Alterations in epidermal sulfated proteoglycan production following topical application of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate to mouse skin.

Topical application of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin causes marked changes in epidermal cell growth and differentiation. In the present studies we characterized the production of sulfated proteoglycans in the epidermis following treatment with TPA since these macromolecules are important structural and functional components of the tissue. We found that 35S-sulfate was readily incorporated into mouse epidermal proteoglycans. Sepharose CL-4B column chromatography revealed one major peak of sulfated proteoglycans in this tissue (Kav = 0.4-0.5). Approximately 65% of these proteoglycans were heparan sulfate and 10-20% chondroitin sulfate. Using specific monoclonal antibodies and flow cytometry, we found that the epidermal cells produced chondroitin-4-sulfate, chondroitin-6-sulfate and chondroitin-O-sulfate. Within 24 hr of application of TPA to mice, an increase in glycosaminoglycan content of the epidermis was observed. This was associated with a decrease in 35S-sulfate uptake into the tissue. Although TPA had no effect on the size or relative distribution of the epidermal sulfated proteoglycans, an increase in chondroitin-4-sulfate expression was observed in treated skin. Changes in the production of proteoglycans following TPA treatment may underlie structural alterations that occur in the epidermis during tumor promotion.     

Modulation of phorbol ester-elicited events in mouse epidermis by dietary n-3 and n-6 fatty acids

Because arachidonic acid-derived eicosanoids are potent modulators of hyperproliferation and inflammation during skin tumor promotion with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) (17, 18), it was hypothesized that dietary modification of epidermal fatty acids might modulate TPA-induced biochemical events in mouse skin. Semipurified diets containing 10% total fat composed of corn oil (CO) or a combination of CO and menhaden oil (MO) or coconut oil (CT) were fed to SENCAR mice for 4 weeks. Fatty acid composition of epidermal phospholipids generally reflected fatty acid composition of dietary oils fed to the mice. Since fatty acid-derived eicosanoids are thought to be essential in tumorigenesis, we compared the effects of dietary fats on prostaglandin E (PGE) production in epidermis treated with a single dose of TPA. TPA-induced PGE production in mouse epidermis from mice fed the MO diet was significantly reduced compared to PGE production in epidermal homogenates from mice fed the CO or CT diets. Type of dietary fats did not appear to modulate TPA-induced vascular permeability, however hyperplasia was slightly elevated in skins of mice fed MO. The subcellular distribution of protein kinase C, the plasma membrane receptor for TPA predominantly located in the cytosol (80%), was altered in epidermis from mice fed the MO diet compared to preparations from mice fed CO or CT diets which exhibited normal protein kinase C distribution. Our results suggest that n-3 rich dietary lipids modulate TPA-elicited events in mouse skin to a greater extent than diets containing higher proportions of saturated or n-6 fatty acids.     

Responses of Preneoplastic Epidermal Cells to Tumor Promoters and Growth Factors: Use of Promoter-Resistant Variants for Mechanism Studies

The JB6 mouse epidermal cell model system is being used to study the mechanism of promotion of transformation. Promotion of anchorage independence in JB6 cells occurs in response to second-stage but not first-stage promoters, and is inhibited by inhibitors of second-stage not first-stage promotion. A number of variants that are resistant to the phorbol diester TPA have been derived. Some are resistant to plateau density mitogenic stimulation by TPA; others are resistant to promotion of anchorage independence by TPA. Some of the mitogen-resistant variants were promotable by TPA, thus ruling out a requirement for TPA mitogenesis in promotion of transformation in JB6 cells. TPA promotable clones were also sensitive to mezerein and EGF while the TPA nonpromotable variants were also resistant to mezerein and EGF, suggesting that sensitivity to promoters in these JB6 cells is determined at a level distal to receptor binding. Promotion sensitivity did not require available EGF receptors since two TPA promotable variants were EGF receptorless. The mitogenic response of JB6 cells to TPA may however be mediated by EGF since four of four mitogen-resistant variants showed low to zero levels of EGF binding. Tumor promoting phorbol esters produce specific changes in cellular gangliosides. Certain of these changes occur in promotable but not nonpromotable variants of JB6 cells, suggesting that ganglioside changes may be involved in the process of promotion of transformation.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin ornithine decarboxylase and protein kinase C by polyphenolics from grapes

