Two mechanisms of spermidine/spermine N1-acetyltransferase-induction

The changes in activity of spermidine/spermine N1-acetyltransferase (SAT), a rate-limiting enzyme in polyamine degradation, were investigated to understand the mechanism of the induction of this enzyme in bovine lymphocytes. The activity of SAT was induced by stimulation with phytohemagglutinin (PHA), calcium ionophore A23187, sodium n-butyrate, or methylglyoxal bis(guanylhydrazone) (MGBG). When the cells were treated with a combination of PHA with either MGBG or butyrate, the increase in SAT was synergistic. However, the treatment of cells with both PHA and A23187 did not cause more induction of the enzyme activity than the stimulatory effects of each agent alone. The elevation in SAT caused by PHA or A23187 was inhibited by the simultaneous addition of 25 microM H-7, a protein kinase C inhibitor; the induction of the enzyme activity by MGBG or butyrate was slightly enhanced in the presence of H-7. In cells treated with a high concentration of O-tetradecanoylphorbol 13-acetate, which results in the breakdown of protein kinase C, PHA and A23187 did not give the maximum response, and MGBG slightly enhanced the enzyme activity. Dibutyryl cyclic AMP inhibited PHA-induced enzyme activity, but it stimulated MGBG- or butyrate-induced activity. Exposure to PHA or A23187 but not to MGBG or butyrate significantly increased the ornithine decarboxylase activity and DNA synthesis. These results showed that there were two different mechanisms of SAT induction. One is dependent on protein kinase C. The other one is independent of protein kinase C and is enhanced by cyclic AMP.     

Ornithine decarboxylase induction in cells stimulated to proliferate differs from that in continuously dividing cells

Ornithine decarboxylase activity increases at least 4–5-fold before DNA synthesis both in synchronous cycling cells and in quiescent cells stimulated to proliferate. The purpose of our experiments was to test whether the transient peaks of ornithine decarboxylase activity in both growth situations were biochemically regulated in a similar manner. We found that the regulation of this particular enzyme activity is distinct in two ways. Firstly, the addition of 2mm-hydroxyurea will block the induction of ornithine decarboxylase in continuously dividing Chinese-hamster ovary cells, while having no effect on ornithine decarboxylase induction in stimulated quiescent cells. Hydroxyurea added after the induction occurs has no effect on the enzyme activity. The apparent half-life of the enzyme is not altered in cells treated with hydroxyurea. Hydroxyurea does not affect the enzyme directly, since incubation of cell homogenates with this drug results in no loss of measurable ornithine decarboxylase activity and hydroxyurea does not markedly alter general RNA- or protein-synthesis rates. The inactivation of ornithine decarboxylase activity by hydroxyurea does not resemble the loss of activity observed with a 90min treatment with spermidine. Thiourea, a less potent inhibitor of ribonucleoside diphosphate reductase, will also inhibit ornithine decarboxylase activity, but to a lesser extent. Secondly, the expression of ornithine decarboxylase in quiescent cells stimulated to proliferate is biphasic as these cells traverse G₁ and enter S phase, whereas only one peak of activity is apparent in synchronous cycling G₁-phase cells. The time interval between the first peak of ornithine decarboxylase activity and the onset of DNA synthesis is approx. 5h longer in non-dividing cells stimulated to proliferate than in continuously dividing cells. The results suggest that the regulation of ornithine decarboxylase activity is different in the two growth systems in that the induction of ornithine decarboxylase in continuously dividing cells occurs closer in time to DNA synthesis and is dependent on deoxyribonucleoside triphosphates.     

Hydroxyurea inhibits ODC induction, but not the G1 to S phase transition.

Chinese hamster ovary cells, selected in mitosis and plated into medium containing hydroxyurea, can progress through G₁ and enter S phase although bulk DNA synthesis is prevented. As the cells progress through G₁ in the presence of hydroxyurea, ornithine decarboxylase activity remains low while general protein synthesis appears unaffected. After hydroxyurea is removed, ornithine decarboxylase activity increases, but only after approximately 20% of the DNA has been replicated. These results suggest that ornithine decarboxylase induction is not essential for cellular progression into S phase but is required for the completion of DNA synthesis.     

