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.     

In vitro induction of human skin ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

A method was developed for the in vitro induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) of ornithine decarboxylase (ODC) activity in human skin punch biopsy samples. Addition of TPA to 1 ml serum-free minimum essential medium containing a single 3-mm human skin punch biopsy sample obtained from a surgical specimen resulted in an induction of ODC activity with a peak activity at 5 hours after TPA addition. In vitro induction of human epidermal ODC activity was dependent on the TPA concentration in the medium; about a twofold increase in ODC activity was observed 6 hours after the addition of 0.1 microM TPA, and about a fivefold increase in ODC activity was observed with 1 microM TPA. TPA also caused about a fivefold to sixfold increase in ODC activity in 3-mm skin punch biopsy samples from healthy volunteers. Human skin punch biopsy samples remained responsive to TPA induction of ODC activity even when stored in serum-free medium at 4 degrees C for 24 hours. A similar degree of induction of ODC activity by TPA was observed whether whole unfractionated human epidermis or a soluble epidermal extract was used for ODC assays. Increased ODC activity was the result of the increase in enzymatically active ODC protein, quantitated by a [3H]difluoromethylornithine-binding assay. Thus human skin, like mouse skin, is responsive to TPA for ODC induction.     

Effects of retinoic acid and juvenile hormone on the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate

The tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), a highly active comitogen in phytohemagglutinin-treated bovine lymphocytes, induces an 11-fold increase in ornithine decarboxylase activity over cultures treated with the lectin alone. This synergistic action of TPA could be antagonized by the simultaneous addition of the acyclic sesquiterpene, insect juvenile hormone III. Retinoic acid (vitamin A acid), an inhibitor of the tumor-promoting action of TPA in mice, was also an effective antagonist but required administration to lectin-activated lymphocytes 1 hr prior to TPA. These data suggest that metabolic activation of retinoic acid is required in order to exert its antagonistic action. Comparison of the responses in the lymphocytes and mouse skin suggests that the lymphocytes provide an excellent system for studying the molecular processes through which phorbol esters and retinoids influence the growth and differentiation of both normal and premalignant cells.     

Tumor promoter-induced refractory state against ornithine decarboxylase induction by 12-O-tetradecanoylphorbol-13-acetate in mouse epidermis

More than one application of the potent tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), to mouse skin at intervals of more than 48 h led to a larger induction of ornithine decarboxylase (EC 4.1.1.17; ODC) than did a single application. In contrast, at intervals of less than 24 h, the first application of TPA appeared to induce a refractory state; the second application of TPA did not induce ODC. The extent of the inhibitory effect caused by the first application of TPA was dependent on the dose. The abilities of a series of phorbol esters to induce the refractory state correlated with their promoting abilities. However, both mezerein and ethylphenylpropiolate, potent hyperplastic agents with little or no promoting properties, induced the refractory state. On the other hand, pretreatment with TPA caused a refractory effect on ODC induction by mezerein but potentiated ODC induction by ethylphenylpropiolate. The epidermal cells escaped from the refractory state by repeated application of TPA at intervals of 24 h as well as at intervals of twice a week; that is, there was a full induction of ODC activity following a second application within 24 h of a prior application. TPA did not elicit production of detectable ODC-antizyme activity in mouse epidermis. Mixing of a soluble extract from mouse epidermis in the refractory state with that from TPA-stimulated epidermis gave essentially additive ODC activity. Elimination of ODC induction by topical application of retinoic acid or injection of cycloheximide concurrent with the first application of TPA did not restore the ability of a second application of TPA to induce ODC. These results suggest that the refractory effect on ODC induction by TPA does not result from feedback regulation of ODC.     

Inhibition of the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate in mouse skin by sphingosine sulfate

We investigated the effect of sphingosine sulfate on the inductionof ODC (ornithine decarboxylase) activity by TPA (12-O-tetradecanoylphorbol-13-acetate)in mouse skin. When applied topically to the shaved skin ofSENCAR mice at dosages of 10–40 µunol per animal,30 min before the superficial application of 8.5 nmol of TPA,sphingosine sulfate dramatically inhibited the induction ofODC activity by the tumor promoter. Significant inhibition ofTPA-induced ODC activity was observed at 4, 6 and 8 h afterTPA treatment in separate studies. The results indicate thatsphingosine sulfate is an effective inhibitor of ODC inductionby TPA in mouse skin.     

