Effects of butylated hydroxyanisole on ornithine decarboxylase activity and its gene expression induced by phorbol ester tumor promoter

Butylated hydroxyanisole (BHA) is a phenolic antioxidant that has been found to suppress the activity of skin tumor promoters. In this study, we investigated the effect of BHA on the activity of ornithine decarboxylase (ODC, an indicator of tumor promotion) and its gene expression induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in mouse skin. TPA-induced ODC activity was markedly inhibited by the topical application of 55 mumol of BHA (the inhibition rate at 6 h was about 80%). In Northern and dot-blot analysis, the TPA-induced increase in ODC mRNA was shown to be markedly reduced by the same dose of BHA (the inhibition rate at 4 h was about 60%). These results suggest the involvement of a decrease in ODC gene expression in the mechanism of the inhibition of ODC activity by BHA.     

Effect of ultraviolet A on ornithine decarboxylase and metallothionein gene expression in mouse skin

BACKGROUND: Ultraviolet A (UVA) is known to induce the expression of many stress responsive genes due to the generation of reactive oxygen species (ROS). However, UVA's role in inducing metallothionein (MT) gene expression has not been studied. Furthermore, our group demonstrated that UVA enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated induction of ornithine decarboxylase (ODC) activity in mouse skin (1). METHODS: We examined the interaction of UVA, TPA and antioxidants on the induction of MT and ODC mRNA in mouse skin. Female CD-1 mice were exposed to UVA (19 J/cm2) and total RNA was isolated from the skin. Northern blot analysis for MT and ODC mRNAs was performed. ODC activity in mouse epidermis was also determined in some experiments. RESULTS: UVA induced MT mRNA in mouse skin; however, it did not increase ODC mRNA. 1,4-Diazabicylo-[2,2,2]-octane (DABCO), a singlet oxygen scavenger, reduced UVA-mediated induction of MT mRNA by 40%. The data suggest that ROS produced by UVA exposure may contribute to its ability to induce MT mRNA. UVA slightly enhanced TPA-mediated ODC mRNA induction, while it enhanced ODC enzyme activity 70%. UVA additively intensified TPA-mediated MT mRNA induction. alpha-Tocopherol pretreatment inhibited the induction of ODC enzyme activity by TPA treatment combined with UVA exposure (TPA + UVA); however, alpha-tocopherol had less of an inhibitory effect on ODC mRNA induction by TPA + UVA. Curcumin, a plant pigment, dramatically inhibited both TPA- and TPA + UVA-induced expression of ODC and MT genes. CONCLUSIONS: These results demonstrate that UVA can induce MT gene expression and enhance TPA-induced ODC and MT gene expression. The data further suggest that these effects are partially mediated by ROS.     

Magnitude of ornithine decarboxylase induction by epidermal mitogens: Effect of the assay technique

The polyamines, putrescine, spermidine and spermine, as well as ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, are increased in the blood, urine and skin of psoriasis patients. A study of the association between stimulated epidermal cell proliferation and induction of ODC activity was done using both an intact, living cell ODC assay and the routinely used homogenized cell supernate assay. 10(-6) to 10(-8) mol/l of the tumor promoter, tetradecanoyl phorbol acetate (TPA), 10(-3) mol/l 8-BrcAMP, 10(-6) mol/l cholera toxin and 10(-3) mol/l MIX, which are epidermal cell mitogens, were used to stimulate basal cells in short-term suspension cultures, freshly plated monolayers, or up to 8-day-old cultures. The results showed that the ODC enzyme must be assayed in supernates from disrupted epidermal cells in order for significant ODC induction by hyperplastic agents to be observed.     

Effects of cholera toxin on ornithine decarboxylase activity in mouse skin

The subcutaneous injection of cholera toxin into adult mice resulted in a sustained increase in cyclic AMP levels in mouse epidermis after a lag period of about 2 hr. An increase in ornithine decarboxylase activity occurred between 7 and 10 hr, which was maintained for at least 10 hr. The increase in decarboxylase activity was localized to the area of epidermis visually affected by cholera toxin and was unaffected by hypophysectomy, suggesting a direct effect of the toxin on the epidermal cells. The subcutaneous injection of cholera toxin also led to an increase in cyclic AMP levels in newborn mouse skin. In contrast to adult mice, newborn mouse skin contained high basal activities of ornithine decarboxylase in both the epidermal and dermal fractions. The activity in both fractions was markedly decreased following cholera toxin injection. The ability of cholera toxin to induce both epidermal and dermal ornithine decarboxylase activity developed between 10 and 21 days after birth.     

