Synergistic induction of ornithine decarboxylase by diacylglycerol, A23187, and cholera toxin in guinea pig lymphocytes

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetyl-glycerol (OAG), A23187, and cholera toxin, ornithine decarboxylase activity was induced synergistically, peaking at 6 h. Addition of 12-O-tetradecanoyl-phorbol 13-acetate (TPA), A23187, and dibutyryl cAMP caused the same kind of induction. Cholera toxin potentiated the ability of A23187 to induce ornithine decarboxylase, but not that of OAG. Dibutyryl cAMP augmented the induction caused by A23187 but not by TPA. These results suggest that both the activation of Ca++-sensitive, phospholipid-dependent protein kinase (protein kinase C) and the increase in intracellular levels of Ca++ and cAMP are necessary for this induction. cAMP may potentiate the induction by modulating a Ca++ messenger system other than that for protein kinase C activation.     

Synergistic stimulation of S-adenosylmethionine decarboxylase activity by Ca2+ ionophore A23187, cholera toxin and 1-oleoyl-2-acetylglycerol

The Ca2+ ionophore A23187 induced S-adenosylmethionine decarboxylase in guinea-pig lymphocytes, and cholera toxin stimulated the induction synergistically. The activator of protein kinase C, 1-oleoyl-2-acetylglycerol, did not induce S-adenosylmethionine decarboxylase activity but potentiated the enzyme activity induced by A23187 or by A23187 and cholera toxin. The addition of both A23187 and cholera toxin induced S-adenosylmethionine decarboxylase, but the further addition of 1-oleoyl-2-acetylglycerol or 12-O-tetradecanoylphorbol 13-acetate did not potentiate the enzyme induction in protein kinase-C-down-regulated cells that had been treated with 12-O-tetradecanoylphorbol 13-acetate for 18 h. These results suggest that a Ca2+-dependent pathway, other than that for protein kinase C, is essential for the induction of S-adenosylmethionine decarboxylase and that a cAMP-dependent pathway and also protein kinase C are involved in the potentiation of the induction.     

A possible naturally occurring tumor promoter, teleocidin B from Streptomyces.

Dihydroteleocidin B, a derivative of teleocidin B, when painted on mouse skin, caused marked induction of ornithine decarboxylase within 4 hrs. This induction of ornithine decarboxylase was inhibited by painting the skin with 13-cis-retinoic acid one hour before dihydroteleocidin B. Dihydroteleocidin B induced cell adhesion of human promyelocytic leukemia cells (HL-60) to the surface of culture flasks, and inhibited terminal differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide. Its effective dose for these actions was comparable to that of the potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate. Teleocidin B seems to be a new type of promoter of carcinogenesis.     

Enhanced papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene.

We have studied the induction of papilloma formation in response to skin tumor promotion in transgenic mice overexpressing the human ornithine decarboxylase gene and in their nontransgenic littermates. The transgenic animals displayed a basal epidermal ornithine decarboxylase activity that was nearly 20 times higher than in their nontransgenic littermates. A single topical application of 12-O-tetradecanoylphorbol-13-acetate induced a much more profound and longer-lasting increase in transgene-derived ornithine decarboxylase activity in comparison with the endogenous enzyme activity. Initiation of skin tumorigenesis with a single topical application of dimethylbenz[a]antracene followed by twice-weekly application of 12-O-tetradecanoylphorbol-13-acetate resulted in the appearance of first papillomas both in nontransgenic and transgenic animals by week 7. However, after 11 weeks of 12-O-tetradecanoylphorbol-13-acetate application, the number of papillomas per animal was almost 100% higher in the transgenic animals than in their nontransgenic littermates. These results indicate that an overexpression of epidermal ornithine decarboxylase confers a growth advantage on skin tumors in vivo.     

