Treatment with alpha-difluoromethylornithine plus a spermidine analog leads to spermine depletion and growth inhibition in cultured L1210 leukemia cells

Of the three biological polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), the relevance of Spm to cell proliferation has yet to be defined because of our general inability to deplete it selectively in intact cells. In the present study, Spm depletion was accomplished by treating cultured L1210 cells for 96 hr with alpha-difluoromethylornithine (DFMO) and an analog of Spd such as aminopropylcadaverine, N4-methylSpd, N4-ethylSpd, or homoSpd. DFMO, a specific inhibitor of ornithine decarboxylase, halts continued polyamine biosynthesis and the Spd analog serves as a functional substitute for Spd. Thus, while the Spd analog fulfills the role(s) of Spd in cell proliferation, Spm becomes steadily depleted. In cells treated with DFMO plus the analog, aminopropylcadaverine, Spm pools decline steadily and growth inhibition occus after 48 hr (when Spm pools decline to 60% of control). By 96 hr, Spm is approximately 15% of control and growth is less than 30%. Prevention studies with exogenous polyamines confirm a causal relationship between Spm depletion and growth inhibition. The critical levels of polyamines for cell proliferation to take place were found to be 30% of control for Spd and 60% for Spm. The use of DFMO plus a Spd analog is proposed as a system for studying the cellular consequences of Spm depletion. Spd depletion can be achieved for comparison purposes by treating cells with DFMO alone.     

Phorbol esters stimulate spermidine/spermine N1-acetyltransferase activity in mitogen-stimulated bovine lymphocytes

Phorbol 12-myristate-13-acetate (PMA) is shown to induce spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, in bovine lymphocytes. When PMA and phytohemagglutinin (PHA) were added simultaneously, the enzyme activity was stimulated synergistically. The ability of phorbol esters to stimulate the enzyme activity was consistent with their tumor-promoting ability. Phorbol, which is not a tumor promotor, was incapable of stimulating the enzyme activity. Phorbol diacetate weakly stimulated the activity of the acetylase. Phorbol dibutyrate had a similar stimulatory effect to PMA. These results suggest that the spermidine/spermine N1-acetyltransferase may play an important role in changes in polyamine levels in phorbol ester-treated cells and that the increase in the enzyme activity may have some relationship to the control of cell growth and differentiation by phorbol esters.     

Interaction of insulin and phorbol esters on the regulation of DNA synthesis in rat hepatoma cells

Insulin and phorbol esters stimulated DNA synthesis in rat H4 hepatoma cells. Insulin and phorbol ester induction of thymidine incorporation was dose-dependent, with a maximal 4.2- and 3.0-fold increases at concentrations of 1 x 10(-9)M and 1 microM, respectively. Phorbol esters in combination with increasing concentrations of insulin resulted in additive effects, but only at submaximal insulin concentrations. The combination failed to increase thymidine incorporation above the maximal effects produced by insulin alone. When cells were pretreated with phorbol esters for 24h to produce protein kinase-C (PKC) deficiency, basal DNA synthesis was depressed. Pretreatment with phorbol esters abolished the effects of phorbol esters to induce DNA synthesis but did not impair the magnitude of insulin-induced DNA synthesis. Thus, although phorbol ester-activatable PKC-activity was necessary for basal DNA synthesis, it was not necessary for insulin-induction of DNA synthesis in H4 cells.     

Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters. Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.

To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate 13-acetate (PMA) blocked a normally occurring (20-24 h) peak of serum-stimulated thymidine incorporation in normal and keloid cells. In keloid fibroblasts PMA induced a delayed peak of DNA synthesis. When indomethacin was added with PMA the delayed peak appeared in normal fibroblasts. The ED50 for inhibition of the 20-24-h peak was 1 nM, whereas the delayed peak required a 50-fold-higher PMA concentration. In both cell types PMA induced prostaglandin E2 (PGE2) synthesis, and exogenous PGE2 caused 50% inhibition of the 20-24-h peak. When PMA and indomethacin were added with PGE2 the delayed peak was inhibited 90% in normal fibroblasts, whereas inhibition of keloid cells was the same as with PGE2 alone. Normal and keloid fibroblasts had the same number of phorbol ester binding sites. However, in normal cells, phorbol 12,13-dibutyrate bound with greater affinity, and down-regulation of phorbol ester binding occurred to a greater extent. These findings suggest that altered expression of protein kinase C isozymes or another molecule that binds phorbol esters may play a role in abnormal growth regulation of keloid cells.     

