Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E-cadherin.

Gap junctional intercellular communication (GJIC) of cultured mouse epidermal cells is mediated by a gap junction protein, connexin 43, and is dependent on the calcium concentration in the medium, with higher GJIC in a high-calcium (1.2 mM) medium. In several mouse epidermal cell lines, we found a good correlation between the level of GJIC and that of immunohistochemical staining of E-cadherin, a calcium-dependent cell adhesion molecule, at cell-cell contact areas. The variant cell line P3/22 showed both low GJIC and E-cadherin protein expression in low- and high-Ca2+ media. P3/22 cells showed very low E-cadherin mRNA expression. To test directly whether E-cadherin is involved in the Ca(2+)-dependent regulation of GJIC, we transfected the E-cadherin expression vector into P3/22 cells and obtained several stable clones which expressed high levels of E-cadherin mRNA. All transfectants expressed E-cadherin molecules at cell-cell contact areas in a calcium-dependent manner. GJIC was also observed in these transfectants and was calcium dependent. These results suggest that Ca(2+)-dependent regulation of GJIC in mouse epidermal cells is directly controlled by a calcium-dependent cell adhesion molecule, E-cadherin. Furthermore, several lines of evidence suggest that GJIC control by E-cadherin involves posttranslational regulation (assembly and/or function) of the gap junction protein connexin 43.

Skin tumor-promoting activity of benzoyl peroxide, a widely used free radical-generating compound.

Benzoyl peroxide, a widely used free radical-generating compound, promoted both papillomas and carcinomas when it was topically applied to mice after 7,12-dimethylbenz[a]anthracene initiation. Benzoyl peroxide was inactive on the skin as a complete carcinogen or as a tumor initiator. A single topical application of benzoyl peroxide produced a marked epidermal hyperplasia and induced a large number of dark basal keratinocytes, effects similar to those produced by the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate. Benzoyl peroxide, like other known tumor promoters, also inhibited metabolic cooperation (intercellular communication) in Chinese hamster cells. In view of these results caution should be recommended in the use of this and other free radical-generating compounds.

Tumor initiation by N-acyloxy derivatives of piperidine and N-arylacetamides.

Four N-Acetoxy-N-arylacetamides previously found to be local sarcomagens in the rat have been found to be initiators of tumorigenesis in mouse skin. The order of activity was: N-acetoxy-2-acetamidophenanthrene greater than N-acetoxy-4-acetamino-stilbene similar to N-acetoxy-2-acetamido-fluorene greater than N-acetoxy-4-acetamidobiphenyl. Two substituted N-benzoyloxypiperidines previously shown to yield nitrenium ions on solvolysis in methanol also had initiating activity in mouse skin.

Acetic acid, a potent stimulator of mouse epidermal macromolecular synthesis and hyperplasia but with weak tumor-promoting ability.

The effects of a single application of various dose levels of acetic acid or the weak tumor promoter, phorbol-12,13-ditetradecanoate, on the incorporation of tritiated thymidine (3H-TDR), 3H-cytidine, and 3H-leucine into DNA, RNA, and protein of mouse epidermis, respectively, were determined and compared with histologic changes in the skin. Treatment with either 500 or 833 mumoles acetic acid induced a sequential and sustained stimulation of RNA, protein, and DNA synthesis, which was followed by extensive epidermal hyperplasia similar to that reported for the strong promoter and irritant, 12-O-tetradecanoyl-phorbol-13-acetate. A dose-response relationship between the amount of acetic acid and the rate of DNA synthesis was found between the dose levels of 33 to 833 mumoles of acetic acid per application. The latter dose induced the maximum activation of 3H-TDR into DNA at 723% of control at 2 days, whereas 33 mumoles stimulated DNA synthesis earlier and peaked at 210% of control at 3 hours. Phorbol-12,13-ditetradecanoate also stimulated macromolecular synthesis in a similar sequence, though to a lesser degree. No observable inflammation and only a slight hyperplastic response were noted with phorbol-12,13-ditetradecanoate. Weekly applications of 667 mumoles of acetic acid produced a maximal tumor response of 0.73 papilloma/mouse after 32 weeks of promotion. However, a weekly dose of 677 mumoles of acetic acid was essentially inactive when given in two divided doses. When croton oil was administered twice weekly at a 0.25%-dose level, 10.2 papillomas/mouse were induced after 32 weeks of promotion. The results showed that the previously considered nonpromoting inflammatory agent, acetic acid, must be a weak promoter. However, there was no correlation between stimulated macromolecular synthesis or hyperplasia and tumor promotion when phorbol esters were compared with acetic acid.

