Calcium, asparagine and cAMP are required for ornithine decarboxylase activation in intact Chinese hamster ovary cells.

Asparagine specifically activated ornithine decarboxylase activity 5–7 fold by 7–8 h in confluent cultures maintained with a salts/glucose medium. When dibutyryl cAMP was added with asparagine, a 40–50 fold stimulation of ornithine decarboxylase activity was produced. Ornithine decarboxylase activation in the salts/glucose medium was not sensitive to actinomycin D. Omission of Ca⁺⁺ and Mg⁺⁺ from the medium abolished the ability of asparagine and/or dibutyryl cAMP to stimulate enzyme activity. Calcium was essential for the asparagine and dibutyryl cAMP mediated stimulation of ornithine decarboxylase activity.     

The relationship between levels and rates of synthesis of polyamines during mammalian cell cycle

The objective of this study was to examine the rate of synthesis and the intracellular levels of polyamines as a function of the HeLa cell cycle. The intracellular levels of ornithine, which were high during mitosis and early G1 phase, decreased rapidly during late G1 phase when the ornithine decarboxylase activity was at its peak. The activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase reached a peak during G1 and decreased rapidly during the S phase. The levels of polyamines were maximum in mitosis and S phase. In constrast, the rate of polyamine synthesis during S phase was 5–10 fold lower than that in mitosis or G1 phase. We have also observed fluctuations in diamine-oxidase activity during the cell cycle. The enzyme activity was high during mitosis and late G1 and low during S phase. Thus, the results of this study suggest an important role for the catabolic enzymes in the regulation of polyamine levels during the mammalian cell cycle.     

Polyamine starvation prolongs the S and G2 phases of polyamine-dependent (arginase-deficient) CHO cells.

This study analyzes the effects of polyamine starvation on cell cycle traverse of an arginase-deficient CHO cell variant (CHO-A7). These cells grow well in serum-free medium, provided that it contains ornithine or polyamines or both. In the absence of ornithine or polyamines or both, the CHO-A7 cells develop severe polyamine deficiency and, as a consequence, grow more slowly. When grown to a stationary phase in the presence of ornithine or putrescine or both, the CHO-A7 cells became arrested in G0/early G1. However, when starved for ornithine and polyamines, they accumulated in the S and G2 phases. Ornithine and polyamine starvation of CHO-A7 cells causes an increase in ornithine decarboxylase activity. When this increase was prevented by treatment with DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, growth was further suppressed, and a greater fraction of cells were found in the S and G2 phases of the cell cycle.     

Cellular effects of ornithine decarboxylase induction in cells maintained with a salts/glucose medium

Cultures preincubated in a growth restricted salts/glucose medium in the presence and absence of ornithine decarboxylase (ODC) activating factors were then incubated under ideal growth conditions to study the influence of these factors on cell growth. Incubation of confluent cultures in a salts/glucose medium alone did not induce ODC or change the other biochemical parameters investigated. However, if cultures were incubated in the salts/glucose medium supplemented with asparagine (ASN) and agents that increase cellular cAMP levels then ODC was induced after 6–8 h. This primary induction in the salts/glucose medium resulted in altered and delayed ODC induction during growth stimulation and also caused a delay in (³H) thymidine incorporation without affecting (³H) uridine and (³H) leucine incorporation. These results demonstrate that incubation of cultures in a salts/glucose media with ASN and dibutyryl cAMP (dBcAMP) causes refractory ODC induction and altered (³H) thymidine incorporation upon growth challenge with complete medium. These effects were not observed when cells were preincubated in a salts/glucose medium alone.     

Evolution of ornithine decarboxylase activity during the cell cycle of Euglena gracilis Z in synchronous culture. Influence of vitamin B-12.

Ornithine decarboxylase activity in Euglena gracilis Z was studied during the normal cell cycle and in vitamin B-12 deficiency. The cells were synchronized by means of alternating periods of light and dark. During the normal cell cycle, ornithine decarboxylase activity was very weak in the dark period, while three peaks of activity were recognized in the light period. The first peak, in the G1 phase, occurred when luminous stimulation started; the second preceded the S phase and the third was found in G2. In B-12-deficient cells, ornithine decarboxylase activity was greatly decreased and only the first peak remained. Elimination of the deficiency by addition of vitamin B-12 to the medium induced a very fast and significant increase in ornithine decarboxylase activity.     