Enhancement by adriamycin of the effects of 12-O-tetradecanoylphorbol-13-acetate on mouse epidermal glutathione peroxidase activity, ornithine decarboxylase induction and skin tumor promotion

Adriamycin (ADR) failed to inhibit and paradoxically enhanced the biological action of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis in vivo and in vitro. In the presence of ADR, the tumor promoter caused a greater sequential rapid increase and prolonged decrease in glutathione (GSH) peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity accompanied by a greater decrease in the ratio of reduced (GSH)/oxidized (GSSG) glutathione in isolated epidermal cells. The ability of ADR to deplete the intracellular level of GSH correlated with its ability to increase basal and TPA-induced ornithine decarboxylase (ODC, L-ornithine carboxylase, EC 4.1.1.17) activities. In vivo, topical ADR treatments also enhanced TPA-induced ODC activity as well as the tumor-promoting ability of TPA in the two-stage system of mouse skin carcinogenesis. Since lipid peroxidation has been associated with ADR toxicity, these data suggest that the enhancement of the tumor-promoting ability of TPA by ADR may be the result of an increased oxidative challenge that overwhelms the GSH-dependent antioxidant protective system of the epidermal cells.     

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.     

Independent mechanisms for tumor promoters phenobarbital and 12-O-tetradecanoylphorbol-13-acetate in reduction of epidermal growth factor binding by rat hepatocytes

Primary cultures of hepatocytes derived from adult Fischer 344 rats were used to test for effects of the liver tumor promoter phenobarbital on several components of the epidermal growth factor (EGF) receptor signal transduction pathway. Phenobarbital had no effect on the binding of 125I-labeled EGF by its hepatocyte receptor at 4 degrees C or on EGF-induced receptor down-regulation. However, pretreatment of hepatocytes with phenobarbital (3 mM) at 37 degrees C caused inhibition of subsequent 125I-labeled EGF binding. This response temporally resembled that of hepatocytes to 12-O-tetradecanoylphorbol-13-acetate (TPA) in that maximal inhibition occurred after 1 h of pretreatment but was reversed after longer pretreatment times. The inhibitory effects of phenobarbital and TPA on EGF binding were additive, suggesting that distinct mechanisms mediated the responses to these two tumor promoters. In addition, treatment with TPA, but not phenobarbital, caused a redistribution of the activity of Ca2+/phospholipid-dependent protein kinase C. In untreated and phenobarbital-treated hepatocytes, 20% of protein kinase C activity was isolated with a membranous fraction, while 75% of the activity was membrane associated in TPA-treated hepatocytes. These results demonstrate that phenobarbital, like TPA and other tumor promoters, can modulate the EGF receptor system but suggest that it does so without directly competing with EGF for binding to its receptor or by activating protein kinase C.     

Inhibitory effects of glutathione level-raising agents and D-{alpha}-tocopherol on ornithine decarboxylase induction and mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

The constituent amino acids of reduced glutathione (GSH), GSHitself, and D--tocopherol inhibited 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced ornithine decarboxylase (ODC, L-omithine carboxy-lyase,EC 4.1.1.17 [EC] ) activity in mouse epidermis in vivo and in vitro.The inhibitory effects of cysteine (Cys), GSH and D--tocopherolon ODC induction were proportional to their abilities to decreasethe incidence of skin tumors in the initiation-promotion protocol.Moreover, the ability of the constituent amino acids of GSHand GSH to inhibit TPA-induced ODC activity correlated wellwith their ability to increase the ratio of GSW/oxidized glutathione(GSSG) in isolated epidermal cells. In vitro, various treatmentswith 1 mM GSH, 1 mM glutamic acid (Glu), 1 mM glycine (Gly),0.4 mM Cys and/or 0.2 mM cystine (CysCys) inhibited dramaticallythe sharp decline in the intracellular ratio of GSH/GSSG causedby 0.1 µM TPA. Since the inhibitory effects of Cys onboth the decrease in the ratio of GSH/GSSG and the inductionof ODC activity by TPA were greatly reduced by the inhibitorsof -glutamyl transpeptidase and -glutamylcysteine synthetase,it is suggested that some of the inhibitory effects of Glu,Cys and Gly on tumor promotion could result from their interferencewith the metabolism of the tripeptide GSH, a natural antioxidantwhich inhibits chemical carcinogenesis. The free radical scavengerD--tocopherol, which did not alter directly the intracellularratio of GSH/GSSG, also prevented completely the decrease inthe ratio of GSH/GSSG caused by TPA. These results, therefore,suggest that GSH level-raising agents and other antioxidantsmight inhibit by diverse means the effects of TPA on GSH metabolismand skin tumor promotion.     

