Effect of 3-isobutyl-1-methylxanthine on prolactin actions on RNA synthesis, casein synthesis, lipid synthesis and ornithine decarboxylase activity in mouse mammary gland explants

The effect of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, was tested on several actions of prolactin in cultured mouse mammary tissues. At concentrations of 0.5 mM and above, IBMX abolished the actions of prolactin on RNA and casein synthesis. IBMX by itself, stimulated ornithine decarboxylase (ODC) activity in a dose-response fashion; but the IBMX at concentrations up to 1 mM had no effect on the magnitude of the prolactin-stimulated ODC activity. IBMX inhibited in a dose-response fashion the rate of [14C]-acetate incorporation into lipids; however, prolactin stimulated lipid biosynthesis in the presence of IBMX concentrations of up to 1 mM.     

Saponin effects of prolactin-like stimulation of ornithine decarboxylase activity in mouse mammary gland explants.

Saponin, a naturally occurring plant glycoside, was found to elicit a prolactin-like stimulation of ornithine decarboxylase (ODC) activity in mouse mammary gland explants. A dose-response activation of ODC was observed with saponin at concentrations between 2 and 10 micrograms/ml. At concentrations of 10 and 15 micrograms/ml, saponin effected a response similar to that of PRL; when tested in concert, PRL and saponin caused a nonadditive response. The time-course of the saponin and PRL effects on ODC activation were not different; a maximum response occurred 2-4 hours after addition of saponin. The saponin and PRL responses were abolished by antibiotics (puromycin and cyclohexamide) that inhibit protein synthesis, but not by actinomycin D which inhibits RNA synthesis. Finally, saponin, by itself, did not affect the rate of milk product formation, but at higher concentrations (above 0.5 microgram/ml) impaired the PRL stimulation of lipid and casein synthesis.     

Effects of Polyamines and Kinetin on In Vitro Frond Senescence in Lemna aequinoctialis 6746

Both polyamines and kinetin could retard the loss of chlorophyll during dark-induced senescence in excised frond of Lernna aequinoctialis 6746. The effect of polyamines on retarding the chlorophyll loss was stronger than that of kinetin. Kinetin remarkably inhibited the loss of soluble proteins and the increase of protease activity, while no similar effects were observed from polyamines. An inhibitor of polyamine biosynthesis, methylglyoxal bis- (guanyl- hydrazone) (MGBG), slightly increased the loss of chlorophyll and soluble proteins. During senescience, both the increase of putrescine (Put) content and the decrease of spermidine (Spd) content were inhibited by kinetin at the concentration of 0.05 mmol/L, but the spermine (Spm) level was not affected by kinetin. The arginine decarboxylase (ADC) activity was dominant in frond of Lemna aequinoctialis 6746. Kinetin slightly increased ADC activity, while it had no marked effect on ornithine decarboxylase (ODC) and s-adenosylmethionine decarboxylase (SAMDC). The possible relationship between polyamines and cytokinins in retarding senescence was also discussed.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

多胺与激动素对稀脉浮萍离体叶状体衰老的影响

多胺与ＫＴ 都可抑制暗诱导衰老的稀脉浮萍（Ｌｅｍ ｎａ ａｅｑｕｉｎｏｃｔｉａｌｉｓ）离体叶状体的叶绿素损失,且多胺的作用大于ＫＴ。ＫＴ 还显著抑制蛋白质的损失与蛋白酶活性的上升,而多胺对此却无大的影响。０．０５ ｍ ｍ ｏｌ／Ｌ的甲基乙二醛二脒基－腙（ＭＧＢＧ）轻微促进叶绿素和蛋白质的损失。０．０５ ｍ ｍ ｏｌ／Ｌ的ＫＴ 可抑制衰老过程中腐胺（Ｐｕｔ）的上升和亚精胺（Ｓｐｄ）的下降,而对精胺（Ｓｐｍ ）无明显影响。在稀脉浮萍中,精氨酸脱羧酶（ＡＤＣ）活性占优势。ＫＴ 可轻微促进ＡＤＣ 活性,而对鸟氨酸脱羧酶（ＯＤＣ）和Ｓ－腺苷甲硫氨酸脱羧酶（ＳＡＭＤＣ）活性无显著影响。讨论了多胺与细胞分裂素在抑制植物叶片衰老过程中作用途径的可能关系     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

The effects of polyamine antimetabolites on polyamine-responsive casein kinase activity

The effects of two inhibitors of ornithine decarboxylase activity, alpha-difluoromethylornithine (DMFO) and (2R,5R) 6-heptyne-2,5 diamine (HDA), and an inhibitor of S-adenosylmethionine decarboxylase, methylglyoxal bis-guanylhydrazone (MGBG), were tested on casein kinase activity and endogenous phosphorylation in the cytosol fractions of mouse thyroid and a rat prostate tumor model, Dunning R 3327 MAT LyLu subline. When tested at 5 mM, spermine, DMFO, HDA, and MGBG stimulated mouse thyroid casein kinase activity by 230%, 14%, 65% and 106%, respectively. Similar responses were observed in prostate tumor cytosol. In mouse thyroid cytosol, spermine stimulates 32P incorporation primarily into 3 proteins (MW: 107, 88, and 56 kDa). At 5 mM, MGBG partially reproduces the effects of spermine; HDA is less effective and DMFO is without effect. Similar effects were observed on 3 proteins in prostate tumor cytosol with molecular weights of 91, 41, and 32 kDa. These data provide additional support for the hypothesis that the observed synergistic inhibitory effect of DMFO and MGBG on cell growth may not be due solely to the inhibition of polyamine biosynthesis. Our findings suggest that MGBG-mediated reduction in the phosphorylation of casein kinase substrate should be considered as one locus of action.     