Ornithine decarboxylase is the rate-limiting enzyme in the biosynthesis of polyamines, which are believed to play an essential role in diverse biological processes including cell proliferation and differentiation. We have previously reported [J. Bomser, K. Singletary, M. Wallig, M. Smith, Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds, Cancer Letters 135 (1999) 151-157] that pre-application of a grape polyphenolic fraction (GPF) to mouse skin epidermis inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, as well as 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated, TPA-promoted mouse skin tumorigenesis. The present studies were designed to further characterize the effect of time and dose of application of GPF on TPA-induced ODC activity and protein expression, and on protein kinase C activity in mouse skin epidermis. In addition, the effect of GPF on ODC kinetics in vitro was examined. Application of 5, 10, and 20 mg of GPF 20 min prior to treatment with TPA resulted in a significant decrease in epidermal ODC activity of 54, 53, 90%, respectively, compared with controls. Yet, ODC protein levels (Western blot) in the 10 and 20 mg GPF groups were significantly increased by 1.8 and 1.9-fold, respectively, compared with controls. A similar response was observed with the ODC inhibitor 2-difluoromethylornithine (DFMO), which served as a positive control. Application of grape polyphenolics (20 mg) at 60 and 30 min prior to treatment with TPA inhibited ODC activity by 62 and 68%, respectively, compared with controls (P<0.05). In contrast, application of grape polyphenolics (20 mg) at 60, 120 and 240 min after treatment with TPA resulted in no significant changes in ODC activity. A similar increase in epidermal ODC protein was observed in these GPF-treated animals, similar to that observed when GPF application preceded TPA. When applied to mouse skin prior to TPA, GPF was associated with a decrease in subsequent PKC activity compared with controls at 10 and 30 min following TPA treatment. The GPF-associated decrease in PKC activity preceded the decrease in ODC activity. In a separate in vitro study, kinetic analyses indicated that GPF is a competitive inhibitor of ODC activity. Collectively these data suggest that the grape polyphenolic fraction is effective as an inhibitor of ODC activity when applied before TPA, and that the magnitude of inhibition is independent of epidermal ODC protein content. In addition, GPF is a competitive inhibitor of ODC activity in vitro. The decrease in TPA-induced ODC activity due to GPF treatment is preceded by an inhibition of TPA-induced PKC activity. Thus, the polyphenolic fraction from grapes warrants further examination as a skin cancer chemopreventive agent that interferes with cellular events associated with TPA promotion.     

The tumor promoter 12-0-tetradecanoylphorbol-13-acetate induces ornithine decarboxylase in proliferating basal cells but not in differentiating cells from mouse epidermis

The cultivation of mouse epidermal cells in medium of reduced calcium concentration (0.02–0.1 mM) selects for basal cell growth. Elevation of medium calcium levels above 0.1 mM results in rapid and well defined differentiative changes. This model was utilized to determine which cell type in mouse epidermis responds to the phorbol ester tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by an induction of the enzyme ornithine decarboxylase (ODC). Previous data had shown that TPA induces ODC in primary mouse epidermal cells only during the first 36 hr after plating in medium containing 1.44 mM Ca²⁺. In contrast, the induction in cells grown in low calcium medium was 2–10-fold greater, and inducibility persisted for at least 4 weeks. The greater inducibility of ODC in low calcium cells is not paralleled by increased thymidine incorporation after TPA treatment, probably because these cells are already proliferating at a maximum rate. When low calcium cells grown in 0.07 mM Ca²⁺ medium were switched to 1.2 mM Ca²⁺, there was a rapid loss of ODC inducibility. These results strongly suggest that the basal cells of the epidermis constitute the major target cells for the induction of ODC by TPA. The induction of ODC by ultraviolet light was not enhanced by growth of cells in low calcium medium, indicating that extracellular calcium concentration per se does not determine ODC inducibility. When epidermal cells grown in 1.2 mM or 0.07 mM Ca²⁺ medium were exposed to both UV light and TPA, there was a significant synergistic effect of combined treatment over the sum of each individual response, suggesting that factors in addition to differentiation determine the extent of ODC induction.     