Cyclo-oxygenase products do not participate in the induction of ornithine decarboxylase in human epidermis

Extensive animal data suggest that prostaglandins are involved in the epidermal induction of ornithine decarboxylase (ODC). We undertook this study to investigate their role during induction of hyperproliferation in human skin. Topical indomethacin (Elmetacin) or vehicle only were applied under occlusion on the backs of healthy volunteers. This was followed 1 h later by Sellotape stripping and biopsies were carried out for the estimation of the levels of ODC. There was no significant difference in the level of ODC in the indomethacin-treated and control sites. Also, test sites were irradiated with 3 MEDs of UVB, and this was immediately followed by the application of indomethacin or vehicle only on the irradiated sites. After 8 h biopsies were taken and the levels of ODC were again similar in both sites. The data indicate that the cyclo-oxygenase products in human epidermis do not contribute to the induction of ODC.     

Independent induction and inhibition of ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in rat epidermis

Changes in the activities of ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) were investigated in rat epidermis after wounding the skin and application of 7,12-dimethylbenz(a)anthracene (DMBA), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and several enzyme inhibitors. Wounding of the skin by vigorous shaving led to a marked induction of ODC activity with a peak at 6 hr. Topical application of a single dose of tetradecanoylphorbol-13-acetate to wounded skin did not affect the activities of ODC and AHH. Application of single large dose (2.5 mg) of DMBA increased AHH activity 7-fold without affecting ODC activity. DL-alpha-difluoromethyl ornithine, a specific irreversible inhibitor of ODC, almost completely abolished ODC activity but did not inhibit DMBA- or TCDD-induced AHH activity. Several potential modifiers, including retinoic acid, indomethacin, 1,3-diamino-2-propranol, alpha-naphthoflavone, and SKF 525 A had unequal effects on ODC and AHH activities. These data indicate that ODC and AHH induction processes in the epidermis are independent biochemical events that are not causally related.     

Effects of butylated hydroxyanisole on ornithine decarboxylase activity induced by ultraviolet-B and PUVA in mouse skin.

The effects of butylated hydroxyanisole (BHA), a representative phenolic antioxidant, on the activity of ornithine decarboxylase (ODC, an indicator of tumor promotion and epidermal hyperproliferation) induced by ultraviolet-B (UVB) or PUVA in mouse skin were investigated. By topical application of BHA (55 mumol), PUVA-induced ODC activity was suppressed by about 60% at both 12 h and 24 h after treatment. In contrast, BHA failed to suppress UVB-induced ODC activity in mouse skin. These results suggest that the induction of ODC activity by UVB or PUVA is mediated by different pathways.     

Regulation of ornithine decarboxylase gene expression, polyamine levels, and DNA synthetic rates by all-trans-retinoic acid in cultured human keratinocytes.

Regulation of ornithine decarboxylase (ODC) gene expression and cell growth by all-trans-retinoic acid in the presence and absence of exogenous putrescine were examined in normal keratinocyte cultures maintained in serum-free medium containing 0.15 mM Ca++. Putrescine and the higher polyamines are negative feedback regulators of ODC synthesis and are essential for cell growth. Human keratinocytes were incubated with and without 1 microM putrescine and the effects of 5 x 10(-7) M retinoic acid on ODC mRNA levels, ODC activity, polyamine levels, and DNA synthetic rates were determined. Northern blot analysis of total RNA isolated from breast reduction keratinocytes treated with retinoic acid up to 24 h showed a time-dependent suppression of ODC mRNA levels that was unaffected by putrescine. ODC activity was suppressed more rapidly in keratinocytes grown in the absence of putrescine; however, at 24 h, ODC activity was suppressed to an equal extent under both culture conditions. The effect of retinoic acid on polyamine levels was determined in the absence of exogenous putrescine. Retinoic acid treatment markedly suppressed putrescine and N1-acetylspermidine levels, whereas spermidine and spermine levels were relatively unaffected. The effect of retinoic acid on DNA synthetic rates, as measured by 3H-thymidine incorporation, was variable. Retinoic acid either stimulated or had little effect on keratinocyte DNA synthetic rates in cells derived from breast reductions and cultured in the absence of putrescine; these effects were not opposed by the presence of exogenous putrescine. In contrast, DNA synthesis in keratinocytes derived from neonatal foreskins was consistently suppressed by retinoic acid, independent of the polyamine status. Our data, therefore, suggest that the effect of retinoic acid on cell growth, as indicated by DNA synthetic rates, does not necessarily parallel its effect on ODC activity and mRNA levels.     

