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Previous studies demonstrated a requirement for arachidonic acid metabolites in tumor development in mouse skin. The goal of this study was to determine whether the arachidonate content of epidermal phospholipids could be altered by increasing dietary levels of linoleate and whether specific metabolites of linoleate and arachidonate have dissimilar biological effects. In a series of tumor studies in which the quantity of dietary linoleate was incrementally increased, a slight reduction in phospholipid levels of arachidonate was observed that correlated with an increased phospholipid level of linoleate and a suppression in tumor yield. A comparison of the arachidonate lipoxygenase metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) with the 13-hydroxyoctadecadienoic acid (13-HODE) lipoxygenase metabolite of linoleate revealed that 12-HETE has biological activities that mimic the phorbol ester tumor promoters, whereas 13-HODE has antithetical effects. Specifically, 12(S)-HETE enhanced the activation of protein kinase C by phorbol esters, mimicked phorbol ester-induced adhesion of keratinocytes to fibronectin and mimicked phorbol ester repression of expression of a differentiation-related gene, keratin-1. 13-HODE blocked 12-HETE-induced cell adhesion and prevented 12-HETE-induced suppression of keratin-1 expression. Overall, these studies suggest that arachidonate and linoleate have opposing functions in the epidermis, particularly with regard to events involved in tumor development. [ABSTRACT FROM AUTHOR]

Interleukin-1 receptor antagonist (IL-1Ra) is an endogenous inhibitor of interleukin-1. The expression of IL-1Ra and interleukin-1alpha (IL-1alpha) was measured in murine epidermis after treatment with tumor promoters and in tumor cell lines. A single treatment with three different tumor promoters (12-O-tetradecanoylphorbol-13-acetate (TPA), anthralin, and thapsigargin) induced IL-1Ra mRNA with different kinetics in mouse skin. The expression of IL-1Ra mRNA also was induced by TPA and IL-1alpha in a dose-related and time-dependent manner in cultured mouse keratinocytes. Expression of IL-1Ra mRNA peaked 6 h after treatment. Both IL-1Ra and IL-1alpha protein and IL-1Ra and IL-1alpha mRNA were measured in various keratinocyte tumor cell lines (C50, MT1/2, HEL30, JWF2, CH72, and BPCC2). The expression of IL-1alpha was increased in papilloma and squamous cell carcinoma cell lines. IL-1Ra protein also was increased in nontumorigenic and papilloma cell lines; however, the expression was dramatically reduced in some carcinoma cell lines. Finally, we detected IL-1alpha and IL-1Ra protein in mouse skin tumors by western blot analysis, and localization was assessed by immunohistochemical analysis. Positive staining for both IL-1alpha and IL-1Ra was observed in the cytoplasm and was most prominent in the suprabasal layer. Although IL-1Ra protein increased in papillomas and carcinomas, IL-1alpha protein was not significantly increased above basal level in most tumors.

An earlier study indicated that increased levels of corn oil in the diet resulted in decreased tumor yield after promotion by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in Sencar mouse epidermis (J Leyton, ML Lee, M Locniskar, MA Belury, TJ Slaga, et al. Cancer Res 51, 907-915, 1991). In the present study we investigated whether corn oil diets could alter the subcellular distribution and activity of protein kinase C (PKC), which is part of an important signaling pathway in carcinogenesis. We used three 15% (wt/wt) fat semipurified diets containing three ratios of corn oil to coconut oil: 1.0%:14.0% (Diet L), 7.9%:7.1% (Diet M), and 15.0%:0.0% (Diet H). The translocation to the membrane fraction of epidermal PKC by 12-O-tetradecanoylphorbol-13-acetate was decreased as the corn oil content of the diet was increased, and this correlates with the decrease in tumor yield. The translocation to the membrane fraction of specific isoforms of PKC was affected by increased dietary corn oil: the largest decreases were in cytosolic PKC-alpha and -beta, and the smallest change was in PKC-epsilon. The other isoforms, PKC-delta and -zeta, were unaffected. The major constituent of corn oil is linoleic acid, which did not affect the binding of phorbol ester to PKC, which suggests that inhibition of such binding was not responsible for the effects of increased dietary corn oil. Products of linoleic acid metabolism, i.e., arachidonic acid and 13-hydroxyoctadecadienoic acid, also did not affect the binding of phorbol ester to PKC. Thus the results of these studies suggest that the subcellular distributions of PKC and its isoforms can be modulated by dietary lipids.

