrated, similar to what has been shown recently in epithelial cells from other tissues, including the palate (5,8). Finally, buccal epithelial cells in serum-free culture undergo a morphodifferentiation similar to in vivo, i.e. squamous differentiation without anucleation, typical of a stratified and parakeratinized epithelium. This experimental system now offers a defined in vitro model for future studies of both normal and pathologic biology of buccal epithelium.
4. 5. 6.
7. 8.
9.
10. 11.
12. 13.
14. 15. 16. 17.
PREFERENCES 1. Barrandon, Y.; Green, H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-a and epidermal growth factor. Cell 50:1131-1137; 1987. 2. Boyce, S. T.; Ham, R. G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal cultures and serum-free serial cultures. J. Invest. Dermatol. 81:33s40s; 1983. 3. Clausen, H.; Vedtofle, P.; Moe, D., et al. Differentiation-dependent
18.
19.
expression of keratins in human oral epithelia. J. Invest. Dermatol. 86:249-254; 1986. Fuchs, E.; Green, H. Changes in gene expression during terminal differentiation of keratinocytes. Cell 19:1033-1042; 1980. GrafstriJm, R. C.; Dypbukt, J. M.; Willey, J. C., et al. Pathobiological effects of acrolein in cultured human bronchial epithelial cells. Cancer Res. 48:1717-1721; 1988. Grafstrlim, R. C. Carcinogenesis studies in human epithelial tissues and cells in vitro: emphasis on serum-free culture conditions and transformation studies. Acta Physiol. Scand. 140: Suppl. (592):93133;, 1990. Jetten, A. M. Multistep process of squamous differentiation in tracheobronchial epithelial cells in vitro; analogy with epidermal differentiation. Environ. Health Perspect. 80:149-160; 1989. Kasperbauer, J. L.; Neel, H. B., III; Scott, R. E. Proliferation and differentiation characteristics of normal human squamous mucosal cells of the upper aerodigestive tract. Ann. Otol. Rhinol. Laryngol. 99:29-37; 1990. Lechner, J. F.; Stoner, G. D.; Yoakum, G. H., et al. In vitro carcinogenesis studies with human tracheobronchial tissues and cells. In: Schiff, L. J., ed. In vitro models of respiratory epithelium. Boca Raton, FL: CRC Press; 1986:143-159. MacCallum, D. K.; Lillie, J. H.; Jepsen, A., et al. The culture of oral epithelium. Int. Rev. Cytol. 109:313-330; 1987. Nickoloff, B. J.; Mitra, R. S.; Riser, B. L., et al. Modulation of keratinocyte motility. Correlation with production of extracellular matrix molecules in response to growth promoting and antiproliferation factors. Am. J. Pathol. 132:543-551; 1988. Oda, D.; Dale, B. A.; Bourkis, G. Human oral epithelial cell culture II. Keratin expression in fetal and adult gingival ceils. In Vitro Cell. Dev. Biol. 26:596-603; 1990. Parkinson, E. K. Defective responses of transformed keratinocytes to terminal differentiation stimuli: their role in epidermal tumour promotion by phorbol esters and by deep skin wounding. Br. J. Cancer 52:479-493; 1985. Reiss, M.; Sartorelli, A. C. Regulation of growth and differentiation of human keratinocytes by type/3 transforming growth factor and epidermal growth factor. Cancer Res. 47:6705-6709; 1987. Roop, D. R.; Cheng, C. K.; Tifferington, L., et al. Synthetic peptides corresponding to keratin subunits elicit highly specific antibodies. J. Biol. Chem. 259:8037-8040; 1984. Roop, D. R.; Krieg, T. M.; Mehrel, T., et al. Transcriptional control of high molecular weight l~eratin gene expression in multistage mouse skin carcinogenesis. Cancer Res. 48:3245-3252; 1988. Shipley, G. D.; Pittelkow, M. R.; Wille, J. J., et al. Reversible inhibition of normal human prokeratinocyte proliferation by type/3 transforming growth factor-growth inhibitor in serum-free medium. Cancer Res. 46:2068-2071; 1986. WiUe, J. J.; Pittelkow, M. R.; Shipley, G. D., et al. Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies. J. Cell. Physiol. 121:31-44; 1984. Willey, J. C.; Maser, C. E., Jr.; Lechner, J. F., et al. Differential effects of 12-O-tetradecanoylphorbol-13-acetate on cultured normal and neoplastic human bronchial epithelial cells. Cancer Res. 44:51245126; 1984.