Induction of ornithine decarboxylase in guinea pig lymphocytes by the divalent cation ionophore A 23187. Effect of dibutyryladenosine 3',5'-monophosphate

The divalent cation ionophore, A23187, at a concentration of 0.25 microgram/ml, enhanced influx of Ca2+, activity of ornithine decarboxylase and incorporation of [3H]thymidine into DNA of guinea pig lymphocytes. Combined treatment of cells with A23187 and dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) augmented these three events. A23187 at a concentration of 0.06 microgram/ml was insufficient for induction of ornithine decarboxylase stimulated neither Ca2+ influx nor [3H]thymidine incorporation, but stimulated Ca2+ efflux. A23187 (0.06 microgram/ml) in combination with Bt2cAMP caused a marked induction of ornithine decarboxylase and stimulation of [3H]thymidine incorporation into DNA. When the time of Bt2cAMP addition was delayed after A23187, the stimulation of ornithine decarboxylase activity decreased. Washout of Bt2cAMP from cell culture earlier than 4 h of incubation caused a reduction in the stimulatory effect of Bt2cAMP. These results suggest that raising concentrations of cytoplasmic Ca2+ and cellular cAMP are important to some initial events leading to induction of ornithine decarboxylase and these biochemical changes are obligatory sequential steps for stimulation of DNA synthesis.     

Relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase induction, and DNA synthesis

The relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis was examined with bovine small lymphocytes stimulated by concanavalin A (Con A). The Na+/H+ antiport activity was activated immediately after addition of concanavalin A; the maximum was reached 1 h after Con A addition and the activation continued at least 6 h. With increasing concanavalin A concentrations, the activities of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis increased in a parallel manner. In the presence of HCO3- in the medium, the internal alkalinization of lymphocytes was not induced by Con A. Ornithine decarboxylase and DNA synthetic activities were not inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a specific inhibitor of the Na+/H+ antiporter. In contrast, in the absence of HCO3- in the medium, the internal pH was alkalinized approximately 0.06 pH units by Con A. EIPA did inhibit the alkalinization of the internal pH or DNA synthesis significantly. Ornithine decarboxylase activity was not inhibited by EIPA. These results indicate that the activation of a Na+/H+ antiporter is not a trigger for cell proliferation, but its activation is important probably through the maintenance of the internal pH optimum, especially in HCO3(-)-free medium.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.     

Hormonal control of liver regeneration

Two peaks in cyclic AMP production in rat livers 4 and 12h after partial hepatectomy (MacManus et al., 1972) were confirmed and a third peak established at 22h, which is the peak of DNA synthesis. The increases in cyclic AMP were prevented by beta-adrenergic blocking agents, propranolol and pindolol, without affecting ornithine decarboxylase induction or DNA synthesis. The alpha-blocking agents, phenoxybenzamine and phentolamine, given at the time of partial hepatectomy, delayed the rise in ornithine decarboxylase normally found 4h after operation, but did not affect DNA synthesis. If the alpha-blocking agents were given at 9-12h or 18h, the onset of DNA synthesis was delayed. Phenoxybenzamine did not affect the induction of ornithine decarboxylase in intact rat livers by glucagon or growth hormone, but did inhibit induction by dexamethasone. The induction of ornithine decarboxylase produced by dexamethasone was inhibited by 17alpha-hydroxy-progesterone; this compound also blocked the induction of ornithine decarboxylase in livers of partially hepatectomized rats.     

Induction of asparagine synthetase during lymphocyte activation by phytohemagglutinin

Asparagine synthetase was increased in cultured mouse spleen lymphocytes after stimulation by phytohemagglutinin. After a lag period of about 24h, the enzyme activity level rose sharply by 48h, reached its maximum at 72h, and decreased thereafter. The time course of the change in the enzyme activity was similar to that of the change in the rate of DNA synthesis. From the results that there was no increase of the activity of asparagine synthetase at the time induction of ornithine decarboxylase would occur (6h), it seems unlikely that asparagine synthesized in the cells contributes to the enhancement of ornithine decarboxylase during the activation of lymphocytes. The increase of asparagine synthetase activity was inhibited by cycloheximide and somewhat by actinomycin D, suggesting de novo enzyme synthesis during the stimulation.     

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.     

An organ culture of adult mouse skin: An in vitro model for studying the molecular mechanism of skin tumor promotion

A simple method to culture explants of adult mouse skin in a modified Eagle's HeLa cell medium was developed in order to further study the biochemical responses to the tumor promoting phorbol esters. The skin explants remained viable for at least 48 hr, as determined by their ability to incorporate ³H-thymidine into DNA as well as to induce epidermal ornithine decarboxylase (EC 4.1.1.17) activity following 12-0-tetradecanoylphorbol-13-acetate addition. The time course of induction of ornithine decarboxylase activity by the tumor promoter was similar to that observed with intact mice. Furthermore, the addition of retinoic acid and indomethacin, the agents that are known to inhibit the induction of ornithine decarboxylase activity by topically applied TPA, also inhibited the induction of ornithine decarboxylase activity by TPA in skin explants.     