Comparative tumor-initiating activity of complex mixtures from environmental particulate emissions on SENCAR mouse skin.

The value of the SENCAR mouse for testing tumorigenic properties of complex mixtures on mouse skin was studied. Seven complex mixtures were obtained as dichloromethane extracts of collected particulate emissions from three diesel-fueled automobiles, a heavy-duty diesel engine, a nonleaded gasoline-fueled automobile, a coke oven battery, and a roofing tar pot. These emissions were applied topically at multiple doses to both male and female SENCAR mice that were subsequently promoted with 12-O-tetradecanoylphorbol 13-acetate. Two statistical analyses were applied to the data to rank the samples and to provide 95% confidence intervals. One analysis used tumor multiplicity data, applied them to a nonlinear Poisson model, and the second analysis used tumor incidence data and applied them to a log-probit model. Both analyses ranked the complex mixtures in similar order. Benzo[a]pyrene content alone could not account for all the tumorigenic activity in each complex mixture, indicating that other components also contribute to the overall tumorigenic activity.

Epidermal cell proliferation and promoting ability of phorbol esters.

Dose-response relationships on the abilities of several phorbol ester tumor promoters to promote skin tumors after 7,12-dimethylbenz[a]anthracene initiation and to bring about edema, inflammation, and epidermal hyperplasia were determined in female Charles River CD-1 mice. The promoting ability of the potent synthetic promoter, phorbol-12,13-dioctanoate (PdiC8), was determined over a dose range of 0.1-10 mug/application. Administration of PdiC8 two times weekly at dosages of 4, 6, 8, and 10 mug gave little variation in tumor response. A dose-dependent tumor response occurred at doses of 1-4 mug PdiC8. Only 1 papilloma was observed when PdiC8 was given twice weekly at a dose of 0.1 or 0.5 mug. A similar dose-response relation was observed for the ability of PdiC8 to stimulate epidermal hyperplasia. Investigations of other phorbol esters revealed an excellent correlation between their promoting ability and their ability to induce epidermal hyperplasia; however, that was not the case for compounds outside the phorbol ester series (i.e., acetic acid, cantharidin, and ethylphenylpropiolate).

Prolonged inhibition of mouse epidermal DNA synthesis by dexamethasone.

The effect of dexamethasone, an anti-inflammatory agent that inhibits skin carcinogenesis, on DNA, RNA, and protein synthesis in mouse epidermis was investigated. Within 1 hour after the topical application of 75 mug dexamethasone to the skin of mice, epidermal DNA synthesis was inhibited drastically and lasted for approximately 5 days, followed by a biphasic stimulation at 7 and 10 days after treatment. Histologically, the skin after dexamethasone treatment revealed only subtle quantitative changes. The dermis was not altered. The epidermal thickness was unchanged, but the nuclei of the epidermal cells were more densely stained and the chromatin was more densely clumped. These changes were present by 12 hours and persisted for 10 days. Both epidermal RNA and protein syntheses were stimulated early, reached a peak of around 180% of controls at 1 hour, but returned to control levels by 3 hours. They were slightly inhibited between 3 and 12 hours after treatment and then returned to control levels. This prolonged inhibition of DNA synthesis by dexamethasone may be related to its anticarcinogenic effect.

Simultaneous appearance of keratin modifications and gamma-glutamyltransferase activity as indicators of tumor progression in mouse skin papillomas.

gamma-Glutamyltransferase (GGT), an enzyme not found in normal adult epidermis, was detected in most skin papillomas larger than 13 mm in diameter and in all squamous carcinomas induced by 7,12-dimethylbenz[a]anthracene initiation and 12-O-tetradecanoylphorbol 13-acetate promotion in noninbred Sencar mice. Furthermore, these GGT-positive lesions were also characterized by a marked decrease or absence of high-molecular-weight components of epidermal keratin. Since these characteristics are common to both carcinomas and large papillomas but are practically undetectable in normal epidermis and small papillomas, GGT activity and lack of high-molecular-weight keratin components seem to be good indicators of tumor progression, i.e., from papilloma to squamous carcinoma.