Stimulation of DNA synthesis in murine fibroblasts by the tumour promoter teleocidin: relationship to phorbol esters and vasopressin

The recently reported tumour promoter teleocidin is a potent mitogen for murine fibroblasts, at nM concentrations. Despite its unrelated structure, teleocidin inhibits binding of phorbol dibutyrate to Swiss 3T3 cells, and is inactive as a mitogen for cells in which the phorbol dibutyrate receptors have been down-regulated. Teleocidin shows synergistic stimulation of DNA synthesis with a wide range of purified growth factors, but fails to synergise with vasopressin, suggesting that it utilises the same mitogenic pathway as this physiological ligand.     

Effects of phorbol esters on steroidogenesis in small bovine luteal cells

The possible influence of an activator of protein kinase C, the tumor-promoting phorbol ester, PMA (phorbol-12-myristate-13-acetate), upon small bovine luteal cell steroidogenesis was investigated in vitro, PMA had no significant effect on basal and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production but markedly modulated the LH-stimulated progesterone and cAMP productions. PMA potentiated the LH-stimulated cAMP accumulation whatever the dose of LH used. It also potentiated the LH-induced progesterone production in the presence of low doses of LH. Paradoxically, in the presence of maximal or submaximal effective doses of LH, PMA exerted a time- and dose-dependent inhibition of progesterone synthesis. Diacylglycerol was able to mimic the effects of PMA on LH-induced steroidogenesis. These observations suggest that the Ca2+- and phospholipid-dependent protein kinase C can modulate the regulation by LH of small bovine luteal cell steroidogenesis at a step before the synthesis of cAMP. They also suggest that the interaction between LH and its receptor is able to trigger a negative regulatory signal which would be only expressed for high doses of LH and in the presence of an activator of PKC.     

Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes

An early event in death of interphase lymphocytes exposed in vivo or in vitro to low doses of gamma-irradiation is the degradation of DNA into nucleosome-sized fragments. Induction of fragmentation required RNA and protein synthesis because actinomycin D and cycloheximide, respectively, are able to inhibit DNA fragmentation in irradiated lymphocytes. Studies adding cycloheximide and actinomycin D at various times postirradiation suggest that once the metabolic process is initiated within an individual cell it proceeds to completion. The reversible RNA synthesis inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole inhibits DNA fragmentation in irradiated thymocytes. When this drug is removed after 6 hr, irradiated thymocytes proceed to fragment their DNA; this suggests that an inducing "signal" that is not simply mRNA persists within the irradiated cell for at least 6 hr after irradiation. In contrast to mitogen-activated T and B lymphoblasts, resting T and B cells show significant DNA fragmentation after exposure to 100 to 500 rad. At 2000 rad, all of the splenic subpopulations die rapidly via a different mechanism. By studying the mechanism of DNA fragmentation induced during the interphase death of lymphocytes, we hope to understand better the extreme sensitivity of resting lymphocytes to radiation and what may be the common final pathway of programmed cell death.     

Inhibition of phorbol ester-accelerated amino acid transport in bovine lymphocytes.

