Phospholipid turnover during cell-cycle traverse in synchronous Chinese-hamster ovary cells. Mitogenesis without phosphoinositide breakdown.

The turnover of phospholipids was investigated in quiescent serum-starved Chinese-hamster ovary (CHO-K1) cells stimulated to progress through the cell cycle by the addition of dialysed bovine serum. A variety of radiolabelling techniques were employed to study the rapid effects of serum on phospholipids and later events during G1 and S phases of the cell cycle. Pulse-labelling studies using [32P]Pi revealed that there was a stimulation of the synthesis rate of all phospholipids investigated during the initial few hours after serum addition. The greatest stimulation (20-fold) was observed in phosphatidylcholine, and the smallest in the polyphosphoinositides (PPIs). Mock stimulation with serum-free medium caused a similar increase in PPI turnover, but little or no effect on turnover of other phospholipids. This effect could be accounted for by a stimulation of the turnover of cellular ATP pools increasing [32P]ATP specific radioactivity. Late G1 and S phases were associated with a decrease in the rate of synthesis of all phospholipids. Phosphatidic acid was the only phospholipid whose labelling fell below that in mock-stimulated cells during the period of the cell cycle. Stimulation of serum-starved cells that had been prelabelled with myo-[2-3H]inositol caused no change in the amounts of inositol trisphosphate, but both serum-stimulated and mock-stimulated cells exhibited similar small decreases in both inositol bisphosphate and inositol monophosphate, of approx. 30% after 30 s. When cells were serum-stimulated in the presence of 10 mM-Li+, there was no increase in the size of the total inositol phosphate pool. We conclude that mitogenic stimulation and cell-cycle traverse cause profound and complex effects on phospholipid turnover in CHO-K1 cells, but there is no evidence for a role of inositol lipid turnover in the proliferative response to serum in this cell line.     

Inhibition by trioxalen (psoralen) plus near-ultraviolet light of the induction of ornithine decarboxylase in Chinese-hamster cells.

Trioxalen (trimethylpsoralen) plus near-u.v. light, a potent inhibitor of DNA and RNA synthesis, inhibits the induction of ornithine decarboxylase in stationary-phase V79 fibroblasts. It does not affect the translation of pre-existing mRNA. The method, in view of its high degree of specificity and precise timing, is a better choice for inhibiting RNA synthesis than the commonly used chemical inhibitors and precursor analogues.     

Mutant strain of Chinese hamster ovary cells with no detectable ornithine decarboxylase activity.

We previously described an arginase-deficient, polyamine-dependent Chinese hamster ovary cell line which grows in serum-free medium. From this strain we isolated a new mutant strain that has no detectable catalytic ornithine decarboxylase activity. The mutant cells contain, however, immunoreactive ornithine decarboxylase-like protein roughly in the same quantity as the parent strain. The mutant and the parent cell line strains also contain similar amounts of ornithine decarboxylase-mRNA hybridizable to a specific cDNA. If polyamines are omitted from the medium, proliferation of the mutant cells is considerably retarded and ceases in 6 to 10 days. Addition of ornithine or alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, has no effect on these cells. Putrescine and spermidine decreased in the mutant cells to undetectable levels during polyamine starvation, whereas spermine was reduced to 1/5th of that found in the control cultures. Polyamines appear to be indispensable for the mutant strain, but this was obvious only after the amount of polyamines, found as impurities in bovine serum albumin used in the medium, was reduced by dialysis to 10(-12) M. Because sera contain polyamines, the ability of the mutant strain to grow in serum-free medium is a great advantage in elucidation of the mechanisms of polyamine function.     

Lipopolysaccharide and serum synergistically stimulate ornithine decarboxylase in Chinese hamster ovary cells

Summary Lipopolysaccharide (LPS), the active component of bacterial endotoxin, caused no significant increase in ornithine decarboxylase (ODC) activity in serum-starved, Chinese hamster ovary fibroblasts. However, concurrent addition of LPS with 10% fetal bovine serum caused a synergistic 30 to 40-fold increase in enzyme activity as compared to the 10 to 20-fold increase seen after addition of serum alone. This synergism was not due to an alteration in the time course of enzyme induction after serum addition. The LPS-induced synergy of ODC induction by serum was inhibited by the concurrent addition of the specific LPS-antagonist, Polymyxin B. This investigation was supported by PHS Grant CA32444, awarded by the National Cancer Institute. A. R. L. G. is a recipient of a USPHS fellowship, GM09226-01, and S. M. T. was supported by NIH training Grant AMO 7282.     

Lipopolysaccharide and serum synergistically stimulate ornithine decarboxylase in Chinese hamster ovary cells

Lipopolysaccharide (LPS), the active component of bacterial endotoxin, caused no significant increase in ornithine decarboxylase (ODC) activity in serum-starved, Chinese hamster ovary fibroblasts. However, concurrent addition of LPS with 10% fetal bovine serum caused a synergistic 30 to 40-fold increase in enzyme activity as compared to the 10 to 20-fold increase seen after addition of serum alone. This synergism was not due to an alteration in the time course of enzyme induction after serum addition. The LPS-induced synergy of ODC induction by serum was inhibited by the concurrent addition of the specific LPS-antagonist, Polymyxin B.     

