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.     

Effect of calf and rat serum on the induction of DNA synthesis and mitosis in primary cultures of adult rat hepatocytes by cyproterone acetate and epidermal growth factor

Summary Induction of hepatocyte DNA synthesis in culture by cyproterone acetate (CPA), a potent hepatomitogen in vivo, was studied. Adult rat hepatocytes were grown on collagen gels in primary culture for 3 to 10 d. Epidermal growth factor (EGF) was used as a model inducer to establish appropriate culture conditions. (a) In serum-free medium EGF stimulated a wave of DNA synthesis in 10 to 30% of the hepatocytes. CPA had only a weak effect. (b) Increasing concentrations of newborn bovine serum (NBS) at 5 to 95% progressively inhibited the stimulatory effect of EGF. A similar inhibition was obtained by adding bovine serum albumin; 20% NBS, however, had a slightly stimulatory effect on the induction of DNA synthesis by CPA. (c) Portal rat serum (RS) at concentration of 5 to 95% markedly stimulated DNA synthesis, a plateau being reached between 20 and 95%. EGF had a distinct enhancing effect on DNA synthesis in the presence of 5 and 20% RS but not at 50 and 95%. CPA stimulated DNA synthesis in the presence of 20, 50, and 95% RS in a synergistic way. (d) Mitoses were found after treatment with EGF or with CPA. These results show that CPA can induce DNA synthesis in cultured hepatocytes and that RS contains factors facilitating the response to CPA. This study was supported by Gesellschaft für Strahlen-und Umweltforschung mbH, München, Germany.     

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.     

Effects of EGF and PMA on the growth and proliferation of IEC-6 cells

Proliferation of an epithelial line (IEC-6) derived from the crypts of rat jejunum was induced with epidermal growth factor (EGF). EGF enhanced synthesis of protein, RNA, and DNA in a dose-dependent manner. Protein synthesis increased within 6-12 hours of exposure to EGF and remained elevated for 72 hours. Maximal 3H-thymidine incorporation occurred 48 hours after addition of EGF. The stimulatory effect of EGF on 3H-thymidine incorporation was two-fold greater in serum-free media than in media containing fetal calf serum (FCS). In contrast to EGF, phorbol-12-myristate-13-acetate (PMA) decreased 3H-thymidine uptake by IEC-6 cells and had no effect on either protein synthesis or RNA synthesis. EGF did not alter protein kinase-C activity in IEC-6 cells whereas PMA induced enzyme activity: activity was translocated from cytosol to membrane. Moreover, the EGF-associated increase in 3H-thymidine uptake was not altered by amiloride. These data suggest protein kinase-C activation may not be involved in the proliferation of IEC-6 cells.     

Morphologic changes in human carcinoma cells (A-431) stimulated by epidermal growth factor: effect of cholesterol and low-density lipoproteins on the ruffling response

Stimulation of A-431 carcinoma cells with epidermal growth factor (EGF) causes dramatic morphologic responses including ruffling, rounding, and bulk-phase pinocytosis. In attempts to explore the mechanisms responsible for changes in plasmalemma topography, we have investigated the effects of exogenous sterols thought to alter membrane fluidity. Light and scanning electron microscopy revealed a time- and concentration-dependent inhibition of ruffling (greater than 90%) by cholesterol. This effect could be duplicated by preincubation of the cells with comparable levels of low-density lipoproteins (LDL). EGF-stimulated bulk-phase endocytosis also is inhibited by treatment with cholesterol. No alteration of EGF binding, kinase stimulation, or internalization was detected in cells incubated in cholesterol-enriched medium (175 micrograms/ml in 0.5% ethanol), nor did cholesterol or LDL have any effect on EGF-stimulated rounding. Morphometry of electron micrographs from cholesterol-treated cells revealed a selective depletion of interdigitating lateral surface membrane that normally appears to be recruited to generate apical ruffles. Thus, the sterol inhibition of ruffling may be due to redistribution of plasmalemma rather than to changes in membrane viscosity. Together with previous observations, these data suggest that EGF-stimulated ruffling and bulk-phase pinocytosis are related phenomena, whereas EGF-stimulated cell rounding is an independent process.     

