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.     

Purification of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase from mouse epidermis

Ornithine decarboxylase was purified at least 1500-fold from mouse epidermis pretreated with five consecutive doses of 12-O-tetradecanoylphorbol-13-acetate and 3-isobutyl-1-methylxanthine at 3- to 4-day intervals. Following DEAE-cellulose chromatography and ammonium sulfate precipitation, ornithine decarboxylase was purified further by affinity chromatography. Ornithine decarboxylase was then radioactively labeled by covalently binding [3H]-alpha-difluromethylornithine to the enzyme following polyacrylamide gel electrophoresis under non-denaturing conditions. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis and silver staining of protein, a band was identified that corresponded to a molecular weight of approx. 56,000, coincident with a peak of radioactivity. This is the first study to purify ornithine decarboxylase from mouse epidermis.     

Inhibition by epidermal extracts of the 12-O-tetradecanoylphorbol-13-acetate induced peak of ornithine decarboxylase activity in the mouse epidermis

The time course of induction of epidermal ornithine decarboxylase (E.C. 4.1.117) (ODC) activity following a single topical application of 17 nmoles of 12-O-tetradecanoylphorbol-13-acetate (TPA) on hairless mouse skin was established. Prior intraperitoneal (i.p.) administration of a crude epidermal extract prepared from hairless mouse epidermis led to a time-dependent, 50% inhibition of the peak level of TAP-induced ODC activity. Maximum inhibition was observed when the extract was injected 1.5 h before TPA treatment. The crude epidermal extract did not affect ODC activity in vitro. Following the administration of epidermal extracts, the inhibition of the TPA-induced ODC-response correlated positively with the presence of epidermal G2-chalone activity (determined by a stathmokinetic method) whereas myocardial, skeletal muscle, or heat-inactivated epidermal extracts with no epidermal G2-chalone activity, had no effect on TPA-induced ODC activity. These results indicate a possible relationship between ODC-activity and the control of mitotic rate by G2-chalone.     

Phosphorylation of high molecular weight proteins in platelets treated with 12-O-tetradecanoylphorbol-13-acetate

Changes in the phosphorylation of three high molecular weight cytoskeletal proteins in platelets (actin binding protein, platelet talin and myosin heavy chain) were investigated after treatment with a phorbol ester. All three showed rapid increases in phosphate incorporation, reaching near-maximal values within three minutes. Phosphopeptide maps of the proteins before and after phorbol treatment revealed a single new site in myosin heavy chain, two new peptides in actin binding protein, and multiple sites in talin. These results point to multiple cytoskeletal targets of protein kinase C and suggest complex mechanisms for reorganizing microfilaments.     

Effects of diverse intracellular thiol delivery agents on glutathione peroxidase activity, the ratio of reduced/oxidized glutathione, and ornithine decarboxylase induction in isolated mouse epidermal cells treated with 12-O-tetradecanoylphorbol-13-acetate

Since the enhancement of the activity of the natural glutathione (GSH)-dependent antioxidant protective system of the epidermal cells appears to inhibit the oxidative challenge presumably linked to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA), we have compared the effectiveness of diverse intracellular thiol delivery agents as inhibitors of the effects of TPA on GSH metabolism and ornithine decarboxylase (ODC; L-ornithine carboxylase, EC 4.1.1.17) induction in isolated mouse epidermal cells. Here we report at a 2-mM concentration, the monoethyl and monomethyl esters of GSH, N-acetyl-L-cysteine, and L-2-oxothiazolidine-4-carboxylate are all significantly more effective than GSH in inhibiting the sharp decline in the intracellular ratio of reduced GSH/oxidized glutathione (GSSG), the prolonged decrease in GSH peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity, and the induction of ODC activity caused by 1 microM TPA. Moreover, diethyldithiocarbamate prevents totally the initial drop in the GSH/GSSG ratio of TPA-treated cells and is the most potent inhibitor of TPA-decreased GSH peroxidase activity in relation with its remarkable 98% inhibition of TPA-induced ODC activity, suggesting that the potential antitumor-promoting activity of this compound in mouse skin may be far superior to that previously demonstrated by GSH in the initiation-promotion protocol.     