Prolactin stimulation of iodide uptake and incorporation into protein is polyamine-dependent in mouse mammary gland explants

Previous studies have demonstrated that the prolactin stimulation of most lactational processes (casein, lactose, and triglyceride synthesis) requires an earlier stimulating effect of prolactin on the synthesis of the polyamines. Spermidine appears to be the specific polyamine required for prolactin to enhance milk product synthesis. Inorganic iodide is present in milk at more than an order of magnitude higher concentration than that of the maternal plasma. Since prolactin stimulates iodide accumulation in milk, the goal of these studies was to determine the role of the polyamines in this hormone response. Two drugs were employed in these studies: DFMO (difluoromethylornithine), which inhibits ornithine decarboxylase, and MGBG [methylglyoxal bis(guanyl-hydrazone)], which inhibits S-adenosyl methionine decarboxylase. In mammary gland explants from midpregnant (10-14 days of pregnancy) mice, MGBG at 100 microM abolished the prolactin stimulation of iodide uptake and incorporation into milk proteins, whereas DFMO caused a concentration-dependent inhibition of the PRL response. Selected sensitivity of the MGBG and DFMO inhibitions was validated by a reversal of the drug inhibitions with the addition of 1 mM spermidine to the culture medium. These data suggest that the polyamine signaling pathway is involved in the prolactin stimulation of iodide uptake into milk.     

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.     

Effect of DL-alpha-hydrazino-delta-aminovaleric acid, an inhibitor of ornithine decarboxylase, on polyamine metabolism in isoproterenol-stimulated mouse parotid glands.

The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.     

Stabilization of ornithine decarboxylase and N1-spermidine acetyltransferase in rat liver by methylglyoxal bis(guanylhydrazone)

The activities of ornithine decarboxylase and spermidine N1-acetyltransferase started to rise in normal rat liver 4 h after the intraperitoneal injection of methylglyoxal bis(guanylhydrazone) (MGBG; 80 mg/kg). Ornithine decarboxylase had its greatest activity 24 h after a single injection of MGBG and the acetyltransferase peaked 8 h after the injection. Measurement of the apparent half-life of ornithine decarboxylase after MGBG treatment revealed a clear decrease in the decay rate of the enzyme in both normal and regenerating rat liver. MGBG slowed the decay of the transferase also in normal rat liver, as well as inhibiting its activity in vitro. The stabilization by MGBG of these two short-lived proteins involved in metabolism of polyamines should lead to their accumulation in liver, thus explaining their increased activities. In the case of ornithine decarboxylase, studies with a specific antibody against mouse kidney ornithine decarboxylase showed that the rise in ornithine decarboxylase activity after MGBG application was not due to the appearance of an immunologically different isozyme.     

Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, ofS-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.     

Prolactin stimulation of Nb 2 node lymphoma cell division is inhibited by polyamine biosynthesis inhibitors

The mitogenic action of prolactin in Nb 2 node lymphoma cells was inhibited by two drugs which interfere with polyamine biosynthesis. At concentrations of 0.5 mM and above alpha-difluoromethyl ornithine (DFMO), which inhibits ornithine decarboxylase and the conversion of ornithine to putrescine, significantly attenuated the mitogenic effect of prolactin. This inhibition was prevented by the addition of putrescine, spermidine, or spermine to the culture medium. At concentrations of 1 microM and above methylglyoxal bis(guanylhydrazone) (MGBG), which inhibits S-adenosylmethionine decarboxylase and hence the conversion of putrescine to spermidine and spermine, abolished the mitogenic action of prolactin. This inhibition was prevented by the addition of spermidine or spermine, but not putrescine, to the culture medium. These studies show that ongoing polyamine biosynthesis is essential for prolactin to express its mitogenic effect in this lymphoma cell line.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the 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. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Polyamine metabolism in some helminth parasites

Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the particulate fraction containing mitochondria, not inhibited by alpha-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.     

Prolactin actions on casein and lipid biosynthesis in mouse and rabbit mammary gland explants are abolished by p-bromphenacyl bromide and quinacrine, phospholipase A2 inhibitors

p-Bromphenacyl bromide (BPB) at concentrations of 50 microM and above and quinacrine (50 microM) abolished the actions of prolactin (PRL) on casein and lipid biosynthesis in cultured mouse mammary gland explants. In cultured rabbit mammary gland explants, 100 microM BPB or quinacrine abolished the PRL stimulation of casein synthesis, while 50 microM BPB or 250 microM quinacrine abolished the PRL stimulation of lipid biosynthesis. Since BPB and quinacrine are known to inhibit the enzyme phospholipase A2 (PLA2), it is possible that ongoing PLA2 activity is essential for prolactin to express its actions on at least certain lactogenic processes.     

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.