Serum-free,chemically defined medium to evaluate the direct effects of growth factors and inhibitors on proliferation and function of neonatal rat cardiac muscle cells in culture

Summary Neonatal rat cardiac myocytes were isolated and cultured to evaluate the effects of growth factors and inhibitors on proliferation, survival, and functions in a serum-free medium. Insulin and transferrin in MCDB 107 nutrient medium elicited DNA and protein synthesis in cells on a fibronectin-coated culture surface in serum-free medium. Insulin was most effective on both DNA and protein synthesis in serum-free culture conditions. The serum-free, hormone-supplemented medium eliminated the contamination of noncardiac myocytes and supported the long-term survival (over 18 d) of cardiac myocytes. Dexamethasone was required to induce optimal contractility with or without insulin and transferrin. Serum contained both negative and positive effectors of DNA and protein synthesis of the cardiac myocytes. Concentrations of serum (above 5%) inhibited DNA and protein synthesis. Low density lipoprotein (LDL) accounted in part for the inhibitory activity. The serum-free culture system provides a useful model to elucidate the role of hormones, growth factors, and drugs in heart cell regeneration and function.     

Epidermal growth factor (EGF) influences DNA synthesis in human fetal kidneys maturing in serum-free organ culture

Human fetal kidney explants (13-17 weeks of gestation) were maintained in serum-free organ culture. The influence of epidermal growth factor (EGF) was determined after 2 and 5 days by evaluating DNA and protein synthesis as well as the activities of five brush border hydrolases. During the studied period the overall morphology was preserved and the analysed parameters remained constant. Only DNA synthesis decreased after 2 days. The addition of EGF to the medium did not change any of the cell activities, except DNA synthesis. In fact, the incorporation of [3H]thymidine was significantly stimulated by 105% in 5-day explants cultured in the presence of the growth factor. These results indicate that EGF directly influences proliferation but not maturation of brush border enzymes in fetal human kidneys in culture.     

Stimulation of DNA synthesis in quiescent rabbit chondrocytes in culture by limited exposure to somatomedin-like growth factors

Cartilage-derived factor (CDF), extracted from fetal bovine cartilage, and multiplication-stimulating activity (MSA) stimulated DNA synthesis in quiescent rabbit costal chondrocytes in culture under serum-free conditions. As described previously, when added in the presence of fibroblast growth factor (FGF) or epidermal growth factor (EGF) a somatomedin-like growth factor, CDF or MSA, synergistically stimulated DNA synthesis in the cultured chondrocytes. The present study showed that exposure of the cells to MSA or CDF for only the initial 5 h was sufficient for transmission of their full stimulatory effect. Furthermore, the limited exposure did not alter the time course of stimulation of DNA synthesis: [3H]thymidine incorporation into DNA began to increase after 16 h and reached a maximum after 24 h. In contrast to the somatomedin-like growth factors, FGF and EGF were required continuously in the culture medium during traverse of the entire G1 phase for stimulation of DNA synthesis, and the mitogenic effects of FGF and EGF in cultured chondrocytes were stronger than those of CDF and MSA. Synergistic stimulation of DNA synthesis by CDF or MSA in the presence of FGF or EGF could be observed as long as FGF or EGF was continuously present, even when CDF or MSA was withdrawn after the first 5 h of culture. These findings suggest that, in contrast to FGF and EGF, somatomedin-like growth factors affect an early distinct stage in the G1 phase of chondrocytes.     

Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells [published erratum appears in J Cell Biol 1990 Mar;110(3):859]

Addition of EGF to human epidermoid carcinoma A431 cells increases the rate of fluid-phase pinocytosis 6-10-fold as measured by horseradish peroxidase uptake (Haigler, H.T., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:82-90). We show here that in the absence of extracellular Na+ or in the presence of amiloride the stimulation of pinocytosis by EGF is substantially reduced. Amiloride had no effect on the endocytosis of EGF itself or of transferrin, demonstrating that the receptor-mediated endocytotic pathway operated normally under conditions that blocked stimulated pinocytosis. Amiloride blocked EGF- stimulated pinocytosis in both HCO3(-)-containing and HCO3(-)-free media. The EGF-stimulated pinocytotic activity can frequently be localized to areas of the cell where membrane spreading and ruffling are taking place. These results demonstrate that (a) EGF induces a distinct amiloride-sensitive endocytotic pathway on A431 cells; (b) occupied EGF receptors do not utilize this pathway for their own entry; (c) endocytosis of occupied EGF receptors is not in itself sufficient to stimulate pinocytosis.     