Cypermethrin exposure leads to regulation of proteins expression involved in neoplastic transformation in mouse skin

Cypermethrin, a synthetic pyrethroid insecticide is shown to exert carcinogenic effects in rodents; however, its underlying mechanism remains elusive. Here, we showed the effect of cypermethrin on protein expression involved in neoplastic transformation in mouse skin. Comparative protein expression profiles between untreated control and cypermethrin-treated mouse skin were explored using 2-DE. A total of 27 spots that were statistically significant (p<0.05) and differentially expressed in response to cypermethrin exposure were identified by MALDI-TOF/TOF and LC-MS/MS. Among them, six up-regulated proteins (carbonic anhydrase 3 (Ca 3), Hsp-27, S100A6, galectin-7, S100A9, S100A11) and one down-regulated protein (superoxide dismutase [Cu-Zn] (Sod 1)) are associated with cancer-related key processes. These selected dysregulated proteins were further validated in 2-DE gels of mouse skin treated with known tumorigens (benzo-[a]-pyrene, 12-O-tetradecanoyl-phorbol-13-acetate and mezerein), respectively. Comparative studies showed that Ca 3, S100A6, S100A9, S100A11 and Sod 1 are specific for stages of development and progression of tumors whereas Hsp-27 and galectin-7 are specific for tumor promotion stage by cypermethrin in mouse skin. Furthermore, these chosen proteins confirmed by Western blotting and immunofluorescence staining were consistent with changes in 2-DE check. This proteomic investigation for the first time provides key proteins that will contribute in understanding the mechanism behind cypermethrin-induced neoplastic transformation.     

Keratin alterations during embryonic epidermal differentiation: a presage of adult epidermal maturation

Differentiation of the epidermis during embryonic rabbit development was found to be accompanied by dramatic changes in keratin proteins. Immunofluorescent labeling with keratin antiserum revealed that the undifferentiated epithelium of 12-d embryos was already committed to making keratin proteins. At 18 d of embryogenesis, the epithelium contained keratin proteins in the molecular weight range of 40,000-59,000. The stratification of the epithelium into two cell layers at 20 d of development coincided with the appearance of a 65-kdalton keratin. When a thick stratum corneum developed at 29 d, several additional keratins became prominent, most notably the large keratins (61- and 64-kdalton) and a 54-kdalton keratin. In addition, the 40-kdalton keratin, which had been present in earlier embryonic epidermis, disappeared. Newborn epidermis resembled that of a 29-d embryonic epidermis, with the exception of the appearance or increase in concentration of two more keratin species (46- and 50-kdalton). In vitro culturing of keratinocytes from 12- and 14-d embryonic skin demonstrated that these cells contained essentially the same keratin profiles as the undifferentiated epithelium of 18-d embryos (40-59 kdalton). Keratinocytes grown from older embryos contained increased amounts of keratin, similar to the in vivo situation, but did not synthesize the high molecular weight keratins. The changes observed during embryonic epidermal differentiation appear to be recapitulated during the sequential maturation steps of adult epidermis.     

