Enhancement of Squamous Cell Development in Cultured Skin by Cyclic Adenine Nucleotide and Prostaglandins

Differentiation

Differentiation (1981) 20 :260-263

0 Springer-Verlag 1981

Short Reports

Enhancement of Squamous Cell Development in Cultured Skin by Cyclic Adenine Nucleotide and Prostaglandins FREDERICK V. SCHAEFER, R. PHILIP CUSTER, and SAM SOROF Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA The present study tests the hypothesis that agents known to elevate the level of intracellular cyclic adenine nucleotide may direct different epithelial cells onto a pathway of epidermoid (squamous) development and differentiation. We report here that the mixture of dibutyryl cyclic AMP (dbcAMP), prostaglandins El, & and Bl (PG E,, E2,B,), and papaverine (pap) enhances the rate of normal squamous cell development in organ-cultured skin of chick embryos. The three components may act synergistically to elevate the level of intracellular cyclic adenine nucleotide. We recently reported that the same group of agents induces abnormal development (squamous metaplasia) and aberrant differentiation (keratin production) in the normally cuboidal epithelium of cultured whole mammary glands of mice [l]. Thus, dbcAMP, PG El, EZ,B1, and pap are effective in enhancing normal squamous cell development and also in inducing squamous metaplasia de novo in the epithelial components of two different organs of embryonic and adult animals of two classes of vertebrates. The combined findings are suggestive that cyclic adenine nucleotide together with the prostaglandins may act generally on diverse types of epithelia to bring about squamous cell development and a differentiation marked by keratin production.

Introduction The molecular mediation of cellular differentiation is a subject at the forefront of present biologic research. The question of how epithelial cells are directed to particular pathways of development and differentiation is unanswered. We recently reported that the mixture of dibutyryl cyclic AMP (dbcAMP), prostaglandins (PG) El, El, and B1, and papaverine (pap) induces the normally cuboidal epithelium of cultured mammary glands to undergo an abnormal cellular development along an epidermoid route (squamous metaplasia), followed by the aberrant differentiation marked by considerable production of keratin [l]. The inducers thus redirect epithelial cells of the mammary gland onto a pathway of development and differentiation that is fundamentally different from that to which the organ is normally committed. The question therefore arises whether these inducers act on a common mechanism that mediates the squamous (epidermoid) development and differentiation of epithelial cells in various normal, abnormal, transformed, and malignant tissues. In search of an answer to part of this question, the present study examined the effects of these inducers on another epithelial organ culture system, namely, normal skin of the chick embryo. Methods Cuhring and Processing of Skin The skin of the short shank region from the legs of 13-day-old white leghorn chick embryos (Truslow Farms, MD) was cut into 2-3 mm squares, spread on Dacron rafts, and floated in

1ml of BGJb medium (Grand Island Biological Co., Grand Island, NY) in 35 mm tissues culture dishes. The basal medium contained 5% fetal bovine serum (inactivated at 56°C for 30 min), 0.1 mg/ml streptomycin sulfate (Sigma Chemical Co., St. Louis, MO), 0.1 mg/ml amphotericin B (Sigma), and 100 IU/ml penicillin (Grand Island Biological Co.) [2-41. The cultures were treated with or without supplements at 36" C in an atmosphere of 95% air -5% C02. The cultured skins were thereafter fixed in acetic acid : ethanol (1 : 3 v/v), stained with alum-carmine to facilitate histologic processing, dehydrated in ethanol, transferred to xylene, embedded in paraffin, sectioned at 5 pm, stained with hematoxylin-eosin for histologic study, and processed by the Masson's trichrome method for the histochemical detection of keratin (bright red color). Scoring of Differentiation in Cultured Skin Normal development and differentiation of skin involves the progression of columnar basal epithelial cells to flattened squamous cells followed by keratin production [5]. The development and differentiation of the surface cells of the cultured skin were scored in terms of the following six stages that are illustrated and numbered in the panels of Figs. 1and 2. On the day of culturing (dayO), the cells (five to six cell thickness) above the basal cell layer were polyhedral (stage 0). At stage 1, the surface of the skin consisted of one to two layers of squamous cells. Stage 2 was marked by at least three to four layers of squamous cells. Parakeratin or keratin was not apparent at this or the earlier stages. At stage 3, parakeratin and degenerating squamous cells were evident. Stage 4 was characterized by the presence of loose keratin and the absence 0301-4681/81/0020/0260/$01.00

Fq. 1. Stages in the development and differentiation of cultured embryonic chick skin. The development and differentiation of the surface cells of the cultured skin were scored in terms of the six numbered and illustrated stages that are also defined in the Methods section. Shown are sections of cultured skin stained with hematoxylin and eosin. Magnification 8OOx

0

1

of visible remains of the uppermost squamous cells. In stage 5 , there was a tightly condensed, intensely acidophilic stratum corneum.

