Arginase as one of the inhibitory principles in the density-dependent as well as plasma membrane-mediated inhibition of liver cell growth in vitro

Liver cells isolated from the adult rat livers under mild conditions were preincubated for 1 day with Williams medium E (WE) containing serum, dexamethasone and insulin, and then the cells (monolayered) were incubated for 2-3 days with WE (1 ml) containing only insulin to measure DNA synthesis and/or mitosis. DNA synthesis of cultured liver cells was dependent on cell densities within a region from 0.1 X 10(6) to 1.0 X 10(6) nuclei/dish (Falcon, diameter 35 mm). The addition of EGF from the beginning of preincubation stimulated DNA synthesis (or replication) as well as cell proliferation in vitro, but the density-dependent inhibition of DNA synthesis was observed similarly in the presence of EGF. In contrast to the low and high density cultures, DNA synthesis in the intermediary density cultures was enhanced by enlarging the medium volume or by adding ornithine (arginase inhibitor). DNA synthesis in low density cultures was inhibited by liver plasma membranes in a concentration-dependent fashion. The inhibition of DNA synthesis by liver plasma membranes in low concentrations (less than 30 micrograms protein/ml) was reduced by adding either extra arginine or ornithine. DNA synthesis of cultured liver cells (low density) was inhibited by replacing arginine in WE with equimolar ornithine and urea or by adding a commercial arginase (bovine liver). These, together with earlier findings indicating the presence of arginase in liver plasma membranes (outer leaflet), seem to support the idea that arginase may be involved in density-dependent as well as plasma membrane-mediated inhibition of DNA synthesis of cultured liver cells. However, this does not exclude possible involvement of other inhibitory principle(s), such as direct cell-to-cell or cell-to-plasma membrane interactions, especially in higher cell densities or larger plasma membrane concentrations.     

Hormonal regulation of DNA synthesis in primary cultures of adult rat hepatocytes : Action of glucagon

Primary cultures of adult rat hepatocytes, grown in modified minimal essential medium (Eagle's) containing 10% calf serum, could be induced into DNA replication by combinations of epidermal growth factor (EGF), insulin and glucagon. The three hormones acted synergistically, and cells began entering DNA synthesis 48 h after hormone addition. The ability of the hormones to stimulate DNA synthesis was enhanced by plating cells at high cell concentrations or by conditioned medium, and was diminished by daily medium change. The contribution of glucagon to DNA synthesis was replaced by cAMP plus 1-methyl, 3-isobutyl xanthine or by adrenergic agents. Evidence is presented which suggests that all three hormones are required on the first day of culture, and that EGF and insulin are also required after the first day. This appears to be a useful system for studies on the hormonal initiation of growth in quiescent cells.     

Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

Control of DNA synthesis and ornithine decarboxylase activity by hormones and amino acids in primary cultures of adult rat hepatocytes

The conditions for stimulation of ornithine decarboxylase (ODC) and DNA synthesis in primary monolayer cultures of non-growing, highly differentiated hepatocytes from adult rats were compared. The syntheses of ODC and DNA were not stimulated by hormones on the 1st day of culture, but they were induced markedly by insulin (10⁻⁸ M) and epidermal growth factor (EGF, 0.1 μg/ml) in cells cultured for 40 h. The effects of insulin and EGF were synergistic, and the ODC activity as well as the DNA synthesis in the presence of these hormones was comparable to that of cultured hepatocytes from partially hepatectomized liver. Other factors had different effects on the two processes. Dexamethasone induced ODC slightly, but it inhibited DNA synthesis strongly. Putrescine inhibited ODC activity, but it had no effect on DNA synthesis. Asparagine and glutamine induced ODC activity, but they inhibited DNA synthesis; their inhibitory effects on DNA synthesis were specific to primary cultured liver cells and were not seen in an established rat liver cell line or in mouse L cells. These results show that although there is some correlation between ODC induction and DNA synthesis, the former is not essential for cell growth. There was no indication of cell division under conditions where maximal ODC induction and DNA synthesis were observed. Cytofluorometry of cells treated with insulin and EGF showed that the DNA content increased from 2 N to 4 N, and to 8 N in some cells. Therefore, under the present culture conditions, mature liver cells could enter G2 phase through S phase, but could not enter M phase.     

