Transforming growth factor is a potent stimulator of testicular ornithine decarboxylase in immature mouse

Administration of transforming growth factor, type alpha (TGF-alpha), to eight-day old mice resulted in 22 fold increase of testicular ornithine decarboxylase activity. Two to seven fold increases of enzyme activities by TGF-alpha were also observed in intestine, kidney, spleen, liver, and heart. The maximal enzymatic responses were reached 2-4 h after TGF-alpha administration in vivo. The induction of tissue ornithine decarboxylase activity was accompanied by an increase in new protein synthesis. These effects were found to be comparable to those in littermates administered with mouse epidermal growth factor but were significantly more pronounced than with bovine growth hormone. Daily administration of TGF-alpha to newborn mice also produced a 1.8 fold increase of testicular weight after 14 days. The present studies therefore show that TGF-alpha is an epidermal growth factor-like mitogen and is likely to be important for the testicular development of immature animal.     

Nerve growth factor-induced reduction in epidermal growth factor responsiveness and epidermal growth factor receptors in PC12 cells: an aspect of cell differentiation.

The rat pheochromocytoma clone PC12 responds to nerve growth factor through the expression of a number of differentiated neuronal properties. One of the most rapid changes is a large, transient increase in the activity of ornithine decarboxylase. These cells also show an increase in ornithine decarboxylase activity in response to the mitogen, epidermal growth factor, but do not respond morphologically as they do to nerve growth factor. Specific, high-affinity epidermal growth factor receptors are present on the cells. When the cells are differentiated with nerve growth factor, the response to epidermal growth factor is markedly diminished and there is a marked reduction in the binding of epidermal growth factor to the cells.     

Lack of ornithine decarboxylase activity in isolated rat liver parenchymal cells

Polyamines are associated with fundamental metabolic and functional steps in cell metabolism. The activity of ornithine decarboxylase, the key enzyme in polyamine metabolism, was followed during the preparation of rat liver parenchymal cells and in the isolated cells during incubation. In experiments in which ornithine decarboxylase was not induced in vivo, enzyme activity dropped to barely measurable values during the preparation. An even more drastic loss of enzyme activity was noted in livers in which ornithine decarboxylase activity was stimulated in vivo 20-40fold by previous injection of bovine growth hormone, or thioacetamide or elevated because of circadian rhythmical changes of the enzyme activity. Within the first 20 min of liver perfusion to disintegrate the tissue, ornithine decarboxylase activity decreased by up to 80%. The presence of bovine growth hormone during cell preparation cannot prevent the loss of enzyme activity. Incubation of the isolated cells for periods of up to 240 min did not restore the enzyme activity. Furthermore, incubation of the cells with bovine growth hormone did not induce ornithine decarboxylase, even though the medium was supplemented with amino acids in physiological concentrations. During normal liver perfusion and in contrast to the situation with isolated cells, there is no loss of enzyme activity but a small rise. Following pretreatment of the animals with bovine growth hormone or thioacetamide the highly stimulated activity of ornithine decarboxylase declined slowly during liver perfusion, but never dropped to values lower than normal for perfusion periods of up to 240 min. Moreover, in the intact perfused organ ornithine decarboxylase remains responsive to bovine growth hormone. The experiments demonstrate that enzymatic tissue dispersion by collagenase in particular or the preparation of isolated cells in general drastically alters the metabolic and functional state of rat liver parenchymal cells.     

Induction of ornithine decarboxylase activity by growth and differentiation factors in FRTL-5 cells

Induction of ornithine decarboxylase has been correlated with the onset of cellular proliferation and cAMP production. Whether the resulting increases in polyamine levels are essential mediators of growth and/or differentiation or are merely incidental remains controversial. We have used FRTL-5 thyroid cells in culture to study the effects of three growth factors on ornithine decarboxylase activity. These factors [TSH, bovine calf serum, and 12-O-tetradecanoylphorbol-13-acetate (TPA)] are thought to act through different intracellular pathways. TSH stimulates cAMP production in thyroid cells, calf serum acts through ill-defined pathways to stimulate growth, and TPA is known to activate protein kinase C. Bovine calf serum and TSH acted synergistically to induce ornithine decarboxylase activity. Activity was maximal when the phosphodiesterase inhibitor, methyl isobutyl xanthine, was included. Individually, neither serum nor TSH was a potent stimulator of the enzyme. Ornithine decarboxylase mRNA was apparent on Northern blots as a doublet following one hour of exposure to these agents. TPA did not stimulate ornithine decarboxylase activity and had an inhibitory effect on enzyme induction by TSH and serum. Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, inhibited growth induced by both TPA and TSH in putrescine-free medium. This effect was not apparent in medium containing 10(-5) M putrescine. The data indicate that, although intracellular levels of cyclic AMP regulate ornithine decarboxylase activity, a component in serum is necessary for significant induction of this enzyme. Factors stimulating growth by non-cyclic AMP-dependent pathways may act without apparently stimulating this enzyme, although polyamines appear to be essential for their growth stimulatory effects.     

Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH-3T3 fibroblasts

Ornithine decarboxylase activity was assessed in serum-deprived quiescent NIH-3T3 murine fibroblasts after exposure to a variety of growth-promoting factors. Ornithine decarboxylase activity increased after treatment with phorbol 12-myristate 13-acetate (PMA), fetal calf serum, bovine pituitary fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and the synthetic diacyglycerol sn-1,2-dioctanolyglycerol but not after treatment with epidermal growth factor, insulin, 4 alpha-phorbol 12,13-didecanoate, sn-1,2-dibutyrylglycerol, or the calcium ionophore A23187. Activity peaked at 3-4 h and returned to basal levels after 8 h. To determine the importance of protein kinase C in this increase, cells were pretreated with PMA for 16 h to make the cells effectively deficient in protein kinase C; this deficiency was documented by direct measurement of enzyme activity and immunoreactivity. The ornithine decarboxylase response to each mitogen was then compared in cells pretreated with PMA or control conditions. PMA pretreatment abolished the increase in ornithine decarboxylase activity due to additional PMA and decreased but did not eliminate the ability of serum, FGF, and PDGF to cause increases in ornithine decarboxylase activity. Similarly, pretreatment with PMA abolished the ability of additional PMA to increase ornithine decarboxylase mRNA levels but did not prevent the increases in these mRNA levels caused by FGF or serum. These data suggest that the increases in ornithine decarboxylase activity and mRNA levels that occur in quiescent fibroblasts in response to serum, FGF, or PDGF are due to activation of at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C.     

Platelet derived growth factor induces ornithine decarboxylase activity in NIH 3T3 cells

Incubation with highly purified human Platelet Derived Growth Factor induced ornithine decarboxylase activity in quiescent NIH 3T3 cells concomitantly with mitogenic stimulation. Pretreatment of cells with a specific ornithine decarboxylase inhibitor, DL-alpha-difluoromethyl-ornithine significantly inhibited the effect of the mitogen on DNA synthesis. These experiments suggest that the mitogenic activity of Platelet Derived Growth Factor, similarly to that of other serum growth factors or tumor promoters, is mediated through rise in polyamine levels.     

Regulation of ornithine decarboxylase activity in rat ovarian cells in vitro.

Incubation of rat ovarian cell suspension with human choriogonadotropin (hCG) caused a marked enhancement of ornithine decarboxylase (EC 4.1.1.17) activity after a lag period of several hours. Even though ovarian ornithine decarboxylase could be induced in minimum essential medium by the hormone alone, supplementation of the medium with various sera greatly enhanced the stimulation of the enzyme activity. All the sera tested (human, fetal calf and horse) were able to stimulate ornithine decarboxylase activity even in the absence of hCG. Maximum stimulation of the enzyme activity by hCG and/or serum occurred in ovarian cell suspensions prepared from 30 to 33-day-old rats. There was a close correlation between the stimulation of ornithine decarboxylase activity and the accumulation fo cyclic AMP in response to the administration of the hormone (in the presence or absence of serum). However, while various sera alone markedly enhanced ovarian ornithine decarboxylase activity in vitro they, if anything, only marginally stimulated the accumulation of cyclic AMP and the secretion of progesterone in ovarian cells in the absence of gonadotropin. A similar dissociation of the stimulation of ornithine decarboxylase activity from the production of cyclic AMP and progesterone was likewise found when the ovarian cells were incubated in an enriched medium (M199) supplemented with albumin and lactalbumin hydrolysate in the absence of the hormone. Under these culture conditions ornithine decarboxylase activity was strikingly enhanced, greatly exceeding the stimulation obtained with various sera, while the accumulation of cyclic AMP and the secretion of progesterone remained virtually unchanged. Specific inhibition (up to 90%) of gonadotropin-induced ornithine decarboxylase activity by difluoromethyl ornithine or 1,3-diamino-2-propanol had little effect on the ability of the ovarian cells to respond to the hormone with increasing production of cyclic AMP and progesterone. While showing that rat ovarian ornithine decarboxylase can be induced in vitro by choriogonadotropin or various sera, our results indicate that the activation of the enzyme involves at least two different mechanisms: (i) One (in response to gonadotropin) involving a prior stimulation of cyclic AMP production, and (ii) another (in response to serum) that is not associated with increases in the accumulation of the cyclic nucleotide.     

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.     

Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.     

