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.     

Ornithine decarboxylase activity and the accumulation of its mRNA during early stages of liver regeneration

The marked and well documented stimulation of hepatic ornithine decarboxylase (ODC; EC 4.1.1.17) in response to partial hepatectomy is at least to some extent attributable to an enhanced accumulation of the enzyme's mRNA. The stimulation of ODC activity was associated with an increased accumulation of two ODC-related mRNA species (2.1 and 2.6 kilobases; kb) as revealed by Northern blot hybridization analyses. The levels of the above-mentioned messages remained elevated for 6 h after partial hepatectomy, at which time the enzyme activity had returned to almost control levels. Furthermore, ODC protein levels remained relatively stable after the first peak of ODC activity, suggesting that posttranslational activity was responsible for the changes in ODC activity after the initial burst. In addition to the two mRNA species typical of mouse cells, rat tissues contained a third hybridizable message (1.6 kb). This smaller poly(A)+ RNA was never seen in samples obtained from mouse or human cells, but was always present in samples obtained from rat tissues. Interestingly this rat-specific message appeared to be expressed in somewhat opposite manner to the other two mRNA species.     

Haemosiderin-like properties of free-radical-modified ferritin.

Evidence was sought that the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mouse epidermal ornithine decarboxylase (ODC, EC 4.1.1.17) activity involves both increased ODC mRNA and ODC protein. Application of 10 nmol of TPA to mouse skin led to a dramatic increase in soluble epidermal ODC activity which paralleled an increase in amount of enzymically active ODC protein as determined by gel electrophoresis of immunoprecipitated difluoromethyl[3H]ornithine-bound ODC. Application of TPA to mouse skin also resulted in an increase in ODC mRNA measured by dot-blot analysis using a radiolabelled cDNA probe. ODC mRNA induction preceded the increase in ODC activity by TPA. TPA-increased ODC mRNA displayed a single major band of 2.1 kilobases in size identified by the Northern blotting procedure.     

促癌物TPA诱导小鼠表皮鸟氨酸脱羧酶mRNA表达和维甲类化合物对该表达的影响

強皮肤促癌物十四烷酰佛波醋酸酯(TPA)局部应用时可触发一系列的生物化学改变,其中最明显的事件之一就是对ODC活性的短暂而急到的诱导,而这种诱导作用与其促癌作用密切相关。利用Northern印迹分析和条带(Slot)印迹分析证明,10nmol/L TPA一次局部处理小鼠背部皮肤可刺激ODC mRNA(2.0kb大小)表达,在4h左右最为明显,随后逐渐降低。10nmol/L TPA多次处理小鼠皮肤(每2天一次,共4次)也有类似的促进作用,但却在6h左右最为明显。在二甲基苯蒽和巴豆油诱发的二阶段小鼠皮肤乳头瘤和癌组织中也观察到了相同大小的ODC mRNA的高水平表达,尤以癌组织最高。新维甲类化合物R8605虽能明显抑制巴豆油诱导的ODC活性,但却未见对TPA诱导的ODC mRNA增加有明显抑制作用。     

Effect of dexamethasone on the activity and expression of ornithine decarboxylase in rat liver and thymus

A single intraperitoneal injection of the synthetic glucocorticoid dexamethasone into rats resulted in a marked stimulation (more than 60-fold) of hepatic ornithine decarboxylase (ODC) at 4 h after the injection, whereas the enzyme activity in thymus was almost totally (about 95%) depressed at the same time. The stimulation of ODC activity in liver was in all likelihood attributable to a greatly enhanced accumulation of mRNA species for the enzyme as revealed by Northern blot and dot-blot hybridization analyses. ODC activity in thymus, in response to dexamethasone, was only 5% of that found in control animals, but this decrease was apparently not accompanied by similar reductions of the levels of ODC message, which was in fact decreased only by 50% at the maximum. In addition to two mRNA species (2.1 and 2.6 kilobases; kb), typical to mouse cells, rat tissues seemed to contain a third hybridizable message for ODC, smaller (1.6 kb) than the above-mentioned species and not seen in samples obtained from mouse or human cells. Interestingly, these smaller poly(A)+ RNA sequences, hybridizable with cDNA complementary to mouse ODC mRNA, were apparently constitutively expressed, as the treatment with glucocorticoid altered the amount of these sequences only slightly.     

