The role of polyamines in somatomedin-stimulated differentiation of L6 myoblasts

The somatomedins are potent stimulators of proliferation and differentiation of cultured myoblasts. In studies on the mechanism(s) of these actions, we have measured the activities of ornithine decarboxylase (ODC), an enzyme associated with rapid cell proliferation, and creatine kinase (CK), a biochemical marker for muscle differentiation, after treatment of L6 myoblast cultures with Multiplication Stimulating Activity (MSA), a member of the somatomedin family of insulinlike growth factors. ODC levels reached a peak 24 hours after MSA addition (before any detectable differentiation of the myoblasts) and then decreased as differentiation commenced and CK activity increased. Addition of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, caused a dramatic decrease in differentiation. Measurement of 3H-thymidine incorporation, DNA content, and cell number established that the effect of DFMO on differentiation was not a simple consequence of its antiproliferative actions. Cellular levels of putrescine and spermidine (but not spermine) decreased substantially following addition of DFMO to the cultures. The inhibitory effects of DFMO were abolished upon addition of exogenous polyamines to the medium. However, addition of polyamines in the absence of MSA or DFMO did not mimic the stimulation of differentiation by MSA. We conclude that polyamines play an essential role in the stimulation of L6 myoblast differentiation by somatomedins, but they are not sufficient to effect this stimulation.     

Polyamines and HeLa-cell DNA replication.

HeLa cells were synchronized for S-phase DNA synthesis by the double thymidine-block procedure. A comparison was made of the polyamine content and S-phase DNA synthesis in cells from control cultures and cultures to which an inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine, was added to the synchronization medium. Control cells showed a peak of synchronous DNA synthesis at 3 h and a maximum concentration of polyamines at 6-9 h after release of the second thymidine block. Cells from cultures containing the inhibitor were severely inhibited in the synthesis of DNA and contained no putrescine and only traces of spermidine while the spermine content was lowered by as much as 80%. Supplementation of cultures containing alpha-difluoromethylornithine with a polyamine, at the time of release of the second thymidine block, replenished the intracellular pool of the administered polyamine and partially restored S-phase DNA synthesis, with a lag of 3-6 h. Almost complete restoration of DNA synthesis in cells depleted of polyamines was achieved by the addition of a polyamine to cultures at least 10 h before release of the second thymidine block. The lag in initiation of synchronous S-phase DNA synthesis was eliminated in these cells. It is concluded that reversal by polyamines of the deficiency in S-phase DNA synthesis, in polyamine-depleted HeLa cells, is a time-dependent process indicative of the necessity for the replenishment of replication factors or their organization into an active replication complex.     

Novel glycosaminoglycan mimetic (RGTA, RGD120) contributes to enhance skeletal muscle satellite cell fusion by increasing intracellular Ca2+ and calpain activity

Glycosaminoglycans (GAG) are classes of molecules that play an important role in cellular processes. The use of GAG mimetics called regenerating agent (RGTA) represents a tool to investigate the effect of GAG moiety on cellular behavior. A first member of the RGTA family (RG1192), a dextran polymers with defined amounts of sulfate, carboxymethyl, as well as hydrophobic groups (benzylamide), was shown to stimulate skeletal muscle repair after damage and myoblast differentiation. To obtain a comprehensive insight into the mechanism of action of GAG mimetics, we investigated the effect on myoblast differentiation of a novel RGTA, named RGD120, which was devoid of hydrophobic substitution and had ionic charge similar to heparin. Myoblasts isolated from adult rat skeletal muscles and grown in primary cultures were used in this study. We found that chronic treatment with RGD120 increased the growth of adult myoblasts and induced their precocious fusion into myotubes in vitro. It also partially overcame the inhibitory effect of the calpain inhibitor N-acetyl-leu-leu-norleucinal (ALLN) on these events. Western blot and zymography analyses revealed that milli calpain was slightly increased by RGD120 chronic treatment. In addition, using fluorescent probes (Indo-1 and Boc-leu-met-MAC), we demonstrated that RGD120 added to prefusing myoblast cultures accelerates myoblast fusion into myotubes, induced an increase of cytosolic free calcium concentration, and concomitantly an increase of intracellular calpain protease activity. Altogether, these results suggested that the efficiency of RGD120 in stimulating myogenesis might be in part explained through its effect on calcium mobilization as well as on the calpain amount and activity.     