The neurite-promoting effect of fibroblast growth factor on PC12 cells

Treatment of PC12 cells with fibroblast growth factor(s) from either brain or pituitary caused neurite outgrowth comparable to that produced by nerve growth factor. The neurite outgrowth was preceded by a substantial rise in the activity of ornithine decarboxylase.     

The effects of nerve growth factor on polyamine metabolism in PC12 cells

Nerve growth factor treatment produces a large increase in the activity of ornithine decarboxylase and a moderate decrease in the activity of S-adenosylmethionine decarboxylase in PC12 cells. These changes are reflected weakly, if at all, in the levels of putrescine, spermidine, and spermine in the cells. The rates of polyamine synthesis are increased somewhat more than the overall levels, but still are not comparable in extent to the increase in the ornithine decarboxylase activity. Inhibitors of ornithine decarboxylase and S-adenosylmethionine decarboxylase have their expected effects on the induction of ornithine decarboxylase and on the activities of both enzymes. Neither inhibitor alone, nor a combination of inhibitors, altered the rate or extent of nerve growth factor-induced neurite outgrowth in the cells.     

A nonmitogenic analogue of epidermal growth factor induces early responses mediated by epidermal growth factor

Cyanogen bromide-cleaved epidermal growth factor (CNBr-EGF) binds to EGF receptors with reduced affinity compared to the native hormone but fails to induce DNA synthesis. However, at similar receptor occupancy, CNBr-EGF is as potent as EGF in activating early cell responses to the hormone. The phosphorylation of membrane proteins, the stimulation of Na+-K+-ATPase as reflected by the ouabain-sensitive uptake of 86Rb of fibroblasts, changes in the organization of microfilaments and in cell-morphology, and the activation of the enzyme ornithine-decarboxylase are all induced by CNBr-EGF as well as EGF Our results are consistent with the notion that EGF-induced phosphorylation could act as a "second messenger" for the action of various EGF-induced responses such as activation of Na+-K+-ATPase, changes in the cytoskeleton and cell morphology, and the activation of the enzyme ornithine decarboxylase. However, the stimulation of phosphorylation of membrane proteins and other early responses are either not required or necessary but insufficient for the induction of DNA synthesis. Suboptimal concentrations of EGF together with CNBr-EGF stimulate DNA synthesis in human fibroblasts. Other growth factors such as insulin, fibroblast growth factor, and prostaglandin F2 alpha, which potentiate the mitogenic response of EGF, do not effect the response to CNBr-EGF. This suggests that the restoration of the mitogenic properties of CNBr-EGF by suboptimal doses of EGF occurs at the level of EGF receptors or during their processing.     

Biochemical properties of the endothelium-derived growth factor: Comparision to other growth factors

Cultured bovine aortic endothelial cells (BAEC) can be maintained at saturation density for several weeks in the absence of serum. These cells retain viability and normal culture morphology, and continuously produce a growth factor for mesenchymally derived cells–the endothelium-derived growth factor (EDGF). The amount and specific activity of EDGF that is produced by BAEC under serum-free conditions remains constant for weeks. The levels of EDGF produced under these serum-free conditions is equivalent to levels produced in medium containing 5% plasma-derived serum. EDGF has been found to be trypsin sensitive, acetone and ammonium sulfate precipitable, and resistant to heat and sodium dodecyl sulfate treatment. Gel filtration on Sephacryl S-200 in the presence of formic acid (1%) yields two major peaks of activity corresponding to proteins of apparent molecular weights of approximately 24,000 and 14,000 daltons. This chromatographic step affords a ten-to 12-fold purification with a combined recovery of greater than 85%. Unlike brain or pituitary fibroblast growth factor, EDGF activity is destroyed by dithiothreitol or periodic acid. EDGF is not a somatomedin since it exhibits no detectable sulfation activity in a porcine cartilage assay. EDGF is not inhibited by antiserum to epidermal growth factor and is capable of stimulating DNA synthesis in a 3T3 variant cell line that is nonresponsive to and lacks receptors for epidermal growth factor. The majority of EDGF activity does not behave like the platelet-derived growth factor during ion exchange chromatography. Antisera prepared in rabbits and in mice to human platelet-derived growth factor has little effect on bivine or human EDGF activity. These biochemical and immunological properties of EDGF indicate that it is distinct from several other well-characterized polypeptide growth factors.     