12-O-tetradecanoylphorbol-13-acetate induces selective desquamation of superficial cells in rat urinary bladder epithelium

Summary 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to affect the proliferation and/or differentiation of several types of cells. We injected TPA directly into the lumen of rat bladder to determine, using scanning and transmission electron microscopy, its effects on the bladder epithelium in vivo. At 1 h after TPA injection (1g/ml), the superficial cells of the epithelium had changed their morphology, and large spherical vacuoles occupied their cytoplasm. In some areas, the underlying intermediate cells were exposed by the desquamation of the superficial cells. During the next few hours, TPA was excreted from the bladder lumen by voluntary micturition, but the desquamation of the superficial cells proceeded further. All the superficial cells were lost from the luminal surface by 24 h after TPA injection. The changes noted were specific for the superficial cells and were not observed in the intermediate or basal cells. After 24h, part of the epithelium had a three-layer structure, indicating that regeneration was taking place. These results demonstrate that TPA selectively affects and desquamates superficial cells in a short period of time. This experimental system may be useful for studying in vivo cell proliferation and/or differentiation.     

Consequences of aberrant ornithine decarboxylase regulation in rat hepatoma cells

DH23A cells, an α-difluoromethylornithine (DFMO)–resistant variant of rat hepatoma tissue culture cells (HTC), contain high levels of very stable ornithine decarboxylase (ODC). In the absence of DFMO, the high ODC activity results in a large accumulation of endogenous putrescine. Concomitant with the putrescine increase is a period of cytostasis and a subsequent loss of viable cells. In contrast, HTC cells with a moderate polyamine content can be maintained in exponential growth. This suggests that a moderate polyamine concentration is necessary for both optimal cell growth and survival. The cytoxicity observed in the DH23A cells is apparently not due to byproducts of polyamine oxidation or alterations in steady state intracellular pH or free [Ca²⁺]. It is possible to mimic the effects of high levels of stable ODC by treatment of cells with exogenous putrescine in the presence of DFMO. This suggests that overaccumulation of putrescine is the causative agent in the observed cytotoxicity, although the mechanism is unclear. These data support the hypothesis that downregulation of ODC may be necessary to prevent accumulation of cytotoxic concentrations of the polyamines. © 1994 Wiley-Liss, Inc.     

Diacylglycerol signals the translocation of CTP:choline-phosphate cytidylyltransferase in HeLa cells treated with 12-O-tetradecanoylphorbol-13-acetate.

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated phosphatidylcholine biosynthesis in HeLa cells was investigated. TPA caused a 3-fold increase in particulate CTP:phosphocholine cytidylyltransferase activity in HeLa cells which correlated with decreased cytidylyltransferase activity in the cytosol. The increase in membrane-associated cytidylyltransferase was confirmed by immunoblotting. Immunoprecipitation studies suggested that TPA had no effect on the phosphorylation state of cytidylyltransferase. Enhanced binding of cytidylyltransferase to diacylglycerol-enriched membranes has previously been shown. Diacylglycerol levels in TPA-treated HeLa cells increased approximately 2-fold (2.29 to 4.02 nmol/mg of protein) after 1 h of TPA treatment. A time course experiment showed a temporal relationship in which production of diacylglycerol appeared to signal translocation of cytidylyltransferase to membranes followed by a stimulation of phosphatidylcholine biosynthesis. Diacylglycerol was further evaluated as a translocator of cytidylyltransferase by depleting HeLa cells of protein kinase C and incubating with dioctanoylglcerol. This treatment increased both membrane-associated cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis approximately 2-fold. A time course experiment with dioctanoylglycerol showed a strong positive correlation (r2 = 0.89) between the amount of particulate cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis. Therefore, TPA stimulates phosphatidylcholine biosynthesis by causing a translocation of cytidylyltransferase from the cytosol to membranes, which appears to be mediated by increased diacylglycerol.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin ornithine decarboxylase and protein kinase C by polyphenolics from grapes