Cyclic AMP-dependent regulation of ornithine decarboxylase activity in Chinese hamster ovary cells maintained with a salts/glucose medium.

Ornithine decarboxylase activity (ODC) increased about 7 fold 6--8 h following 10mM asparagine (ASN) addition to confluent cultures that had been previously serum deprived and then placed in a salts/glucose medium. Optimal concentrations of dibutyryl cAMP (dB cAMP) when incubated with the ASN caused up to a 50 fold increase in the activity of this enzyme after 7--8 h. The enhancement of ODC activity by ASN and dB cAMP was not sensitive to continuous (0--7 h) treatment with actinomycin D but similar treatment with cycloheximide depressed enzyme activity 40--60%. The synergistic stimulation of ODC activity by dB cAMP added with ASN was dose dependent and the dB cAMP stimulation of ODC activity displayed an absolute requirement for ASN when cells were maintained in the salts/glucose medium. The addition of dB cAMP always further enhanced ODC activity above the levels produced by addition of various levels of ASN (1 to 40mM) to the salts/glucose medium. Other agents which elevated cAMP levels such as 1-methyl-3-isobutylxanthine (IBMX) also enhanced ODC activity when administered with ASN. Additionally, treatment with sodium butyrate at concentrations ranging from 0.001mM to 5.0mM did not elevate ODC activity above the activity obtained with ASN alone. Addition of dB cAMP at various times after placing cells in salts/glucose medium with ASN further stimulated ODC activity only when added during the first 3-4 h. These results demonstrate the involvement of cAMP in the ASN mediated stimulation of ODC activity using cells maintained in a salts/glucose medium.     

Changes in ornithine decarboxylase activity and cyclic adenosine-3'-5'-monophosphate concentrations during the cell cycle of synchronized BHK cells.

BHK cells were synchronized by excess thymidine treatment, which resulted in approximately 90% synchrony. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, elevated in early S phase, decreased in G2 + M and G1 phase and then increased during late G1 approximately second round of early S phase. The concentration of cyclic adenosine-3'-5'-monophosphate (cAMP) gradually decreased during S approximately G2 + M phase and then increased during late G1 approximately second round of early S phase, preceding that of ODC activity. The data suggest that ODC activity might be regulated by cellular cAMP level.     

Polyamine biosynthesis and accumulation during the G1 to S phase transition

Ornithine decarboxylase, an important enzyme in growth regulation, is increased in CHO cells in G₁ phase of the cell cycle and decreases as the cells progress into S phase. S-adenosyl-L-methionine decarboxylase activity, which is dependent on either the presence of putrescine or spermidine for the synthesis of spermidine and spermine respectively, shows a maximal increase in late G₁/early S phase which corresponds very closely with the cell cycle phase specific accumulation of spermidine and spermine during S phase. Total culture evaluation of spermidine and spermine, which included extracellular as well as intracellular concentrations, indicated that extracellular accumulations of these polyamines occurred only in G₁ and that entry into S phase was concomitant with intracellular accumulation patterns. Hyperthermia (43°C for 1 hour) in mid-G₁ phase of the cell cycle resulted in rapid decreases in the activities of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase. In these cells, DNA replication was also not detectable until nine hours after mitosis, a time at which there had been recovery of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase activities. Previous data have further indicated a requirement for polyamine reaccumulation before control DNA replication rates are resumed. We therefore suggest that polyamine biosynthesis and intracellular accumulation are both temporal and quantitative prerequisites for transition through S phase.     