Cholera toxin, a potent inducer of epidermal hyperplasia but with no tumor promoting activity in mouse skin carcinogenesis

Intracutaneous injection of cholera toxin into mice induced epidermal hyperplasia to a greater extent than 12-O-tetradecanoylphorbol-13-acetate. It also induced adenylate cyclase and though weakly, ornithine decarboxylase of the epidermis. Cholera toxin, however, showed no tumor promoting activity in mouse skin carcinogenesis. In the single stage promotion, cholera toxin (50 ng) was injected once a week for 10 weeks into the skin of SENCAR mice initiated with 25 micrograms 7,12-dimethylbenz[a]anthracene, but no tumors developed. In the two-stage promotion test, cholera toxin (10-100 ng) was injected for one or two weeks into the initiated skin and then mezerein (4 micrograms) was applied twice a week for 18 weeks, but the toxin did not increase incidence or numbers of papillomas.     

Multivariate flow cytometry of epidermal regeneration provoked by a skin irritant and a tumor promoter.

The DNA content and the changes in cellular and nuclear size of isolated regenerating mouse epidermal basal cells were studied after topical application of the skin irritant cantharidin and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to the back skin of hairless mice. The DNA and protein contents of isolated basal cells were stained with propidium iodide and fluorescein isothiocyanate, respectively, and analysed by flow cytometry using the total protein fluorescence as an estimate of cell size and the DNA fluorescence pulse width as an estimate of nuclear size. Transmission electron microscopy was used to identify cells sorted from regions in the bivariate DNA/protein distributions. The results showed that both chemicals induced an increase in cellular as well as nuclear size of the basal cells. The increase in size was higher in TPA treated than in cantharidin treated animals, and the bivariate DNA/protein distributions of TPA treated cells differed from those of cantharidin treated cells in that two subpopulations of cycling keratinocytes could be identified. These deviations are probably related to the higher proliferative response observed after TPA treatment and the possibility that proliferative subpopulations in epidermis respond differently to TPA. It may reflect mechanisms providing for a growth advantage of initiated cells, important in tumor promotion. About 8% of the cells in the suspensions from treated animals were non-cycling non-keratinocytes, probably infiltrating leukocytes. The results indicate a strong correlation between rapid regenerative cell cycle progression, i.e., reduced G1 transit time and increased cellular and nuclear size.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the Mechanisms Involved in Multistage Carcinogenesis in Mouse Skin

Skin tumors can be effectively induced in mice by the repetitive application of a carcinogen. The relative order of sensitivity to complete carcinogenesis is Sencar > CD-1 > C57BL/6 ≥ BALB/c ≥ ICR/Ha Swiss > C3H. Skin tumors in mice can also be induced by the sequential application of a sub-threshold dose of a carcinogen (initiation phase) followed by repetitive treatment with a weak or noncarcinogenic tumor promoter (promotion phase). The relative order of sensitivity to initiation-promotion is Sencar > > CD-1 > ICR/Ha Swiss ≥ Balb/c > C57BL/6 ≥ C3H ≥ DBA/2. The initiation phase requires only a single application of a carcinogen and is essentially an irreversible step, which probably involves a somatic cell mutation as is evidenced by a good correlation between the carcinogenicity of many chemical carcinogens and their mutagenic activities; the promotion stage, however, is initially reversible, later becoming irreversible. For strains and stocks of mice which respond to initiation-promotion, there is a good correlation between the tumor-initiating activities of polycyclic aromatic hydrocarbons (PAH) and their abilities to bind covalently to DNA. Potent inhibitors and stimulators of PAH tumor initiation appear to effect the level of the PAH diol epoxide bound to specific DNA adducts. However, when the binding of a given PAH to DNA is compared in various stocks and strains of mice, there is no correlation, since in those mice which are able to metabolize PAH, the amounts of carcinogen bound to DNA are similar. The phorbol ester tumor promoters have been shown to have several cellular and biochemical effects on the skin. Of all the observed phorbol ester related effects on the skin, the induction of epidermal cell proliferation, polyamines, prostagladins, and dark basal keratinocytes as well as other embryonic conditions appear to correlate the best with promotion. Mezerein, a weak promoter, was found to induce many cellular and biochemical changes similar to 12-O-tetradecanoylphorbol-13 acetate (TPA), especially epidermal hyperplasia and polyamines; however, it was not a potent inducer of dark cells. We recently found that promotion could be divided into at least two stages. The first stage (I) can be accomplished by limited treatment with TPA or the nonpromoting agents, 4-O-methyl TPA and the calcium ionophore A23187, and the second stage (II) by repetitive applications of mezerein. The dark basal cells appear to be important in the first stage of promotion, since TPA, 4-0-methyl TPA, and A23187 are potent inducers of dark cells. Fluocinolone acetonide (FA) was found to be a potent inhibitor of stage I and II. Retinoic acid (RA) was ineffective in Stage I but was a potent inhibitor of Stage II promotion, whereas tosyl phenylalanine chloromethylketone (TPCK) specifically inhibited Stage I. In addition, FA and TPCK effectively counteracted the appearance of dark basal keratinocytes but had very little effect on polyamines, whereas RA had no effect on dark cells but is a potent inhibitor of TPA-induced ornithine decarboxylase activity and subsequent putrescine formation. These results provide additional evidence for the importance of dark basal keratinocytes (primitive stem cells) in Stage I of promotion and indicate that most of the other cellular and biochemical responses normally associated with promotion (such as polyamines) are actually associated with Stage II of promotion. Although C57BL/6 mice are relatively resistant to initiation-promotion by PAH initiation and phorbol ester promotion, they are fairly sensitive to complete carcinogenesis by PAH. This suggests that the C57BL/6 mice are resistant to phorbol ester tumor promotion. Preliminary experiments suggest that C57BL/6 and Sencar mice respond qualitatively but not quantitatively to a single treatment with TPA.     