The authors are grateful to Dr. F. Bertolero for comments on the manuscript; Dr. J. Lindberg for providing autopsy specimens; Dr. H. Bystedt and Dr. L. ~,on Konow (Dept. of MaxiUofacial Surgery, Sabbatsberg Hospital) for providing surgical specimens; Ms. S. Mattson for assistance with keratin analyses; Ms. B. Silva, Ms. K. Forslind, and Ms. M. SjiJstriim for technical assistance; and Ms. A. L. Marcus and Ms. G. Els~n for typing assistance. This work was supported in part by grants from the Swedish National Board of Laboratory Animals, the Swedish Medical Research Council, the Swedish Natural Science Research Council, the Swedish Fund for Scientific Research Without Animal Experiments, the Swedish Cancer Society, the Swedish Tobacco Company, and Lions Club International, Djurg~den, Stockholm, Sweden.
GROWTH REGULATION OF SERUM-FREE CULTURES OF EPITHELIAL CELLS FROM NORMAL HUMAN BUCCAL MUCOSA KRISTINA SUNDQVIST, YUN LIU, KRISTINA ARVIDSON, KARl ORMSTAD, LENNART NILSSON, RUNE TOFTGARD, ~I~ ROLAND C. GRAFSTROMl Department of Toxicology (K. S., Y. L., R. C. G.), Department of Prosthetic Dentistry (K. A.), and Department of Forensic Medicine (K. 0., L. N.), Karolinska lnstitutet, Box 60400, S-I04 01 Stockholm, Sweden; and Center for Biotechnology (R. T.), Karolinska lnstitutet, Box 60400, Novum, S-141 52 Huddinge, Sweden (Received 9 January 1991; accepted 27 February 1991)
SUMMARY
Human buecal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins 5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings per day on flbronectin/collagen-eoated dishes at clonal density. Studies of markers of proliferation and differentiation in buecal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factor/~-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylpborbol-13-aeetate or an elevated Ca2+ concentration (from 0.l to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area, involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reprodueibly derived from explant cultures of human buecal mueosa specimens and the cells transferred under serum-free conditions. Buccal epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably respond to several agents shown to modulate growth of cells that originate from other types of epithelia. Key words: human buecal mucosa; epithelial cells; serum-free culture; growth and differentiation markers. epithelial growth and differentiation are complex and interdependently regulated (6,7,18). Human buccal mucosa in vivo exhibits a stratified and parakeratinized epithelium that is structurally different from epidermis and bronchial epithelium, as well as adjacent gingiva and palate, indicating that epithelial growth and differentiation may be inherently different in buccal tissue. Therefore, the aims of the present study were to develop procedures for propagation of human buccal epithelial cells under serum-free conditions, to verify the epithelial identity of these cell cultures, and to investigate their growth and differentiation. Herein we describe for cultured buccal epithelial cells, phenotypic characteristics (i.e. morphology and keratin expression), cloning ability, longevity in culture, growth rate, migration, surface area and their ability to express involucrin and to form cross-linked envelopes after exposure to several agents that modulate growth and differentiation, including EGF, cholera toxin, retinoic acid, Caz+, TGF-~, TPA, fetal bovine serum, and extracts of human or bovine pituitaries.