12-O-Tetradecanoylphorbol-13-acetate, a highly active tumor-promoting agent and lymphocyte comitogen, rapidly accelerates the transport of alpha-aminoisobutyric acid in cultured bovine lymphocytes. Structure-activity studies show that the ability of phorbol diesters to accelerate alpha-aminoisobutyric acid uptake runs parallel to their potency as lymphocyte comitogens and as tumor promoters in mouse skin. This phorbol ester-accelerated, amino acid transport is largely insensitive to the inhibition of RNA and protein synthesis by actinomycin D and cycloheximide, respectively, and is insensitive to the inhibition of membrane movement by cytochalasin B and colchicine. Retinoic acid, an antagonist of the tumor-promoting and comitogenic actions of phorbol esters also inhibits the acceleration of amino acid uptake by 12-O-tetradecanoylphorbol-13-acetate; however, the epoxy derivatives of retinoic acid and structurally related analogs, which are potent antagonists of the other aspects of phorbol ester activation of lymphocytes, are inactive in blocking amino acid uptake. Comparative studies in lymphocytes show that this phorbol ester elicits a number of metabolic responses which appear to originate at the cell membrane and that these are differentially antagonized by retinoic acid, the 5,6-epoxide of retinoic acid, and related retinoid analogs.     

Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum-free conditions. TPA induces DNA synthesis measured by [3H]thymidine incorporation in a dose-dependent manner with an ED50 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 +/- 15% for TPA alone to 393 +/- 96% for TPA plus insulin. Down-regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of Mr 80,000, pl 4.5 (termed 80K) and Mr 28,000, pI 5.7-5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED50 of 8 ng/ml TPA. Exposure of mesangial cells to heat-shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13-dibutyrate inhibit [125 I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4 alpha-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role for protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.     

Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells

Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.     

Inhibition of spermidine and spermine synthesis leads to growth arrest of rat embryo fibroblasts in G1

Following growth stimulation of rat embryo fibroblast (REF) cells previously arrested in G₁ by serum deprivation, there occurs a large increase in the synthesis of the polyamines putrescine, spermidine and spermine. Methylglyoxal bis(guanylhydrazone) (MGBG), a potent inhibitor of S-adenosylmethionine decarboxylase can block the accumulation of both spermidine and spermine over a period of several days. Under such conditions REF cells treated with MGBG will approximately double in number and then become growth-arrested again predominantly in the G₁ phase of the cell cycle. REF cells therefore appear to contain sufficient spermidine and spermine to progress through one cell cycle before the intracellular levels of these polyamines is reduced sufficiently to arrest growth in the absence of continued polyamine synthesis. Limitation of intracellular polyamine levels is therefore not the mechanism by which deprivation of serum growth factors arrests cell growth. While continued growth is nevertheless dependent on polyamine synthesis, this cell type is capable of limited proliferation in its absence. Addition of spermidine or spermine to MGBG-arrested REF cells results in a rapid resumption of proliferation demonstrating that either polyamine can fulfill the role played by these polyamines in the growth process. Low levels of spermidine and spermine therefore arrest this cell type at a resriction point in G₁ at which it is decided whether the intracellular level of these polyamines is sufficiently high to enable a cell to enter into and complete a new cell cycle. This polyamine-sensitive restriction point is considered to be analogous to the restriction point(s) in G₁ at which serum and nutrient limitation act.     

Heterogeneity of the regulation of phospholipase C by phorbol esters in T lymphocytes

In many cells, protein kinase C (PKC) activation inhibits cellular phospholipase C thereby preventing receptor-mediated phosphatidylinositol (PI) metabolism. In T lymphocytes, the T cell antigen receptor (Ti)/CD3 complex regulates PI hydrolysis and we have examined the consequences of PKC activation on Ti/CD3-mediated PI metabolism in human peripheral blood-derived T lymphocytes (T lymphoblasts) and the leukemic T cell line Jurkat. In Jurkat cells, PI metabolism after Ti/CD3 stimulation, is inhibited by PKC activation. PKC activation also inhibits calcium-induced PI metabolism in permeabilized Jurkat cells. In marked contrast, PI metabolism after Ti/CD3 stimulation in T lymphoblasts, is not inhibited by PKC activation. Moreover, in permeabilized T lymphoblasts PI metabolism can be induced by calcium in synergy with guanine 5'-O-(3-thiotrisphosphate) via a PKC-insensitive mechanism. The different effect of PKC stimulation on PI metabolism in Jurkat cells and T lymphoblasts reveals heterogeneity of PLC regulation in T lymphocytes. The data also indicate that the role of PKC as a regulator of Ti/CD3 signal transduction can differ depending on cell type.     