Further characterization of skin tumor promotion and progression by mezerein in SENCAR mice.

This study evaluated the skin tumor-promoting activity of mezerein in SENCAR mice. The effect of initiation dose of 7,12-dimethylbenz(a)anthracene (DMBA) on tumor promotion by mezerein was examined. Excellent dose-response relationships were observed for initiation with DMBA at 0.2-20 micrograms per mouse with mezerein as a complete promoter. None of the mezerein-only promotion groups had papilloma responses similar to those of the corresponding groups receiving two-stage promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) followed by mezerein, even when a 40-micrograms initiating dose of DMBA was used. The effect delaying promotion with mezerein for 10 weeks was also examined in mice initiated with either 0.2, 2, 20, or 40 micrograms of DMBA per mouse. The 10-week delay led to a slight increase in the number of papillomas per mouse in some but not all treatment groups. Again, none of the delayed-mezerein-treatment groups had papilloma responses similar to those of the corresponding two-stage promotion (TPA-mezerein) groups at any corresponding initiating dose of DMBA. Finally, the progression of papillomas to carcinomas during promotion with mezerein was examined in groups of mice initiated with either 2 or 20 micrograms of DMBA. Higher ratios of carcinomas to papillomas were observed in mice promoted with mezerein than in mice receiving TPA promotion or two-stage promotion (TPA-mezerein). However, the presence of two to four times more papillomas in some mezerein-treated groups did not lead to greater numbers of carcinomas than in the groups with fewer papillomas. The data do not support the idea that spontaneous stage I promotion can be induced by delaying mezerein treatment for 10 weeks. Furthermore, the data suggest that the higher ratio of carcinomas to papillomas observed with mezerein promotion may be a function of the lower tumor burdens obtained after promotion with this compound rather than a specific property of the chemical.

Lack of involvement of 6-hydroxymethylation in benzo[a]pyrene skin tumor initiation in mice.

The skin tumor-initiating activities of benzo[a]pyrene (BP), 6-hydroxymethylbenzo[a]pyrene (6-OH-CH2-BP), and 6-methylbenzo[a]pyrene (6-CH3-BP), as well as the effects of 7,8-benzoflavone (7,8-BF), quercetin, and 1-benzylimidazole on their activity, were determined in outbred female CD-1 mice by use of a two stage system of tumorigenesis. The skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP was 12.5 and 20%, respectively, of the activity of BP, 7,8-BF had little effect on the skin tumor-initiating activity of 6-OH-CH2-BP and 6-CH3-BP. However, a dose-dependent inhibition of BP tumorigenesis by 7,8-BF was noted. Quercetin and 1-benzylimidazole also inhibited BP skin tumor-initiating activity. These findings indicated that direct hydroxymethylation of BP is not an important pathway in the activation of BP in mouse skin tumor initiation.

Inhibition of phorbol ester-induced tumor promotion in mice by vitamin A analog and anti-inflammatory steroid.

The effects of a vitamin A analog, TMMP ethyl retinoate [or ethyl-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-trans-2,4,6,8-nonatetraenoate] (abbreviated Ro 10-9359), and an anti-inflammatory steroid, fluocinolone acetonide (or 6 alpha, 9 alpha-difluoro-11 beta, 16 alpha, 17,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic 16,17-acetal) (abbreviated FA), given alone or together were studied in a two-stage carcinogensis system. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was used as the tumor promoter in a 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin system. Two stocks of female mice, CD-1 and Sencar, which differ in their degrees of sensitivity to skin carcinogenesis, were used. A dose-dependent inhibition of carcinogenic expression, as determined by a decreased number of papillomas per animal, was observed in each mouse stock with the use of both FA and Ro 10-9359 when given alone. When FA and Ro 10-9359 were given together, an enhanced effect on the lowering of tumor incidence was noted. FA effectively inhibited tumor formation in the sensitive mouse stock even when the steroid was given 1 day prior to TPA treatment under conditions of unusually high doses of initiator (DMBA) and/or promoter (TPA). These results suggest that both anti-inflammatory steroids and retinoids inhibit tumor promotion and can be effectively used as a combination regimen for increased chemopreventive response.