INTRODUCTION Cultures of human oral epithelial cells have been commonly obtained using methods applied to other epithelia, e.g., epidermis, including the use of feeder cells and fibroblast-conditioned and serum-supplemented media (10). For several types of human epi° thelial cells, serum-free cell growth based on the MCDB 153 medium formulation (2) have resulted in the identification of various mitogenie factors, including epidermal growth factor (EGF), cholera toxin (an agent that elevates the intraeellular content of adenosine 3',5'-monophosphate), and retinoids (6). Also, factors that induce growth arrest or terminal squamous differentiation or both, including serum fractions, Ca2+, transforming growth factor-~l, (TGF-~), and the tumor-promoting agent 12-O-tetradeeanoylphorbol-13-acetate (TPA) have been identified (6). Induction of squamous differentiation commonly involves changes in many cellular functions including a decrease in cell migratory activity, an increase of the cell surface area, and synthesis of involucrin and a cross-linked envelope (6, ] ]). Overall, comparisons of serum-free cultures of epithelial cells from various human tissues have indicated a certain degree of tissue specificity; further, the cellular programs that modulate
MATERIALSANDMETHODS Culture conditions. Human buccal tissue was obtained from donors without clinical evidence of pathology at autopsy or surgery.
1 To whom correspondence should be addressed. 562
CULTURED HUMAN BUCCAL EPITHELIAL CELLS
A
t::
Fro. l. Characteristics of primary cultures of normal human buccal epithelial cells. A, culture dish showing outgrowth of cells and satellite colony (after removal of explant). X 1. B, phase contrast micrograph of outgrowth. >90. C, ceils showing rnierovilIi (SEM ) 9 8 % were epithelial cells as determined by immunostaining for keratin) were generally obtained, although during the third or later transfers of explants, some fibroblastic cells were occasionally seen (not shown). Mostly small, polygonal cells typical of epithelial cells were observed (Fig. 1 B). Scanning and transmission elec-
tron microscopic analysis of primary epithelial cell cultures demonstrated typical microvilli and microprojections (Fig. 1 C-E) with interdigitations between adjacent cells (Fig. 1 E). Epithelial cells from the outgrowths showed few tonofilaments and desmosomes (Fig. 1 E).
Fie. 5. Ultrastruetures in buccal epithelial cells exposed to 1 mM Ca2+. Section showing cells with numerous tonofilament bundles (F), desmosomal junctions between cells (D), and microvilli (M). Transmission electron mierograph, ×33 000.
567
CULTURED HUMAN BUCCAL EPITHELIAL CELLS Immunoblot analyses of keratin extracts from confluent cultures demonstrated expression of five major keratin subunits (Fig. 2) Based on relative mobility and reactivity with the AE1 and AE3 antibodies these were tentatively identified as keratins K5, K6, K14, K16/17, and K19. The expression of K6 was confirmed by immunoblot analyses using a subunit specific antibody (16). Over 100 buccal specimens were established in explant culture and greater than 90% produced outgrowths. Usually, good outgrowth of epithelial cells was obtained from autopsy specimens harvested within 6 h after death, although ceil growth was occasionally obtained from tissue received as late as 72 h after death. The initial three explant outgrowths contained mitotically active ceils exhibiting up to 6% cloning efficiency when subcultured on fibronectin/ collagen-coated dishes. Ceils at higher density (>5 X 103 cells/ cm2) could be subcultured 3 to 5 times in BEG-2 medium with 4 to 6 population doublings per passage occurring during a period of about 2 mo. Using buccal epithelial cells derived in chemically defined BEG1 medium, extracts of either human or bovine pituitaries were mitogenic at concentrations between 10 and 30 ttg/ml (Fig. 3), indicating no species difference between these extracts. Determinations of growth rate, planar ceil surface area, and migration of buccal epithelial cells derived in BEG-2 medium and grown at clonal density in the presence of various mitogens are shown in Table 1. Subcultured cells replicated at about 0.8 population doublings per day during an 8-day clonal growth assay without significant differences among the three different BEG-media. The presence of EGF markedly enhanced migration and mean planar surface area; these effects were partially counteracted by PEX. Cholera toxin significantly enhanced growth in all three BEG media. In addition, the ceil surface area and migration were decreased; these effects were more pronounced in the absence of EGF (with or without the simultaneous presence of PEX). Retinoic acid stimulated growth, decreased cell surface area, and enhanced migration of cells. These responses were not affected by EGF with or without PEX. The effects of fetal bovine serum, Ca2+, TGF-/~, and TPA on colony forming efficiency growth rate, ceil surface area, migration, involucrin