UVB radiation induces phosphorylation of the epidermal growth factor receptor, decreases EGF binding and blocks EGF induction of ornithine decarboxylase gene expression in SV40-transformed human keratinocytes

Abstract We previously demonstrated that epidermal growth factor (EGF) induces a several-fold increase in ornithine decarboxylase (ODC) activity and the steady-state level of ODC mRNA in cultured SV40-transformed human keratinocytes (1). Pretreatment of cell cultures with ultraviolet B (UVB) radiation resulted in a reduction of EGF-induced ODC activity. To determine whether UVB inhibits the accumulation of ODC mRNA by EGF, cells were pretreated with 20 mJ/cm² UVB or sham-irradiated and then incubated with 100 ng/ml EGF. Northern blot analysis revealed that UVB irradiation entirely blocked the EGF induction of ODC mRNA. Since the binding of EGF to its plasma membrane receptor is the first step in initiating a biological response, the effect of UVB on EGF binding was evaluated. UVB treatment of cultured keratinocytes resulted in an immediate and dose-dependent reduction of EGF binding. Scatchard analysis revealed thai the reduction of EGF binding was due to a 52% decrease in the number of available receptors, from 6.2 × 10⁴/cell to 3.0 × 10⁴/cell. However, UVB decreased the EGF-binding affinity very little (Kd = 0.60 nM in control and Kd=0.75 nM in UVB-treated Z114 cells). In addition, UVB did not alter the rate of EGF internalization. These data suggest that UVB blocks the signal transduction pathway of EGF that is involved in regulation of ODC gene expression. Immunoblot analysis of extracts from irradiated cells showed that UVB induced tyro-sine phosphorylation of EGFR and that the quantity of EGFR protein was unaffected by UVB treatment. Phosphorylation of EGFR may be responsible for decreased binding of EGF to its receptor.     

Detection of ornithine decarboxylase gene expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin using in situ hybridization.

The localization of ornithine decarboxylase gene expression in mouse skin by tumour promoter, 12-O-tetradecanoylphorbol-13-acetate, was investigated using in situ hybridization. After 4 h of treatment with tumour promoter, the grains representing ornithine decarboxylase mRNA increased remarkably in number in the epidermis, especially in the follicular region. When the cells containing more than 5 grains were counted as positive, the number of positive cells increased by about 52-fold in the follicular epidermis, by about 19-fold in the interfollicular epidermis and by 4-fold in the dermis, as compared with controls. These results indicate that the epidermal cells are mainly responsible for the activation of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate.     

Sphingosine inhibits phorbol ester-induced inflammation, ornithine decarboxylase activity, and activation of protein kinase C in mouse skin

Tumor promoting phorbol esters, such as 12-0-tetradecanoylphorbol-13-acetate (TPA), when applied topically to mouse skin cause inflammation and hyperplasia. The major cellular phorbol ester receptor is a calcium and phospholipid dependent protein kinase, protein kinase C (PK-C). PK-C is directly activated by TPA and most of the responses of cells to TPA appear to be mediated by PK-C. This suggests that PK-C may play a key role as a mediator of inflammation and growth in TPA treated mouse skin. Sphingosine has been reported to be a potent inhibitor of PK-C in vitro and in intact leukocytes. We therefore have investigated the effects of sphingosine upon TPA-induced inflammation, hyperplasia, induction of ornithine decarboxylase (ODC) activity and ODC mRNA, and activation of PK-C in mouse skin. The results demonstrate that sphingosine is a potent inhibitor of all of the TPA-induced responses examined. These data are compatible with the hypothesis that PK-C is a major mediator of the phorbol ester response in mouse skin. Furthermore, PK-C inhibitors may have therapeutic potential in inflammatory skin diseases such as psoriasis.     