Induction of ornithine decarboxylase in guinea-pig lymphocytes. Synergistic effect of diacylglycerol and calcium

Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and trifluoperazine inhibited ornithine decarboxylase induction in lymphocytes activated with phytohemagglutinin or inophore A23187. W-7, a more potent calmodulin antagonist than W-5, suppressed ornithine decarboxylase induction in a higher extent than did W-5. These results suggest that calmodulin may play an important role in ornithine decarboxylase induction in the activated lymphocytes. However, the extent of ornithine decarboxylase induction was greater in cells pretreated with Clostridium phospholipase C and then incubated with ionophore A23187 than in cells incubated with ionophore A23187 without the pretreatment. Moreover, combined treatment of cells with ionophore A23187 and tumor promotor, phorbol 12-myristate 13-acetate, caused synergistic induction of ornithine decarboxylase activity. These results, taken together, suggest that both activations of Ca2+-activated phospholipid-dependent protein kinase by diacylglycerol and of calmodulin-dependent function resulted from an elevation of cytosolic Ca2+ concentration may operate in the induction of ornithine decarboxylase in the activated lymphocytes.     

Inhibition of phorbol ester-caused synthesis of mouse epidermal ornithine decarboxylase by retinoic acid

The mechanisms by which topically applied retinoic acid to mouse skin inhibits tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced epidermal ornithine decarboxylase activity were analyzed. Retinoic acid inhibition of the induction of epidermal ornithine decarboxylic activity was not the result of nonspecific cytotoxicity, production of a soluble inhibitor of ornithine decarboxylase, or direct effect on its activity. In addition, inhibition of TPA-caused increased ornithine decarboxylase activity does not appear to be due to enhanced degradation and/or post-translational modification of ornithine decarboxylase by transglutaminase-mediated putrescine incorporation. We found that retinoic acid inhibits the synthesis of ornithine decarboxylase caused by TPA. Application of 10 nmol TPA to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase activity which was paralled by increased [3H]difluoromethylornithine binding and an increased incorporation of [35S]methionine into the enzyme. Application of 17 nmol retinoic acid 1 h prior to application of 10 nmol TPA to skin resulted in inhibition of the induction of activity which accompanied inhibition of [3H]difluoromethylornithine binding and [35S]methionine incorporation into ornithine decarboxylase protein as determined by the tube-gel electrophoresis of the enzyme immunoprecipitated with monoclonal antibodies to it. Inhibition of ornithine decarboxylase synthesis was not the result of the inhibitory effect of retinoic acid on general protein synthesis. The results indicate that retinoic acid possibly inhibits TPA-caused synthesis of ornithine decarboxylase protein selectively.     

Ornithine decarboxylase activity in foetal rat brain cells and in the mouse embryo fibroblasts C3H/10T1/2 CL8 cells: differences in response to medium change and to the tumor promoter TPA

12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced ornithine decarboxylase (ODC, EC 4.1.1.17) in normal, preneoplastic and malignant rat brain cells in culture, but treatment with phorbol, acetate or medium shift resulted in a similar response. Medium shift induced ODC activity in C3H/10T1/2 CL8 cells 4 and 12 hr after treatment. TPA induced only the 12 hr peak. ODC induction in C3H/10T1/2 CL8 cells was completely inhibited by cycloheximide and actinomycin D. Addition of alpha-amanitin abolished the 12 hr peak, but the TPA induced ODC activity was only partly inhibited. ODC induction by TPA was lower in C3H/10T1/2 CL8 cells initiated with 3-methyl-cholanthrene (MCA). ODC increased with TPA up to 10(-7) M and decreased at higher concentrations of TPA.     