Phosphorylation of keratin polypeptides

The phosphorylation of keratin polypeptides was examined in calf snout epidermis. When slices of epidermis were incubated in the medium containing ³²P_i, the radioactivity was incorporated into several proteins. The predominant phosphorylated proteins migrated in SDS-polyacrylamide gels with apparent molecular weight between 49000 and 69000 and coincided with keratin polypeptides. The extent of keratin phosphorylation was not altered in the presence of dibutyryl cyclic AMP or reagents which elevate intracellular cyclic AMP. When homogenates of epidermis were incubated with [γ-³²P]ATP, keratin polypeptides were the predominant species phosphorylated as was also observed in epidermal slices. The presence of cyclic AMP or heat-stable inhibitor of cyclic AMP-dependent protein kinase in the reaction mixture did not affect the phosphorylation of keratin polypeptides, although the phosphorylation of exogenously-added histone was stimulated and inhibited, respectively, by these additions. Keratin polypeptides extracted from calf snout epidermis by 8 M urea were phosphorylated by incubation with [γ-³²P]ATP and cyclic AMP-dependent protein kinase form calf snout epidermis or bovine heart. No proteins were phosphorylated without the addition of the enzymes. The presence of cyclic AMP in the reaction mixture stimulated the keratin phosphorylation, and further addition of heat-stable protein kinase inhibitor reduced this stimulation.     

Chronic exposure to ionizing radiation as a tumor promoter in mouse skin.