The hairless SKH-1 mouse strain has a higher skin tumor incidence, shorter tumor latency, and higher tumor yield in response to ultraviolet (UV) irradiation than the SENCAR strain. In this study we assessed the differences in UV susceptibility of both strains by measuring DNA photodamage and epidermal proliferation after one UV treatment and after 1, 3, 6, and 9 wk of chronic UV irradiation. Induction rates for cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4) PDs] were significantly greater in the SKH-1 strain than the SENCAR strain, but no strain differences in repair kinetics were detected for CPDs or (6-4) PDs. With chronic UV exposure we observed the following: (i) there was an equal amount of DNA photodamage in both strains; (ii) the number of (6-4) PDs was significantly greater than the CPDs after 6 wk; (iii) there were a significantly greater number of epidermal cells (1.5-fold increase) in the SKH-1 strain; (iv) the number of cycling cells, as measured by 5-bromo-2'-deoxyuridine (BrdU), were located both basally and suprabasally and were significantly greater in the SKH-1 strain; and (v) the number of cells immunoreactive to p53 was equivalent in both strains, but immunoreactive cells were located suprabasally in the SKH-1 strain after 9 wk of UV. These results show that the etiologic role of UV in tumorigenesis is dependent on events other than the amount of DNA photodamage in mouse epidermis.

Differentiation of cultured keratinocytes is controlled by the calcium concentration of the medium and is marked by the expression of differentiation-specific keratins. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) alters the normal differentiation program and suppresses keratin (K) 1 expression. Based on reported similarities in the effects of TPA and the arachidonic acid metabolite 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), we hypothesized that 12(S)-HETE might suppress K1 expression in mouse keratinocytes. We also investigated the effect of pretreatment with 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE) because others have reported that 13(S)-HODE prevents 12(S)-HETE-induced events. In our study, 100 nM 12(S)-HETE mimicked the effect of 500 nM TPA in suppressing K1 mRNA expression within 24 h of calcium-induced differentiation. Pretreatment with 100 nM 13(S)-HODE blocked the 12(S)-HETE effect but not the TPA effect. A role for protein kinase C (PKC) was suggested for both TPA and 12(S)-HETE based on the loss of response with the PKC inhibitors bryostatin-1 or RO-31-8220. Both TPA and 12(S)-HETE stimulated keratinocyte PKC activity. Pretreatment with 13(S)-HODE blocked the 12(S)-HETE-induced increase in PKC activity. Immunoblotting showed that whereas TPA caused a rapid, partial translocation of the PKC alpha isozyme, it had no effect on the distribution of PKC delta. Conversely, 12(S)-HETE had no effect on the distribution of PKC alpha but caused a complete translocation of PKC delta. Pretreatment with 13(S)-HODE prevented 12(S)-HETE-elicited translocation of PKC delta. We conclude that 12(S)-HETE mimics the effect of TPA on K1 mRNA and that the effect is mediated through different isoforms of PKC.

The effects of dietary spray-dried yogurt powder product (YPP) and two strains of lactic acid bacteria on the initiation and promotion stages of carcinogenesis were investigated using the 7,12-dimethylbenz[a]anthracene (DMBA)-12-O-tetradecanoylphorbol-13-acetate (TPA) mouse skin carcinogenesis model. In two independent studies, Sencar mice were fed a diet containing 86%, 43%, or 0% YPP or the 0% YPP diet supplemented with viable cultures of Lactobacillus acidophilus or bifidobacteria. Animals were initiated with a single topical application of DMBA (10 nmol). Promotion began three weeks later with twice weekly treatment of TPA (1 microgram/200 microliters acetone). During the initiation study (Study 1) the experimental diets were fed for four weeks before and one week after DMBA treatment. All mice were then switched to the AIN-76 diet. For the promotion study (Study 2) the experimental diets were begun one week after initiation and fed during the remainder of the study. Gross appearance of tumors was assessed weekly. No statistically significant differences in body weight or food disappearance were observed among the diet groups during the studies. For Studies 1 and 2, final histologically verified papilloma incidence and multiplicity and carcinoma incidence were not statistically different. These data suggest that different levels of YPP or lactic acid bacteria fed during the initiation or promotion stage of carcinogenesis do not significantly affect chemically induced skin tumor development.

