Inhibition of mouse skin tumor promotion and of promoter-induced epidermal polyamine biosynthesis by methylglyoxal bis(butylamidinohydrazone)

Effects of methyiglyoxal bis(butylamidinohydrazone) (MGBB),a reversible inhibitor of ornithine decarboxylase (ODC) andS-adenosylmethionine decarboxylase (AdoMetDC), on 12-0-tetradecanoylphorbol-13-acetate(TPA)-induced increases of ODC and AdoMetDC activities, ODCmRNA level and polyamine contents in mouse skin were investigatedin connection with tumor formation. Formation of papillomasby applications of TPA to 7,12-dimethylbenz[a]anthracene (DMBA)-initiatedmouse skin was effectively inhibited by simultaneous topicalapplications of MGBB. MGBB also dose-dependently inhibited theability of TPA to induce increases of ODC activity, ODC mRNAlevel and the accumulation of putrescine and spermidine in mouseskin. Induction of AdoMetDC activity was not affected by thedrug. These inhibitory effects of MGBB on ODC induction andtumor promotion were more evident in multiple application experimentsthan with a single application of the drug.     

Ornithine decarboxylase activity and DNA synthesis after treatment of cells in culture with 12-O-tetradecanoylphorbol-13-acetate.

The ability of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce the enzyme ornithine decarboxylase (ODC) and to stimulate DNA synthesis was studied in four different cell types in vitro. The effects of this agent on each cell type were different: (a) in hamster embryo cells, TPA induced ODC but had no effect on DNA synthesis; (b) TPA induced ODC and stimulated DNA synthesis in BALB/c 3T3 mouse cells; (c) it did not induce ODC in human fibroblasts but did stimulate DNA synthesis; and (d) it induced neither ODC nor DNA synthesis in rat embryo fibroblasts. In contrast to the effects of TPA, ODC was induced and DNA synthesis was stimulated in all cell types by fresh serum-containing medium. Treatment of the cells with a combination of fresh medium and TPA resulted in an approximate summation of the effects of treatment with each agent alone. These results emphasize the differences in the responses of various cells to TPA. They also show that in some cells, at least, the induction of ODC and stimulation of DNA synthesis following TPA treatment can be regulated independently.     

Inhibitory effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced increase in ornithine decarboxylase mRNA in mouse epidermis

Application of 5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA)to the skin of female CD-1 mice led to a rapid increase in theconcentration of epidermal ornithine decarboxylase (ODC) mRNAfrom an undetectable level in control mice to a high maximumlevel at 4–5 h after TPA administration. The concentrationof epidermal ODC mRNA then decreased rapidly during the next5 h. The time course for TPA-induced increases in epidermalODC enzyme activity paralleled very closely the time coursefor TPA-induced increases in ODC mRNA. Topical administrationof 1, 3 or 10µmol curcumin together with 5 nmol TPA inhibitedby 66, 81 and 91% respectively TPA-induced increases in epidermalODC enzyme activity measured 5 h later. In a parallel study,TPA-induced increases in the concentration of epidermal ODCmRNA was inhibited by 54, 85 and 82% respectively. Intraperitonealinjection of 10 or 30 µmol curcumin 1h before topicalapplication of 5 nmol TPA inhibited TPA-induced increases inepidermal ODC enzyme activity by 75 or 89% respectively. Ina parallel study, the induction of epidermal ODC mRNA was inhibitedby 53 and 65% respectively. The results indicate that curcumininhibits TPA-induced increases in epidermal ODC enzyme activityby inhibiting the synthesis and/or enhancing the breakdown ofODC mRNA.     