C-rasH and ornithine decarboxylase are induced by oestradiol-17β in the mouse uterine luminal epithelium independently of the proliferative status of the cell

Oestradiol-17 β (E₂) treatment of the ovariectomized mouse results in a synchronised wave of cell proliferation in the uterine luminal epithelium. At the peak of DNA synthesis the mRNA level of the c-ras^H protooncogene and ornithine decarboxylase were significantly increased. Progesterone treatment completely inhibits the E₂ induced wave of DNA synthesis but does not greatly influence the level of these 2 mRNAs. Thus in the uterine luminal epithelium E₂ regulates the level of ornithine decarboxylase and c-ras^H independently of cell proliferation.     

Induction of ornithine decarboxylase in guinea-pig lymphocytes and its relation to phospholipid metabolism

Treatment of lymphocytes with exogenous phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3.) derived from Clostridium perfringens at concentrations similar to those which induced ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity produced diacylglycerol and phosphatidate. A divalent cation ionophore, A23187, and phytohemagglutinin induced not only diacylglycerol formation, but also ornithine decarboxylase activity. Dibutyryl cAMP inhibited both diacylglycerol formation and ornithine decarboxylase induction to a similar extent in phytohemagglutinin-stimulated lymphocytes, but stimulated them somewhat in ionophore A23187-activated lymphocytes. This suggests that the activation of intracellular phospholipase C and the formation of diacylglycerol is involved in ornithine decarboxylase induction in 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.     

Biphasic effects of dibutyryl cyclic adenosine 3',5'-monophosphate on synergistic stimulation of DNA synthesis by diacylglycerol, and the ionophore A23187 in guinea pig lymphocytes

When guinea pig lymphocytes were cultured with 1-oleoyl-2-acetylglycerol (OAG) and the ionophore A23187 for 8 h, [3H]-thymidine incorporation into the acid-insoluble fraction of the cells was stimulated synergistically. Further addition of dibutyryl cAMP caused a biphasic effect on the synergistic stimulation. Dibutyryl cAMP augmented the synergistic stimulation when A23187 was at the concentration of 0.075 micrograms/ml, but inhibited it when the ionophore was at 0.25 micrograms/ml. At the higher concentration of A23187, dibutyryl cAMP stimulated the [3H]thymidine incorporation when culture was for 4 h, but inhibited it when culture was for 8 h. The results were the same when 12-0-tetradecanoylphorbol-13-acetate (TPA) was used instead of OAG. Butyrate could replace dibutyryl cAMP for stimulation of [3H]thymidine incorporation in combination with TPA and A23187, but not with OAG and A23187 at the lower ionophore concentration. Dibutyryl cAMP but not butyrate stimulated ornithine decarboxylase induction caused by TPA and A23187. These results suggest that the effect of dibutyryl cAMP on DNA synthesis induced by OAG and A23187 was biphasic and depended on the concentration of A23187 and on the time of culture, and that the stimulation mechanism of butyrate is different from that of dibutyryl cAMP.     

Purification,properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis

S-Adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis (guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32 000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [³H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme syntheis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic. There was no further incraese in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occuring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.     

Regulation of polyamine biosynthesis in normal and transformed hamster cells in culture

Several aspects of polyamine biosynthesis were compared in low-passage hamster embryo fibroblasts and transformed hamster fibroblasts. Earlier studies had demonstrated a larger and longer-lasting induction of ornithine decarboxylase activity in transformed cells than in hamster embryo fibroblasts. The increases in intracellular polyamine concentrations after serum stimulation were much greater in chemically transformed HE68BP cells than in normal hamster fibroblasts. Treatment of confluent cultures with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, greatly potentiated ornithine decarboxylase induction by fresh medium in HE68BP cells, but not in hamster fibroblasts. A similar synergistic effect was observed when transformed cells, but not normal cells, were treated with the combination of insulin and promoter. HE68BP cells were capable of growth in medium containing serum concentrations as low as 0.5%, whereas only concentrations of 5% or more supported the growth of hamster embryo fibroblasts. Low serum concentrations induced ornithine decarboxylase in HE68BP cells but not in normal cells, and a given serum concentration always produced a greater induction of ornithine decarboxylase in transformed than in normal cells.Another enzyme involved in polyamine synthesis, $\text{S-}\text{adenosyl-}\text{L}\text{-methionine}$ decarboxylase was induced in normal and transformed cells by serum-containing medium or tetradecanoylphorbol acetate, but in contrast to ornithine decarboxylase, no synergistic effect was seen in transformed cells exposed to the combination of fresh medium and the tumor promoter. A macromolecular inhibitor of ornithine decarboxylase was readily detected in hamster fibroblast cultures treated with high concentrations of putrescine, but little or none of this inhibitor was found in HE68BP cultures. In both cell types, however, serum induction of ornithine decarboxylase was inhibited under conditions of excess putrescine.The results demonstrate several differences between normal and transformed hamster cells in the regulation of polyamine synthesis.