To investigate the effect of various levels of corn oil and coconut oil on ultraviolet (UV) light-induced skin tumorigenesis and ornithine decarboxylase (ODC) activity, Sencar and SKH-1 mice were fed one of three 15% (weight) fat semipurified diets containing three ratios of corn oil to coconut oil: 1.0%:14.0%, 7.9%:7.1%, and 15.0%:0.0% in Diets A, B, and C, respectively. Groups of 30 Sencar and SKH-1 mice were fed one of the diets for three weeks before UV irradiation; then both strains were UV irradiated with an initial dose of 90 mJ/cm2. The dose was given three times a week and increased 25% each week. For Sencar mice (irradiated 33 wks for a total dose of 48 J/cm2), tumor incidence reached a maximum of 60%, 60%, and 53% for Diets A, B, and C, respectively, with an overall average of one to two tumors per tumor-bearing animal. For the SKH-1 mice (irradiated 29 wks for a total dose of 18 J/cm2), all diet groups reached 100% incidence by 29 weeks, with approximately 12 tumors per tumor-bearing mouse. No significant effect of dietary corn oil/coconut oil was found for tumor latency, incidence, or yield in either strain. The effect of increasing corn oil on epidermal ODC activity in chronically UV-irradiated Sencar and SKH-1 mice was assessed. Three groups of mice from each strain were fed one of the experimental diets and UV irradiated for six weeks. Sencar mice showed no increase in ODC activity until six weeks of treatment, when the levels of ODC activity in the UV-irradiated mice fed Diet A were significantly higher than those in mice fed Diet B or Diet C: 1.27, 0.55, and 0.52 nmol/mg protein/hr, respectively. In the SKH-1 mice, ODC activity was increased by the first week of UV treatment, and by three weeks of treatment a dietary effect was observed; ODC activity was significantly higher in mice fed Diet C (0.70 nmol/mg protein/hr) than in mice fed Diet A (0.18 nmol/mg protein/hr). Although there was no significant effect of dietary corn oil/coconut oil on UV-induced tumor incidence, the data indicate that chronically UV-irradiated hairless SKH-1 mice are more susceptible to UV-induced skin carcinogenesis than Sencar mice and that this susceptibility is correlated with increased in ODC activity, a parameter of cell proliferation.

The effects of spray-dried yogurt powder product (YPP), bifidobacteria, and Lactobacillus acidophilus were studied during the initiation and promotion phases of carcinogenesis using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced mouse mammary carcinogenesis model. In two separate studies, Sencar mice were fed a diet consisting of 86%, 43%, or 0% YPP or 0% YPP, but with added cultures of bifidobacteria or L. acidophilus. When the animals were 55-63 days old, DMBA was administered by intragastric gavage at 1 mg/mouse and continued once a week for six weeks. During the initiation study, the test diets were fed for four weeks before and during DMBA administration. One week after the final DMBA treatment, all animals were switched to a basal diet based on the AIN-76 formulation. For the promotion study, the diets were introduced one week after the final dose of DMBA and fed for the remainder of the study. Palpable tumor development was monitored weekly throughout the studies. For the initiation study, mice fed 86%, 43%, or 0% YPP or 0% YPP supplemented with bifidobacteria or L. acidophilus had a histologically verified mammary tumor incidence of 15%, 35%, 19%, 30%, and 20%, respectively. The histologically verified tumor incidence for the promotion study was 48%, 58%, 36%, 59%, and 43% in the mice fed diets consisting of 86%, 43%, or 0% YPP or 0% YPP supplemented with bifidobacteria or L. acidophilus, respectively. The data indicate that neither the initiation nor the promotion phase of carcinogenesis is significantly affected by diets composed of 86% YPP, 43% YPP, 0% YPP, or 0% YPP supplemented with bifidobacteria or L. acidophilus.

We investigated the signal transduction pathways leading to the 12-O-tetradecanoylphorbol-13-acetate (TPA)- and interleukin-1 alpha (IL-1)-induced IL-1 alpha mRNA in mouse keratinocytes. Induction of IL-1 alpha mRNA by TPA or IL-1 alpha was followed by increases in cell-associated IL-1 alpha protein measured by enzyme-linked immunosorbent assay. Although protein kinase C (PKC) was involved in TPA-induced IL-1 alpha mRNA, down-regulation of PKC did not block the induction of this gene by TPA. The autocrine induction of IL-1 alpha was not mediated through PKC or cAMP. IL-1 alpha did activate mitogen-activated protein kinase. Genistein, a tyrosine kinase inhibitor, blocked both IL-1 alpha-induced mitogen-activated protein kinase activation as well as IL-1 alpha mRNA expression. Genistein, at an unsaturating dose, plus a serine/threonine kinase inhibitor, H7, completely blocked the autocrine induction of IL-1 alpha suggesting that expression of this gene is regulated by tyrosine kinase(s) in combination or independently with serine/threonine kinase(s). In addition, both TPA and IL-1 alpha caused increases not only in the phosphorylation of c-Jun and c-Fos protein but also in the transactivating activity of AP-1 nuclear transcription factor. Neither TPA nor IL-1 alpha induced NF-kappa B binding activity, as assessed by electrophoretic mobility shift analysis. This study suggests that the activation of AP-1 may be a common event through which TPA and IL-1 alpha induce IL-1 alpha mRNA.