Mechanism-based cancer prevention approaches: targets, examples, and the use of transgenic mice.

Humans are exposed to a wide variety of carcinogenic insults, including endogenous and man-made chemicals, radiation, physical agents, and viruses. The ultimate goal of carcinogenesis research is to elucidate the processes involved in the induction of human cancer so that interventions may be developed to prevent the disease, either in the general population or in susceptible subpopulations. Progress to date in the carcinogenesis field, particularly regarding the mechanisms of chemically induced cancer, has revealed several points along the carcinogenesis pathway that may be amenable to mechanism-based prevention strategies. The purpose of this review is to examine the basic mechanisms and stages of chemical carcinogenesis, with an emphasis on ways in which preventive interventions can modify those processes. Possible ways of interfering with tumor initiation events include the following: i) modifying carcinogen activation by inhibiting enzymes responsible for that activation or by direct scavenging of DNA-reactive electrophiles and free radicals; ii) enhancing carcinogen detoxification processes by altering the activity of the detoxifying enzymes; and iii) modulating certain DNA repair processes. Possible ways of blocking the processes involved in the promotion and progression stages of carcinogenesis include the following: i) scavenging of reactive oxygen species; ii) altering the expression of genes involved in cell signaling, particularly those regulating cell proliferation, apoptosis, and differentiation; and iii) decreasing inflammation. In addition, the utility for mechanism-based cancer prevention research of new animal models that are based on the overexpression or inactivation of specific cancer-related genes is examined.

Mouse skin tumor-initiating activity of 5-, 7-, and 12-methyl- and fluorine-substituted benz[a]anthracenes.

As an approach for elucidation of structure activity relationships that underlie the exceptionally large difference in carcinogenic activity between benz[a]anthracene and 7,12-dimethylbenz[a]anthracene (7,12-DMBA), 11 methyl- and/or fluorine-substituted benz[a]anthracenes were evaluated for tumor-initiating activity on mouse skin. Outbred CD-1 and outbred Sencar mice received a single topical application of the hydrocarbons followed by twice weekly applications of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate for 16-26 weeks. 7,12-DMBA was almost two orders of magnitude more active as a tumor-initiator than 7- and 12-methylbenz[a]anthracene. Methyl substitution at the 7- and 7,12-positions of benz[a]anthracene was significantly more effective in the enhancement of tumorigenic activity than fluorine substitution at these positions. Although 7-fluorobenz[a]anthracene, 12-fluorobenz[a]anthracene, and 7,12-difluorobenz[a]anthracene had only 0.15, 0.26, and less than 0.005 times the tumor-initiating activity of their respective methyl-substituted derivatives, they were severalfold more active than benz[a]anthracene. 7-Fluorobenz[a]anthracene was slightly less active than 12-fluorobenz[a]anthracene, whereas 7-methylbenz[a]anthracene was about twofold more than 12-methylbenz[a]anthracene. For 7,12-di-substituted benz[a]anthracenes, 7-methyl-12-fluorobenz[a]anthracene was more than twice as tumorigenic as 7-fluoro-12-methylbenz[a]anthracene, but each was individually more active than 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene, respectively. Both fluorinated compounds were much less active than 7,12-DMBA. Substitution of fluorine or methyl at the 5-position of 7-methylbenz[a]anthracene and substitution of fluorine at the 5-position of 12-methylbenz[a]anthracene dramatically reduced their tumorigenic activity.

Effect of trichloropropene oxide on the ability of polyaromatic hydrocarbons and their "K-region" oxides to initiate skin tumors in mice and to bind to DNA in vitro.

