Inhibition Of Ornithine Decarboxylase Induction Research Articles

Shared Biosynthesis of the Saliniketals and Rifamycins in Salinispora arenicola is Controlled by the sare1259-Encoded Cytochrome P450

Saliniketals A and B are unusual polyketides from the marine actinomycete Salinispora arenicola that inhibit ornithine decarboxylase induction. The structural similarities between the saliniketals and the ansa chain of the potent rifamycin antibiotics, which co-occur in the fermentation broth, suggest a common origin between the two compound classes. Using PCR-directed mutagenesis, chemical complementation studies, and stable isotope feeding experiments, we showed that the saliniketals are byproducts of the rifamycin biosynthetic pathway diverging at the stage of 34a-deoxyrifamycin W. Our results suggest that a single enzyme, the cytochrome P450 monooxygenase encoded by sare1259, catalyzes multiple oxidative rearrangement reactions on 34a-deoxyrifamyin W to yield both the saliniketal and rifamycin structural classes.

Effect of Green Tea Extract on the Induction of Ornithine Decarboxylase and the Activation of Extracellular Signal-Regulated Kinase in Bladder Carcinoma ECV304 Cells

According to several studies, green tea and individual catechins can inhibit the induction of ornithine decarboxylase (ODC), the key enzyme in the biosynthesis of polyamines. It has been suggested that the inhibition of ODC induction may offer an explanation to the anticancer and chemopreventive activities of green tea. In the present study, however, treatment of bladder carcinoma ECV304 cells with green tea extract (GTE) was not able to reduce the induction of ODC by fetal calf serum. Actually, in the absence of serum, GTE provoked a dose-dependent and remarkable induction of ODC activity. The induction of ODC, which could be elicited also by (-)-epigallocatechin 3-gallate, a major green tea component, required an early activation of extracellular signal-regulated kinase 1 and 2 (ERK), and both events appeared to be dependent on an alteration of the status of cellular thiol groups. Pretreatment with specific ERK or ODC inhibitors was able to prevent a late caspase activation but hardly affected the loss of cell viability provoked by GTE. In conclusion, to our knowledge, this is the first study showing that GTE can promote ODC induction in a tumor cell line.

Signaling pathways leading to the induction of ornithine decarboxylase: Opposite effects of p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK inhibitors

Treatment of serum-starved, human ECV304 cells with histamine or ATP elicited a transient induction of ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis, to an extent similar to that provoked by phorbol myristate acetate or serum re-addition. All these agents also provoked an increase in active phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK. The involvement of p44/42 MAPK and p38 MAPK in the induction of ODC was investigated by using selective inhibitors. U0126 and PD98059, two specific p44/42 MAPK kinase inhibitors, prevented the induction of ODC elicited by any stimulus employed, whereas SB203580 and SB202190, which are widely used as p38 MAPK inhibitors, enhanced ODC induction in a way that appeared dependent on p44/42 MAPK activation. By using inhibitors of other key signaling proteins that may lead to activation of p44/42 MAPK, we provide evidence that protein kinase C, but not phosphoinositide 3-kinase, is involved in histamine-stimulated ODC induction. These results show that the p44/42 MAPK pathway, but not p38 MAPK, is essential for ODC induction stimulated either by agonists of G-protein-coupled receptors, phorbol esters, or serum, and suggest that the inhibition of ODC induction may be an important event in the antiproliferative response to p44/42 MAPK pathway inhibitors.

Bioactive constituents of Thuja occidentalis.

An ethyl acetate-soluble extract of the combined leaves and twigs of Thuja occidentalis was found to inhibit 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase (ODC) in cultured mouse epidermal ME 308 cells. Bioassay-guided fractionation of this extract led to the isolation of six active constituents (1-6), namely, (+)-7-oxo-13-epi-pimara-14,15-dien-18-oic acid (1), (+)-7-oxo-13-epi-pimara-8,15-dien-18-oic acid (2), (+)-isopimaric acid (3), (1S,2S,3R)-(+)-isopicrodeoxypodophyllotoxin (4), (-)-deoxypodophyllotoxin (5), and (-)-deoxypodorhizone (6). Compounds 1 and 4 are new natural products, and their structures and stereochemistry were determined using spectroscopic methods. Compounds 1-6 were evaluated for inhibition of the transformation of murine epidermal JB6 cells, inhibition of ornithine decarboxylase induction with murine epidermal ME 308 cells, and cytotoxic activity against KB cells.

