Changes of serum-induced ornithine decarboxylase activity and putrescine content during aging of IMR-90 human diploid fibroblasts

The roles of ornithine decarboxylase (ODC, EC 4.1.1.17) and polyamines in cellular aging were investigated by examining serum-induced changes of these parameters in quiescent IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL) and in human progeria fibroblasts. Serum stimulation caused increases of ODC and DNA synthesis in IMR-90 human diploid fibroblasts, with maximal values occurring, respectively, 10 hr and 22 hr after serum stimulation. Both serum-induced ODC activity and DNA synthesis in IMR-90 cells were found to be inversely related to their PDL. Maximal ODC activity and DNA synthesis in young cells (PDL = approximately 18-22) were, respectively, five-fold and six-fold greater than that in old cells (PDL = approximately 50-55), which in turn were comparable or slightly higher than that in progeria fibroblasts. Polyamine contents (putrescine, spermidine, and spermine) in quiescent IMR-90 cells did not show significant PDL-dependency. The putrescine and spermine contents in quiescent progeria cells were comparable to those in quiescent IMR-90 cells. The spermidine content in quiescent progeria cells, however, was extremely low, less than half of that in quiescent IMR-90 cells. Serum stimulation caused a marked increase in putrescine content in young cells but not in old cells or in progeria cells. The spermidine and the spermine content in IMR-90 cells, either young or old, and in progeria cells did not change significantly after serum stimulation. Our study indicated that aging of IMR-90 human diploid fibroblasts was accompanied by specific changes of polyamine metabolism, namely, the serum-induced ODC activity and putrescine accumulation. These changes were also observed in progeria fibroblasts derived from patients with Hutchinson-Gilford syndrome.     

Ornithine decarboxylase and thymidine kinase activity in tissues of prolactin-treated rats: effect of hypophysectomy

The activities of ornithine decarboxylase and thymidine kinase were determined in tissues of young intact and hypophysectomized rats at various times after treatment with prolactin. In both types of animals, ornithine decarboxylase activity increased in liver, kidney, spleen and adrenal of prolactin treated rats. Thymidine kinase activity increased only in liver and spleen of intact rats. Increase in the kinase activity was smaller, and occurred later than the change in ornithine decarboxylase. In hypophysectomized animals, thymidine kinase activity increased in spleen, but not in liver, following prolactin treatment.     

Dramatic attenuation of hypusine formation on eukaryotic initiation factor 5A during senescence of IMR-90 human diploid fibroblasts

Deoxyhypusine synthase catalyzes the conversion of lysine to deoxyhypusine residue on the eukaryotic initiation factor 5A (eIF-5A) precursor using spermidine as the substrate. Subsequent hydroxylation of the deoxyhypusine residue completes hypusine formation on eIF-5A. Hypusine formation is one of the most specific polyamine-dependent biochemical events in eukaryotic cells. Although changes in polyamine metabolism have been demonstrated in human diploid fibroblasts during senescence (Chen and Chang, 1986, J. Cell. Physiol., 128:27–32.), it is unclear whether or not polyamine-dependent hypusine formation itself is an age-dependent biochemical event. In the present study, hypusine-forming activity was measured by a radiolabeling assay in cells whose polyamines have been depleted by prior treatment of α-difluoromethyl ornithine (DFMO). In addition, an in vitro cross-labeling assay was developed for simultaneous measurement of the deoxyhypusine synthase activity and protein substrate (eIF-5A precursor) amount. We showed that the hypusine-forming activity in low-passage presenescent IMR-90 cells [population doubling level (PDL) = 15–23, termed young cells] was prominently induced by serum whereas little or no hypusine-forming activity could be detected in late-passage senescent cells (PDL = 46–54, termed old cells). The striking difference in hypusine-forming activity between young and old cells was due to changes in both deoxyhypusine synthase activity and eIF-5A precursor amount in IMR-90 cells during senescence. However, Northern blot analysis showed no significant difference in the eIF-5A messenger RNA (mRNA) between young and old cells, suggesting that the age-dependent attenuation of eIF-5A precursor protein may be regulated at either translational or posttranslational level. J. Cell. Physiol. 170:248–254, 1997. © 1997 Wiley-Liss, Inc.     

An inhibitor of ornithine decarboxylase in lactogen-deprived Nb2 node rat lymphoma cells

A previous study has shown that the activity of ornithine decarboxylase in cultured Nb2 node rat lymphoma cells falls to undetectable levels when cells become quiescent following incubation in lactogen (prolactin)-deficient medium. In the present study, it was found that addition of extracts of the lactogen-deprived, quiescent cells to extracts of log-phase cells markedly reduced the ornithine decarboxylase activity of the latter, the inhibitory activity being proportional to the amount of quiescent cell extract added. Evidence is presented that the ornithine decarboxylase-inhibitory activity in the quiescent cell extracts is due to an antizyme-like, polypeptide factor with an Mr of approx. 28,000. The activity of the inhibitor appears to be directed rather specifically against ornithine decarboxylase, since the activities of S-adenosylmethionine decarboxylase, thymidine kinase and uridine kinase were not affected. The Nb2 cell ornithine decarboxylase inhibitor may have an important role in modulating the cellular levels of ornithine decarboxylase as they change in response to the withdrawal and restoration of extracellular mitogenic lactogens.     