expression, and Ca ionophore-induced formation of cross-linked envelopes of buccal epithelial cells are shown in Table 2 and Fig. 4. Each of these agents was growth-inhibitory and decreased both the colony forming efficiency and the clonal growth rate. Fetal bovine serum also markedly increased surface area, decreased migration, and increased involucrin expression and the ability of ceils to form cross-linked envelopes (Table 2). Moreover, ceils exhibited multiple nuclei and vacuoles, typical of cells undergoing squamous differentiation (not shown). The concentration of 100 ttM Ca2+ in BEG-1 medium clearly resulted in the most rapid growth, whereas higher concentrations in a dose-response fashion decreased the growth rate (Fig. 4). A concentration of 1 mM Ca2+ inhibited migration and caused the formation of cohesive cell aggregates without significantly affecting the mean surface area (Table 2). Moreover, this elevated Ca2+ concentration enhanced severalfold the ability of cells to express involucrin and to form crosslinked envelopes. Transmission electron microscopic analysis showed that Ca2+-exposed cells were stratified and contained numerous desmosomal junctions and an extensive network of tonofilaments (Fig. 5). TGF-/~ significantly inhibited growth of buccal epithehal cells at 0.1 pM; 50% growth inhibition occurred at 0.6 pM
(Fig. 4). Cells exposed to TGF-/~ exhibited an increased surface area, whereas migration, involucrin expression, and the ability of cells to form cross-linked envelopes was similar to that of unexposed controls (Table 2). When the effect of 1 nM TPA was assayed, involucrin expression was increased, whereas migration, surface area, and cross-linked envelope formation were insignificantly affected (Table 2). Although the mean surface area was not altered, TPA-exposed cells exhibited various phenotypical changes, including squamous morphology and formation of long cytoplasmic extensions from and between ceils (not shown). No clonal growth was obtained at 10 nM TPA whereas this concentration caused a 10fold increase in the ability of cells to form cross-linked envelopes. DISCUSSION Growth factor-enriched, modified MCDB 153 medium efficiently cultivates epithelial ceils from explants of human buecal mueosa. Consistently, at least three consecutive outgrowths containing virtually pure populations of epithelial cells can be obtained. Transfer cultures can easily be passaged 3 or more times, and these cells exhibit up to 6% eloning ability when subcultured on fibronectin/ collagen-coated dishes. Cells exhibit typical epithelial characteristics, including cobblestone-like or squamous morphology, microvilli, desmosomes, tonofilaments, interdigitations, formation of cross-linked envelopes, and expression of involucrin and various keratin subunits. The expression of keratins K5, K6, K14, K16/ 17, and K19, as shown in this study, is common to many epithelial cells in vitro when grown as submerged cultures (4). A recent report also demonstrated that the same keratin subunits are expressed in eultures of both fetal and adult human gingival cells (12). Because keratins K6 and K16 are not expressed in buecal epithelial in vivo (3), K6 expression in cultured cells was confirmed by a subunit specific antibody (cf. Fig. 2), demonstrating that bueeal cells express a similar set of keratin subunits as cultured epidermal and gingival ceils. Investigation of growth regulation of human buccal epithelial cells now demonstrates that EGF, cholera toxin, and retinoic acid are mitogens at defined conditions, i.e. in the absence of serum and, if required, in the absence of pituitary extract (cf. Table 1). Each of the agents exhibits a distinctive mitogenic ability in that EGF significantly increases the cell yield of primary cultures, the cell migratory ability, and surface area; cholera toxin increases clonal growth and decreases both migration and surface area, whereas retinoic acid increases clonal growth and migration and decreases surface area. Except for EGF, enhanced growth by these various treatments is correlated to a decrease in cell surface area (cf. Table 1). Stimulation of migration by EGF probably relates to its mitogenic effect, as previously shown with serum and feeder-cell-dependent epidermal cells (1), although the partly divergent effects of the other mitogens on various growth-related parameters indicate that cell proliferation may be stimulated without the concomitant enhancement of migration. Fetal bovine serum inhibits growth and induces squamous differentiation of buccal epithelial cells as demonstrated by inhibition of clonal growth and migration and by an increase in surface area, involucrin expression, and cross-linked envelope formation. Such serum-dependent effects are not detected when oral epithelial cells are cultured with feeder cells in other media (10). Both growth rate and migration are also markedly decreased by elevation of the
568
SUNDQVIST ET AL.