Phosphatidylinositol 3-Kinase Is Required for the Induction of Ornithine Decarboxylase in Leukemia Cells Stimulated to Growth

The involvement of phosphatidylinositol 3-kinase (PI3K) in the induction of ornithine decarboxylase (ODC) was investigated by using specific PI3K inhibitors. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, treatment with LY294002 inhibited cell growth and provoked a complete block of the induction of ODC activity (IC50∼2 μM) and ODC protein. Some reduction in the accumulation of ODC mRNA was also observed, whereas ODC turnover was not affected significantly. Wortmannin, another specific inhibitor of PI3K, structurally unrelated to LY294002, also inhibited ODC induction with an IC50of about 10 nM. These results indicate that PI3K activity is required for the induction of ODC, possibly affecting both ODC mRNA level and translation. Since p70 S6 kinase (p70S6K) is considered an important mediator of PI3K action in several experimental systems, the effect of rapamycin, which can lead to selective inhibition of p70S6K, was also investigated. Rapamycin inhibited p70S6Kactivity and produced ODC inhibiting effects similar to those elicited by LY294002. However, LY294002 and wortmannin at concentrations which inhibited almost completely PI3K activity did not decrease p70S6Kactivity, suggesting that p70S6Kdoes not mediate the PI3K effects on ODC, but may lie on a separate pathway in this experimental model.

Leishmania donovani: Cellular control of ornithine decarboxylase in promastigotes

Ornithine decarboxylase, a key enzyme in polyamine biosynthesis, is essential for normal cell growth and proliferation. Furthermore, the inhibition of this enzyme is a potential way of controlling such growth. In order to shed light on the role of ornithine decarboxylase in regulation of Leishmania growth we examined the activity of this enzyme during the life cycle of these organisms. Exponentially growing Leishmania promastigotes were resuspended at a density of 3 × 10 6 cells/ml. 2 × 10 7 cells were withdrawn 24 hr later at different time intervals for induction studies and ornithine decarboxylase activity was measured. Ornithine decarboxylase showed a growth related pattern in L. donovani promastigotes. Induction studies showed that ornithine decarboxylase activity rapidly increased in late log phase cells when resuspended in fresh medium. A biphasic induction curve was observed similar to that observed in mammalian cells. The first peak was observed at 6 hr and the second at 16 hr. Cycloheximide and Actinomycin D inhibited induction at 16 hr by 65–68%. Polyamines at a level not inhibitory to growth (10 μM) inhibited ornithine decarboxylase induction by 30–40% late in the induction period. Putrescine and spermidine both inhibited the first peak of induction. Putrescine suppressed ornithine decarboxylase activity by 39% at 16 hr whereas spermidine by only 29%. The half life of ornithine decarboxylase in promastigote forms grown in the presence of cycloheximide was > 6 hr. These studies indicate that although the Leishmanial ornithine decarboxylase follows a similar induction pattern to that previously reported in the mammalian cells, it is less susceptible to exogenous polyamines and is comparatively stable. This lack of ornithine decarboxylase regulation and turnover may be exploitable in the development of various therapeutic agents to inhibit Leishmanial growth.

Post-transcriptional inhibition of ornithine decarboxylase induction by zinc in a difluoromethylornithine resistant cell line

Addition of Zn 2+_ to cell medium inhibited the induction of ornithine decarboxylase (ODC) activity in ODC overproducing L1210-DFMO r cells. A significant effect was observed at a concentration as low as 0.01 mM, however a more marked inhibition was caused by the addition of 0.1 mM Zn 2+. The inhibition of the induction of ODC activity was accompanied by a proportional decrease in the content of immunoreactive ODC protein, whereas the level of ODC mRNA, detemined by a solution hybridization RNase protection assay, was not affected signigicantly. Instead, some acceleration of ODC turnover was observed. the addition of 0.1 mM Co 2+ or Mn 2+, but not of other divalent metal ions, also inhibited ODC induction; differently from Zn 2+ however, these metals affected cell viability and/or cell growth. Removal of endogenous Zn 2+ by a chelator also provoked a strong decrease of ODC induction, which was reversed by Zn 2+. However, addition of Zn 2+ in excess of the chelator proved to be markedly inhibitory. These results indicate that both a restricted Zn 2+ availability and an enhanced presence of the metal can inhibit the induction of ODC in L1210-DFMO r cells.