Molecular mechanisms of the synergistic induction of ornithine decarboxylase by asparagine and glucagon in primary cultured hepatocytes

In primary cultures of adult rat hepatocytes maintained in a salts/glucose medium, a more than 100-fold increase in ornithine decarboxylase (EC 4.1.1.17) activity was caused by asparagine and glucagon in a synergistic manner. The synthesis rate of ornithine decarboxylase was determined by [35S]methionine incorporation into the enzyme protein, and the amount of ornithine decarboxylase-mRNA was measured by hybridization with a cloned rat liver ornithine decarboxylase-cDNA. The synthesis rate of ornithine decarboxylase was stimulated more than 20-fold by asparagine and glucagon together, but the amount of ornithine decarboxylase-mRNA was increased only 3-4-fold, indicating that translational stimulation was involved in the induction process. Asparagine alone stimulated the synthesis of ornithine decarboxylase without substantial effect on the amount of ornithine decarboxylase-mRNA, whereas glucagon alone increased the amount of ornithine decarboxylase-mRNA about 3-fold without a detectable change in either enzyme activity or enzyme synthesis. Asparagine, at least in part, also suppressed degradation of ornithine decarboxylase.     

Ribonucleotide reductase activity and deoxyribonucleoside triphosphate metabolism during the cell cycle of S49 wild-type and mutant mouse T-lymphoma cells

We investigated deoxyribonucleoside triphosphate metabolism in S49 mouse T-lymphoma cells synchronized in different phases of the cell cycle. S49 wild-type cultures enriched for G1 phase cells by exposure to dibutyryl cyclic AMP (Bt2cAMP) for 24 h had lower dCTP and dTTP pools but equivalent or increased pools of dATP and dGTP when compared with exponentially growing wild-type cells. Release from Bt2cAMP arrest resulted in a maximum enrichment of S phase occurring 24 h after removal of the Bt2cAMP, and was accompanied by an increase in dCTP and dTTP levels that persisted in colcemid-treated (G2/M phase enriched) cultures. Ribonucleotide reductase activity in permeabilized cells was low in G1 arrested cells, increased in S phase enriched cultures and further increased in G2/M enriched cultures. In cell lines heterozygous for mutations in the allosteric binding sites on the M1 subunit of ribonucleotide reductase, the deoxyribonucleotide pools in S phase enriched cultures were larger than in wild-type S49 cells, suggesting that feedback inhibition of ribonucleotide reductase is an important mechanism limiting the size of deoxyribonucleoside triphosphate pools. The M1 and M2 subunits of ribonucleotide reductase from wild-type S49 cells were identified on two-dimensional polyacrylamide gels, but showed no significant change in intensity during the cell cycle. These data are consistent with allosteric inhibition of ribonucleotide reductase during the G1 phase of the cycle and release of this inhibition during S phase. They suggest that the increase in ribonucleotide reductase activity observed in permeabilized S phase-enriched cultures may not be the result of increased synthesis of either the M1 or M2 subunit of the enzyme.     

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

Evolution of ornithine decarboxylase activity during the cell cycle of Euglena gracilis Z in synchronous culture Influence of vitamin B-12

Ornithine decarboxylase activity in Euglena gracilis Z was studied during the normal cell cycle and in vitamin B-12 deficiency. The cells were synchronized by means of alternating periods of light and dark.During the normal cell cycle, ornithine decarboxylase activity was very weak in the dark period, while three peaks of activity were recognized in the light period. The first peak, in the G₁ phase, occurred when luminous stimulation started; the second preceded the S phase and the third was found in G₂. In B-12-deficient cells, ornithine decarboxylase activity was greatly decreased and only the first peak remained. Elimination of the deficiency by addition of vitamin B-12 to the medium induced a very fast and significant increase in ornithine decarboxylase activity.     

Regulation of phosphodiesterase and ornithine decarboxylase by cAMP is cell cycle independent.

Cyclic AMP (cAMP) causes growth arrest in G1 and induction of cAMP phosphodiesterase and decrease of ornithine decarboxylase in S49 mouse lymphoma cells. Dibutyryl cAMP treatment of partially synchronized cells causes similar changes in activities of both enzymes, regardless of position in the cell cycle. This suggests that cAMP regulation of these enzymes is not mediated by growth perturbation.     

Cell cycle dependent gene expression in quiescent stimulated and asynchronously cycling arterial smooth muscle cells in culture.