In Vitro effects of thyroxine and insulin on myoblasts from chick embryo skeletal muscle

The role of insulin and l-thyroxine (L-T₄) in stimulating myoblast proliferation and differentiation was investigated in vitro. A superphysiological concentration of insulin or a physiological concentration of L-T₄ was added to cultures of myoblasts from 11-day-old chick embryo thigh muscle, grown in serum-free DM-153 medium. While the addition of insulin resulted in an increase in the total number of cells, in the extent of fusion, and in the creatine phosphokinase (CPK) activity, myotubes changed into globular structures which tended to degenerate rapidly. On the other hand, while the addition of L-T₄ had less effect on myogenesis, myotubes retained their differentiated state longer. Furthermore, the two hormones exhibited synergistic effects. An increase in the initial cell density resulted in an increase in the amount of protein and CPK activity, irrespective of the presence or absence of the hormones. This suggests that the effect of insulin and L-T₄ on myogenesis is not a differentiation-specific effect, but rather an indirect result of cell proliferation.     

Effects of the somatomedins and insulin on myoblast differentiation in vitro

The effects of insulin and the somatomedins on differentiation of rat myoblasts were investigated in experiments on cells cloned from Yaffe's L6 line. Incubation for 48 hr with either insulin or Temin's multiplication stimulating activity (MSA), a member of the somatomedin family, caused a dramatic increase in myoblast fusion. This stimulation of differentiation is not a simple consequence of the increased cell density resulting from the effects of these hormones on myoblast proliferation, and the increase in fusion is not an effect common to all mitogens (FGF inhibits the process). Other somatomedins (human somatomedin C and insulin-like growth factor I), were as effective as MSA in stimulating differentiation. The somatomedins were active at concentrations in the range of their levels in fetal blood, in contrast to insulin, which was inactive at concentrations below 10^?7, M. Growth hormone (GH) had no effect on muscle differentiation. In serum-free medium MM-1 (in which myoblasts maintain apparently normal morphology and metabolic activity), the very high levels of insulin required to stimulate differentiation could be replaced entirely by physiological levels (1.0 μg/ml) of MSA, further supporting our view that insulin at high concentrations serves primarily as an analogue of the somatomedins in stimulating the growth and development of muscle cells.     

Cycloheximide elicits in human fibroblasts a response characteristic for initiation of cell proliferation

Earlier I found that a variety of stimuli to proliferation of cultured human fibroblasts caused an increase in the rate of putrescine transport into the cells. This paper reports the effects of cycloheximide on putrescine transport in stationary and growing cultures. Cycloheximide in concentrations that inhibited protein synthesis caused increased putrescine transport in serumstarved and density-inhibited cultures. Similar effects were found with pactamycin, also an inhibitor of protein synthesis. Actinomycin D in concentrations that suppressed messenger RNA (mRNA) synthesis, did not cause increased putrescine transport. When both serum and cycloheximide were added to serum-starved cultures, the increase in putrescine transport was greater than when serum alone was added. However, cycloheximide had an inhibitory effect when added 1–2 h after addition of serum. These results suggest that one or more rapidly metabolizing proteins may be important in the regulation of putrescine transport and initiation of cell growth.     

Lysosomal thyroid hormone 5''-deiodinase

The transglutaminase-mediated insertion of putrescine into casein was inhibited competitively by alpha-difluoromethylornithine (alpha-DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase. Preincubation of the amine acceptor (casein) or the enzyme itself with the inhibitor did not affect enzyme activity. Alpha-DFMO is a poorer substrate for transglutaminase (Km = 2.10 mM) than putrescine (Km = 0.17 mM). The inhibitory effect was also found with fibronectin as amine acceptor.     

Differential regulation of putrescine uptake in Trypanosoma cruzi and other trypanosomatids.