We have tested chronic exposure to 90Y beta radiation for its action as a complete tumor promoter, a stage I tumor promoter, or a stage II tumor promoter in SENCAR mouse skin. In skin initiated with a single application of 7,12,dimethylbenz[a]anthracene (DMBA, 10 nmol), chronic exposure to beta radiation as a complete promoter (0.5 Gy, twice/week, 13 weeks) produced no tumors and, when added to a complete chemical promoter (TPA), reduced tumor frequency about 30%. A similar result was observed when beta radiation was tested as a stage II promoter. DMBA-initiated mice that received chemical (12-O-tetradecanoylphorbol-13-acetate, TPA) stage I promotion followed by 13 weeks of beta-radiation exposure (0.5 Gy, twice/week) as stage II promotion produced essentially no tumors, and combining the same chronic beta-radiation exposure with chemical (mezerein) stage II promotion reduced tumor frequency about 20% when compared to a similar group that was not irradiated. Chronic beta-radiation exposure was tested two ways as a stage I tumor promoter in initiated skin that was subsequently treated with mezerein as a stage II promoter. Stage I promotion was shown to proceed with the passage of time, indicating this process occurs naturally in the absence of chemical or physical stimulation. Hyperthermia, previously shown to be a potent inhibitor of chemically stimulated stage I promotion, had no effect on the natural process, indicating at least some differences in mechanism between the two processes. The natural process was, in fact, inhibited by chemical tumor promoters, but not by radiation. In addition to the increase resulting from this natural process, tumor frequency was further increased slightly but significantly (12-15%, P less than or equal to 0.05) when chronic radiation exposure was given as a stage I promoter (0.5 Gy, twice/week, 13 weeks) subsequent to initiation, in spite of the expected 20% reduction resulting from this dose. Exposure of initiated animals to radiation (0.5 or 1.0 Gy, twice/week, 2 weeks) in addition to TPA as stage I promotion produced a similar increase in tumor frequency (P less than 0.02). At higher radiation doses, however, tumor frequency was reduced compared to unirradiated controls. In a third test as a stage I promoter, beta radiation (0.5 Gy twice/week, 4 weeks) was given prior to initiation with N-methyl-N'-nitro-N-nitrosoguanidine in animals subsequently promoted by TPA (twice/week, 13 weeks), and again the radiation slightly but significantly (P less than 0.03) increased tumor frequency compared to the unirradiated control group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Staurosporine, a potent protein kinase C inhibitor, fails to inhibit 12-O-tetradecanoylphorbol-13-acetate-caused ornithine decarboxylase induction in isolated mouse epidermal cells

Staurosporine, a most potent protein kinase C inhibitor, actually inhibited protein kinase C activity obtained either from cytosol or particulate fraction of mouse epidermis. Staurosporine at the concentrations which exert protein kinase C inhibition, however, failed to inhibit, but markedly augmented 12-O-tetradecanoylphorbol-13-acetate (TPA)-caused ornithine decarboxylase (ODC) induction in isolated mouse epidermal cells. Staurosporine by itself induced ODC activity as TPA does. Mechanism of ODC induction seems different between these two compounds. Another protein kinase C inhibitor, H-7, inhibited both staurosporine- and TPA-caused ODC induction.     

An organ culture of adult mouse skin: An in vitro model for studying the molecular mechanism of skin tumor promotion

A simple method to culture explants of adult mouse skin in a modified Eagle's HeLa cell medium was developed in order to further study the biochemical responses to the tumor promoting phorbol esters. The skin explants remained viable for at least 48 hr, as determined by their ability to incorporate ³H-thymidine into DNA as well as to induce epidermal ornithine decarboxylase (EC 4.1.1.17) activity following 12-0-tetradecanoylphorbol-13-acetate addition. The time course of induction of ornithine decarboxylase activity by the tumor promoter was similar to that observed with intact mice. Furthermore, the addition of retinoic acid and indomethacin, the agents that are known to inhibit the induction of ornithine decarboxylase activity by topically applied TPA, also inhibited the induction of ornithine decarboxylase activity by TPA in skin explants.     

Identification of murine type I keratin 9 (73 kDa) and its immunolocalization in neonatal and adult mouse foot sole epidermis