Inhibitory Effects of a Calcium Antagonist on Ornithine Decarboxylase Induction in Pancreatic Cancer Cell Lines

The calcium channel blocker verapamil has been previously shown to augment the chemosensitivity of pancreatic adenocarcinoma cell lines to doxorubicin by mechanisms other than changes in the intracellular accumulation, retention, or metabolism of doxorubicin. Because of our interest in polyamine biosynthesis and metabolism and the known involvement of calcium in the induction of ornithine decarboxylase (ODC) by serum refeeding of cultured cells, the effects of verapamil on the serum-stimulated ODC activity in two hamster pancreatic adenocarcinoma cell lines were examined. In plateau phase well-differentiated (WD) PaCa and poorly differentiated (PD) PaCa cells, a dose-dependent inhibition of the 4-h serum induction of ODC was seen at concentrations of 1, 5, and 10 microM verapamil. At the higher concentrations of verapamil, the inhibition of ODC induction was comparable to that achieved with 5 mM alpha-difluoromethylornithine (DFMO, a specific enzyme inhibitor of ODC) and greater than that seen with 2 mM EGTA plus calcium-depleted serum. Log phase PD PaCa cells, included for comparison, showed less ODC induction with serum and lesser degrees of inhibition of the response to serum refeeding with verapamil, DFMO, and calcium depletion. No direct inhibition of the ODC enzyme was found when verapamil was added at the time the activity was measured. Based on our present data, a possible influence of intracellular calcium pools in the verapamil effect on ODC activity is unclear. Nevertheless, the present findings suggest that verapamil's effects on cytotoxicity may be mediated (at least in part) by inhibition of the serum-mediated induction of ODC.

The potent anti-tumor-promoting agent isoliquiritigenin

A topical application of a chalcone derivative, 4,2',4'-trihydroxychalcone (isoliquiritigenin) inhibited epidermal ornithine decarboxylase (ODC) induction and ear edema formation, i.e. inflammation, caused by a topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) in CD-1 mice. In addition, isoliquiritigenin potently inhibited 7,12-dimethylbenz[alpha]anthracene (DMBA)-initiated and TPA-promoted skin papilloma formation. This inhibitory effect of isoliquiritigenin was not due to any damage inflicted on the initiated cells but due to its anti-tumor-promoting action. Isoliquiritigenin also inhibited epidermal ODC induction and skin tumor promotion caused by 7-bromomethylbenz[alpha]anthracene (BrMBA), a non-TPA type of tumor-promoting agent, in DMBA-initiated mice. Isoliquiritigenin inhibits neither 12-lipoxygenase nor cyclooxygenase in epidermal subcellular fractions. This compound, however, inhibited TPA-stimulated prostaglandin E2 (PGE2) production in intact epidermal cells. ODC induction caused by TPA was inhibited by a topical application of cyclooxygenase inhibitor, indomethacin. Inhibition of ODC induction by indomethacin was counteracted by a topical application of PGE2, while inhibition caused by isoliquiritigenin was not overcome by PGE2. The results suggest that a mechanism other than the inhibition of PGE2 production is involved in the anti-tumor-promoting action of isoliquiritigenin. Isoliquiritigenin failed to inhibit phospholipase A2 activity of platelet sonicates, but inhibited platelet 12-lipoxygenase and 5-lipoxygenase in polymorphonuclear leukocytes. Therefore, it might be possible that isoliquiritigenin exerts its anti-tumor-promoting action through the lipoxygenase inhibition by acting on cells other than the target epidermal cells. Our present results, in combination with our previous data, demonstrate that some chalcone derivatives and flavonoids which show a potent lipoxygenase inhibitory action act on a common step in the skin tumor promotion caused by two different types of tumor-promoting agents, i.e. TPA and BrMBA, and suggest that these compounds show promise as drugs to prevent tumor promotion.

Induction of ornithine decarboxylase by nerve growth factor in PC12 cells: dissection by purine analogues.