Putrescine uptake in Trypanosoma cruzi epimastigotes is 10 to 50-fold higher than in Leishmania mexicana or Crithidia fasciculata. Polyamine transport in all these trypanosomatids is an energy-dependent process strongly inhibited by the presence of 2,4-dinitrophenol or KCN. Putrescine uptake in T. cruzi and L. mexicana was markedly decreased by the proton ionophore carbonylcyanide m-chlorophenylhydrazone but it was not affected by ouabain, a Na(+)-K+ pump inhibitor. The depletion of intracellular polyamines by treatment of parasite cultures with alpha-difluoromethylornithine elicited a marked induction of putrescine uptake in L. mexicana and C. fasciculata by increasing considerably the Vmax of this process. Conversely, the uptake of putrescine in T. cruzi was essentially unchanged by the same treatment. The differential regulation of putrescine transport in T. cruzi might be related to some distinctive features of polyamine metabolism in this parasite.     

Involvement of Putrescine in the Development of Kindled Seizure in Rats

During the development of kindling by daily electrical stimulations applied to the left amygdala of rats, concentrations of the polyamines putrescine, spermidine, and spermine were measured in the left amygdala and the remainder of the cerebrum. A significant increase of putrescine concentration appeared first at the left amygdala in prekindled rats and then propagated to the remainder of the cerebrum with the development of kindling. This increase in putrescine concentration in the left amygdala was higher in prekindled rats than in fully kindled rats and lasted for at least 24 h after the final kindling stimulation. The concentrations of spermidine and spermine were slightly increased in a fully kindled state. To clarify the role of putrescine in kindling, the development of amygdaloid kindling was examined in rats after microinjections of alpha-difluoromethylornithine, a specific inhibitor of polyamine synthesis, and putrescine into the ipsilateral amygdala. Pretreatment with alpha-difluoromethylornithine (50 nmol) for 10 days accelerated both the development of behavioral kindling and the propagation of the afterdischarge from the left amygdala to the frontal cortex. In contrast, pretreatment with putrescine (200 nmol) for 10 days retarded the development of kindling. These results suggest that the increase in putrescine concentration in the kindled brain has an inhibitory effect on the development of kindling.     

The role of polyamine depletion and accumulation of decarboxylated S-adenosylmethionine in the inhibition of growth of SV-3T3 cells treated with alpha-difluoromethylornithine.

The effects of alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, on cell growth rate, polyamine content and the content of decarboxylated S-adenosylmethionine in SV-3T3 transformed mouse fibroblasts were studied. DL-alpha-Difluoromethylornithine at 1 mM or higher concentrations decreased the growth rate by over 90% after 2 or more days of exposure, but the cells remained viable, although quiescent for at least 9 days. Addition of 10 microM-spermidine or -spermine or 50 microM-putrescine at any time throughout this period completely reversed the inhibition of growth. Treatment with alpha-difluoromethylornithine decreased putrescine and spermidine contents by more than 98% and that of spermine by 60%, but cells exposed to exogenous polyamines did not require complete replenishment of the polyamine pools to resume growth. In fact, a virtually normal growth rate was obtained in cells lacking putrescine, having 2% of normal spermidine content and 156% of normal spermine. These results suggest that the well-known increase in putrescine and spermidine in cells stimulated for growth is not essential for this to occur and that mammalian cells can utilize spermine as their only polyamine. A substantial reversal of the growth-inhibitory effect of alpha-difluoromethylornithine was produced by a number of polyamines not normally found in mammalian cells, including the spermidine analogues aminopropylcadaverine and sym-homospermidine, which were partially converted into their respective spermine analogues by addition of an aminopropyl group within the cell. The spermine analogue sym-norspermine was also effective, but the maximal growth rate produced by these unphysiological polyamines was only 60-70% of that produced by the normal polyamines. These results indicate that spermidine and spermine have the optimal length for activation of the cellular processes critically dependent on polyamines and should help in identifying these processes. Exposure to alpha-difluoromethylornithine leads to an enormous rise in the concentration of decarboxylated S-adenosylmethionine, which reached a peak at 530-fold after 3 days of exposure and steadily declined to 140-fold after 11 days. This increase was abolished by addition of exogenous polyamines, which rapidly decreased the activity of S-adenosylmethionine decarboxylase. The increase in decarboxylated S-adenosylmethionine is unlikely to be solely responsible for the decrease to the same extent by spermine, sym-norspermidine and sym-homospermidine, which produce 97%, 16% and 60% of the control growth rate, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Triiodothyronine influences quail myoblast proliferation and differentiation*