In a previous study (Cancer Res 51, 907, 1991) in which we found an inverse relationship between quantity of dietary corn oil and saturated fat, in a constant 15% fat diet, on the tumor promotion stage of skin carcinogenesis, it was not clear whether one or both types of fat played a modulatory role. The purpose of the present study therefore was to compare the effect of 1) increasing corn oil in corn oil-only diets and 2) increasing saturated fat, with a constant level of 5% corn oil, on tumor promotion. In the first study, the effects of five levels of dietary corn oil (5%, 10%, 15%, 20%, and 25%) on the incidence and rat of papilloma and carcinoma development were determined in female Sencar mice fed these diets one week after initiation with 7,12-dimethylbenz[a]anthracene and three weeks before the start of promotion with 12-O-tetradecanoylphorbol-13-acetate. A papilloma incidence of 100% was reached first in the 5% corn oil group, at 10 weeks, followed by the 10% group at 13 weeks and the 15% and 20% group at 16 weeks. The highest corn oil group achieved a 90% incidence. There were marked differences in latency of carcinoma development among the diet groups. At Week 29, the cumulative carcinoma incidence was 56% and 32%, respectively, in the 5% and 10% corn oil groups, whereas the incidence in the two highest corn oil (20% and 25%) groups was only 8% and 4%, respectively. In the second study, the effects of diets containing 5% corn oil and increasing levels of coconut oil (5%, 10%, 15%, and 20%) on the incidence and rat of papilloma and carcinoma development were determined, as described above. No significant difference in latency or incidence of papillomas or carcinomas was noted among these saturated fat diet groups. It thus appears that higher levels of dietary corn oil are associated with a reduced cancer incidence in this model system.

Increased expression of interleukin-1 (IL-1) in skin elicits a variety of responses, including inflammation and epidermal hyperplasia, which are also characteristic events elicited by tumor promoters. The goal of this study was to investigate whether various classes of tumor promoters increase expression of IL-1 alpha and whether phorbol ester-induced IL-1 alpha expression can be blocked by antitumor promoters. Northern analysis of mRNA isolated from the dorsal skins of SENCAR mice treated with 1 microgram of 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA) showed that a single application of TPA produced a significant increase in IL-1 alpha mRNA at 6 h that decreased by 24 h after treatment. Two treatments of TPA at 48-h intervals induced, at 6 h, twice as much IL-1 alpha mRNA as one treatment. Of the other promoters tested, anthralin (22.6 micrograms), mezerein (2 micrograms), calcium ionophore A23187 (120 micrograms), and benzoyl peroxide (20 mg) induced IL-1 alpha mRNA with different kinetics and to different extents. On the other hand, the non-tumor promoting phorbol ester analogue 4 alpha-12-O-tetradecanoylphorbol-13-acetate had little effect on the expression of IL-1 alpha mRNA. The effects of various antitumor promoters on TPA-induced IL-1 alpha mRNA expression were also assessed. Fluocinolone acetonide, mepacrine, and 5,8,11,14-eicosatetraynoic acid were the most effective inhibitors, and each produced about 80% inhibition. Other antitumor promoters such as retinoic acid, N-tosyl-L-phenylalanine chloromethyl ketone, and butylated hydroxytoluene inhibited approximately 35%, 65%, and 50% of TPA-induced IL-1 alpha mRNA expression, respectively. Therefore, this study suggests a possible role of IL-1 alpha in the promotion stage of skin carcinogenesis.

Increasing levels of dietary corn oil have been correlated with inhibition of 12-O-tetradecanoylphorbol-13-acetate-(TPA) promoted skin tumorigenesis in mice (Leyton et al. Cancer Res. 51, 907-915, 1991). This study was undertaken to assess the effects of dietary corn oil on several events associated with tumor promotion. Three semipurified diets containing 15% (wt/wt) total fat with increasing levels of linoleate (0.8%, 4.5%, and 8.4%) supplied by corn oil were fed to mice for at least four weeks. Although incorporation of linoleate into epidermal phosphatidylcholine increased with increasing amounts of dietary corn oil, the elongated desaturated product of linoleate, arachidonate, was similar or decreased slightly in mice fed the three diets. Minimal activity of delta 6-desaturase, the rate-limiting enzyme in the conversion of linoleate to arachidonic acid, was found in the epidermis compared with the liver, suggesting that linoleate is not converted to arachidonic acid in the skin. Subcellular distribution of protein kinase C was altered in mice fed 0.8% linoleate, where 69% of protein kinase C activity was in the cytosol compared with 78% and 74% for groups fed 4.5% and 8.4% linoleate, respectively. Activation of partially purified protein kinase C isolated from mouse epidermis by linoleate was significantly lower (p < 0.01) than that isolated by arachidonic acid. TPA-induced vascular permeability was significantly greater (p < 0.05), whereas hyperplasia 48 hours after TPA treatment was significantly lower, in mice fed the 8.4% linoleate diet. However, TPA induction of ornithine decarboxylase activity did not appear to be significantly modified by dietary linoleate. These data suggest that cellular processes associated with carcinogenesis are affected by the level of dietary linoleate.