Growth signal transduction: rapid activation of covalently bound ornithine decarboxylase during phosphatidylinositol breakdown

We have previously shown that treatment of T lymphocytes with mitogenic ligands induces a rapid activation of ornithine decarboxylase (ODC) through a mechanism that is independent of protein synthesis but requires energy and an intact cytoskeleton. Here we show by immunoprecipitation experiments and by chemical analyses that ODC is covalently linked to the cell membrane by inositol. Treatment of sonicated cells with a phosphatidylinositol-specific phospholipase C from B. thuringiensis caused a rapid 3-fold increase in ODC activity. Similar treatment of intact cells had no effect, suggesting that the ODC is attached to the cytoplasmic surface of the membrane. We conclude that ODC release and activation occur by a novel mechanism involving phosphatidylinositol breakdown following ligand-receptor interaction.     

Reproduction of murine encephalomyocarditis virus in enucleated Krebs 2 ascitic carcinoma cells]

Enucleated cells of ascites carcinoma Krebs 2 (cytoplasts) were prepared. The cells were centrifugated for this purpose in a discontinuous Ficoll density gradient containing cytochalasine B. The cytoplasts formed turbid bands in Phycol solution at the density of 1.037--1.053 g/ml. The cytoplasts failed to synthesize RNA, but proteins were synthesized for several hours. Encephalomyocarditis virus replicated in the ascites carcinoma cytoplasts, but its yield was 10 to 100-fold lower as compared to that in the intact cells. Apparently the reduction of the virus yield was not due to the low efficiency of early stages of the virus-cytoplast interaction. Viral infection resulted in inhibition of the synthesis of cellular proteins in the cytoplasts as well as in the intact cells.     

Interaction of Zn2+ and Eu3+ with bovine liver glutamate dehydrogenase.

Stepwise increments of the concentration of 2-difluoromethylornithine (DFMO), a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (ODC), resulted in a selection of cultured human IgG-myeloma cells (Sultan cell line) capable of growing in the presence of up to 3 mM-DFMO. This capacity was associated with 10-fold increase in ODC activity in the dialysed extracts of drug-resistant myeloma cells, markedly enhanced synthesis rate for ODC enzyme molecules, as revealed by a 20 min [35S]methionine labelling of cellular proteins, followed by specific immunoprecipitation and SDS/polyacrylamide-gel electrophoresis, dose-dependently increased expression of ODC mRNA in resistant cells (effective dose causing 50% inhibition), dose-dependent amplification of ODC gene sequences in a 9-kilobase-pairs EcoRI genomic DNA fragment, and (v) a 10-fold increase in the ED50 (effective dose causing 50% inhibition) for the anti-proliferative action of DFMO in these myeloma cells. These results represent one of the few gene amplifications described in cultured human cells.     

Zinc can influence ornithine decarboxylase activity in rat thymus cells

Summary The thymus of young rats contained a high basal activity of ornithine decarboxylase (ODC). Treatment with zinc sulphate caused a slight increase of thymic ODC activity within 6 hours and a more marked enhancement (three-fold) in the spleen 24 h after treatment. In spite of the high activity of thymic ODCin vivo, ODC was not detectable in primary cultures of rat thymocytes, but was early and largely induced after treatment with Concanavalin A (Con A). The presence of 0.1 mM zinc in the medium increased the response of ODC to Con A. This effect of zinc in mitogen activated thymocytes may be due to the stabilization of ODC, which was found to decay with a half life of 65 min after the block of protein synthesis with cycloheximide. On the contrary in absence of zinc the half life of the enzyme was 40 min, as in the rat thymus in vivo.Zinc alone, at 0.1 mM concentration, did not affect ODC activity in resting thymocytes during the early times, but the metal was able to cause an increase of the enzyme activity after 4–6 days of culture. Other heavy metals such as mercury, cadmium and copper provoked a late increase of ODC activity, but their action was evident only at dosages which were toxic for the cells.     

Effects of acetylated polyamines on ornithine decarboxylase in rat HTC cells

N-Monoacetylputrescine and N⁸-monoacetylspermidine, metabolites of the naturally occurring polyamines, activate the enzyme ornithine decarboxylase (ODC). When added to cultures of hepatoma (HTC) cells growing in log phase, in concentrations of 5×10^?5M and 2.5×10^?7M respectively, these substances cause a 3 to 5-fold increase in the activity of ODC with a peak effect at one hour. This previously undescribed stimulating effect is in sharp contrast to the well established suppressing effects of nonacetylated polyamines on ODC activity.     

Superinduction of ornithine decarboxylase by halogenated ribofuranosylbenzimidazoles.