The influence of triiodothyronine (T3) on avian myoblast proliferation and differentiation was studied in secondary cultures using plating densities of 2500 and 7000 cells/cm². Culture media were depleted of T3 (control myoblasts) and increasing amounts were then added to concentrations of 0.6, 3 and 15 nM T3 (treated myoblasts). Independent of the cell density, T3 induced a dose-related decrease in myoblast proliferation measured by cell number, doubling time and ³H-thymidine incorporation. However, with the lower plating density, this influence was delayed, occurring only after the third day of culture for 0.6 nM T3-treated myoblasts and simultaneous with the onset of myosin heavy chain accumulation. Moreover, when myoblasts were exposed to BrdU for 48 h, the T3 growth inhibitory effect disappeared, thus showing that this effect was clearly linked to differentiation. In addition, we have shown that T3 induced an early fusion of myoblasts: 65% of the maximal value of the fusion index was reached on day 3 in the T3-treated cells in comparison to 25% in the control myoblasts. This hormone also enhanced accumulation of muscle-specific proteins (connectin, acetylcholine receptors, myosin heavy chain), tested by cytoimmunofluorescence, ELISA, binding experiments and Western blot. All these results show that T3 increased myoblast differentiation through a pathway including myoblast withdrawal from the cell cycle. The influence of T3 could partly explain its previously reported positive effect on the number of muscle fibers.     

Overexpression of ornithine decarboxylase increases myogenic potential of H9c2 rat myoblasts

Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines—such as putrescine, spermidine and spermine—are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion—by means of α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase—abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells.     

Altered polyamine metabolism in Chinese hamster cells growing in a defined medium

Chinese hamster cells (line CHO) maintained in McCoy's 5A medium (modified) supplemented with insulin (10 micrograms/ml), transferrin (5 micrograms/ml), and ferrous sulfate (1.1 microgram/ml) proliferate at rates similar to cultures growing in the McCoy's medium supplemented with 10% fetal bovine serum. Colony-forming ability is similar in cultures supplemented with either serum or the combination of growth factors. By 6 hours after replacement of serum with growth factors, ornithine decarboxylase (ODCase) activity increases, reaching a maximum value by 24 hours after serum replacement. This maximum is cell density dependent and can exceed a 30-fold increase over enzyme activity in cultures supplemented with serum. The increased enzyme activity is due to a decrease in the turnover rate of the enzyme, based on protein synthesis inhibition studies, and an accumulation of active enzyme molecules rather than an activation of existing molecules, since the catalytic activity of ODCase, determined using the radiolabeled form of alpha-difluoromethylornithine (an enzyme-activated, irreversible inhibitor of ODCase) in concert with supplements. Intracellular putrescine and spermidine levels are substantially decreased when cultures are maintained in medium supplemented with insulin, transferrin, and ferrous sulfate, rather than serum, which is the sole source of exogenous ornithine. Titration of cultures growing in the defined medium with ornithine leads to a decrease in ODCase activity and an increase in intracellular putrescine and spermidine levels. Putrescine- and spermidine-dependent S-adenosyl-L-methionine decarboxylase activities are similar in cultures maintained in either medium. These data demonstrate that some, but not all, aspects of polyamine biosynthesis are affected by the availability of ornithine, the first substrate in the pathway.     

Effect of polyamines on the activity of malarial alpha-like DNA polymerase

DNA polymerase from the malarial parasite Plasmodium falciparum required Mg2+ for activity, Putrescine (1 mM) caused a twofold increase in enzyme activity in the presence of a suboptimal concentration of MgCl2 (2 mM). Spermidine (1.5-2.0 mM) or spermine (0.1-0.3 mM) increased the activity of malarial DNA polymerase, in the presence of 2 mM MgCl2, by factors of 6 and 3-5, respectively. The activity of DNA polymerase from calf thymus or from NIH 3T3 cells transformed by the ras oncogene were not stimulated by these polyamines to the same extent. These findings suggest that in malaria-infected erythrocytes, polyamines, at physiological concentrations, serve as a cofactor for the parasitic alpha-like DNA polymerase. Malarial parasites grown in cultured human erythrocytes did not synthesize DNA after treatment with alpha-difluoromethylornithine, which caused polyamine depletion in the infected cells. DNA synthesis was resumed after adding putrescine to the polyamine-depleted cultures. DNA synthesis was also initiated when actinomycin D was added along with putrescine to polyamine-depleted cells. It thus appears that polyamines are essential for the translation of the DNA polymerase mRNA and that polyamines play an important role in regulating the cell cycle of the malarial parasite.     