Immunohistochemical localization of ornithine decarboxylase in the rat ovary

Summary The present report describes the immunocytochemical localization of ornithine decarboxylase in the prepubertal rat ovary after administration of human chorionic gonadotropin (HCG). Numerous ornithine decarboxylase immunoreactive cells appeared in the thecal layer as well as in the interstitial gland tissue after treatment with HCG. The granulosa cells, the ovum and the ovarian stroma were devoid of immunoreactive ornithine decarboxylase. In contrast to the ovary of HCG-treated rats, the ovary of prepubertal rats given the vehicle alone contained only a few weakly immunoreactive cells.     

Amplification of ornithine decarboxylase gene in response to polyamine deprivation in Chinese hamster ovary cells

We have isolated from an arginase-deficient polyamine-dependent Chinese hamster ovary cell line a new mutant strain that has greatly increased ornithine decarboxylase activity. This enables the cells, in the absence of ornithine, to decarboxylate lysine into cadaverine (diaminopentane) that is further converted into N-(3-aminopropyl)cadaverine and N,N'-bis(3-aminopropyl)cadaverine. These unusual polyamines can support the growth of the cells without added polyamines derived from ornithine. Immunoreactive ornithine decarboxylase-like protein was clearly increased in the mutant cells but could not solely account for the greatly increased enzyme activity. Southern blot analysis of DNA hybridized to a plasmid carrying ornithine decarboxylase-cDNA revealed at least a 32-fold amplification of the ornithine decarboxylase gene. Ornithine decarboxylase-mRNA concentration was also highly increased in the cells. The half-life of the enzyme and the Km for ornithine were not altered from those of the parental cell line.     

Regulation of ornithine decarboxylase by ODC-antizyme in HTC cells.

Low concentrations of putrescine (10^?5M) blocked ornithine decarboxylase (ODC) in rat hepatoma (HTC) cells in culture, but the lower homologue of putrescine, 1, 3 diaminopropane, had no effect on ornithine decarboxylase at 10^?5M. Higher concentrations of both putrescine and 1, 3 diaminopropane induced approximately the same amount of soluble ODC antizyme type inhibitor. When concentrated dialyzed supernatants of cells grown in 10^?5M putrescine were treated with 250 mM NaCl and chromatographed on a superfine Sephadex G-75 column, both ODC and inhibitor were recovered. Spermidine, spermine and cadaverine also induced the inhibitor suggesting a low specificity of induction by amines.     

Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.

N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.     

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.     

Effect of sodium arsenite on the induction and turnover of ornithine decarboxylase activity in erythroleukemia cells

Sodium arsenite proved effective in preventing the induction of ornithine decarboxylase (ODC) activity elicited by dilution of Friend erythroleukemia cells in fresh medium. A 50 per cent inhibition was produced at approximately 1 microM arsenite and complete inhibition was obtained at concentrations above 10 microM. However, addition of arsenite 5 h after cell dilution, i.e. when ODC was already induced, appeared to stabilize the enzyme. The half-life of ODC activity, measured after cycloheximide treatment, increased almost six-fold after addition of sodium arsenite. Agents known to provoke oxidative alteration of the thiol-redox status in cells, also caused a similar effect on the induction and stability of ODC.     

Synthesis and secretion of active lipoprotein lipase in Chinese-hamster ovary (CHO) cells.

Cultured Chinese-hamster ovary cells (CHO cells) were found to produce and secrete a lipase, which was identified as a lipoprotein lipase by the following criteria. Its activity was stimulated by serum and apolipoprotein CII, and was inhibited by high salt concentration. The lipase bound to heparin-agarose and co-eluted with 125I-labelled bovine lipoprotein lipase in a salt gradient. A chicken antiserum to bovine lipoprotein lipase inhibited the activity and precipitated a labelled protein of the same apparent size as bovine lipoprotein lipase from media of CHO cells labelled with [35S]methionine. The lipase activity and secretion were similar in growing cells and in cells that had reached confluency. Hence, lipoprotein lipase appears to be expressed constitutively in CHO cells and is not linked to certain growth conditions, as in pre-adipocyte and macrophage cell lines. At 37 degrees C, but not at 4 degrees C, heparin increased the release of lipase to the medium 2-4-fold. This increased release occurred without depletion of cell-associated lipase activity, suggesting that heparin enhanced release of newly synthesized lipase.     

Cellular origin of prolonged induction of ornithine decarboxylase in the rat ovary

The temporal changes of ornithine decarboxylase (ODC) activity were investigated in the immature rat ovary following a single subcutaneous injection of pregnant mare serum gonadotropin (PMSG). A dose-response relationship was established. Maximal ODC activity was obtained at a dose of 25 IU of PMSG. This increase in ODC activity was accompanied by an increase of ovarian weight before reaching a maximum. A 250-fold increase of ODC activity was observed 1 day following a single dose of PMSG (50 IU). The enzyme specific activity only returned to the control level 4-5 days after hormone treatment. Immunoreactive ODC in immature, PMSG-primed rat ovaries and in heavily luteinized rat ovaries was localized utilizing the immunoperoxidase method and an antibody to ODC. Immunoreactive enzyme was confined to the cytoplasm of the granulosa cells but was not present in luteal cells. Thecal cells showed only weak immunostaining. This study provides clear evidence that the granulosa cell is the unique source of ODC activity in response to PMSG treatment. Furthermore, these data support the concept that polyamines play a role in granulosa cell proliferation and hence follicular development.