Effects of lipoxygenase and cyclooxygenase inhibitors on the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate and isoproterenol in mouse tissues in vivo

I.p. administration of 12-O-tetradecanoylphorbol-13-acetate(TPA) (400 µg/kg) caused a remarkable increase in ornithinedecarboxylase (ODC) activity in CD-1 mouse liver (8.3-fold),spleen (17.8-fold), kidney (4-fold), lung (7.7-fold) and brain(2.7-fold). TPA induced an increase in ODC activity in liver,spleen and kidney in a dose-dependent manner (100–800µg/kg). The putrescine contents of these tissues werealso increased by TPA injection. BW755C, an inhibitor of cyclooxygenaseand lipoxygenase, prevented the TPA-induced increase in ODCactivity in liver, spleen and kidney in a dose-dependent manner.AA861-a selective lipoxygenase inhibitor, also showed the inhibitionof TPA-induced increase in ODC activity in these tissues. Significantinhibition was observed either by BW755C or AA861 at the doseof 30 mg/kg. On the other hand, indomethacin, a selective cyclooxygenaseinhibitor, enhanced the TPA-induced increase in ODC activityin these tissues dose-dependently. Significant enhancement wasobserved at 3 mg/kg for liver and spleen and 1 mg/kg for kidney.The subcutaneous administration of isoproterenol (1 mg/kg) causedan increase in ODC activity in both liver (11-fold) and spleen(3.4-fold). Both AA861 and BW755C failed to inhibit the isoproterenol-inducedincrease in ODC activity in these tissues. These results indicatethat product(s) of lipoxygenase pathway play an important rolein ODC induction caused by TPA in liver, spleen and kidney,while the lipoxygenase pathway does not play an essential rolein the isoproterenol-induced increase in ODC activity.     

Effects of 12-O-tetradecanoylphorbol-13-acetate and mezerein on epidermal ornithine decarboxylase activity, isoproterenol-stimulated levels of cyclic adenosine 3':5'-monophosphate, and induction of mouse skin tumors in vivo.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the antileukemic agent mezerein are diterpene esters of plant origin with certain structural similarities. Both compounds, when applied topically to mouse skin, were equipotent on a molar basis in inducing hyperplasia, inflammation, and ornithine decarboxylase activity, as well as in reducing cyclic adenosine 3':5'-monophosphate accumulation in response to beta-adrenergic stimulation. In contrast, mezerein was much less effective as a tumor promoter; the phorbol ester at 8.5 nmol/application yielded 78-fold more tumors than did 8.5 nmol mezerein per application to similarly initiated SENCAR mice. The superiority of the phorbol ester was nearly as great in CD-1 mice.     

Identification of epidermal cell subpopulations with increased ornithine decarboxylase activity following treatment of murine epidermis with 12-O-tetradecanoylphorbol-13-acetate

Ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway, has been used as a marker for the hyperplasia that occurs following exposure of mouse epidermis to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Using flow cytometry in combination with polyclonal antibodies to ODC, we examined the levels of ODC-associated immunoreactive protein present within mouse epidermal cells at 4 and 24 h after a single topical application of TPA, as well as following chronic exposure to TPA and in papillomas. Basal levels of ODC-specific antibody binding were detectable in acetone-treated CD-1 mouse epidermis and were increased 3-fold at 4 h after TPA treatment. The amount of ODC antibody binding detected after exposure to 17 nmol TPA twice weekly for 3 weeks was similar to that detected within cells isolated from papillomas and was 2.5-fold higher than in cells isolated at 4 h after a single topical treatment of mice with TPA. These observations support the hypothesis that specific subpopulations of keratinocytes constitutively express high levels of ODC following chronic exposure to TPA. The novel method for ODC detection described in these studies provides a means to identify, isolate, and further characterize epidermal cells that may give rise to papillomas and carcinomas.     

Induction of mouse brain ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate is independent of TPA receptor concentration

Ornithine decarboxylase (ODC, E.C. 4.1.1.17) activity was measured in a 35,000 X g brain supernatant fraction, prepared 5 h after intracisternal injection of 12-O-tetradecanoylphorbol-13-acetate (TPA) into developing mouse brain. TPA-dependent induction of ODC activity was maximal on days 5 and 9 postnatally while on day 7, the developmental (endogenous) level of ODC in brain was high and, concurrently, the ability of TPA to induce ODC was reduced. Both TPA-dependent and developmental increases in mouse brain ODC activity were significantly reduced by intracisternal injection of retinoic acid (RA). The efficacy of TPA in elevating ODC activity at postnatal ages 1-220 days-old was independent of both soluble-and particulate-associated TPA receptor concentration. These observations suggest that although TPA receptor activation may be an obligatory event in ODC induction, TPA receptor activation and its concentration per se, are not sufficient determinants for ODC induction and tumorigenesis. Furthermore, the endogenous mechanism of ODC induction is distinct from that of the TPA-dependent increase in ODC enzyme activity.     