The foot sole epidermis of the fore and hind feet of the adult mouse contains an acidic (type I) mRNA-encoded 73-kDa keratin polypeptide which cannot be detected in any other skin site of the mouse integument. Western blot analysis using an antibody specific for the 64-kDa keratin 9 of human and bovine callus-forming epidermis [A. C. Knapp et al. (1986) J. Cell Biol. 103, 657-667] demonstrates that the 73-kDa keratin represents the murine analog of keratin 9 of man and cow. Concomitant investigations in two related rodent species indicate that the size of this keratin varies more among species than that of any other orthologous keratin. Histological examination of adult mouse foot sole skin reveals an extremely thick and undulated epidermis covering the apical portion of the six footpads, whereas the epidermal-dermal junction of the lateral walls of these nodular protuberances as well as that of the remainder of the foot sole skin is essentially flat. If sections of adult foot sole skin are investigated by indirect immunofluorescence with the keratin 9-specific antibody, intense cytoplasmic staining is restricted to the apical rete pegs of the footpad epidermis in which virtually all suprabasal cells express keratin 9. However, we also observed keratin 9-negative cell columns ascending straight above the tips of the dermal papillae and separating the keratin 9-positive rete pegs from each other. At the transition from the strongly undulated apical epidermis to the flat epidermis of the lateral walls of the footpads, keratin 9-positive cells loose their coherence and gradually disappear toward the inter-footpad epidermis. This intimate relationship between the morphogenesis of epidermal ridges and inter-ridges and the expression of keratin 9 is also visible in foot sole epidermis of neonatal mice. Here we observed the appearance of keratin 9-positive suprabasal cells concomitant with the onset of pronounced folding of the apical footpad epidermis by about Day 3 after birth. Our findings confirm the view that the expression of keratin 9 is characteristic of a highly specialized pathway of epidermal differentiation. We propose a hypothesis for keratin expression in skin sites which are subject to pronounced mechanical wear and tear.     

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis

Studies were conducted to determine the effects of the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured human epidermal cells for comparison with known effects on mouse keratinocytes. In contrast to its effect on mouse cells, TPA did not stimulate human epidermal cell DNA synthesis. TPA stimulated differentiation in human keratinocytes resulting in sloughing of many cells by the 3rd day after exposure. Quantitative assays revealed that 50% of the TPA-exposed population was composed of cornified cells as opposed to 8% in untreated controls. A morphologically distinct cell type (TT cell) emerged after TPA treatment which was triangular in shape, did not stratify, appeared to proliferate rapidly and at most TPA concentrations became the predominant cell type within 1–2 weeks. Cultures composed predominantly of TT cells formed few cornified envelopes, grew well in the absence of TPA and formed colonies at low cell input. In contrast to its effect on keratinocytes, TPA enhanced TT colony formation 3–4-fold and decreased the doubling time of TT cells. Studies were performed to determine the origin of TT cells. Immunofluorescent staining indicated that TT cells lacked the keratinocyte antigens keratin, pemphigus and pemphigoid. Tonofilaments and desmosomes were not seen by electron microscopy. The lack of both melanosomes and standard histochemical DOPA oxidase staining indicated that TT cells were probably not of melanocyte origin. Tests used to identify Langerhans cells were negative. Whereas TT cells, as well as dermal fibroblasts, yielded positive immunofluorescence with antibodies to vimentin, TT cells gave a weak histochemical leucine aminopeptidase reaction, while the reaction of fibroblasts exposed to TPA was strong. Treatment of human dermal fibroblasts with TPA did not yield TT cells. The endothelial cell antigen factor VIII-associated protein was absent by immunofluorescence. These results suggest that the primary effect of TPA on cultured human epidermis is to accelerate terminal differentiation in the keratinocyte population and to stimulate growth of an as yet unidentified cell type.     

Pro/antioxidant Status in Murine Skin Following Topical Exposure to Cumene Hydroperoxide Throughout the Ontogeny of Skin Cancer