Polyamine depletion results in impairment of polyribosome formation and protein synthesis before onset of DNA synthesis in mitogen-activated human lymphocytes

Lymphocytes, exposed to mitogens in culture, show enhanced protein and RNA synthesis before the onset of DNA synthesis. Inhibition by DL-alpha-difluoromethylornithine of polyamine synthesis in phytohaemagglutinin-activated human lymphocytes resulted in a suppression of protein synthesis, which was evident before the initiation of DNA synthesis. The mitogen-induced increase in the incorporation of [3H]thymidine into DNA was subsequently inhibited in parallel with the activity of thymidine kinase in the polyamine-depleted cells. Ultraviolet absorbance measurement of the ribosomes after sucrose gradient centrifugation revealed a suppression of polyribosome formation that coincided with the decrease in the rate of protein synthesis. The disturbance in the polysomal profiles did not appear to be due to a shortage of mRNA, since the synthesis of poly(A)-rich mRNA was reduced less than that of rRNA after inhibiting polyamine synthesis. Entry of both the pre-existing and newly synthesized ribosomal subunits into polysomal structures was found to be impaired. These results thus suggest an important role for polyamines in the initiation step of protein synthesis.     

Inhibition of DNA synthesis by phorbol esters through protein kinase C in cultured rabbit aortic smooth muscle cells

In cultured rabbit aortic smooth muscle cells (SMC), 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis in the presence of plasma-derived serum to a small extent, but inhibited markedly the rabbit whole blood serum (WBS)-, platelet-derived growth factor (PDGF)- and epidermal growth factor-induced DNA synthesis. Phorbol-12,13-dibutyrate (PDBu) mimicked this antiproliferative action of TPA, but 4 alpha-phorbol-12,13-didecanoate was inactive in this capacity. Prolonged treatment of the cells with PDBu caused the partial down-regulation of protein kinase C. In these protein kinase C-reduced cells, WBS still induced DNA synthesis, but TPA did not inhibit the WBS-induced DNA synthesis. We have previously shown that protein kinase C is involved at least partially in the PDGF-induced DNA synthesis in rabbit aortic SMC. The present results together with this earlier observation suggest that protein kinase C has not only a proliferative but also an antiproliferative action in rabbit aortic SMC.     

Treatment of rabbit neutrophils with phorbol esters results in increased ADP-ribosylation catalyzed by pertussis toxin and inhibition of the GTPase stimulated by fMet-Leu-Phe

The effects of pretreatment of rabbit neutrophils with phorbol 12-myristate 13-acetate on the ability of pertussis toxin to catalyze ADP-ribosylation and of fMet-Leu-Phe to activate a high-affinity GTPase in these cell homogenates were examined. The addition of phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol 12,13-didecanoate, to intact cells was found to stimulate by more than 100% the pertussis toxin-dependent ribosylation of a 41 kDa protein (either the alpha-subunit of the 'inhibitory' guanine nucleotide-binding protein N or a closely analogous protein) and to inhibit by more than 60% the activation by fMet-Leu-Phe of the GTPase of the neutrophil homogenates. The addition of fMet-Leu-Phe to intact cells increases the ADP-ribosylation catalyzed by pertussis toxin of the 41 kDa protein. On the other hand, the exposure of neutrophil homogenates to fMet-Leu-Phe results in a decreased level of ADP-ribosylation. This decreased ribosylation reflects a dissociation of the GTP-binding protein oligomer that is not followed by association, possibly because of the release of the alpha-subunit into the suspending media. The implications of these results for the understanding of the mechanism of inhibition of cell responsiveness by phorbol esters and the heterologous desensitization phenomenon are discussed. Prominent among these are the possibilities that (i) the rate of dissociation of the Ni oligomer is affected by the degree of its phosphorylation by protein kinase C, and/or (ii) the dissociated phosphorylated alpha-subunit (the 41 kDa protein) is functionally less active than its dephosphorylated couterpart.     