Ca2+concentration from 0.1 to i mM, which causes cells to undergo squamous differentiation evident from increased expression of involucrin and formation of cross-hnked envelopes, stratification of cells, and the increased presence of desmosomes and tonofilaments. TGF-/3 decreases clonal growth rate and significantly increases the cellular surface area but does not affect migration, involucrin expression, or cross-linked envelope formation, indicating that this agent is primarily a growth-inhibitory agent for buccal epithelial cells, as previously shown in epidermal cells (12). Moreover, growth inhibitory effects and induction of differentiation by nanomolar concentrations of TPA in buccal cells agree with the hypothesis that certain tumor-promoting agents inhibit growth and induce differentiation of normal cells as part of the selection for growth of preneoplastic cells (13,19). In summary, selective serum-free culture conditions for maintenance of explants and for serial transfer and clonal growth of human buccal epithelial cells have now been established. Proliferating buccal epithelial cell cultures generally consist of comparatively smaller cells that exhibit a high degree of motility. In contrast, cells that undergo squamous differentiation exhibit a comparatively larger mean surface area, show less migratory activity, increased expression of involucrin, and form cross-linked envelopes. Defined physiologic factors such as EGF, retinoic acid, Ca 2+, and TGF-~ regulate the growth behavior of buccal epithelial cells without the presence of confounding serum factors. Moreover, interactive effects among various agents is clearly demonstrated by EGF-dependent inhibition of the growth-modulating effects of cholera toxin. Overall, regulation of cell proliferation and differentiation of buccal epithelial cells seems to be highly integrated, similar to what has been shown recently in epithelial cells from other tissues, including the palate (5,8). Finally, buccal epithelial cells in serum-free culture undergo a morphodifferentiation similar to in vivo, i.e. squamous differentiation without anucleation, typical of a stratified and parakeratinized epithelium. This experimental system now offers a defined in vitro model for future studies of both normal and pathologic biology of buccal epithelium.
4. 5. 6.
7. 8.
9.
10. 11.
12. 13.
14. 15. 16. 17.
PREFERENCES 1. Barrandon, Y.; Green, H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-a and epidermal growth factor. Cell 50:1131-1137; 1987. 2. Boyce, S. T.; Ham, R. G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal cultures and serum-free serial cultures. J. Invest. Dermatol. 81:33s40s; 1983. 3. Clausen, H.; Vedtofle, P.; Moe, D., et al. Differentiation-dependent
18.