The potent epoxide hydrase inhibitor, 1,1,1-trichloro-2,3-propene oxide (TCPO), enhanced the tumor-initiating ability of benzo[alpha]pyrene (BP) and 3-methylcholanthrene (MCA) but had no effect on 9,10-dimethyl-1,2-anthracene (DMBA) initiation in a two-stage system of tumorigenesis in female Charles River CD-1 mice. The tumor-initiating ability of dibenz[alpha,h]-anthracene (DBA) was decreased by prior topical treatment with 10 mumoles of TCPO. The tumor latency period of BP and MCA was decreased by TCPO but had no effect on DMBA or DBA. Topical treatment with 10 mumoles of TCPO did not initiate tumors in a two-stage system in mouse skin nor did it cause any histopathologic changes in the skin. The "K-region" epoxides of BP, DMBA, and MCA were weak tumor initiators when compared to the parent compounds. TCPO only slightly increased or had no effect on the tumor-initiating activity of the above epoxides. Pretreatment with Croton oil 18 hours prior to initiation with BP-4,5-epoxide also slightly enhanced the tumorigenic response in mouse skin. DBA-5,6-epoxide, when tested as a complete carcinogen at high doses (1 mg daily/10 days), was found to be a weak carcinogen but with activity comparable to that of DBA. TCPO only slightly increased the in vitro epidermally mediated covalent binding of the above parent polycyclic hydrocarbons to DNA.

Promotion of carcinomas by repeated abrasion in initiated skin of mice.

Repeated abrasion of the skin of Sencar mice initiated with 100 nmol of dimethylbenzanthracene resulted in the appearance of carcinomas in addition to papillomas. These results strongly support the argument that repeated epidermal regeneration is a sufficient stimulus for the promotion of both benign and malignant neoplasms in the skin of mice.

Glucocorticoid receptor levels in mouse skin after repetitive applications of 12-O-tetradecanoylphorbol-13-acetate and mezerein.

Female Sencar mice were given twice weekly topical applications of 0.2 ml of acetone, 4 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA), 2 micrograms of mezerein, or 4 micrograms of TPA followed by 2 micrograms of mezerein. Whole skin cytoplasmic glucocorticoid receptor content and dissociation constants were determined at various times after the last application of the agents. After four to 12 applications of acetone, the mean glucocorticoid receptor level was 0.49 +/- 0.02 pmol/mg cytosol protein, and the dissociation constant was 6.0 +/- 0.17 nM. The glucocorticoid receptor level was markedly reduced and remained low for at least 72 hr after repetitive applications of TPA. The mean receptor level was 0.113 +/- 0.011 (S.E.) pmol/mg cytosol protein, and the Kd was 2 to 4 times the control value. In contrast, the glucocorticoid receptor level was not significantly reduced after repetitive applications of mezerein. If four applications of mezerein were preceded by four applications of TPA, then the glucocorticoid receptor level was similar to that after TPA alone. It was also demonstrated that the glucocorticoid receptor level in papillomas was extremely low (0.022 pmol/mg cytosol protein) but was much higher in newborn skin (0.52 pmol/mg cytosol protein).

Keratin modifications in epidermis, papillomas, and carcinomas during two-stage carcinogenesis in the SENCAR mouse.

To elucidate the role of keratin modification in tumor promotion, we investigated the keratin polypeptide patterns of mouse epidermis, papillomas, and carcinomas throughout an initiation-promotion experiment. The epidermal keratin modifications induced by repetitive 12-O-tetradecanoylphorbol-13-acetate treatments in both initiated and noninitiated mouse skin were essentially identical to those observed with a single 12-O-tetradecanoylphorbol-13-acetate application. These changes were even more pronounced in epidermal papillomas. In addition, the keratins of the papillomas displayed greater charge heterogeneity, particularly among the high-molecular-weight keratins (Mr 60,000 to 62,000). As the experiment progressed, there appeared to be a selective loss of one group of high-molecular-weight keratins (Mr 62,000) in some of the papillomas. Interestingly, the carcinomas that appeared at this time had significant reduction in both groups of high-molecular-weight keratins. In fact, the keratin profiles of carcinomas were very similar to the patterns observed in basal cells after a single 12-O-tetradecanoylphorbol-13-acetate treatment of adult epidermis. This may indicate that the program of keratin expression of a carcinoma becomes permanently fixed at a basal cell pattern. Changes in keratin patterns may serve as a biochemical marker of malignant progression in mouse epidermis.