Dissociation of tumour-promoter-induced effects on prostaglandin release, polyamine synthesis and cell proliferation of 3T3 cells.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate induces tumour promotion, inflammation, cell proliferation and prostaglandin release. Recent reports suggest that the prostaglandins released by 12-O-tetradecanoylphorbol 13-acetate (TPA) initiate a cascade of events leading to polyamine synthesis and cell proliferation. In experiments designed to test this contention, it was found that addition of TPA (1 microM to 1 nM) to confluent mouse 3T3 fibroblasts successively caused the release of prostaglandins E2 and I2, induction of the enzyme ornithine decarboxylase (EC 4.1.1.17), stimulation of [3H]thymidine incorporation into DNA, and cell proliferation. Pretreatment of the cells with the anti-inflammatory steroid dexamethasone (1 microM) or the non-steroidal anti-inflammatory drug indomethacin (1 microM) inhibited TPA-induced prostaglandin release. However, dexamethasone enhanced the other effects of TPA, whereas indomethacin was ineffective. Addition of prostaglandin E2 to the cultures did not induce ornithine decarboxylase activity and cell proliferation. Pretreatment of the cells with 1,3-diaminopropane (1 mM) or alpha-methylornithine (5 mM), inhibitors of polyamine synthesis, decreased TPA-induced ornithine decarboxylase activity without affecting DNA synthesis. TPA stimulated [3H]thymidine incorporation into DNA, even when the ornithine decarboxylase activity was completely blocked. These data suggest that the proliferative effect of TPA on 3T3 cells is independent of prostaglandin release and polyamine synthesis.     

Prolactin is a tumor promoter in rat liver

Since prolactin, like the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, induces ornithine decarboxylase and plasminogen activator activities, biochemical markers of a trophic response, this hormone might likewise promote neoplasia. To test this theory, rats were initiated with a hepatocarcinogen followed by six weeks of ovine prolactin. This regimen caused hepatomegaly and the development of enzyme-altered foci. Promotion with prolactin for 23 weeks further increased the numbers of enzyme-altered foci. We suggest that prolactin is an endogenous tumor promoter for chemically initiated cells.     

Involvement of two intracellular messenger systems, protein kinase C and cyclic AMP, in the regulation of c-fos gene expression in human promyelocytic leukemia (HL-60) cells

Incubation of human promyelocytic leukemia (HL-60) cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, caused a marked increase in c-fos mRNA in a dose-dependent manner. Phorbol-12,13-dibutyrate and 1-oleoyl-2-acetyl-glycerol, other protein kinase C-activating agents, were also active in this capacity. 4 alpha-Phorbol-12,13-didecanoate, known to be inactive for protein kinase C, was ineffective. 8-Bromo-cyclic AMP (8-Br-cAMP) also increased c-fos mRNA in a dose-dependent manner. This action of 8-Br-cAMP was mimicked by prostaglandin E2, which is known to raise the cyclic AMP level in HL-60 cells. c-fos mRNA increased within 15 min and reached a maximal level 45 min after the stimulation of the cells by TPA or 8-Br-cAMP. The simultaneous stimulation of the cells by TPA and 8-Br-cAMP at the respective doses giving maximal elevation of c-fos mRNA increased this mRNA in an additive manner. These results suggest that in HL-60 cells expression of the c-fos gene is regulated independently by two different intracellular messenger systems, protein kinase C and cyclic AMP.     

Induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol 13-acetate in hamster fibroblasts. Relationship between levels of enzyme activity, immunoreactive protein, and RNA during the induction process

Phorbol ester tumor promoters and growth factors rapidly stimulate ornithine decarboxylase activity in the transformed hamster fibroblast line HE68BP. We report here a close correspondence between the time courses and magnitudes of induction of ornithine decarboxylase activity and immunoreactive ornithine decarboxylase protein following treatment of HE68BP cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) and/or refeeding with fresh medium. Cycloheximide addition to induced cells caused a rapid fall in the levels of both ornithine decarboxylase activity and ornithine decarboxylase protein. Northern blot analysis of RNA isolated from HE68BP cells indicated that treatment with TPA and fresh medium increased the amount of two species of mRNA of lengths 2.4 and 2.1 kilobase. This increased accumulation of ornithine decarboxylase mRNA corresponded temporally to that observed at the protein level, with a 15-fold maximal induction 7 h after treatment followed by a rapid decline in hybridizable RNA. These data indicate that stimulation of ornithine decarboxylase activity by TPA or refeeding involves changes in levels of ornithine decarboxylase mRNA as well as changes in the rate of synthesis of ornithine decarboxylase protein.     