Reagents In addition to the above listings, dbcAMP, pap, dibutyryl cyclic GMP, 5'-adenylic acid, sodium butyrate, prolactin (P), and aldosterone (A) were obtained from Sigma Chemical Co. Insulin (I) and hydrocortisone (H) were purchased from Calbiochem (La Jolla, CA). PG El, E2, and BIwere kindly donated by Dr. J. E. Pike of The Upjohn Co. (Kalarnazoo, MI).

Results

Fig. 2. Diagrammatic representation of the stages of squamous cell development and differentiation in cultured embryonic chick skin.The stages, numbered in each panel, correspond to those in Fig. 1

Embryonic chick shank skin in organ culture can develop and differentiate normally and fully in medium containing only 5% fetal bovine serum [2-41. Thedreshly cultured embryonic chick skin is six to seven cells thick, consisting of a one cell layer of columnar basal cells above which are five to six layers of polyhedral cells. At day 5 , the cultured skin is fully differentiated with stratified squamous cells that are overlaid by a condensed stratum corneum (Figs. 1and 2). Six stages of skin development can be grouped into two periods. The first P e r i d involves the proliferation and development of the columnar basal cells to squamous Cell morphology with early parakeratin production (Figs. 1and 2, stages 0-3). The second

262

F. V. Schaefer et al.: Squamous Cell Development in Cultured Skin Table 1. Acceleration of squamous cell development in cultured

embryonic chick skin Series 1 2

--t--l 5 6

DAYS IN CULTURE Fig. 3. Enhancement of squamous cell development in cultured embryonic chick skin. Chick embryonic skin was cultured as in the

Methods section for different durationsin medium containing 5% fetal or bovine serum without further additions (control) supplemented with dbcAMP M), pap M), and PG El, E2, Plotted are the means of the integral BI, each at 5 pg/ml (-A-A-). values of the stages of development and differentiation, and the standard errors of the means (vertical limits). Each point in the curves is derived from 20-84 replicate cultures. No linearity in the scale of stages 0-5 is implied by the vertical axis in the graph

*-*,

period involves the profuse production of loose keratin and its condensation to a cornified layer (stages 4 and 5). Exposure of the cultured skin to a mixture of dbcAMP, pap, and PG El, &, and B1to basal medium containing 5% fetal bovine serum (enhancing medium) significantly enhances the rate of development during the first period (stages 0-3), i.e., the development and differentiation of the basal cells to squamous morphology with production of parakeratin (Fig. 3). By day 3 in culture (during the first period), development of skin in the enhancing medium is accelerated by 1.5 days beyond that of control cultures in the basal medium containing only fetal bovine serum (Table 1). The second phase of skin development and differentiation, involving the progression of loose keratin to comified keratin (stage 4 and 5), appears to be relatively unaffected by enhancing medium. Thus, skin fragments that are cultured with or without the enhancing supplements are equally developed and differentiated by day 5 (Fig. 3). The explants retain their intact morphology at least until day 9. The individual enhancing supplements act synergistically to accelerate skin development. Table 1shows the mean values of the integral stages of skin development achieved by various supplements at day 3 in culture. DbcAMP alone, or the mixture of the three prostaglandins, or pap, or dibutyryl cyclic GMP, or the combination of dibutyryl cyclic GMP, pap, and PG El, &, B1is each moderately to weakly stimulatory (series 3-7). The full enhancing effect is achieved only by the combination of dbcAMP, pap, and PG El, &, B1 (series 2), being significantly different from that of dbcAMP alone (P 5 0.0oOS). The control substances, sodium butyrate, 5’-adenylic acid, or insulin, produce insignificant effects compared to that of the basal medium containing 5% fetal bovine serum (series 1, 8-10). The hormone mixture, insulin, prolactin, aldosterone, and hydrocortisone, which induces lobuloalveolar development and lactogenesis in cultured whole mammary glands of mice [6-81, is mildly inhibitory (series 11). The level of mitosis of the basal cells of the skin was not significantly affected by the presence of the enhancing reagents

3 4 5 6 7 8 9 10 11

Supplementsn None DbcAMP + Pap + PG El, &, B1 (enhance) DbcAMP Pap PG El, &, Bi DbcGMP DbcGMP + Pap + PG El, E2, B1 Sodium butyrate 5’-AMP I