Stimulation of HTC hepatoma cell growth in vitro by hepatic stimulator substance (HSS) : Interactions with serum, insulin, glucagon, epidermal growth factor and platelet derived growth factor

Hepatic stimulator substance (HSS), a partially purified extract of weanling or regenerating adult rat liver, is an organ-specific stimulator of liver growth in vivo and in vitro. The HTC hepatoma cell line is particularly responsive to HSS. The present experiments show that HSS will stimulate HTC cells in the complete absence of serum, although graded doses of fetal cal serum (FCS), from 0.1 to 5.0%, will increase the degree of stimulation in a dose-dependent manner. In contrast, when HSS is absent, increasing doses of FCS above 0.5% inhibit DNA synthesis. Much of this inhibition is removed by prior dialysis of the FCS and maximum enhancement of the HSS-induced stimulation occurs with only 0.1–0.5% of the dialysed FCS. Sera from older animals have less or even negative effect. Evidence is presented to show that the enhanced stimulation by HSS in the presence of serum is not due to insulin, glucagon, epidermal growth factor (EGF), or platelet derived growth factor (PDGF) and that HSS does not act via a shared receptor for one of these hormones. These experiments provide further evidence that HSS is a unique stimulator of liver growth and lend support to a model of organ-specific growth control.     

Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

Modulation of the mitogenic response of an epidermal growth factor-dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor-beta

The stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGR receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up-regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high-affinity receptors (less than 500) are required for EGF-dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF-stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin-like growth factors I or II (IGF-I, IGF-II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF-I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor-beta (TGF-beta) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF-beta receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF-beta) significantly altered 125I-EGF binding characteristics in MK cells, suggesting a point of action distal to 125I-EGF binding.     

Density-dependent growth control of adult rat hepatocytes in primary culture

Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.     

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.     

Presence of epidermal growth factor receptors and influence of epidermal growth factor on proliferation and aging in cultured smooth muscle cells.

Epidermal growth factor (EGF) at nanomolar concentrations stimulated DNA synthesis in confluent, serum-starved cultures of calf aorta and human uterine smooth muscle cells. Stimulation of DNA synthesis in lens epithelial cells was studied for comparison. L and D-ascorbic acid potentiated the effect of serum and EGF on DNA synthesis in calf aorta cells. In contrast L-ascorbic acid had minimal potentiating effect with serum and no effect with EGF present along with serum on DNA synthesis in human uterine smooth muscle and rabbit lens epithelial cells. EGF and ascorbic acid increased cell number when added to stationary phase cultures. Specific binding of 125I-labelled EGF to smooth muscle cells was demonstrated. Receptor concentration in calf-aorta smooth muscle cells was higher in dense cultures compared to sparse cultures. The time course of binding and dissociation of 125I-labelled EGF was similar in "dense" and "sparse" cultures. Human uterine smooth muscle cells in culture exhibited a finite lifespan. There was no stimulation of DNA synthesis in response to serum and EGF in cells of high population doubling level (PDL); although 125I-labeled EGF binding was higher in old cells (high PDL) compared to young cells (low PDL). This increase in binding was shown to be due to changes in the concentration of receptors without changes in their affinity for EGF.     

Protein synthesis during induction of DNA replication in thyroid epithelial cells: evidence for late markers of distinct mitogenic pathways

The synthesis of specific protein has been investigated in primary cultures of dog thyroid epithelial cells, which can be induced to progress into G1 phase, in the presence of insulin, by different types of mitogens: thyrotropin (TSH) acting through cyclic adenosine monophosphate (cAMP), epidermal growth factor (EGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or 10% serum. EGF, TPA, or serum specifically induce [35S] methionine labeling of protein 1 (Mr approximately 80,000). The effect of EGF on protein 1 labeling and DNA replication is dependent on insulin. The level of protein 1 labeling as well as that of DNA synthesis is higher when TSH or TSH + serum are added together with EGF. It peaks in mid-G1. TSH alone, in the presence of insulin, stimulates DNA replication without inducing protein 1 synthesis, which thus represents a cell-cycle-dependent event that is not obligatory in mitogenic activation through cyclic AMP. Among the eight proteins whose synthesis is stimulated by TSH, only the labeling of protein 7, molecular weight ratio (Mr approximately 38,000), correlates with the DNA synthetic activity of the cells. The present authors identified protein 7 as cyclin/proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase-delta. The effect of TSH on cyclin synthesis is already detectable when most of the cells are in late G1, but its stimulation by EGF or EGF + serum is delayed and detected only after extending the labeling period to the S-phase. These data support the view that the cAMP-mediated mitogenic pathway remains partly distinct from the better known pathways induced by growth factors and tumor promoters, even at late stages of the G1-phase.     

Synergistic stimulatory effect of glucocorticoid, EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes.

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 microM) and/or for 20 h with dexamethasone (1 microM) before the end of incubation. The incorporation of [3H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2-3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [32P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.     