Tumor-promoting phorbol ester amplifies the inductions of tyrosine aminotransferase and ornithine decarboxylase by glucocorticoid

In adrenalectomized rats, the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the inductions of tyrosine aminotransferase (TAT) and ornithine decarboxylase by glucocorticoids, even with sufficient concentration of glucocorticoids to have a maximal effect, whereas it had no effect on TAT activity and increased ornithine decarboxylase activity only slightly in the absence of glucocorticoids. Phorbol derivatives and components of TPA such as 4 beta-phorbol, phorbol 12-tetradecanoate, phorbol 13-acetate, and 4-O-methylphorbol 12-tetradecanoate 13-acetate, which have no tumor-promoting activity or ability to activate protein kinase C, did not have any effect on TAT induction by glucocorticoid. TPA enhanced the induction of TAT by various glucocorticoids but had no effect on induction of TAT by glucagon or insulin and did not enhance the induction of glucose-6-phosphate dehydrogenase by 17 beta-estradiol. These results suggest that TPA specifically enhances the induction of TAT and ornithine decarboxylase by glucocorticoids. Similar effects of TPA on TAT induction by glucocorticoid were observed in primary cultures of adult rat hepatocytes. Another activator of protein kinase C, rac-1,2-dioctanoylglycerol, was also found to have similar effects on the cells.     

Effects of Ca2+ ionophore A23187, 1,2-dioctanoylglycerol, and dibutyryl cAMP on the activity and expression of ornithine decarboxylase in guinea pig lymphocytes

The Ca2+ ionophore A23187 induced small increases in ornithine decarboxylase activity and ornithine decarboxylase mRNA in guinea pig lymphocytes. 1,2-Dioctanoylglycerol potentiated the A23187-induced ornithine decarboxylase activity and the accumulation of mRNA for this enzyme. Dibutyryl cAMP also potentiated the enzyme activity, but had little effect on the accumulation of mRNA. 1,2-Dioctanoylglycerol and 12-O-tetradecanoylphorbol-13-acetate potentiated ornithine decarboxylase activity that had been increased by treatment with both A23187 and dibutyryl cAMP with a consistent increase in the ornithine decarboxylase mRNA. However, dibutyryl cAMP augmented ornithine decarboxylase activity that had been increased by the combination of A23187 and 1,2-dioctanoylglycerol without affecting the ornithine decarboxylase mRNA level. These results suggest that the protein kinase C and cyclic AMP pathways are involved in the enhancement of ornithine decarboxylase activity in guinea pig lymphocytes, but that the mechanisms of the enhancement differ for each pathway, the former increasing the ornithine decarboxylase mRNA level, but not the latter.     

In vivo induction of rat hepatic ornithine decarboxylase and plasminogen activator by 12-O-tetradecanoypphorbol 13-acetate

Rapid and substantial elevations in ornithine decarboxylase and plasminogen activator have been linked to tumor promotion in mouse epidermis and in vitro. Systemic administration of 12-O-tetradecanoylphorbol 13-acetate (TPA) rapidly increased both enzymic activities in rat liver. Pretreatment with either cycloheximide or actinomycin D attenuated both enzyme inductions. It is concluded that: (1) systemic TPA rapidly induces plasminogen activator and ornithine decarboxylase activities in rat liver; and (2) both inductions reflect de novo enzyme synthesis.     