Distinction of G0 cells from senescent cells in cultures of non-cycling human fetal lung fibroblasts by anti-MAP-1 monoclonal antibody staining

On staining with a monoclonal antibody raised against microtubule-associated protein-1 (MAP-1), dot-like structures were seen in the nuclei of interphase cells, but not in those of non-cycling G0-arrested cells. Dots were also not seen in the nuclei of non-cycling senescent human cells (IMR-90). A SV40-DNA-transformed subline of IMR-90 with a limited growth potential showed progressive decrease of cells with nuclei containing dots in the final stage of their lifespan. The dots appeared in G0-arrested IMR-90 cells when these cells were incubated in medium of high osmotic pressure for 3 min. In contrast, no dots appeared in senescent cells or X-ray-irradiated young cells when they were incubated in medium of high osmotic pressure. Thus irreversibly non-cycling cells could be distinguished from G0-phase cells on the level of whole cultures. The results suggest that senescent cells lose their division potential by entering an irreversible cell-cycle stage differing from G0.     

Age dependency of the metabolic conversion of polyamines into amino acids in IMR-90 human embryonic lung diploid fibroblasts

When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000-dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000-dalton protein. We have investigated these two polyamine-associated biochemical events in IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL). We found that (1) the metabolic labeling of the 18,000-dalton protein was about two-fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six-fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4-fold higher in the young cells as compared to the old cells, our data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR-90 cells was about eight-fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000-dalton protein were also observed in cell strains derived from patients with premature aging disease.     

A marked increase of fucosylation of glycoproteins in IMR-90 human diploid fibroblasts during senescence in vitro

Possible changes of glycoproteins in IMR-90 human embryonic lung fibroblasts during senescence in vitro were studied by the metabolic labeling technique using radioactive precursors for carbohydrate moieties of glycoproteins. IMR-90 fibroblasts at three different population doubling level (PDL) were incubated with [3H]fucose and [3H]glucosamine for various periods of time. The radioactively labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The results indicated a marked increase, by more than eight-fold on per mg protein basis, of labeling by [3H]fucose in old IMR-90 fibroblasts (PDL = 45) as compared to young (PDL = 22) and middle-age (PDL = 30) IMR-90 fibroblasts. In contrast, no significant difference in [3H]glucosamine labeling was observed in young and old IMR-90 cells.     

Biphasic increase in ornithine decarboxylase and its relationship with thymidine kinase and DNA synthesis in liver: Effects of dietary protein and amino acid mixture

In rats, feeding protein free diet for 4 days followed by starvation and then high protein diet induced a biphasic ornithine decarboxylase (EC 4.1.1.17) activity, prolonged thymidine kinase (EC 2.7.1.21) activity and DNA synthesis. In contrast feeding a diet containing casein-equivalent amino acid mixture induced a monophasic ornithine decarboxylase activity, short-lived thymidine kinase activity and DNA synthesis. To maintain prolonged thymidine kinase activity and DNA synthesis high protein diet must be given in the early part of the prereplicative period.     

Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.     

Amiloride added together with bumetanide completely blocks mouse 3T3-cell exit from G0/G1-phase and entry into S-phase.

In this study we tested the hypothesis that stimulation of univalent-cation fluxes which follow the addition of growth factors are required for cell transition through the G1-phase of the cell cycle. The effect of two drugs, amiloride and bumetanide, were tested on exit of BALB/c 3T3 cells from G0/G1-phase and entry into S-phase (DNA synthesis). Amiloride, an inhibitor of the Na+/H+ antiport, only partially inhibited DNA synthesis induced by serum. Bumetanide, an inhibitor of the Na+/K+ co-transport, only slightly suppressed DNA synthesis by itself, but when added together with amiloride completely blocked cell transition through G1 and entry into S-phase. Similar inhibitory effects of the two drugs were found on the induction of ornithine decarboxylase (ODC) (a marker of mid-G1-phase) in synchronized cells stimulated by either partially purified fibroblast growth factor (FGF) or serum. To test this hypothesis further, cells arrested in G0/G1 were stimulated by serum, insulin or FGF. All induced similar elevations of cellular K+ content during the early G1-phase of the cell cycle. However, serum and FGF, but not insulin, released the cells from the G0/G1 arrest, as measured by ODC enzyme induction. This result implies that the increase in cellular K+ content may be necessary but not sufficient for induction of early events during the G1-phase. The synergistic inhibitory effects of amiloride and bumetanide on the two activities stimulated by serum growth factors, namely ODC induction (mid-G1) and thymidine incorporation into DNA (S-phase), suggested that the amiloride-sensitive Na+/H+ antiport system together with the bumetanide-sensitive Na+/K+ transporter play a role in the mitogenic signal.     