1. The effect of dichlororibofuranosylbenzimidazole (DiCl-RB), an inhibitor of hnRNA synthesis and casein kinase-2 activity, on ornithine decarboxylase (ODC) was investigated in a difluoromethylornithine (DFMO) resistant, ODC overproducing cell line. 2. In cells growing in the absence of DFMO, DiCl-RB provoked a marked, but transient increase in ODC activity and immunoreactive ODC content. 3. The ODC response to DiCl-RB was prevented by cycloheximide and was not due to stabilization of the enzyme. 4. The dibromo derivative analogue (DiBr-RB) exerted similar effects on ODC, but was effective at lower concentrations. 5. The halogenated ribofuranosylbenzimidazoles were ineffective in cells growing in the presence of DFMO and containing higher levels of ODC protein.     

Regulation of the activity and synthesis of malic enzyme in 3T3-L1 cells

Malic enzyme activity in differentiated 3T3-L1 cells was about 20-fold greater than activity in undifferentiated cells. A new steady-state level was achieved about 8 days after initiating differentiation of confluent cultures with a 2-day exposure to dexamethasone, isobutylmethylxanthine, and insulin. This increase in enzyme activity resulted from an increase in the mass of malic enzyme as detected by immunotitration of enzyme activity with goat antiserum directed against purified rat liver malic enzyme. Malic enzyme synthesis was undetectable in undifferentiated cells and increased to about 0.2% of soluble protein in differentiated cells, suggesting that the increase in enzyme mass was due primarily to an increase in enzyme synthesis. Thyroid hormone, a potent stimulator of malic enzyme activity in hepatocytes in culture and in liver and adipose tissue in intact animals, decreased or increased malic enzyme activity in differentiating 3T3-L1 cells by about 40% when it was removed or added to the medium, respectively. Insulin, another physiologically important regulator of malic enzyme activity in vivo, had no effect on the initial rate of accumulation of malic enzyme activity in the differentiating cells and caused a 30 to 40% decrease in the final level of enzyme activity in the fully differentiated cells. Cyclic AMP, a potent inhibitor of malic enzyme synthesis in hepatocytes in culture, inhibited this process in 3T3-L1 cells by 30%. Malic enzyme is like several other enzymes in that the large increase in its concentration which accompanies differentiation of 3T3-L1 cells is due to increased synthesis of enzyme protein. However, the hormonal modulation of malic enzyme characteristic of liver and adipose tissue in intact animals does not appear to occur in differentiated 3T3-L1 cells, suggesting that differentiated 3T3-L1 cells may not be an appropriate model system in which to study the hormonal modulation of malic enzyme that occurs in liver and adipose tissue of intact animals.     

Cell-cycle-dependent regulation of ornithine decarboxylase activity in the unicellular green alga Chlamydomonas reinhardtii

Polyamines play an important role in the control of cell growth and cell division. In the unicellular green alga Chlamydomonas reinhardtii as in animal cells, biosynthesis of the 3 commonly occurring polyamines (putrescine, spermidine and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. Therefore, we have investigated the regulation of ODC activity during the cell cycle of Clamydomonas reinhardtii using synchronized cultures. A 2.5–3-fold increase in ODC activity was observed during the transition to the cell division phase. This up-regulation of ODC activity was not due to an increased level of ODC-mRNA as revealed by northern-blot analyses, but correlated with an increased half-life of this particular enzyme (from 1.1 to 3.2 h). Addition of the DNA topoisomerase II inhibitor nalidixic acid during the second half of the growth period caused a transient decrease of ODC activity and a considerable delay of cell divisions. After cell division, a down-regulation of ODC activity was observed which was faster in the dark than in the light and also correlated with changes of the ODC half-life.     

Juvenile hormone stimulation of ornithine decarboxylase activity during vitellogenesis in Drosophila melanogaster

Summary The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, becomes elevated in intact female Drosophila melanogaster shortly after adult eclosion. This activity reaches a peak at 24 h following eclosion, and then drops to lower levels by 48 h. This pattern is not observed in males, consistent with the hypothesis that polyamine synthesis is involved in ovarian maturation in Drosophila. Abdomens isolated within 2 h of adult eclosion do not display elevated ODC activity or ovarian maturation. However, a 250-ng dose of the juvenile hormone analog methoprene (ZR-515) applied in acetone to these abdomens, recovers ovarian maturation and causes a 5–10 fold increase in enzyme activity over controls treated with acetone alone. The same dose of the inactive precursor methyl farnesoate caused no such increase, whereas a 500-ng dose of the newly discovered natural Drosophila JHB₃ stimulated a four-fold response. The response to methoprene was dose-dependent, showing stimulatory activity at a dose as low as 10 ng. This stimulation by JHA is rapid, occurring between 1 and 3 h following hormone treatment, reminiscent of JH induction of fat body vitellogenin synthesis in Drosophila. Elevated ODC activity appeared to be localized in the adult fat body. During embryogenesis, ODC activity remained undetectable until just prior to hatching, when a large increase was detected. We postulate that JH may, either directly or indirectly, regulate polyamine biosynthesis in vivo, and that this synthesis may be required for the production of macromolecules during Drosophila vitellogenesis or embryogenesis.Abbreviations JH juvenile hormone - JHA juvenile hormone analog - ODC ornithine decarboxylase - SAMDC S-adenosyl-methionine decarboxylase - JHB ₃ juvenile hormone III bisepoxide     

Effects of S-adenosyl-1,8-diamino-3-thio-octane and S-methyl-5''-methylthioadenosine on polyamine synthesis in Ehrlich ascites-tumour cells.

The rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are negatively regulated by the polyamines spermidine and spermine. In the present work the spermidine synthase inhibitor S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and the spermine synthase inhibitor S-methyl-5'-methylthioadenosine (MMTA) were used to evaluate the regulatory role of the individual polyamines. Treatment of Ehrlich ascites-tumour cells with AdoDATO caused a marked decrease in spermidine content together with an accumulation of putrescine and spermine. Treatment with MMTA, on the other hand, gave rise to a marked decrease in spermine, with a simultaneous accumulation of spermidine. A dramatic increase in the activity of AdoMetDC, but not of ODC, was observed in MMTA-treated cells. This increase appears to be unrelated to the decrease in spermine content, because a similar rise in AdoMetDC activity was obtained when AdoDATO was given in addition to MMTA, in which case the spermine content remained largely unchanged. Instead, we show that the increase in AdoMetDC activity is mainly due to stabilization of the enzyme, probably by binding of MMTA. Treatment with AdoDATO had no effects on the activities of ODC and AdoMetDC, even though it caused a precipitous decrease in spermidine content. The expected decrease in spermidine-mediated suppression of ODC and AdoMetDC was most probably counteracted by the simultaneous increase in spermine. The combination of AdoDATO and MMTA caused a transient rise in ODC activity. Concomitant with this rise, the putrescine and spermidine contents increased, whereas that of spermine remained virtually unchanged. The increase in ODC activity was due to increased synthesis of the enzyme. There were no major effects on the amount of AdoMetDC mRNA by treatment with the inhibitors, alone or in combination. However, the synthesis of AdoMetDC was slightly stimulated in cells treated with MMTA or AdoDATO plus MMTA. The present study demonstrates that regulation of neither ODC nor AdoMetDC is a direct function of the polyamine structure. Instead, it appears that the biosynthesis of the polyamines is feedback-regulated by the various polyamines at many different levels.     

The development of competence in resting B cells. The induction of cyclic AMP and ornithine decarboxylase activity after direct contact between B and T helper cells.

In the present communication, an experimental approach is utilized that facilitates the study of biochemical processes induced in B cells after their interaction with Th cells. In this approach, Th cell clones are stimulated for 18 h upon anti-CD3-coated plates, fixed with paraformaldehyde, and added at a 2 to 3:1 ratio to small, resting B cells (isolated from Percoll gradients). Th cells not stimulated on anti-CD3-coated plates, but fixed with paraformaldehyde, serve as controls for these experiments. The activated, fixed Th cells induce a transient, sixfold increase in B cell levels of cAMP, as well as an increase in B cell expression of ornithine decarboxylase (ODC) activity. This enzyme initiates the synthesis of polyamines and has been shown to be increased as cells enter the growth phase. In addition, previous studies have shown that the cellular levels of ODC activity are controlled by a multi-tiered regulatory cascade. To examine this aspect, polyclonally stimulated B cells were studied. Such cells demonstrated a gradual increase in ODC mRNA levels that peaked between 6 and 15 h and can be partially explained by a three- to fourfold increase in mRNA stability but not by changes in the enzyme affinity for substrate. The increase in ODC mRNA occurs in the absence of protein synthesis, suggesting that the ODC gene is a member of the immediate/early gene family. Finally, the early increase in ODC mRNA was enhanced in cells in which cAMP levels were artificially elevated, suggesting the possibility that the cAMP-dependent signaling pathway participates during the regulation of this gene expression. The significance of these experimental results concerning the process of B cell activation is discussed.     

A stable, inducible, dose-responsive ODC overexpression system in human cell lines

ODC is a labile protein subject to rapid turnover, and a conditional expression system providing long-term overexpression may be helpful in further understanding the biochemical properties of this enzyme and elucidating aspects of the polyamine biosynthetic pathway that have otherwise been difficult to study. HEK293 and LNCaP cell lines were engineered to stably and inducibly overexpress ODC using a Tet-on inducible construct. Clones from both cell lines were characterized by evaluating ODC mRNA expression, ODC activity, intracellular and extracellular polyamine levels, SSAT activity and growth kinetics. The ODC-inducible cell lines were time- and dose-responsive providing a mechanism to increase ODC and putrescine accumulation to a desired level in a flexible and controllable manner. The findings demonstrate that LNCaP ODC overexpressing cells maintained over a 100-fold increase in ODC activity and over a 10-fold increase in intracellular putrescine after 6 h. ODC induction at the highest levels was accompanied by a slight decline in intracellular spermidine and spermine levels and this observation was supported by the finding that SSAT activity was induced over 40-fold under these conditions. Growth rate remained unaffected following at least 12 h of ODC overexpression. Similar results were observed in the HEK293 ODC overexpressing cells.