19.
expression of keratins in human oral epithelia. J. Invest. Dermatol. 86:249-254; 1986. Fuchs, E.; Green, H. Changes in gene expression during terminal differentiation of keratinocytes. Cell 19:1033-1042; 1980. GrafstriJm, R. C.; Dypbukt, J. M.; Willey, J. C., et al. Pathobiological effects of acrolein in cultured human bronchial epithelial cells. Cancer Res. 48:1717-1721; 1988. Grafstrlim, R. C. Carcinogenesis studies in human epithelial tissues and cells in vitro: emphasis on serum-free culture conditions and transformation studies. Acta Physiol. Scand. 140: Suppl. (592):93133;, 1990. Jetten, A. M. Multistep process of squamous differentiation in tracheobronchial epithelial cells in vitro; analogy with epidermal differentiation. Environ. Health Perspect. 80:149-160; 1989. Kasperbauer, J. L.; Neel, H. B., III; Scott, R. E. Proliferation and differentiation characteristics of normal human squamous mucosal cells of the upper aerodigestive tract. Ann. Otol. Rhinol. Laryngol. 99:29-37; 1990. Lechner, J. F.; Stoner, G. D.; Yoakum, G. H., et al. In vitro carcinogenesis studies with human tracheobronchial tissues and cells. In: Schiff, L. J., ed. In vitro models of respiratory epithelium. Boca Raton, FL: CRC Press; 1986:143-159. MacCallum, D. K.; Lillie, J. H.; Jepsen, A., et al. The culture of oral epithelium. Int. Rev. Cytol. 109:313-330; 1987. Nickoloff, B. J.; Mitra, R. S.; Riser, B. L., et al. Modulation of keratinocyte motility. Correlation with production of extracellular matrix molecules in response to growth promoting and antiproliferation factors. Am. J. Pathol. 132:543-551; 1988. Oda, D.; Dale, B. A.; Bourkis, G. Human oral epithelial cell culture II. Keratin expression in fetal and adult gingival ceils. In Vitro Cell. Dev. Biol. 26:596-603; 1990. Parkinson, E. K. Defective responses of transformed keratinocytes to terminal differentiation stimuli: their role in epidermal tumour promotion by phorbol esters and by deep skin wounding. Br. J. Cancer 52:479-493; 1985. Reiss, M.; Sartorelli, A. C. Regulation of growth and differentiation of human keratinocytes by type/3 transforming growth factor and epidermal growth factor. Cancer Res. 47:6705-6709; 1987. Roop, D. R.; Cheng, C. K.; Tifferington, L., et al. Synthetic peptides corresponding to keratin subunits elicit highly specific antibodies. J. Biol. Chem. 259:8037-8040; 1984. Roop, D. R.; Krieg, T. M.; Mehrel, T., et al. Transcriptional control of high molecular weight l~eratin gene expression in multistage mouse skin carcinogenesis. Cancer Res. 48:3245-3252; 1988. Shipley, G. D.; Pittelkow, M. R.; Wille, J. J., et al. Reversible inhibition of normal human prokeratinocyte proliferation by type/3 transforming growth factor-growth inhibitor in serum-free medium. Cancer Res. 46:2068-2071; 1986. WiUe, J. J.; Pittelkow, M. R.; Shipley, G. D., et al. Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies. J. Cell. Physiol. 121:31-44; 1984. Willey, J. C.; Maser, C. E., Jr.; Lechner, J. F., et al. Differential effects of 12-O-tetradecanoylphorbol-13-acetate on cultured normal and neoplastic human bronchial epithelial cells. Cancer Res. 44:51245126; 1984.
The authors are grateful to Dr. F. Bertolero for comments on the manuscript; Dr. J. Lindberg for providing autopsy specimens; Dr. H. Bystedt and Dr. L. ~,on Konow (Dept. of MaxiUofacial Surgery, Sabbatsberg Hospital) for providing surgical specimens; Ms. S. Mattson for assistance with keratin analyses; Ms. B. Silva, Ms. K. Forslind, and Ms. M. SjiJstriim for technical assistance; and Ms. A. L. Marcus and Ms. G. Els~n for typing assistance. This work was supported in part by grants from the Swedish National Board of Laboratory Animals, the Swedish Medical Research Council, the Swedish Natural Science Research Council, the Swedish Fund for Scientific Research Without Animal Experiments, the Swedish Cancer Society, the Swedish Tobacco Company, and Lions Club International, Djurg~den, Stockholm, Sweden.