Thymidine triphosphate synthesis in senescent WI38 cells : Relationship to loss of replicative capacity

The effect of in vitro age on thymidine triphosphate (TTP) synthesis was assessed in WI38 cultures according to the following measurements: (1) thymidine kinase activity of broken cell preparations; (2) in situ incorporation of [3H]thymidine into acid-soluble material; and (3) total intracellular TTP content as determined by an enzymatic assay. All three parameters were maximal in exponentially proliferating populations and minimal in quiescent monolayers; no significant differences between young and old cultures were observed despite the reduced replicative capacity of the latter. The addition of serum to density-arrested cultures induced both TTP synthesis and DNA replication after a lag of approx. 12 h; although a greater percentage of young cells initiated replication as compared with old, pool sizes expanded to a similar extent in both populations. Pool expansion did not require entry into S phase; the pool sizes of control and cytosyl arabinoside-treated cultures were comparable. These findings suggest that senescent cells retain the ability to synthesize TTP, even though they are incapable of replicating DNA. Because TTP synthesis is a cell cycle-dependent event that normally begins in late G1, senescent cells might be blocked in the latter portion of the prereplicative phase and not in G0 as are quiescent cells.     

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.     

Ornithine decarboxylase from Saccharomyces cerevisiae. Purification, properties, and regulation of activity

Ornithine decarboxylase has been purified 1,500-fold to homogeneity from a spe2 mutant of Saccharomyces cerevisiae which lacks S-adenosylmethionine decarboxylase and is derepressed for ornithine decarboxylase. The ornithine decarboxylase is a single polypeptide (Mr = 68,000) and requires a thiol and pyridoxal phosphate for activity. Addition of 10(-4) M spermidine and 10(-4) M spermine to the growth medium reduces the activity of the enzyme by 90% in 4 h. However, immunoprecipitation studies showed that the extracts of polyamine-treated cells contain as much enzyme protein as normal cell extracts. This loss of ornithine decarboxylase activity is probably due to a post-translational modification of enzyme protein because we found no evidence for any inhibitor of activity in the polyamine-treated cells.     

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.     

Increased level of cAMP-dependent protein kinase in aging human lung fibroblasts

Regulation of the expression of cAMP-dependent protein kinase in cellular aging was studied using the IMR-90 diploid human lung fibroblasts. The level of cAMP-dependent protein kinase present in cell extracts was monitored by 1) photoactivated incorporation of 8-N3-[32P]cAMP into the 47,000- and 54,000-dalton regulatory subunits of the type I and type II cAMP-dependent protein kinases, respectively; 2) cAMP-dependent phosphorylation of histone II AS catalyzed by the catalytic subunit of the kinase; and 3) fractionation and analysis of the type I and type II cAMP-dependent protein kinase by DEAE-Sephacel column chromatography. Our results showed an approximately two- to threefold increase in the level of the type I cAMP-dependent protein kinase and a somewhat smaller increase in the type II kinase in extracts of the "old" IMR-90 cells (population doubling greater than 48) as compared to that of the "young" cells (PDL 22-27). The timing of the increase in cAMP-dependent protein kinase coincided with a significant decrease in the proliferative potential of the cells. This result together with previously demonstrated effects of cAMP in the control of cell growth and differentiation and the increased expression of cAMP-dependent protein kinase during terminal differentiation of the murine preadipocytes (3T3-L1) and myoblast (L-5, L-6, and C2C13) suggests that regulation of the levels of cAMP and cAMP-dependent protein kinase plays a significant role in the control of cell growth and differentiation.     

The effects of 24R,25-dihydroxycholecalciferol and of 1 alpha,25-dihydroxycholecalciferol on ornithine decarboxylase activity and on DNA synthesis in the epiphysis and diaphysis of rat bone and in the duodenum.

The effect of cholecalciferol metabolites on ornithine decarboxylase activity and on DNA synthesis in developing long bones was investigated in vitamin D-depleted rats. In the epiphysis there was a 6.4-fold increase in ornithine decarboxylase activity 5 h after a single injection of 24R,25-dihydroxycholecalciferol but not of 24S,25-dihydroxycholecalciferol or other vitamin D metabolites. In comparison, in the diaphysis and duodenum, 1 alpha,25-dihydroxycholecalciferol, but not other vitamin D metabolites, caused a 3-3.5-fold increase in the enzyme activity. The enzyme activity in the tissues examined attained a maximal value at 5 h after the injection of the metabolites. The activity of ornithine decarboxylase in the epiphysial region increased dose-dependently as the result of a single injection of 24R,25-dihydroxycholecalciferol and attained a maximal value at a dose between 30 and 3000 ng. In addition, administration of 24R,25-dihydroxycholecalciferol, but not 24S,25-dihydroxycholecalciferol or other metabolites, caused within 24 h a 1.7-2.0-fold increase in [3H]thymidine incorporation into DNA of the epiphyses of tibial bones. In comparison, 1 alpha,25-dihydroxycholecalciferol caused a 1.5-fold increase in [3H]thymidine incorporation into DNA of the diaphyses and of the duodenum. The present data indicate that 24R,25-dihydroxycholecalciferol is involved in the regulation of epiphyseal growth, whereas 1 alpha,25,dihydroxycholecalciferol stimulates the proliferation of cells in the diaphysis of long bones and in the intestinal mucosa.