A transient increase in amino acid transport modulated by insulin in differentiating muscle cells.

During synchronous differentiation of embryonic chick muscle cells in cultures, the Na-dependent uptake of an amino acid analog, alpha-amino isobutyric acid (AIB) undergoes in abrupt, transient increase. The increase in AIB uptake is concomitant with the rapid fusion of mononucleated myoblasts, and precedes the accumulation of muscle-specific proteins. Subsequently, Na-dependent AIB transport diminishes markedly during postfusional differentiation of myotubes. The rate of AIB uptake is increased by insulin both before and after myoblast fusion. This stimulation by insulin is restricted to the Na-dependent component of total AIB uptake but is apparently not the result of insulin-mediated increase in the trans-membrane Na gradient.     

Correlation between transglutaminase activity and polyamine levels in human neuroblastoma cells. Effect of retinoic acid and alpha-difluoromethylornithine

The human neuroblastoma cell line SK-N-BE can be induced to differentiate by retinoic acid (RA) or by alpha-difluoromethylornithine (DFMO). The former inducer produces neurite outgrowth, 60% reduction of growth rate, overexpression of neural antigens, and enhanced gamma-aminobutyric acid (GABA) and acetylcholinesterase levels. In contrast, DFMO causes cell body elongation, complete growth inhibition, and higher binding of antibodies directed against neuroectodermal antigens. Polyamine metabolism is also differently affected by the two agents. In particular a large spermine catabolism is induced by RA, while DFMO treatment leads to a small increase in the level of this compound. The neural differentiation induced by RA is accompanied by a marked increase in transglutaminase activity and its induction is paralleled by a transient increase of putrescine and spermidine. The putrescine and spermidine depletion determined by DFMO is accompanied instead by a large inhibition of transglutaminase activity. The inhibiting effect of DFMO treatment on transglutaminase is reversed by the addition of 1 mM putrescine to the culture medium. In the presence of both RA and DFMO a mixed morphological and biochemical pattern is observed. The possibility that the expression of transglutaminase associated to cellular differentiation may be modulated by the level of its substrates is also discussed.     

The role of hormones and prostanoids in the in vitro proliferation and differentiation of human myoblasts

Fetal human myoblasts have been employed to examine the role of hormonal factors in human myogenesis. The results show that human myoblast proliferation is stimulated by insulin, hydrocortisone, and prostaglandin F2 alpha (PGF2 alpha). Exposure of human myoblasts preparing to differentiate to either PGE2 or isoproterenol results in the precocious initiation of differentiation (i.e., cell fusion and increase in creatine kinase activity). Three antagonists of prostanoid synthesis, indomethacin, aspirin, and DL-6-chloro-alpha-methylcarbozole-2-acetic acid, inhibit cell number increase with complete inhibitions of proliferation at 5 X 10(-5) M indomethacin and 6 X 10(-4) M aspirin. Reversal of the indomethacin-imposed block is achieved by prostaglandin F2 alpha. The same antagonists of prostanoid synthesis, when added to older cultures, depress prostaglandin E (PGE) levels and inhibit human myoblast differentiation. During differentiation, PGE is present in both the intracellular compartment (0.47 to 0.66 pmol/microgram DNA) and the culture medium (1.83 to 4.53 nmol PGE). The results suggest a role for prostanoids in the regulation of both human myoblast proliferation and differentiation. They also demonstrate that the active cyclooxygenase products are produced endogenously by the in vitro myogenic population. The findings are discussed within the context of what is known of the relationship between growth factor and prostanoid actions and the roles of these two categories of hormones in the regulation of myogenesis.