I+P+A+H

No. of

cultures

Average stages of developmentb

41 75

2.0 f 0.1 3.6 f 0.1

48 31 32

3.1 f 0.1 2.7 f 0.1 2.7 f 0.1 2.6 It 0.1 2.8 f 0.1

30 29

36 17 12 21

2.2 f 0.1 2.4 f 0.2 1.8 f 0.3 1.3 & 0.2

Fragments of metatarsal skin from 13-day-old chick embryos were cultured for three days as described in the text. Medium containing 5% fetal bovine serum was supplemented with the listed agents at the following concentrations: dibutyryl cyclic AMP (dbcAMP), dibutyryl cyclic GMP (dbcGMP), and 5’-adenylic acid (5‘-AMP), each at M; papaverine (pap) at M; prostaglandins El, Ez, and Bl(PG El, &, Bl), insulin (I), prolactin (P), aldosterone (A), and hydrocortisone (H), each at 5 pg/rnl; and sodium butyrate at 2 x 10-4 M

Stages of development are defined in the Methods section and Figs. 1and 2. Listed are the averages of the integral values of the stages of development and differentiation, and the standard errors of the means

compared to those in control medium, both frequencies at day 3 being 0.5 f 0.1% (16 mitoses per 3,240 cells versus 24 mitoses per 4,387 cells, respectively). This observation is consistent with the accompanying lack of difference in the thickness of the cultured skin (21 k 1 p)in the two media.

Discussion Cyclic adenine nucleotide, operating together with the prostaglandins El, &, and B1 and pap, accelerate normal squamous cell development in cultured embryonic chick skin. The three types of enhancing constituents may act synergistically to elevate the level of intracellular cyclic AMP by three separate means, first by the entry of exogenous cyclic adenine nucleotide, second by stimulating the synthesis of intracellular cyclic AMP through the action of the prostaglandins on adenyl cyclase [9, lo], and third through the inhibition by pap of the degradation of cyclic AMP by phosphodiesterases [ll]. Agents that elevate the level of intracellular cyclic AMP have been variously reported to stimulate or to inhibit cell growth in cultured mammary gland [12], epidermis [13], and other organs [13, 141. In particular, dbcAMP has been indicated to elevate [13, 151 or to depress [16-201 the growth of cultured keratinocytes with [15, 191 or without [13, 201 accompanying markers of differentiation. The diversity of these responses may result from alterations brought about by the use of dissociated cells and the attending tissue disorganization, problems that are avoided with organ culture. In the present study, the level of mitosis of the basal cells of the cultured skin was not significantly affected by the presence of

F. V. Schaefer et al.: Squamous Cell Development in Cultured Skin

the squamous cell-inducing reagents, compared to those in control medium. Substances that presumably elevate the level of intracellular cyclic AMP also elicit normal differentiation in cell cultures of fibroblasts [21-231, skeletal muscle [24], melanoma [25], and other systems [14]. There are conflicting reports in regard to keratinocytes [13, 15, 19, 201. Further, high concentrations of PG El and B1can apparently redirect the developmental program in chick back skin from feather formation to keratinization [26, 271. Lastly, theophylline, a phosphodiesterase inhibitor like pap, under certain conditions has been indicated to be a necessary supplement to dbcAMP in the induction of keratohyaline granules in cultured human keratinocytes [20]. Cyclic adenine nucleotide acting together with certain prostaglandins may be the natural inducers or regulators of the normal and metaplastic development of diverse kinds of epithelia to squamous cells with differentiation marked by keratin production in vivo. First, cyclic adenine nucleotide operating synergistically with certain prostaglandins are potent inducers of squamous metaplasia and keratin production in cultured whole mammary glands of mice [l].Second, the same substances accelerate normal squamous cell development in cultured embyronic chick skin (this report). Cyclic adenine nucleotide and specific prostaglandins have thus been found to enhance normal squamous cell development and to inducesquamous metaplasia de novo followed by keratin production in two different organs from embryonic and adult animals of two classes of vertebrates. Acknowledgementr. We thank Ms. Gail Nussbaum and Mrs. Grace Kroetz for technical assistance, and Dr. J. Pike and the Upjohn Company for a gift of prostaglandins. This study was supported in part by NIH grants CA-21522, CA-30036, CA-05945, CA-06927, and RR-05539, and an appropriation from the Commonwealth of Pennsylvania. Preliminary results were presented at the annual meetings of the American Association for Cancer Research [28]and the American Society of Biological Chemists [29].