Organic peroxides used in the chemical and pharmaceutical industries have a reputation for being potent skin tumor promoters and inducers of epidermal hyperplasia. Their ability to trigger free radical generation is critical for their carcinogenic properties. Short-term in vivo exposure of mouse skin to cumene hydroperoxide (Cum-OOH) causes severe oxidative stress and formation of spin-trapped radical adducts. The present study was designed to determine the effectiveness of Cum-OOH compared to 12-O-tetradecanoylphorbol-13-acetate (TPA) in the induction of tumor promotion in the mouse skin, to identify the involvement of cyclooxygenase-2 (COX-2) in oxidative metabolism of Cum-OOH in keratinocytes, and to evaluate morphological changes and outcomes of oxidative stress in skin of SENCAR mice throughout a two-stage carcinogenesis protocol. Dimethyl-benz[a]anthracene (DMBA)-initiated mice were treated with Cum-OOH (32.8 micro mol) or TPA (8.5 nmol) twice weekly for 20 weeks to promote papilloma formation. Skin carcinoma formed only in DMBA/Cum-OOH-exposed mice. Higher levels of oxidative stress and inflammation (as indicated by the accumulation of peroxidative products, antioxidant depletion, and edema formation) were evident in the DMBA/Cum-OOH group compared to DMBA/TPA treated mice. Exposure of keratinocytes (HaCaT) to Cum-OOH for 18 h resulted in expression of COX-2 and increased levels of PGE(2). Inhibitors of COX-2 efficiently suppressed oxidative stress and enzyme expression in the cells treated with Cum-OOH. These results suggest that COX-2-dependent oxidative metabolism is at least partially involved in Cum-OOH-induced inflammatory responses and thus tumor promotion.     

RasGRP1 represents a novel non-protein kinase C phorbol ester signaling pathway in mouse epidermal keratinocytes

The mouse skin model of carcinogenesis has been instrumental in our appreciation of the multistage nature of carcinogenesis. In this system, tumor promotion is a critical step in the generation of tumors and is usually achieved by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Although it is generally assumed that protein kinase C (PKC) is the sole receptor for TPA in this system, we sought to evaluate whether non-PKC pathways could also contribute to the effects of phorbol esters in skin. We documented expression of the high affinity non-PKC phorbol ester receptor and Ras activator RasGRP1 in mouse primary keratinocytes. Overexpression of RasGRP1 in keratinocytes increased the level of active GTP-loaded Ras. TPA treatment further elevated this Ras activation in a PKC-independent manner and induced the translocation and down-regulation of RasGRP1. Overexpression of RasGRP1 in keratinocytes also caused apoptosis. Finally, induction of keratinocyte differentiation by elevation of extracellular calcium suppressed expression of endogenous RasGRP1, whereas overexpression of RasGRP1 inhibited expression of the differentiation markers keratins 1 and 10 induced by high calcium in the medium. Taken together, our results demonstrate that RasGRP1 is an additional diacylglycerol/phorbol ester receptor in epidermal keratinocytes and suggest that activation of this novel receptor may contribute to some of the phorbol ester- and Ras-mediated effects in mouse epidermis.     

Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation

We report here the isolation and characterization of three antisera, each of which is specific for a single keratin from one of the three different pairs (K1/K10, K14/K5, K16/K6) that are differentially expressed in normal human epidermis and in epidermal diseases of hyperproliferation. We have used these antisera in conjunction with monospecific cRNA probes for epidermal keratin mRNAs to investigate pathways of differentiation in human epidermis and epidermal diseases in vivo and in epidermal cells cultured from normal skin and from squamous cell carcinomas in vitro. Specifically, our results suggest that: (a) the basal-specific keratin mRNAs are down-regulated upon commitment to terminal differentiation, but their encoded proteins are stable, and can be detected throughout the spinous layers; (b) the hyperproliferation-associated keratin mRNAs are expressed at a low level throughout normal epidermis when their encoded proteins are not expressed, but are synthesized at high levels in the suprabasal layers of hyperproliferating epidermis, coincident with the induced expression of the hyperproliferation-associated keratins in these cells; and (c) concomitantly with the induction of the hyperproliferation-associated keratins in the suprabasal layers of the epidermis is the down-regulation of the expression of the terminal differentiation-specific keratins. These data have important implications for our understanding of normal epidermal differentiation and the deviations from this process in the course of epidermal diseases of hyperproliferation.