Purine analogues were used to probe the mechanism by which nerve growth factor (NGF) and other agents regulate cellular ornithine decarboxylase (ODC) activity. Exposure of cultured rat pheochromocytoma PC12 cells to NGF causes a 10-50-fold induction of ODC activity within 4-6 h. We recently found that purine analogues block this induction as well as other, but not all, actions of NGF and have provided evidence that the inhibitory actions of the analogues may be due in part to the suppression of an NGF-activated protein kinase activity (Volonté, C., Rukenstein, A., Loeb, D. M., and Greene, L. A. (1989) J. Cell Biol. 109, 2395-2403). The present results show that the purine analogues also suppress the induction of ODC mRNA. One of the analogues used was 6-thioguanine (6-TG). Although 6-TG was effective when applied simultaneously with NGF, if NGF was administered for as little as 1-3 min before 6-TG, ODC induction was unimpaired. This suggests that 6-TG blocks an early step in the NGF mechanism, and that once this step is triggered, the ODC induction pathway is no longer sensitive to this analogue. In contrast, another purine analogue, 2-aminopurine (2-AP), effectively inhibited ODC induction even if applied only during the last hour of a 5-h exposure to NGF. It is hypothesized that this increased period of sensitivity to 2-AP may be due to its broader range (as compared to 6-TG) as an inhibitor of protein kinase activities. Epidermal growth factor (EGF) and cAMP derivatives also induce ODC activity in PC12 cells, and these effects were suppressed by 6-TG and 2-AP at concentrations similar to those that affect responses to NGF. However, short term (less than 30 min) pretreatment with EGF or a cAMP derivative did not protect induction of ODC activity by these agents from inhibition by 6-TG. This suggests that there are both convergent and divergent elements in the mechanistic pathways used by NGF, cAMP analogues, and EGF to induce ODC.

Trypanosoma brucei brucei: Regulation of ornithine decarboxylase in procyclic forms and trypomastigotes

Ornithine decarboxylase (ODC) activity was measured in procyclic forms of Trypanosoma brucei brucei grown in semidefined medium. ODC activity rapidly increased in late log-phase cells which were resuspended in fresh medium. A biphasic induction curve similar to that observed in mammalian cells was observed over an 18-hr period. ODC activity increased 4.5-to 25-fold over control levels measured at zero time. Actinomycin D and cycloheximide inhibited induction by >90%. Polyamines at a level not inhibitory to growth (10 μ M) inhibited ODC induction, but only by 30–50%, late in the induction period. Putrescine inhibited the first peak of induction and suppressed activity at 14 hr by 75%. Polyamine analogs such as bis(ethyl)spermidine were not effective suppressors of ODC activity. The half-life of ODC in procylic forms grown in the presence of cycloheximide was >6 hr, while that of bloodstream trypomastigotes in mice treated with cycloheximide was 5 hr. A single dose of the ODC inhibitor dl-α-difluoromethylornithine given to infected rats or mice suppressed trypanosome ODC activity >90% for more than 7 hr. These studies indicate that although trypanosome ODC increases rapidly under log growth conditions, it is less susceptible to fluctuation and external control than the enzyme from mammalian sources. The latter may be a factor in the clinical efficacy of ODC inhibitors.

Effects of Timing of Administration and Dose of Difluoromethylornithine on Rat Colonic Carcinogenesis

During azoxymethane (AOM)-induced colonic carcinogenesis in rats, biphasic induction of ornithine decarboxylase (ODC) activity occurs in the colonic mucosa. The relative contributions of these two phases of ODC induction to the carcinogenesis process were examined by studying the effects of the specific ODC inhibitor, difluoromethylornithine (DFMO), administered during either the initial phase of ODC increase or during both phases continuously. The effects of 1% and 0.25% DFMO administered continuously were also compared. Continuous oral administration of DFMO at 1% (approximately 8 mg/g body wt/wk) and 0.25% (approximately 2 mg/g body wt/wk) produced 93% inhibition of ODC induction by AOM in the right and left colons throughout the study. Despite suppression of ODC activity to near-normal levels, colon tumor incidence at 26 weeks in the right colon was not affected by either initial-phase or continuous administration of DFMO. In contrast, tumor incidence in the left colon was reduced from 35% to 5% by DFMO given continuously at doses of both 1% and 0.25% over the entire 26 weeks (P less than .05). No significant reduction in left colon tumor incidence resulted from the short initial 11-week course of DFMO although the tumor incidence was reduced (15% vs. 35%). Results suggest that the second ("post-initiation") phase of ODC induction may be of particular importance in carcinogenesis.