The expression of a set of cell cycle dependent (CCD) genes (c-fos, c-myc, ornithine decarboxylase (ODC), and thymidine kinase (TK)) was comparatively studied in cultured arterial smooth muscle cells (SMC) during exit from quiescence and exponential proliferation. These genes, which were not expressed in quiescent SMC, were chronologically induced after serum stimulation. c-fos mRNA were rapidly and transiently expressed very early in the G1 phase; c-myc and ODC peaked a few hours after serum stimulation and then remained at an intermediary level throughout the first cell cycle; TK mRNA and activity then appeared at the G1/S boundary and peak in G2/M phases. Except for c-fos, the other genes were also expressed in asynchronously cycling SMC (ACSMC); their expression was studied in elutriated subpopulations representative of cell cycle progression. c-fos mRNA were undetectable in any sorted subpopulations, even in the pure early G1 population. Despite a slight increase as the cell cycle advanced, c-myc and ODC genes were expressed throughout the ACSMC cell cycle. A faint TK activity was found in G1 subpopulations and increased in populations enriched in other phases; in contrast, TK mRNA remained highly expressed in all elutriated subpopulations. This study demonstrates significant modulations in CCD gene expression between quiescent stimulated and asynchronously cycling SMC in culture. This suggests that the events occurring during the emergence of SMC from quiescence are probably different from those in the G1 phase of ACSMC.     

Stimulation of Ornithine Decarboxylase Activity in Neural Cell Culture: Potential Role of Insulin

Abstract: Age-dependent decreases in the levels of ornithine decarboxylase activity were observed in the optic lobes, cerebral hemispheres, and midbrain-diencephalon of 6–17-day-old chick embryos. In dissociated cell cultures from chick embryonic brains a similar pattern of declining ornithine decarboxylase activity with time in culture was observed. Ornithine decarboxylase activity in the dissociated brain cell cultures was stimulated by changing the culture medium. The peak stimulatory effect was shown to occur 12 h after changing the medium. Although serum-free medium stimulated ornithine decarboxylase activity slightly, the presence of serum in the medium was the primary stimulatory factor. Both fetal calf serum and heat-inactivated fetal calf serum produced dose-dependent stimulation of ornithine decarboxylase activity. Dialyzed fetal calf sera stimulated ornithine decarboxylase, but to a lower level than that produced by nondialyzed sera. Insulin (0.5–10 μg/ml) stimulated ornithine decarboxylase activity in a dose-dependent manner in serum free medium. In addition, 10² M-L-asparagine stimulated ornithine decarboxylase activity in serum-free medium.     

Cell cycle parameters of Chinese hamster ovary cells during exponential, polyamine-limited growth.

We have previously shown that Chinese hamster ovary cells made polyamine deficient by treatment with alpha-methylornithine, an inhibitor of ornithine decarboxylase, grow exponentially in culture at low densities at one-half the rate observed in untreated (control) cultures. In this study, the cell cycle of polyamine-limited cells was examined by using thymidine autoradiography, mitotic index analysis, and fraction labeled mitoses analysis. We found that the longer doubling time of inhibitor-treated cultures was a consequence of increases in the lengths of the G1 and S phases. The expansion of the S phase was proportional to the increase in doubling time (twofold), whereas the G1 phase was lengthened by slightly more than a factor of 2. The lengths of the G2 and M phases were essentially unchanged. Putrescine stimulated the growth of inhibitor-treated cultures and restored the cell cycle parameters to those of untreated cells.     

Regulation of ornithine decarboxylase and other cell cycle-dependent genes during senescence of IMR-90 human diploid fibroblasts