The induction of ornithine decarboxylase in human epidermis is independent of lipoxygenase and cyclo-oxygenase pathways.

In vivo studies in rodents suggest that prostaglandins and/or leukotrienes are involved in the epidermal induction of ornithine decarboxylase (ODC). Recently, we have shown that, in human epidermis, prostaglandins are not involved in this process. Here we report the role of leukotrienes in epidermal ODC induction in human skin. Topical flufenamic acid (Dignodolin), vehicle, or nothing was applied under plastic occlusion to three sites on the backs of healthy volunteers. This was followed 1 h later by Sellotape stripping. After renewed application and occlusion for 8 h, biopsies were carried out for the estimation of ODC levels. There were no significant differences in the levels of ODC between the flufenamic acid treated and control sites. To confirm this finding, test sites were irradiated with 3 MED of UVB. This was immediately followed by the application of flufenamic acid, vehicle, or nothing to the three irradiated sites. After 8 h, biopsies were taken, and the levels of ODC were again similar in the flufenamic acid- and the vehicle-treated sites. The data indicate that, following Sellotape stripping or UVB irradiation, neither lipoxygenase not cyclooxygenase products contribute to the in vivo induction of ODC in human epidermis.     

Sunscreens block the induction of epidermal ornithine decarboxylase by ultraviolet-B radiation: a new way of evaluating sunscreen efficacy in vivo

Two proprietary sunscreen preparations containing para-aminobenzoic acid and sulisobenzone, respectively, were tested for their ability to block the induction of ornithine decarboxylase by medium wavelength ultraviolet radiation (UV-B) in the epidermis of the hairless mouse. Both preparations were effective, the sunscreen treated animals requiring more radiation for ornithine decarboxylase induction. The UV dose-response gradients were reduced by a mean factor of 7–35 (sulisobenzone) and 15 (PABA). These figures correlate well with other in vivo sunscreen assays. This new method provides a simple and reproducible way of evaluating sunscreens in vivo.     

Effects of cyclosporin and ultraviolet radiation on growth and ornithine decarboxylase activity in cultured human epidermal keratinocytes

Both cyclosporin (CyA) and ultraviolet radiation are effective in the treatment of psoriasis, but their precise mechanisms of action are uncertain. We investigated their effects on ornithine decarboxylase (ODC) activity, ODC gene expression, and cellular proliferation stimulated by epidermal growth factor (EGF), in cultured normal human epidermal keratinocytes. CyA (5 μg/ml) inhibited ODC activity, ODC mRNA level, and cell growth induced by 50ng/ml EGF. Ultraviolet B (10 mJ/cm²) irradiation suppressed the induction of ODC, ODC mRNA, and cell proliferation stimulated by EGF, but ultraviolet A (0-15 J/cm²) irradiation inhibited neither EGF-stimulated ODC activity nor cell proliferation. These findings indicate that reduction of ODC activity in CyA- or ultraviolet B-treated human keratinocytes may contribute to the antiproliferative mechanism of these agents. These results also suggest that the regulation of ODC activity by ultraviolet B and A irradiation may be mediated by different signal transduction pathways.     

The induction of epidermal ornithine decarboxylase following tape stripping is inhibited by a topical vitamin D3 analogue (MC903)

The efficacy of MC903, a vitamin D3 analogue, in reducing hyperproliferation as determined by levels of ornithine decarboxylase (ODC) was investigated in a double-blind study of 15 patients with chronic plaque psoriasis. The lesions of psoriasis were treated for 8 weeks with MC903 in one of two different cream bases or with a placebo cream. Biopsies were taken before and after treatment. In addition an uninvolved area of skin was treated during the last 3 weeks and this as well as control areas were then sellotape stripped and biopsied after 8 h. Clinical improvement was seen in eight out of 11 patients treated with MC903 but there was no reduction in the level of ODC in psoriatic lesions after 8 weeks of treatment. The levels of ODC in the tape-stripped uninvolved skin after 3 weeks of treatment with MC903 averaged 22.5 +/- 4.2 pmol/min/mg protein as compared to 58.6 +/- 12.6 pmol/min/mg protein (P = 0.004). The trauma-induced induction of ODC activity was markedly inhibited by the application of MC903.