Increased efficiency of translation of ornithine decarboxylase mRNA in mitogen-activated lymphocytes

Ornithine decarboxylase (ODC) mRNA was elevated ninefold by 6 h following concanavalin A (ConA) stimulation of bovine lymphocytes. Comparison of the increases in ODC mRNA and ODC activity revealed a fivefold discrepancy, which is consistent with a change in efficiency of translation of ODC mRNA. In resting cells, 45% of the total ODC mRNA was associated with particles sedimenting at about 40 S, and therefore was not translated. The untranslated ODC mRNA in resting cells could be completely shifted into polysomes by a 15-min treatment of the cells with appropriate concentrations of cycloheximide. In activated cells, the proportion of ODC mRNA in untranslated material was reduced to 18%. This shift in distribution of ODC mRNA occurred between 6 h and 12 h following mitogen stimulation with no increase in the cellular level of this message. The rate of synthesis of ODC protein was found in increase twofold between 6 h and 12 h, paralleling the increase in the amount of ODC mRNA associated with polysomes. Thus, in this time frame, a decrease in the amount of untranslated ODC mRNA with a corresponding increase in the amount associated with polysomes leads to an increase in the biosynthesis of ODC with no change in the cellular level of the message. These changes in translational efficiency were not observed with actin mRNA.     

Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.     

Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells. Evidence for protein kinase C-dependent and -independent pathways

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.     

Downregulation of T cell growth factor production by ornithine decarboxylase and its product putrescine: D,L-alpha-difluoromethylornithine suppresses general protein synthesis but augments simultaneously the production of interleukin-2

Treatment of EL-4 lymphoma cells with tetradecanoylphorbol-acetate (TPA), a well-known activator of protein kinase C, induces the production of the T cell growth factor interleukin-2 (IL-2) and the expression of IL-2-specific mRNA within 4-8 h. This system is an ideal model for studies on the induction of a differentiated function in a homogeneous lymphoid cell population by a defined signal. TPA induces also an increase of ornithine decarboxylase (ODC) activity and elevates the intracellular concentrations of putrescine and polyamines within 4-8 h. A similar increase of intracellular putrescine and polyamine concentrations can be achieved by administration of 2 mM putrescine to the culture medium. However, putrescine cannot induce the production of IL-2 in the absence of TPA and cannot reconstitute the IL-2 production in cultures with PGE2 or cyclosporine A, i.e., two well-known immunosuppressive substances which inhibit ODC activity. Putrescine has rather a counter-regulatory effect as concluded from the observation that the TPA-induced TCGF production and IL-2-specific mRNA expression are augmented (superinduced) by the ODC inhibitor D,L-alpha-difluoromethylornithine (DFMO) and again suppressed after the administration of putrescine or polyamines to DFMO-treated cultures. The glycolytic activity, general protein synthesis [( 3H]leucine incorporation), and the cell cycle progression from G2/M to G1, in contrast, are inhibited by DFMO and reconstituted by putrescine. This demonstrates that the cells are able to sacrifice to a large extent several vital functions including their general protein synthesis and to devote themselves at the same time to a fulminant production of their functionally most relevant protein IL-2. This process is downregulated by ODC and its product putrescine. A correlation between increased IL-2 production and accumulation of cells in the G2/M phase was also observed in cultures treated with hydroxyurea or with a combination of amethopterin and adenosine.     

Transient elevation of ribonucleotide reductase activity, M2 mRNA and M2 protein in BALB/c 3T3 fibroblasts in the presence of 12-O-tetradecanoylphorbol-13-acetate

A rapid elevation of ribonucleotide reductase activity was observed with BALB/c 3T3 fibroblasts within 1/2 to 1 hour treatment with 0.1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA). This increase in activity was transient, and returned to about normal levels within 24 to 48 hours. Northern analysis of the two components of ribonucleotide reductase showed a slight transient elevation of M1 mRNA and a marked transient elevation of M2 mRNA after 1/2 hour TPA treatment. As a positive control, ornithine decarboxylase message levels were also observed to be transiently elevated following identical treatment with TPA. Western blot analysis with M1 and M2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the transient elevation of the M2 but not the M1 protein during treatment with 0.1 microM TPA. This first demonstration that the tumor promotor, TPA, can cause rapid and transient alterations in ribonucleotide reductase suggests that the enzyme, particularly the M2 component, may play an important role in the critical events involved in the process of tumor promotion.     

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.     

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).