Axotomy-induced ornithine decarboxylase activity in the mouse dorsal root ganglion is inhibited by the vinca alkaloids

Vinca alkaloids were used to study the role of retrograde axon transport (RT) in activating neuron perikaryal repair response to nerve transection. Mouse lumbar dorsal root ganglia (DRG) (L4-L6) were excised 48 hours after unilateral transection of the sciatic nerve and ornithine decarboxylase (ODC) activity determined. ODC activity in DRG ipsilateral to nerve transection was increased 10-20 fold over contralateral values. Typical ODC activities in ipsilateral and contralateral DRG samples were 6.18 +/- 1.4 and 0.31 +/- 0.09 pmol 14CO2 released/h/3DRG, respectively. Systemic administration of single doses of either vincristine (1 mg/kg) or vinblastine (5 mg/kg) immediately prior to axotomy attenuated ODC induction in ipsilateral DRG by 39% and 47%, respectively. A direct inhibition of ODC activity in the DRG appears unlikely since only high concentrations of vinblastine (0.5-1.0 mM) were able to inhibit ODC activity in vitro. We suggest vinca alkaloids inhibit ODC induction as a consequence of disrupting retrograde axonal transport. Interruption of this intracellular communication mechanism may be etiologically linked to the the distal axon degeneration which follows repetitive exposure to vinca alkaloids and other agents that induce toxic axonal neuropathy.

Testosterone suppression of ornithine decarboxylase activity in rat Sertoli cells.

The effect of testosterone on the induction of Sertoli cell ornithine decarboxylase (ODC) activity was investigated in highly purified cultures derived from testes of 20-day-old rats. Sertoli cell cultures were maintained in serum-free Ham's F-10 medium, with refeeding on days 2 and 4. Before refeeding on day 4, ODC activity was 7.4 U (1 U = 1 pmol 14CO2 released/mg protein.60 min at 37 C). After a medium change, ODC activity increased (41.6 U at 10 h; 180.0 U at 24 h) and then returned to near-basal activity (26.3 U) after 48 h. Simultaneous addition of testosterone (150 ng/ml; 5.2 X 10(-7) M) with the day 4 medium change suppressed the increase in ODC induction (7.53 U at 10 h, 91.6 U at 24 h). Addition of testosterone 24 h before refeeding resulted in greater inhibition of ODC induction (6.9 U at 10 h; 45.4 U at 24 h) than when added simultaneously. Other androgens, 5 alpha-dihydrotestosterone, androsterone, and 5 alpha-androstane-3 alpha,17 beta-diol, also suppressed induction of ODC, whereas 17 beta-estradiol was ineffective, illustrating the steroid specificity of the effect. Coadministration of the antiandrogens hydroxyflutamide or cyproterone acetate partially blocked the testosterone effect. Induction of ODC activity by ovine FSH (10 ng/ml) was also suppressed when Sertoli cells were cultured in the presence of testosterone (150 ng/ml) from the time of isolation. However, induction of ODC activity by (BU)2cAMP was uncompromised by testosterone. These results suggest that testosterone may repress ODC activity and, hence, polyamine biosynthesis in the Sertoli cell, but that cAMP, acting through the trophic hormone FSH, can overcome this suppression of putrescine biosynthesis. Intratesticular and hypophyseal modulation of polyamine biosynthesis may influence not only cellular processes in the Sertoli cell itself but also its role in spermatogenesis.

Inhibition of ornithine decarboxylase induction by retinobenzoic acids in relation to their binding affinities to cellular retinoid-binding proteins.

Retinobenzoic acids induce differentiation of human promyelocytic leukemia cells (HL-60). Like retinoic acid, 14 retinobenzoic acids inhibited the induction of ornithine decarboxylase (ODC) by teleocidin in mouse skin. The mechanism(s) of inhibition of ODC induction by 7 retinobenzoic acids, Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, and Ch 55 was compared with those by all-trans-retinoic acid and the arotinoid compound 19. Application of 114 nmol of Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, or Ch 55, 10 min before 11.4 nmol of teleocidin, resulted in 76.7%, 82.0%, 76.2%, 28.3%, 48.4%, 58.6%, and 85.1% inhibition of ODC induction, respectively. Since all-trans-retinoic acid and compound 19 were also inhibitory, we determined whether retinobenzoic acids bind to cellular retinoic acid-binding protein (CRABP) isolated from bovine adrenal glands. Am 80 and Am 580 inhibited the specific binding of 3H-retinoic acid to CRABP, but also showed less affinity than authentic unlabeled retinoic acid and compound 19. Am 81, Am 590, Am 68, Sa 80, and Ch 55 at up to 10 microM were not effective competitors of the binding of either 3H-retinoic acid or 3H-retinol. These results suggest that the inhibition of ODC induction can be mediated by pathways that do not involve CRABP or the cellular retinol-binding protein.

