Retinoids antagonize the induction of ornithine decarboxylase activity by phorbol esters and phospholipase C in rat tracheal epithelial cells

In this study we examined the action of phorbol esters, several phospholipases and retinoids on the induction of ornithine decarboxylase (ODC) activity in rat tracheal epithelial cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induces ODC activity in these cells in a dose-and time-dependent manner. This induction is inhibited by cycloheximide indicating a requirement for protein synthesis. Tracheal epithelial 2C5 cells contain two binding sites for phorbol esters, one with a high affinity KD,1 = 4.58 nM and one with a low affinity KD,2 = 344.8 nM. The ability of several phorbol esters to induce ODC correlates well with the described efficacy with which they bind to the receptor and is in agreement with the concept that phorbol ester receptors are involved in the induction of ODC. There is strong evidence that the phorbol ester receptor is the protein kinase C for which diacylglycerol is the physiological ligand. Treatment of cells with phospholipase C generates diacylglycerol and induces ODC activity in a dose- and time-dependent manner. Treatment with phospholipase A2 or D has no effect on ODC activity. These results support the concept that activation of protein kinase C is related to the induction of ODC activity. The induction of ODC by TPA as well as by phospholipase C is inhibited by retinoids. Specific cytosolic binding proteins for retinoids might be involved in at least some of the responses to these compounds. To examine whether the binding proteins are involved in the inhibition of ODC we determined the presence of these binding proteins and the structure-activity relationship of retinoids. Both retinol and retinoic acid-binding proteins can be detected in 2C5 cells, their levels are 1.06 and 3.36 pmoles/mg protein, respectively. The ability of several retinoids to inhibit ODC induction correlates well with their binding activity and support a role for these binding proteins in the action of retinoids on ODC induction.     

The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC₅₀ was estimated to be about 2.63 × 10^?7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.     

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 diamines on ornithine decarboxylase activity in control and virally transformed mouse fibroblasts.

1. The induction of ornithine decarboxylase activity in mouse 3T3 fibroblasts or an SV-40 transformed 3T3 cell line by serum was prevented by addition of the naturally occurring polyamines putrescine (butane-1,4-diamine) and spermidine. Much higher concentrations of these amines were required to fully suppress ornithine decarboxylase activity in the transformed SV-3T3 cells than in the 3T3 fibroblasts. 2. Synthetic alpha omega-diamines with 3--12 carbon atoms also prevented the increase in ornithine decarboxylase activity induced by serum in these cells. The longer chain diamines were somewhat more potent than propane-1,3-diamine in this effect, but the synthetic diamines were less active than putrescine in the 3T3 cells. There was little difference between the responses of 3T3 and SV-3T3 cells to the synthetic diamines propane-1,3-diamine and heptane-1,7-diamine. 3. These results are discussed in relation to the control of polyamine synthesis in mammalian cells.     

Induction of ornithine decarboxylase activity in mouse tissues by phorbol ester is effectively blocked by methylglyoxal bis(butylamidinohydrazone)

Ornithine decarboxylase (ODC) was induced in the liver, lung and brain of the mouse injected intraperitoneally with 12-O-tetradecanoylphorbol 13-acetate (TPA), showing maximal enzyme activity four hours after the injection. The increase of ODC activity was due to the enhanced syntheses of mRNA and protein. The induction of ODC activity by TPA was specifically blocked by methylglyoxal bis(butylamidinohydrazone) (MGBB), a competitive inhibitor of ODC and S-adenosylmethionine decarboxylase, but not by the analog methylglyoxal bis(guanylhydrazone) (MGBG).     

Differential effect of retinoic acid on growth regulation by phorbol ester in human cancer cell lines.

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and all-trans-retinoic acid (trans-RA) are potent regulators of growth of cancer cells. In this study, we investigated the effect of TPA and trans-RA alone or their combination on proliferation of human breast cancer ZR75-1 and T47D and lung cancer H460 and H292 cell lines. trans-RA caused various degrees of growth inhibition of these cell lines. However, TPA showed inhibition of proliferation of H460 and H292 cells and induction of ZR75-1 cell growth. Although trans-RA did not significantly regulate the growth inhibitory effect of TPA, it completely prevented its growth stimulating function. The divergent effects of TPA were associated with specific disruption of cell cycle events, an induction of G(0)/G(1) arrest in H460 and H292 cells and inhibition of G(0)/G(1) arrest with increase of S phase in ZR75-1 cells. Induction of G(0)/G(1) arrest was accompanied by induction of p21(WAF1) and ERK activity, whereas inhibition of G(0)/G(1) arrest was associated with enhanced activity of JNK and AP-1 but not ERK. trans-RA did not affect TPA-induced p21(WAF1) expression. However, it inhibited TPA-induced AP-1 activity in ZR75-1 cells and the constitutive AP-1 activity in H460 and H292 cells. Thus, trans-RA modulates TPA activity through its interaction through TPA-induced JNK/AP-1 pathway but not TPA-induced ERK/p21(WAF1) pathway.     

Effect of diabetes on the induction of ornithine decarboxylase by refeeding.

Refeeding of starved rats that had previously been schedule-fed increased ornithine decarboxylase activity 140-fold in liver and six-fold in skeletal muscle within three hours. In diabetic rats, refeeding caused a smaller increase in enzyme activity in liver and none at all in muscle. When insulin was administered together with food to the diabetic rats, ornithine decarboxylase in muscle increased to levels greater than those observed in refed controls. The activity of the enzyme in liver also increased; however, the increase was still less than that observed in refed control rats. The data indicate that the induction of ornithine decarboxylase in liver and muscle following food ingestion is altered in diabetes. In addition, they suggest that insulin, or a factor dependent on insulin, modulates the activity of ornithine decarboxylase in skeletal muscle.     