Regulation of ornithine decarboxylase gene expression in mouse epidermis and epidermal tumors during two-stage tumorigenesis

Topical treatment of mouse skin with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) results in an array of biochemical alterations, one of the earliest being a more than 200-fold transient induction of epidermal ornithine decarboxylase (ODC) activity. There is an excellent correlation between the induction of epidermal ODC activity and changes in the level of immunoreactive ODC protein following a single TPA treatment to skin. Both ODC activity and protein levels peak at 4.5 h after TPA treatment and rapidly fall to basal levels by 24 h. Cycloheximide treatment of mice in which ODC had been previously induced by TPA indicated a similar rapid turnover of both ODC catalytic activity and protein levels. Northern blot analysis of polyadenylated RNA isolated from mouse epidermis after a single TPA treatment revealed the stimulation of one species of ODC mRNA of 2.0 kilobases with a maximum at 3.5 h declining by 16 h. The same-sized species of ODC mRNA was detected 4.5 h after multiple biweekly treatments with TPA as well as in mouse papillomas and carcinomas not treated with TPA for at least 1 week. Southern blot analysis of EcoRI or BamHI digests of DNA derived from mouse liver, papillomas, or carcinomas revealed no ODC gene amplification or rearrangement during neoplastic progression. These observations indicate that the induction of epidermal ODC activity following TPA treatment results in a transient increase in the steady state levels of ODC mRNA and in the rate of synthesis of ODC protein, in contrast to epidermal tumors where the levels of ODC mRNA and protein are constitutively elevated.     

The tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid differ in toxicity, mitogenic activity and induction of gene expression

Okadaic acid (OA) and 12-O-tetradecanoylphorbol-13-acetate (TPA)are both potent tumor promoters in a mouse skin carcinogenesisexperiment. OA was much more toxic than TPA for murine embryocell lines such as Swiss 3T3 cells or C3H10T? cells. TPA isa potent mitogen for 3T3 cells; in contrast OA was unable tostimulate DNA synthesis in these cells. TPA induces a familyof primary response genes, the TPA induced sequence (TIS) genes,in a wide variety of cells. Although OA induced modest levelsof TIS mRNA expression, the time course of the induction ofTIS1 and TIS8 mRNA was delayed when compared to induction byTPA or peptide mitogeas such as fibroblast growth factor (FGF).In addition TPA-mediated down-regulation of protein kinase Cattenuated TIS gene induction by OA, but not by FGF.     

Dissociation between induction of ornithine decarboxylase and oxidative burst by phorbol esters in a macrophage cell line

In order to investigate the correlation between stimulationof superoxide generation and induction of ornithine decarboxylase(ODC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) we haveused the macrophage cell line J774.16 and a clone derived fromthis line that, by contrast with the parental line, is unableto generate superoxides in response to TPA. No difference wasobserved between the normal and the defective cells, with respectto ODC induction by TPA over a wide range of TPA concentrations(0.2–5.0 µg/mI). Similar results were obtained comparingresident and caseinate-elicited mouse peritoneal macrophages.Although resident macrophages did not generate superoxides inresponse to TPA, they did not differ from superoxide-generating,caseinate-elicited macrophages with respect to ODC induction.These data suggest a dissociation between the stimulation ofthe oxidative burst by TPA and a growth factor-like effect suchas ODC induction.     

Heterogeneity of ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin and in epidermal tumors

One of the earliest events after treatment of mouse skin with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is the induction of ornithine decarboxylase (ODC). Using an immunoperoxidase technique with a rabbit antiserum specific for ODC, the localization of cells containing high levels of ODC following TPA treatment was determined. CD-1 female mice treated with multiple topical applications of TPA and killed 4.5 h after the last TPA treatment exhibited a heterogeneous localization of ODC in this hyperplastic epidermis. The cells which exhibited intense immunostaining were found predominantly in the suprabasal cells lining the hair follicles. This specific ODC staining in cells surrounding hair follicles was inhibited by pretreatment of mice with either retinoic acid or cycloheximide 1 h before TPA treatment. The induction of ODC-specific staining after TPA treatment in hyperplastic mouse skin was transient, since no staining was observed 16 or 24 h after TPA treatment. In contrast, benign papillomas produced by two-stage tumorigenesis contained some cells demonstrating high levels of ODC a week after the last TPA application. These results indicate that both normal mouse epidermal cells as well as tumor tissue display cellular heterogeneity of ODC expression.     