Induction of ornithine decarboxylase in rat liver by phorbol ester is mediated by prostanoids from Kupffer cells

Administration of phorbol 12-myristate 13-acetate (PMA) to rats in vivo resulted in the induction of ornithine decarboxylase activity in the liver which could be blocked by preinjection of indomethacin, a cyclooxygenase inhibitor. In vitro administration of PMA to primary cultures of rat parenchymal cells did not lead to an induction of ornithine decarboxylase activity. It was investigated to what extent non-parenchymal liver cells could play an intermediary role in the expression of the PMA effect on ornithine decarboxylase activity in parenchymal liver cells. Addition of conditioned medium from PMA-activated Kupffer cells to cultured parenchymal cells led to the induction of ornithine decarboxylase activity in parenchymal cells. This effect was not observed with conditioned medium from untreated Kupffer cells or from Kupffer cells treated with PMA plus indomethacin. Conditioned media from PMA-treated or untreated endothelial liver cells were ineffective in the induction of ornithine decarboxylase activity in parenchymal liver cells. Prostaglandin D2, the main eicosanoid produced by Kupffer cells, was able to stimulate the synthesis of ornithine decarboxylase in parenchymal liver cells (up to 40-fold) in a dose-dependent way. Prostaglandin (PG) D2 appeared to be a more potent inducer of ornithine decarboxylase activity in parenchymal cells than PGE1 and PGE2. It is concluded that intercellular communication inside the liver mediated by prostaglandins derived from activated Kupffer cells may form a mechanism to induce synthesis of specific proteins in parenchymal cells.     

Induction of ornithine decarboxylase activity by growth and differentiation factors in FRTL-5 cells

Induction of ornithine decarboxylase has been correlated with the onset of cellular proliferation and cAMP production. Whether the resulting increases in polyamine levels are essential mediators of growth and/or differentiation or are merely incidental remains controversial. We have used FRTL-5 thyroid cells in culture to study the effects of three growth factors on ornithine decarboxylase activity. These factors [TSH, bovine calf serum, and 12-O-tetradecanoylphorbol-13-acetate (TPA)] are thought to act through different intracellular pathways. TSH stimulates cAMP production in thyroid cells, calf serum acts through ill-defined pathways to stimulate growth, and TPA is known to activate protein kinase C. Bovine calf serum and TSH acted synergistically to induce ornithine decarboxylase activity. Activity was maximal when the phosphodiesterase inhibitor, methyl isobutyl xanthine, was included. Individually, neither serum nor TSH was a potent stimulator of the enzyme. Ornithine decarboxylase mRNA was apparent on Northern blots as a doublet following one hour of exposure to these agents. TPA did not stimulate ornithine decarboxylase activity and had an inhibitory effect on enzyme induction by TSH and serum. Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, inhibited growth induced by both TPA and TSH in putrescine-free medium. This effect was not apparent in medium containing 10(-5) M putrescine. The data indicate that, although intracellular levels of cyclic AMP regulate ornithine decarboxylase activity, a component in serum is necessary for significant induction of this enzyme. Factors stimulating growth by non-cyclic AMP-dependent pathways may act without apparently stimulating this enzyme, although polyamines appear to be essential for their growth stimulatory effects.     

Inhibition of polyamine biosynthesis in Acanthamoeba castellanii and 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by chitosanoligosaccharide

Growth of Acanthamoeba castellaniiwas inhibited by chitosanoligosaccharide (up to 20 mg ml^–1) from the shells of crabs but was reversed by the polyamines, putrescine or spermidine, at 0.8 mM. Chitosanoligosaccharide strongly inhibited the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate, a key enzyme of polyamine biosynthesis, which is enhanced in tumour promotion.     

Prostaglandins and skin tumor promotion: inhibition of tumor promoter-induced ornithine decarboxylase activity in epidermis by inhibitors of prostaglandin synthesis.

Application of 12-O-tetradecanoylphorbol-13-acetate to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase (EC 4.1.1.17; L-ornithine carboxy-lyase) activity. The degree of induction was remarkably depressed by prior treatment of skin with indomethacin, acetylsalicylic acid or flufenamic acid, inhibitors of prostaglandin synthesis. In contrast, dexamethasone, a steroidal anti-inflammatory drug, was ineffective. The inhibition of tumor promoter-induced ornithine decarboxylase activity by the non-steroidal anti-inflammatory drugs was completely counteracted by treatment with prostaglandin E₁ and E₂ but not with prostaglandin F_1α or F_2α.