References 1. Schaefer FV, Custer RP, Sorof S (1980) Induction of abnormal development and differentiation in cultured mammary glands by cyclic adenine nucleotide and prostaglandins. Nature 286:807 2. Wessells NK (1%1) An analysis of chick epidermal differentiation in situ and in vitro in chemically defined media. Dev Biol 3: 355 3. Sugimato M, Endo H (1%9) Effect of hydrocortisone on keratization of chick embryonic skin cultured in a chemically defined medium. Nature 222: 1270 4. Wilkhoff LJ, Peckham JC, Dulmadge EA. Mowry RW, Chopra DP (1976) Evaluation of vitamin A analogues in modulating epithelial differentiation in 13-day chick embryo metatarsal skin explants. Cancer Res 36: 964 5. Bloom W, Fawcett DW (1968) Skin. A textbook of histology. W B Saunders, New York. p 479 6. Ichinose RR, Nandi S (1966) Influence of hormones on lobulo-alveolar differentiation of mouse mammary glands in vitro. J Endocrinol 35 :331 I . Wood BG, Washburn LL,Mukherjee AS, Banerjee MR (1975) Hormonal regulation of lobulo-alveolar growth, functional differentiation and regression of whole mouse mammary gland in organ culture. J Endocrinol 65: 1 LI

263 8. Tonelli QJ, Custer RP, Sorof S (1979) Transformation of cultured mouse mammary glands by aromatic amines and amides and their derivatives. Cancer Res 39 :1784 9. Samuelsson B, Granstrom E, Green K, Hamberg M, Hammarstrom SA (1975) Prostaglandins. Annu Rev Biochem 44:669 10. Samuelsson B, Granstrom E, Green K, Hamberg M, Hammarstrom SA (1978) Prostaglandins and thromboxanes. Annu Rev Biochem 47: 997 11. Prasad KN, Kumar S (1974) Cyclic AMP and the differentiationof neuroblastoma cell in culture. In: Clarkson B, Baserga R (4s) Control of proliferation in animal cells. Cold Spring Harbor Laboratory, New York, p 581 12. Taylor-Papadimitriou J, Purkis P, Fentiman IS (1980) Cholera toxin and analogues of cyclic AMP stimulate the growth of cultured human mammary epithelial cells. J Cell Physiol 102: 317 13. Green H (1978) Cyclic AMP in relation to proliferation of the epidermal cell: a new view. Cell 15: 801 14. Friedman DL (1976) Role of cyclic nucleotides in cell growth and differentiation. Physiol Rev 56: 652 15. Marcel0 CL (1979) Differential effects of CAMPand cGMP on in vitro epidermal cell growth. Exp Cell Res 120:201 16. Delescluse C, Colburn NH, Duell EA, Voorhees JJ (1974) Cyclic AMP-elevating agents inhibit proliferation of keratizing guinea pig epidermal cells.Differentiation 2: 343 17. Harper RA, Flaxman BA, Chopra DP (1974) Mitotic response of normal and psoriatic keratinocytes in vitro to compounds known to affect intracellular cyclic AMP. J Invest Dermatol 62: 384 18. Flaxman BA, Harper RA (1975) In vitro analysisof the control of keratinocyte proliferation in human epidermis by physiologic and pharmacologic agents. J Invest Dermatol 65 :52 19. Delescluse C, Fukuyama K, Epstein WL (1976) Dibutyryl cyclic AMP-induced differentiation of epidermal cells in tissue culture. J Invest Dermatol66: 8 20. Chopra DP (1977) Effects of theophylline and dibutyryl cyclic AMP on proliferation and keratinization of human keratinocytes. Br J Dermatol %: 255 21. Johnson GS, Friedman RM, Pastan I(1971) Restoration of several morphological characteristics of normal fibroblasts in sarcoma cells treated with adenosine-3' : 5'-cyclic monophosphate and its derivatives. Proc Natl Acad Sci USA 68: 425 22. Hsie AW, Puck lT (1971) Morphological transformation of Chinese hamster cells by dibutyryl adenosine cyclic 3' :5'-monophosphate and testosterone. Prof Natl Acad Sci USA 68:358 23. Sheppard JR (1971) Restoration of contact-inhibited growth to transformed cells by dibutyryl adenosine 3' :5'-cyclic monophosphate. Proc Natl Acad Sci USA 68:1316 24. Aw EJ, Holt PG, Simons PJ (1973) Myogenesis in vitro. Enhancement by dibutyryl CAMP. Exp Cell Res 83: 436 25. Johnson GS, Pastan I (1972) N6,02'-dibutyryl adenosine 3',5'-monophosphate induces pigment production in melanoma cells. Nature 237: 267 26. Kischer CW (1967) Effects of specific prostaglandins on development of chick embryo skin and down feather organ in vitro. Exp Biol 16:203 27. Kischer CW (1969) Accelerated maturation of chick embryo skin treated with a prostaglandin (PGB,): an electron microscopic study. Am J Anat 124:491 28. Schaefer FV, Custer RP, Sorof S (1981) Induction of squamous cell development by cyclic adenine nucleotide and prostaglandins in two cultured organs. Proc Am Assoc Cancer Res 22: 47 29. Schaefer FV, Custer RP, Sorof S (1981) Induction of squamous cell development in two cultured organs by cyclic adenine nucleotide and prostaglandins. Fed Proc 40:1809

Received May 1981/Accepted August 1981