The calcium-dependence of the stimulation of neonatal rat hepatocyte DNA synthesis and division by epidermal growth factor, glucagon and insulin

A low concentration (10(-11) mol/l) of epidermal growth factor (EGF) and/or an equimolar (10(-14) mol/l) mixture of glucagon and insulin stimulated DNA synthesis in hepatocytes in 4-day-old primary cultures of neonatal rat liver. EGF seems to have acted by inducing quiescent hepatocytes to begin cycling, while the glucagon-insulin combination seems to have acted mainly by shortening the cell cycle time. Incubation in low calcium medium blocked untreated hepatocytes in the G1 phase of their cycle and prevented EGF and the glucagon-insulin mixture from stimulating DNA synthesis. Nevertheless, hepatocytes in calcium-deficient medium did respond to these agents, as they reached a late stage of prereplicative development before being blocked: in fact, they initiated DNA synthesis soon after the addition of calcium. EGF, but not the glucagon-insulin combination, also enabled the already cycling hepatocytes (but not the newly activated ones) to overcome the block imposed by the extracellular calcium deficiency after a delay of several hours.     

Growth stimulation and apoptosis induced in cultures of neonatal rat liver cells by repeated exposures to epidermal growth factor/urogastrone with or without associated pancreatic hormones

Summary In untreated primary cultures of neonatal rat liver kept in high-calcium (1.8 mmol/l), foetal bovine serum (10%v/v)-containing minimal essential medium (FBSMEM), the absolute numbers of hepatocytes did not change between day 4 and day 9 because ongoing cell loss was counterbalanced by proliferation of a discrete sub-population of the cells. By contrast, the number of stromal cells increased linearly with time. Growth of hepatocytes and stromal cells was differently affected by the daily addition, between day 4 and day 8 of culture, of fresh medium to which peptide mitogen(s) in concentrations ranging from 10^-14 to 10^-8 mol/l had been added. Epidermal growth factor/urogastrone (EGF/URO) with or without equimolar mixtures of glucagon and insulin, induced first hyperplasia of hepatocytes and stromal cells and then apopotosis (degeneration and death) of the progeny of the stimulated cells. By contrast, equimolar mixtures of glucagon and insulin caused a progressive increase in the number of hepatocytes and stromal cells unbalanced by any increase in cell death. At subphysiological concentrations glucagon, in synergism with EGF/URO and/or some other unknown heat-stable component of serum, acted as a trophic factor for hepatocytes. By contrast, insulin alone did not enhance growth of hepatocytes, but rather blocked the mitogenic effects of EGF/URO. The three hormones exerted neither mitogenic nor apoptotic effects when administered in a low calcium (0.01 mmol/l) FBS-MEM medium.These results reveal that EGF/URO may control the size of cell populations in neonatal liver by calcium-dependent mechanisms that make it unlikely to be a promoter of hepatocyte tumours. They also show that glucagon acts as a positive trophic regulator for hepatocytes.     

Synergistic stimulatory effect of glucocorticoid,EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 μM) and/or for 20 h with dexamethasone (1 μM) before the end of incubation. The incorporation of [³H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2–3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [³²P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.     

Hepatocyte DNA replication: effect of nutrients and intermediary metabolites

Isolated adult rat liver parenchymal cells maintained in serum-free medium are stimulated by insulin and epidermal growth factor (EGF) to undergo DNA synthesis. Pyruvate, lactate, and, to a lesser extent, several other intermediary metabolites strikingly enhance DNA synthesis both under serum-free culture conditions and in the presence of dialyzed rat serum. High concentrations (2-50 mM) of these low-molecular-weight metabolites are necessary to produce optimal stimulation. Both alanine (greater than 2 mM) and glutamine (greater than 4 mM) are inhibitory under similar conditions. Glucose, although not required for hepatocyte maintenance or stimulation in the presence of insulin and EGF, acts synergistically with pyruvate to enhance DNA synthesis in a complete mixture.     

Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.     

The influence of epidermal growth factor and insulin on proliferation and DNA synthesis in ciliates Tetrahymena pyriformis

The influence of the epidermal growth factor (EGE) (10(-8) M), insulin (10(-6) M) and EGF (10(-8) M) in combination with insulin (10(-6) M) on proliferation and DNA synthesis in the nuclei of ciliates Tetrahymena pyriformis GL was studied. Insulin and EGF, known to stimulate growth of many types of mammalian cells revealed a mitogen influence on the unicellular eukaryotes. This effect involves stimulation of DNA synthesis, rising synchronization of cell division (upon the influence of EGF), and increase in cell number during the exponential growth. The mitogen effect may be evoked by cell progression in G1-phase under the action of growth factors and, consequently by earlier entry of cells into S-phase of the first cell cycle. Insulin repressed division of cells that entered into the generative cycle. These cells were delayed in late S-phase and G2-phase of the cycle. Part of these cells perished, while other cells could successively overcome the cell block to start their division by the 4th hours of cultivation. A collateral cytotoxic effect of insulin was found, being most prominent in early periods of Tetrahymena cultivation.