Inhibition of ornithine decarboxylase induction of interferon (alpha + beta) and its reversal by dibutyryl adenosine 3',5'-monophosphate.

Interferon (alpha + beta) given to C3H/HeN mice intraperitoneally inhibited increases in the activities of adenylate cyclase and ornithine decarboxylase after partial hepatectomy. The inhibition of ornithine decarboxylase was prevented by administration of dibutyryl cAMP. Core (2'-5')oligo(adenylate), i.e. A2'p5'A2'p5'A or (A2'p)2A, as well as interferon inhibited the increases in these two enzymes caused by partial hepatectomy. The inhibition by (A2'p)2A of ornithine decarboxylase activity was reversed by dibutyryl cAMP. These results suggested that the activity of interferon was similar to that of (A2'p)2A and that the inhibition of ornithine decarboxylase induction caused by these agents resulted from the inhibition of adenylate cyclase activity.

Inhibition of mammary carcinoma growth by retinoidal benzoic acid derivatives.

The growth of many breast carcinoma cell lines is inhibited by vitamin A, and derivatives as well as synthetic retinoids. New retinoidal derivatives have recently been synthesized. These retinoidal benzoic acid derivatives displayed enhanced potency in their ability to reverse hamster tracheal keratinization and inhibit ornithine decarboxylase induction in mouse epidermis. We therefore screened a series of analogues of these compounds for their ability to inhibit human breast carcinoma cell proliferation utilizing three estrogen receptor-positive and two estrogen receptor-negative cell lines. The compound (E)-4-2-(5,6,7,8)tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)prop enyl benzoic acid (Ro 1374-10) was approximately 2-3 orders of magnitude more potent than all-trans-retinoic acid in inhibiting breast carcinoma cell proliferation while the compound SRI-6409-40, which differs from Ro 1374-10 only by the position of a methyl group, was 50-fold more potent than Ro 1374-10. All of the compounds tested displayed were inactive against the estrogen receptor-negative breast carcinoma lines.

Evaluation of structurally-related phenols for anti-photocarcinogenic and photoprotective properties.

Abstract— Butylated hydroxytoluene (BHT) has previously been shown to provide protection against UV‐mediated erythema, carcinogenesis, and induction of epidermal ornithine decarboxylase (ODC). Butylated hydroxytoluene is but one compound of a class of organic chemicals, the phenols, that demonstrates antioxidant properties. On the basis of structural characteristics, the phenolic compounds butylated hydroxyanisole (BHA), propyl gallate, and vanillin were compared to BHT for antiphotocarci‐nogenic activity. SKH‐Hr‐1 female mice were fed diets containing 0.5% (wt/wt) BHT, BHA, vanillin or 0.3% propyl gallate. The animals received constant, daily (5 days/week) suberythemic levels of UVB radiation from Westinghouse BZS‐WLG lamps. Radiation was discontinued when 25 sunburn units (SBUs) had been delivered at week eleven. Animals were evaluated weekly for tumor latency and multiplicity. Of the four phenols tested, only BHT conveyed significant inhibition of both carcinogenic parameters—increasing latency by 5.2 weeks and reducing tumor multiplicity by 60%. Vanillin significantly reduced tumor multiplicity (48%). BHA and propyl gallate were without effect. Because ODC has been considered a requisite step in carcinogenesis, ODC activity was assayed to determine whether its induction might serve as a rapid screen for potential anti‐carcinogenic phenols. Animals were fed the respective phenol‐containing diets for two weeks, exposed to 0.45 J/cm/ of UVB from FS‐20 lamps, and epidermal extract assayed for ODC activity 28 h post‐irradiation. Both BHT and BHA significantly inhibited ODC induction, the former being most effective. Vanillin and propyl gallate were without effect. These data indicate that neither antioxidant or lipophilic properties, nor the tert‐butyl substituent common to both BHT and BHA, are sufficient determinants of anti‐photocarcinogenic activity. Nor does it appear that inhibition of ODC induction is an adequate indicator of such activity.

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.

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.