Aging of IMR-90 human diploid fibroblasts in vitro is accompanied by significant changes of polyamine metabolism, most notably, a 5-fold decrease of serum-induced activity of ornithine decarboxylase, the key enzyme in the biosynthesis of polyamines (Chen, K. Y., Chang, Z. F., and Liu, A. Y.-C. (1986) J. Cell. Physiol. 129, 142-146). In this paper, we employed Northern blot hybridization and affinity radiolabeling techniques to investigate the molecular basis of this age-associated change of ornithine decarboxylase activity. Since the induction of ornithine decarboxylase by serum is a mid-G1 event, we also examined expressions of other cell cycle-dependent genes that are induced before and after the mid-G1 phase to determine if their expressions may also be age-dependent. Our results demonstrated a 3-fold decrease of the amount of active ornithine decarboxylase molecules that can be labeled by alpha-difluoromethyl[3H]ornithine in senescent IMR-90 cells (population doubling level (PDL) = 52) as compared to young cells (PDL = 22). However, the levels and kinetics of induction of ornithine decarboxylase mRNA in both young and senescent IMR-90 cells were found to be identical throughout a 24-h time period after serum stimulation. The time course and the magnitude of the expression of c-myc, an early G1 gene, were quite similar in young and senescent IMR-90 cells and appeared to be PDL-independent. In contrast, the expression of thymidine kinase, a late G1/S gene, was significantly reduced in senescent IMR-90 cells. Levels of thymidine kinase mRNA and thymidine kinase activity in senescent IMR-90 cells were 6- and 8-fold less than those in young cells, respectively. Based on these data, we proposed that impairment of cell cycling in senescent IMR-90 cells may occur at the late G1/S phase and that decreases of ornithine decarboxylase activity and putrescine accumulation during cell senescence may contribute to this impairment.     

Lipid biosynthesis and its coordination with cell cycle progression

The activation of cell cycle regulators at the G1/S boundary has been linked to the cellular protein synthesis rate. It is conceivable that regulatory mechanisms are required to allow cells to coordinate the synthesis of other macromolecules with cell cycle progression. The availability of highly synchronized cells and flow cytometric methods facilitates investigation of the dynamics of lipid synthesis in the entire cell cycle of the heterotrophic dinoflagellate Crypthecodinium cohnii. Flow cytograms of Nile red-stained cells revealed a stepwise increase in the polar lipid content and a continuous increase in neutral lipid content in the dinoflagellate cell cycle. A cell cycle delay at early G1, but not G2/M, was observed upon inhibition of lipid synthesis. However, lipid synthesis continued during cell cycle arrest at the G1/S transition. A cell cycle delay was not observed when inhibitors of cellulose synthesis and fatty acid synthesis were added after the late G1 phase of the cell cycle. This implicates a commitment point that monitors the synthesis of fatty acids at the late G1 phase of the dinoflagellate cell cycle. Reduction of the glucose concentration in the medium down-regulated the G1 cell size with a concomitant forward shift of the commitment point. Inhibition of lipid synthesis up-regulated cellulose synthesis and resulted in an increase in cellulosic contents, while an inhibition of cellulose synthesis had no effects on lipid synthesis. Fatty acid synthesis and cellulose synthesis are apparently coupled to the cell cycle via independent pathways.     

Regulation of ornithine decarboxylase activity by amino acids, cyclic AMP and luteinizing hormone in cultured mammalian cells

Any one of five amino acis (alanine, asparagine, glutamine, glycine, and serine) is an essential requirement for the induction of ornithine decarboxylase (EC 4.1.1.17) in cultured chinese hamster ovary (CHO) cells maintained with a salts/glucose, medium. Each of these amino acids induced a striking activation of ornithine decarboxylase in the presence of dibutyryl cyclic AMP and luteinizing hormone. The effect of the other amino acids was considerably less or negligible. The active amino acids at optimal concentrations (10 mM) induced only a 10-20 fold enhancement of enzyme activity alone, while in the presence of dibutyryl cyclic AMP, ornithine decarboxylase activity was increased 40-50 fold within 7-8 h. Of the hormones and drugs tested, luteinizing hormone resulted in the highest (300-500 fold) induction of ornithine decarboxylase with optimal concentrations of dibutyryl cyclic AMP and asparagnine. Omission of dibutyryl cyclic AMP reduced this maximal activation to one half while optimal levels of luteinizing hormone alone caused no enhancement of ornithine decarboxylase activity. The induction of ornithine decarboxylase elicited by dibutyryl cyclic AMP, amino acid and luteinizing hormone was diminished about 50% with inhibitors of RNA and protein synthesis. The specific amino acid requirements for ornithine decarboxylase induction in chinese hamster ovary cells was similar to the requirements for induction in two other transformed cell lines. Understanding the mechanism of enzyme induction requires an identification of the essential components of the regulatory system. The essential requirement for enzyme induction is one of five amino acids. The induction of ornithine decarboxylase by dibutyryl cyclic AMP and luteinizing hormone was additive in the presence of an active amino acid.