Human Harvey-ras is biochemically different from Kirsten- or N-ras

The biochemical effects of the human H-, N- and K-ras oncogenes were studied. We analysed the induction of c-fos mRNA and protein by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in exponentially growing NIH3T3 fibroblasts transformed by transfection with ras oncogenes. We found that H-ras has the unique ability to inhibit c-fos induction by TPA. In contrast, normal c-fos expression was induced by TPA in fibroblasts transformed by N- or K-ras or by the ras-unrelated oncogenes dbl and trk. The inhibition of c-fos induction by H-ras was not due to alteration in the binding of TPA to the transformed cells or to the selection of idiosyncratic clones. These results provide clear evidence that H-ras is functionally different from K- or N-ras.     

Palmitoylcarnitine reverses 12-O-tetradecanoylphorbol-13-acetate-induced refractory state for the TPA-caused ornithine decarboxylase induction in mouse epidermis

When a single topical application of 12-O-tetradecanoylphorbol-13-acetate(TPA) was performed 12 h before the second application, ornithinedecarboxylase (ODC) induction by the second application of TPAwas markedly suppressed (refractory state). However, at intervalsof 96 h between the first and the second application, the ODCactivity induced by the second application of TPA was higher(enhanced state) than the activity induced by the single application.When various antitumor promoting agents, i.e. p-bromophenacylbromide, nordihydroguaiaretic acid, quercetin, 1-tosylamide-2-phenylethylchloromethyl ketone, retinoic acid and palmitoylcarnitine, wereapplied concurrently with the first TPA application, the ODCinduction in the refractory state was restored only by palmitoylcarnitine,but not by other anti-tumor promoting agents. None of theseanti-tumor promoting agents affected the ODC induction in theenhanced state. Stearoylcarnitine also had the restorative effectbut was less effective than palmitoylcarnitine. Acetylcarnitineand palmitic acid were not effective. Pretreatment of mice withTPA 12 h or 96 h before the second TPA application resultedin the reduction or the increase in the V_max values of ODC bothfor ornithine and pyridoxal-5'-phosphate, respectively. Palmitoylcarnitinerestored these reduced V_max values to the control values. Twelvehours after TPA treatment, the epidermal protein kinase C activityof both cytosol and particulate fractions decreased moderately.At 96 h after TPA application, protein kinase C activities ofboth cytosol and particulate fractions were fully or at leastpartially restored to the control levels. Protein kinase C activitiesboth in the cytosol and the particulate fractions tended tobe restored by palmitoylcarnitine, but the effect was not alwaysreproducible. The TPA-induced refractory state and the enhancedstate for ODC induction appear to result from the changes inthe protein kinase C activities caused by TPA. However, it isnot known whether such changes in the protein kinase C activitiesare the major causes for the TPA-induced refractory and/or enhancedstate for ODC induction and whether or not the restorative effectof palmitoylcarnitine is due to its modulating action on proteinkinase C activity.     

Protein kinase C isotypes required for phorbol-ester induction of stromelysin-1 in rat fibroblasts

The phorbol-ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of the metalloproteinase stromelysin in fibroblasts in vivo and in several cultured cell lines. Rat-1 and Rat-2 fibroblasts, however, do not respond to TPA stimulation by induction of stromelysin gene activity, although collagenase promoter-mediated activity is induced threefold by TPA treatment in these cells. We determined that rat fibroblasts expressed protein kinase C (PKC) α, PKCδ, PKCϵ, and PKCζ but neither the mRNA nor the protein for PKCβ. When Rat-2 fibroblasts were stably transfected with an expression vector producing PKCβ, however, TPA treatment of these variants resulted in a 3.1-fold induction of stromelysin promoter-mediated luciferase activity compared with a 1.3-fold induction in parental Rat-2 cells (P < 0.002). Transient transfection of PKCϵ produced a small but significant increase in TPA-stimulation of both stromelysin- and collagenase-mediated gene expression. These results suggest that there are PKC isotype-specific signaling pathways that can differentially regulate matrix metalloproteinase gene expression. © 1996 Wiley-Liss, Inc.     