Inhibition by 1 alpha, 25-dihydroxyvitamin D3 of induction of epidermal ornithine decarboxylase caused by 12-O-tetradecanoylphorbol-13-acetate and teleocidin B

Topical application of 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3], an active form of vitamin D3, markedly inhibited induction of ornithine decarboxylase caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) and teleocidin B in mouse skin. The degree of inhibition was dependent on the dose and time of application of 1 alpha, 25(OH)2D3. Application of 1 micrograms 1 alpha, 25(OH)2D3 within 30 min before or after treatment with 10 micrograms TPA caused about 72% inhibition of ODC induction at 4 hr. Similar degrees of inhibition were obtained with dose ratios of 1 alpha, 25(OH)2D3 to TPA of 1:3, 1:10, and 1:30. The dose required for 50% inhibition was 0.063 micrograms, or 0.15 nmol, which is about one-half that of retinoic acid, a known inhibitor of induction of ODC activity by TPA. 1 alpha, 25(OH)2D3 had a specific inhibitory effect, in which 100 times higher doses or more of other derivatives of vitamin D3, such as 1 alpha-hydroxyvitamin D3, 25-hydroxyvitamin D3, 24R,25-dihydroxyvitamin D3, and vitamin D3, were required to inhibit ODC induction by TPA. 1 alpha, 25(OH)2D3 did not inhibit epidermal hyperplasia induced by TPA.     

12-O-tetradecanoylphorbol-13-acetate actions on macromolecular synthesis, ornithine decarboxylase, and cellular differentiation of the rat embryonic visceral yolk sac in culture

We have reported previously that 12-O-tetradecanoylphorbol-13-acetate (TPA) disrupts the morphology and functional development of the rat embryonic visceral yolk sac (VYS) maintained in a whole-embryo culture system. The TPA-mediated disruption of the VYS is characterized by the abnormal progressive separation of the cellular layers that comprise the VYS and appears to be related to late-stage promotion. The present study further characterizes this effect of TPA on the VYS of rat conceptuses in vitro. VYS ornithine decarboxylase levels were not induced but rather were initially depressed by TPA treatment. There was no major effect of TPA treatment on VYS hemoglobin content, as measured by absorbance at 414 nm and polyacrylamide gel electrophoresis. Changes in VYS hemoglobin synthesis during the culture period, measured by [14C]leucine incorporation with subsequent autoradiography, was likewise not a major effect of TPA treatment. VYS DNA synthesis and VYS RNA synthesis, measured by [3H]thymidine and [3H]uridine incorporation, respectively, were unchanged by TPA treatment. VYS protein synthesis, measured by [3H]leucine incorporation, was initially increased by TPA treatment but returned to control values by the end of the culture period. This increase in [3H]leucine incorporation was not due to a TPA-mediated change in the secretory function of the VYS. The data suggest that the tumor promoter-induced disruption of the VYS is not associated with cellular proliferation, ornithine decarboxylase induction, or alterations in differentiation. Effects on the cell surface, altering cell-cell interactions and/or communication might best explain these actions of TPA.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-mediated epidermal ornithine decarboxylase induction and skin tumor promotion by new lipoxygenase inhibitors lacking protein kinase C inhibitory effects

Both 2,3,5-trimethyl-6-(12-hydroxy-5, 10-dodecadiynyl)-l, 4-benzoquinone(AA861) and 3, 4, 2‘, 4’-tetrahydroxychalcone inhibited12-lipoxygenase of mouse epidermis. The IC₅₀ of AA861 and 3,4, 2‘, 4’-tetrahydroxychalcone for epidermal 12-lipoxygenasewere 1.9 and 0.2 µM, respectively. These agents showedvery weak inhibitory actions on epidermal cyclooxygenase, withthe potency of inhibition for cydooxy-genase less than 1/50of that for lipoxygenase. Induction of epidermal ornithine decarboxylaseby 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10 nmol/mouse)was potently inhibited by these agents in a dose-dependent manner(1–30µmol/mouse). TPA (5 nmol/mouse)-induced skintumor formation was also strongly suppressed by these agents(15 µmol/mouse). Both AA861 and 3, 4, 2‘, 4’-tetrahydroxy-chalconefailed to inhbit partially purified epidermal protein kinaseC activity. These results support the proposed involvement oflipoxygenase product(s) of arachidonic acid in TPA-induced skintumor promotion.