Involvement of cAMP and calcium in the induction of ornithine decarboxylase activity in an osteoblast cell line

The role of cAMP and calcium in the induction of ornithine decarboxylase (ODC, E.C.4.1.1.17) activity in the osteogenic sarcoma cell line, UMR 106-01, was studied, with particular interest for parathyroid hormone (PTH). PTH and forskolin dose-dependently induced the ODC activity and the cAMP production. Protein synthesis is involved in the effect of PTH and forskolin on ODC activity but not on cAMP production. Using quin2 we showed that 20 nM PTH and 10 microM forskolin increased the intracellular ionized calcium concentration ([Ca2+]i), thereby offering the possibility for calcium to play a role as cellular mediator in the action of PTH and forskolin in bone. Data obtained with A23187 showed that solely an increase of the [Ca2+]i is not sufficient to stimulate basal or potentiate PTH- and forskolin-induced ODC activity. However, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced ODC activity point to a specific role for calcium. Moreover, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced cAMP production indicate that the involvement of calcium in the induction of ODC activity is primarily located at another site than the adenylate cyclase. These data indicate that calcium is involved in the control of basal ODC activity. Furthermore, these data suggest that both cAMP and calcium are involved in the induction of ODC activity by PTH and forskolin. More precisely, ODC activity in UMR 106-01 cells can be induced by PTH and forskolin via a calcium-dependent cAMP messenger system.     

Regulation of ornithine decarboxylase mRNA translation by polyamines. Studies using a cell-free system and a cell line with an amplified ornithine decarboxylase gene

The translational control of ornithine decarboxylase (ODCase) by polyamines has been studied using a cellular as well as a cell-free system. A mutant L1210 cell line, in which ODCase represents 4-5% of all soluble protein synthesized, was isolated by stepwise selection for resistance to the ODCase inhibitor 2-difluoromethylornithine (DFMO). The exceptionally high expression of ODCase in these cells was due to amplification of the ODCase gene. When the cells were grown in the absence of DFMO, dramatic increases in cellular putrescine and spermidine levels occurred. These increases were accompanied by a rapid decrease in ODCase synthesis. The change in ODCase synthesis was not associated with an alteration in the amount of ODCase mRNA, demonstrating a translational control in these cells. The effects of polyamines on ODCase mRNA translation were also studied in rabbit reticulocyte lysates using mRNA isolated from the DFMO-resistant cells. Low concentrations of spermidine stimulated synthesis of ODCase and that of total protein, when added to gel-filtered lysates. Notably, optimal stimulation of ODCase synthesis was achieved at a spermidine concentration lower than that required for an optimal rate of total protein synthesis. Higher concentrations of spermidine were inhibitory, and their effects of ODCase synthesis were stronger than on protein synthesis in general, resulting in a decrease in the fraction of protein synthesis accounted for by ODCase. The present results demonstrate that at least part of the feedback regulation of ODCase exerted by the polyamines is due to direct inhibition of ODCase mRNA translation.     

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.     

Two variants of ornithine decarboxylase activity in the mouse liver. The nature of enzyme induction

Ornithine decarboxylase activity in mouse liver is predominantly located in the cell nuclei. After injection of some inducing agents (thioacetamide, diethylnitrosamine, hydrocortisone) the enzyme leaves the nucleus for cytosol. A circadian rhythm of ornithine decarboxylase activity has been observed in nucleus and cytosol, the decrease of enzyme activity in the nucleus being accompanied by its increase in cytosol. The enzyme obtained from intact mice with a minimal level of ornithine decarboxylase activity in the cytosol differs in ion-exchange properties, pH-optimum and Km for ornithine from the thioacetamide stimulated (nucleus enzyme).     

Genetic evidence that a phorbol ester tumor promoter stimulates ornithine decarboxylase activity by a pathway that is independent of cyclic AMP-dependent protein kinases in CHO cells

Ornithine decarboxylase (ODC) inductions by cholera toxin and by the phorbol ester tumor promoter, TPA, were compared in wild-type Chinese hamster ovary (CHO) cells and in mutant cells having altered cyclic AMP-dependent protein kinase activity. The aim of these studies was to determine whether cyclic AMP-dependent protein kinase is involved in these inductions. The time course and the magnitude of ODC inductions by either 100 ng/ml cholera toxin or 100 ng/ml TPA were similar in wild-type cells with a maximum at 3-4 hours after treatment and a return to unstimulated levels by 8 hours. Induction of ODC by cholera toxin was suppressed more than 80% in the four protein kinase mutants studied (10215, 10248, 10260, and 10265), strongly implicating a cyclic AMP-dependent kinase step in the mechanism of induction. Similar results were found with the cyclic AMP analog 8-Br-cyclic AMP and the phosphodiesterase inhibitor, methyl-isobutylxanthine. The induction of ODC by TPA, on the other hand, was only partially inhibited (approximately 50%) in three of four mutants. Lower ODC activity in two mutants stimulated by cholera toxin or TPA whose kinetics were studied in more detail could not be ascribed to a reduced affinity (Km) of ornithine for the enzyme, but appeared to be due to reduced catalytic activity (Vmax) in the extracts. These results suggest that the induction of ODC by TPA proceeds by a mechanism which is only partially dependent on an intact cyclic AMP-dependent protein kinase activity.