Transient inhibition of DNA synthesis by 5-fluorodeoxyuridine leads to overexpression of dihydrofolate reductase with increased frequency of methotrexate resistance

Transient but incomplete suppression of DNA synthesis by a single exposure of an asynchronous population of cells to 5-fluoro-2'-deoxyuridine (FdUrd) increases the frequency of appearance of methotrexate (MTX)-resistant colonies. This increase was greater than 10-fold following a 6-h incubation of cells with 3 microM FdUrd prior to selection in MTX, an interval one-half the normal L1210 cell cycle time. During this period of exposure to FdUrd, DNA synthesis decreased to 25% of control rates and cells accumulated at the G1/S interface. The 6-h incubation with FdUrd resulted in greater than a 2.5-fold increase in the dihydrofolate reductase protein level in the treated cell population, which was accounted for, at least in part, by increased de novo synthesis of the enzyme as assessed by [35S]methionine labeling. This increase in dihydrofolate reductase was associated with a decrease in growth inhibition by MTX. A brief reversal (2 h) of FdUrd-induced DNA synthesis inhibition by the addition of thymidine eliminated the amplification of dihydrofolate reductase and the enhanced emergence of MTX-resistant clones. Beyond this, an analysis of clones that survive MTX selection indicates that the dihydrofolate reductase gene copy in cells spontaneously resistant to 50 nM MTX and those which resulted after the additional pretreatment with FdUrd for 6 h are comparable with a 2-4-fold amplification of enzyme in most clones. These studies demonstrate that FdUrd enhancement of dihydrofolate reductase expression can have a profound effect upon the incidence and expression of MTX resistance and that dihydrofolate reductase gene amplification may be another basis for antagonism between these agents.     

Parallel activities of fatty acid methyl esters and analogous phorbol diesters toward mouse lymphocytes

Linear saturated fatty acid methyl esters were comitogenic with lectins for mouse lymphocytes, the degree of comitogenicity being strongly dependent on the length of the acyl group, and maximal for methyl tetradecanoate. Lesser effects were found for analogs with 10, 12 or 16 acyl carbon atoms, whereas those with fewer than 10 or more than 16 were inactive. Analogous structure-function relationships have been described for various membrane-active and tumor-promoting phorbol diesters, where there is a similar dependence on ester acyl group length for many activities. The fatty acid esters may therefore represent simple model compounds for studying mechanistic aspects of phorbol diester activity.     

Induction of competence and progression signals in human T lymphocytes by phorbol esters and calcium ionophores

We have investigated the induction of competence (IL-2 responsiveness) and progression in human T lymphocyte proliferation triggered by phorbol ester and calcium ionophore. The degree of proliferation induced with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore ionomycin was dependent on the duration of exposure to these agents, with more than 6 h required for obtaining maximum proliferation. Following brief exposure to both agents for 30 min, which did not cause significant proliferation, T cells became competent to proliferate in response to exogenous interleukin 2 (IL-2). These competent T cells also progressed to DNA synthesis following incubation with PDB in the absence of ionomycin. Induction of competence to proliferate in response to either PDB or IL-2 was blocked by EGTA, suggesting that transmembrane Ca2+ flux was obligatory at this stage. Since other phorbol esters and synthetic diacylglycerols also stimulated DNA synthesis in competent cells, it is likely that progression was triggered by activation of protein kinase C. Following a brief exposure to PDB and ionomycin, subsequent incubation with PDB induced gene expression and secretion of IL-2 and augmented the expression of IL-2 receptors in the competent cells. Thus, we have demonstrated that Ca2+ mobilization is required for rendering T cells competent to express functional IL-2 receptors, to produce IL-2 in response to subsequent incubation with PDB, and that sustained activation of protein kinase C seems necessary for IL-2 production and subsequent progression of competent T cells to DNA synthesis.