Evidence for structural dissociation of two biologic actions of growth hormone

The effects of purified growth hormone and its CNBr fragments on somatomedin induction and on the stimulation of hepatic and renal ornithine decarboxylase (l-ornithine carboxylase, EC 4.1.1.17) activity in rats have been investigated. At the doses tested, none of the CNBr fragments induced somatomedin as evidenced by lack of an effect on sulfate, leucine, and thymidine incorporation into cartilage of hypophysectomized rats. However, the largest fragment, consisting of two peptides corresponding to Residues 6–124 and 150–179 linked by a disulfide bridge, stimulated both renal and hepatic ornithine decarboxylase activity in hypophysectomized rats and the activity of the hepatic enzyme in intact animals. A smaller CNBr fragment corresponding to Residues 125–149 slightly stimulated the activity of renal ornithine decarboxylase but failed to increase activity of the hepatic enzyme. A similar slight stimulation of the activity of the renal, but not the hepatic, enzyme was produced by a large carboxyl-terminal fragment (molecular weight 8000) prepared by proteolytic cleavage of partially purified ovine growth hormone. Circular dichroic spectra of the CNBr fragments demonstrated that the largest fragment retained much of the ordered secondary structure of intact growth hormone while two smaller CNBr fragments were devoid of ordered secondary structure. These observation indicate that different biological activities of growth hormone may be dissociated by fragmentation of the parent molecule.     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Differential inhibition of nerve growth factor and epidermal growth factor effects on the PC12 pheochromocytoma line

Tests have been made of the action of the methyltransferase inhibitors 5'-S-methyl adenosine, 5'-S-(2-methyl-propyl)-adenosine, and 3-deaza- adenosine +/- L-homocysteine thiolactone, on nerve growth factor (NGF)- dependent events in the rat pheochromocytoma line PC12. Each of these agents inhibited NGF-dependent neurite outgrowth at concentrations of the order of millimolar. Slow initiation of neurite outgrowth over several days and more rapid regeneration of neurites (congruent to 1 d) were blocked, as was the priming mechanism necessary for genesis of neurites. The inhibitions were reversible in that PC12 cells maintained for several days in the presence of inhibitors grew neurites normally after washout of these agents. Other NGF-dependent responses of the PC12 line (i.e., induction of ornithine decarboxylase activity [over 4 h], enhancement of tyrosine hydroxylase phosphorylation [over 1 h], and rapid changes in cell surface morphology [30 s onward]) were inhibited by each of the agents. In contrast, corresponding epidermal growth factor-dependent responses in ornithine decarboxylase activity, phosphorylation, and cell surface morphology were not blocked, but instead either unaffected or enhanced, by the methylation inhibitors. These inhibitors did not act by blockade of binding of NGF to high- or low-affinity cell surface receptors, though they partially inhibited internalization of [125I]NGF. The inhibition of rapidly-induced NGF- dependent events and the differential inhibition of responses to NGF and epidermal growth factor imply that the methyltransferase inhibitors specifically block one of the first steps in the mechanistic pathway for NGF.     

Ornithine decarboxylase activity during rat spermatogenesis in vivo and in vitro: Selective effect of hormones and growth factors

We have established the patterns of ornithine decarboxylase activity (an enzyme related to cell growth, differentiation, and proliferation) during rat testicular development and studied the effect of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-type β (TGF-β), and a serum-free, hormone/growth factor-supplemented medium (TKM) on ornithine decarboxylase (ODC) activity in Sertoli-spermatogenic cell cocultures and cultured seminiferous peritubular cells prepared from sexually immature rats (20–22 days old). Results were correlated with timing of ODC activities during rat testicular development. We have found that: (1) although EGF, alone or combined with PDGF and TGF-β, and TKM stimulated ODC activity in Sertoli-spermatogenic cell cocultures after 6 and 24 h of stimulation, PDGF exerted an inhibitory effect, and (2) cultured peritubular cells stimulated with EGF, PDGF, TGF-β (and their combinations), and TKM displayed an increase in ODC activity after 6 h of stimulation, but ODC activities for most of these treatments declined considerably 24 h after stimulation. Light microscopic autoradiographic studies of [³H]thymidine labeled samples demonstrated that (1) clones of spermatogenic cells traverse S phase synchronously, (2) Sertoli cells are not significantly radiolabeled, probably because of contact inhibition achieved by high cell plating density, and (3) peritubular cells are significantly [³H]thymidine labeled in the presence of TKM, a culture medium that facilitates spermatogenic cell long-term viability and differentiation. We conclude that TKM and EGF have stimulatory effects on the biochemical pathway that precedes synchronous DNA synthesis in spermatogonia and preleptotene spermatocytes, and that ODC activity is a sensitive marker for monitoring these events.     

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.     

The permissive effect of cortical steroids on the induction of brain ornithine decarboxylase by nerve growth factor

The intraventricular administration of nerve growth factor causes a marked increase in the activity of ornithine decarboxylase in rat brain. This increase is much smaller in adrenalectomized rats. Dexamethasone and corticosterone, administered either systemically or intraventricularly, are able to restore the ability of nerve growth factor to induce ornithine decarboxylase. The steroids must be given at least three hours before the nerve growth factor to be fully effective.