The leucine zipper domain of avian cMyc is required for transformation and autoregulation

Small deletions of 7 to 48 amino acids have been generated in the leucine zipper domain of the avian cMyc protein and the mutant cMyc proteins expressed using an avian retroviral vector. Retrovirally encoded cMyc protein transforms primary chick embryo fibroblasts and leads to abnormal regulation of the endogenous c-myc gene. Deletion of the most C-terminal leucine of the zipper motif confers a partial phenotype affecting some but not all parameters of transformation. Complete loss of transforming activity results from deletion of further leucine residues, including one which is not part of the heptad repeat. In cMyc transformed cells endogenous c-myc mRNA is expressed at a low level and is abnormally refractory to induction by serum stimulation. In contrast, a non-transforming cMyc protein which lacks the zipper does not affect normal c-myc expression. These results demonstrate that the leucine zipper domain of avian cMyc is required for both transformation and autoregulation, and suggests that essential leucine residues within the motif may be spaced differently from those in the zippers of Fos and Jun.     

Inhibition by indomethacin and 5,8,11,14-eicosatetraynoic acid of the induction of rat hepatic ornithine decarboxylase by the tumor promoters phenobarbital and 12-O-tetradecanoylphorbol-13-acetate in vivo

The involvement of arachidonate metabolism in the inductionof rat hepatic ornithine decarboxylase (ODC) activity by thetumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) andphenobarbital (PB) was investigated. Pretreatment of the ratswith indomethacin or 5,8,11,14-eicosateraynoic acid dose dependentlyinhibited the induction of ODC by both tumor promoters. Bothinhibitors were more potent inhibitors of PB induction thanTPA induction of ODC. The data are consistent with an involvementof arachidonate cyclooxygenase products in the induction ofrat hepatic ODC by the tumor promoters.     

Acute effects of 12-Otetradecanoylphorbol-13-acetate, teleocidin B, or 2,3,7,8-tetrachlorodibenzo-p-dioxin on cultured normal human bronchial epithelial cells

Effects of teleoddin B, 12-O-tetradecanoylphorboi-13-acetate(TPA), phorbol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and2,7-dichlorodibenzo-p-dioxin (DCDD) on normal human bronchialepithelial cell cultures were assessed by quantitation of cellularmorphology, clonal growth (population doublings per day), cross-linkedenvelope (CLE) formation and the enzymatic activities of arylhydrocarbon hydroxylase (AHH), ornithine decarboxylase (ODC)and plasminogen activator (PA). Toxicity was assessed by clonalgrowth assays. Teleocidin B and TPA had similar effects on growth,morphology and enzyme activities. When the cells were incubatedwith TPA or teleocidin B at concentrations of 1–100 nMfor 6 h, RNA synthesis was unaffected, but DNA synthesis decreasedand squamous differentiation, marked by an increase in cellsurface area and cross-linked envelope formation, was increased.TPA and teleocidin B also increased ODC activity in LHC-0 medium(a maintenance medium without epidermal growth factor) but causeda decrease of ODC activity in LHC-4 (a growth medium containingepidermal growth factor). Finally, TPA and teleocidin B eachcaused an increase of PA and a decrease of AHH activities inboth media. Phorbol, a non-promoting analogue of TPA, had noeffect on growth, morphology or biochemical assays. TCDD (100nM) caused a 15% decrease in cell growth when cells were incubatedin LHC-4, and this was accompanied by an increase in cell surfacearea, PA activity, and CLE formation. TCDD caused an increasein AHH and ODC activities when the cells were incubated in eitherLHC-0 or LHC-4 medium. DCDD did not alter cell growth, and itsmorphological and biochemical effects were similar to thoseof TCDD although less marked. In conclusion, results reportedhere are consistent with the hypothesis that an important propertyof some tumor promoters is their ability to induce terminaldifferentiation in normal, non-initiated epithelial cells.