Inhibition of the tumor-initiating ability of the potent carcinogen 7,12-dimethylbenz(a)anthracene by the weak tumor initiator 1,2,3,4-dibenzanthracene.

ARyl hydrocarbon hydroxylase (AHH) in mouse epidermis was inducible by topical application of several tumor-initiating polycylic aromatic hydrocarbons. The weak tumor initiator 1,2,3,4-dibenazanthracene (1,2,3,4-DBA), at dose level of 200 nmoles, increased AHH activity more than 10-fold over that of the acetone controls at 12 hr after treatment. Administration of the same quantity of the potent initiator 7,12-dimethylbenz(a)anthracene (DMBA) increased AHH activity approximately 4-fold over that of the control at 12 hr after treatment. Simultaneous treatment with 200 or 100 nmoles of DMBA and 1,2,3,4-DBA resulted in AHH activity that was 546 and 732% that of the controls, respectively, 12 hr after treatment: this was less AHH activity than was observed when 1,2,3,4-DBA was administered alone. Doses of 20 nmoles or more of 1,2,3,4-DBA, when given at about the same time as DMBA, effectively inhibited DMBA initiation of skin tumors in a two stage system of tumorigenesis. The results suggest that the weak initiator 1,2,3,4-DBA may program the epidermal AHH system to metabolize the strong carcinogen DMBA to noncarcinogenic intermediate(s).

The effects of antioxidants on skin tumor initiation and aryl hydrocarbon hydroxylase.

Butylated hydroxytoluene, butylated hydroxyanisole, and vitamins C and E are effective inhibitors of 7,12-dimethylbenz(a)anthracene tumor initiation in a two-stage system of tumorigenesis. These antioxidants did not significantly induce epidermal aryl hydrocarbon [benzo(a)pyrene]hydroxylase, nor did they have any effect when added directly to the in vitro aryl hydrocarbon [benzo(a)pyrene]hydroxylase assay. However, butylated hydroxytolene and butylated hydroxyanisole, when topically to mice, inhibited the in vitro, epidermally mediated, covalent binding of radioactive benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA. When butylated hydroxytolene and butylated hydroxyanisole were added in vitro, they did not inhibit the epidermally mediated covalent binding of the hydrocarbons to DNA. The inhibition of polycyclic hydrocarbon tumorigenesis by antioxidants may be related to the ability of antioxidants to prevent the in vivo activation of hydrocarbons to carcinogenic epoxides and/or other electrophilic intermediates or may be related to their ability to increase detoxification of the reactive intermediate that requires intact cells to be operational. In any event, the results suggest that the antioxidants have an indirect effect on the epidermal metabolizing system which leads to a decrease in covalent binding to DNA.

Numerical variation of dark cells in normal and chemically induced hyperplastic epidermis with age of animal and efficiency of tumor promoter.

The percentages of dark keratinocytes was quantitatively assessed in normal epidermis of Sencar mice before and after birth and in adult epidermis after topical application of several compounds of varying promoting efficiency. The percentage of dark keratinocytes reached a maximum at the 19th day of gestation (approximately 40%) and fell abruptly after birth (approximately 3%). Old animals exhibited a very low number of dark basal cells (0.2%). After topical application of the weak promoters resiniferotoxin, anthralin, ethylphenylpropiolate, and 12-deoxyphorbol-13-2,4,6-decatrienoate, the percentage of dark cells in young adult epidermis did not differ markedly from that in control (acetone-treated) specimens. The strong first-stage promoters 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and calcium ionophore A 23187, as well as the strong complete promoter 12-deoxyphorbol-13-deoxyphorbol-13-decanoate, induced the appearance of large numbers of dark keratinocytes, in a percentage similar to that seen after 12-O-tetradecanoylphorbol-13-acetate application (approximately 20%). The similarities between the dark keratinocytes seen after topical application of 12-O-tetradecanoylphorbol-13-acetate or other strong promoters and the dark cells observed in the fetal epidermis before the onset of the adult type of epidermal keratinization indicate that potent and/or first stage tumor promoters can be identified by their ability to induce cells resembling fetal-type dedifferentiated keratinocytes.