Ornithine decarboxylase is the rate-limiting enzyme in the biosynthesis of polyamines, which are believed to play an essential role in diverse biological processes including cell proliferation and differentiation. We have previously reported [J. Bomser, K. Singletary, M. Wallig, M. Smith, Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds, Cancer Letters 135 (1999) 151-157] that pre-application of a grape polyphenolic fraction (GPF) to mouse skin epidermis inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, as well as 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated, TPA-promoted mouse skin tumorigenesis. The present studies were designed to further characterize the effect of time and dose of application of GPF on TPA-induced ODC activity and protein expression, and on protein kinase C activity in mouse skin epidermis. In addition, the effect of GPF on ODC kinetics in vitro was examined. Application of 5, 10, and 20 mg of GPF 20 min prior to treatment with TPA resulted in a significant decrease in epidermal ODC activity of 54, 53, 90%, respectively, compared with controls. Yet, ODC protein levels (Western blot) in the 10 and 20 mg GPF groups were significantly increased by 1.8 and 1.9-fold, respectively, compared with controls. A similar response was observed with the ODC inhibitor 2-difluoromethylornithine (DFMO), which served as a positive control. Application of grape polyphenolics (20 mg) at 60 and 30 min prior to treatment with TPA inhibited ODC activity by 62 and 68%, respectively, compared with controls (P<0.05). In contrast, application of grape polyphenolics (20 mg) at 60, 120 and 240 min after treatment with TPA resulted in no significant changes in ODC activity. A similar increase in epidermal ODC protein was observed in these GPF-treated animals, similar to that observed when GPF application preceded TPA. When applied to mouse skin prior to TPA, GPF was associated with a decrease in subsequent PKC activity compared with controls at 10 and 30 min following TPA treatment. The GPF-associated decrease in PKC activity preceded the decrease in ODC activity. In a separate in vitro study, kinetic analyses indicated that GPF is a competitive inhibitor of ODC activity. Collectively these data suggest that the grape polyphenolic fraction is effective as an inhibitor of ODC activity when applied before TPA, and that the magnitude of inhibition is independent of epidermal ODC protein content. In addition, GPF is a competitive inhibitor of ODC activity in vitro. The decrease in TPA-induced ODC activity due to GPF treatment is preceded by an inhibition of TPA-induced PKC activity. Thus, the polyphenolic fraction from grapes warrants further examination as a skin cancer chemopreventive agent that interferes with cellular events associated with TPA promotion.     

Inhibition of replicon initiation by 12-O-tetradecanoylphorbol-13-acetate

To investigate the inhibition of DNA replication by tumor promoters, we incubated HeLa cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10^?8 to 10^?5 g/ml) and quantified DNA synthesis on alkaline sucrose gradients. TPA was found to selectively inhibit replicon initiation without affecting DNA chain elongation in replicons that had already initiated. No inhibition of DNA synthesis was seen when cells were exposed to the nonpromoting derivative of TPA, 4-α-phorbol 12,13-didecanoate. Superoxide dismutase did not prevent the TPA-induced inhibition of initiation.     

Staurosporine, a potent protein kinase C inhibitor, fails to inhibit 12-O-tetradecanoylphorbol-13-acetate-caused ornithine decarboxylase induction in isolated mouse epidermal cells

Staurosporine, a most potent protein kinase C inhibitor, actually inhibited protein kinase C activity obtained either from cytosol or particulate fraction of mouse epidermis. Staurosporine at the concentrations which exert protein kinase C inhibition, however, failed to inhibit, but markedly augmented 12-O-tetradecanoylphorbol-13-acetate (TPA)-caused ornithine decarboxylase (ODC) induction in isolated mouse epidermal cells. Staurosporine by itself induced ODC activity as TPA does. Mechanism of ODC induction seems different between these two compounds. Another protein kinase C inhibitor, H-7, inhibited both staurosporine- and TPA-caused ODC induction.     

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.     

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.