Pinocytosis in L cells: its dependence on membrane sterol and the cytoskeleton

Pinocytosis in L-cells, grown in serum-free medium, was depressed when cultures were treated with oxygenated derivatives of cholesterol which inhibited sterol synthesis and reduced the sterol concentration of the plasma membranes. Noninhibitory sterols, such as cholesterol or desmosterol counteracted the effects of the inhibitors. Treatment with polylysine increased the rate of pinocytosis in sterol-depleted cells to a level similar to the enhanced rate found in polylysine treated control cells. Drugs which interfere with cytoskeletal systems (microfilaments, microtubules) also depressed pinocytosis but their effect could not be overcome by treatment with polylysine.     

Rate of pinocytic capture of macromolecular substrates by rat yolk sac incubated in serum-free culture medium.

Pinocytic activity was quantified for rat yolk sacs incubated in a medium that was either serum-free or contained 10% (v/v) of calf serum. Absence of serum from the medium caused a small increase in the rate of pinosome formation, as determined by the rates of capture of both 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose. In contrast, the rates of uptake of substrates ingested by adsorptive pinocytosis were greatly enhanced when serum proteins, which compete for the same binding sites on the plasma membrane as used by adsorbing substrates, were absent. Elimination of such competition greatly simplifies the quantitative analysis of the binding process, and permitted a detailed study of the binding to the plasma membrane of formaldehyde-denatured bovine serum albumin, a protein that is rapidly digested within the lysosomal system after its pinocytic capture. Binding obeyed Michaelis-Menten kinetics and showed a dissociation constant of approx. 1 micron, indicating the high affinity of this protein for binding sites on the surface of actively pinocytosing yolk-sac cells.     

Cell-cell contact and growth regulation of pinocytosis in 3T3 cells

In sub-confluent cultures of Balb/c-3T3 cells, pinocytosis rates were increased after exposure to specific growth factors (serum; platelet-derived growth factor, PDGF; epidermal growth factor, EGF). Conversely, as cells became growth-inhibited with increasing culture density, there was a corresponding decline in pinocytosis rate per cell. In order to test whether density-inhibition of pinocytosis was influenced either by the growth cycle or by cell contact independently of growth, cells were induced into a quiescent state at a range of subconfluent and confluent densities. Under such conditions, cell density did not significantly inhibit pinocytosis rate. When confluent quiescent cultures in 2.5% serum were exposed to 10% serum, the resulting round of DNA synthesis was accompanied by enhanced pinocytosis per cell, even though the cells were incontact with one another. Furthermore, in a SV40-viral transformed 3T3 cell line, both the growth fraction and the pinocytosis rate per cell remained unchanged over a wide range of culture densities. These studies indicate that density-dependent inhibition of pinocytosis in 3T3 cells appears to be secondary to growth-inhibition rather than to any direct physical effects of cell–cell contact.     

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.     

Serum-free organ culture of suckling rat jejunum: Effect of regulatory hormones

Summary To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when cultured under hyperbaric conditions for 24 h in serum-free Dulbecco’s modified Eagle’s medium/F12 medium than in RPMI 1640 plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium. Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture. However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after 48 h culture. Dexamethasone increased specific activities of alkaline phosphatase (30%,P<0.001) and lactase (15%,P<0.001), and reduced shedding of alkaline phosphatase into the medium (P<0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the rate of protein or DNA synthesis but did increase villus height (P=0.04) and crypt depth (P=0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II, des-(1–3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity of explants after 24 or 48 h culture. In conclusion, histology, enzyme activity, protein, and DNA synthesis of suckling rat jejunal explants were equivalent or better in serum-free than in serum-containing organ culture systems. Furthermore, biological responsiveness was demonstrated by dexamethasone and insulin altering the explants morphologically or biochemically. None of the IGFs or GH had any biological effects, raising doubts about their direct biological action on the developing intestinal epithelium.     

Protein kinase C-dependent and -independent pathways in the growth factor-induced cytoskeletal reorganization

Human epidermoid carcinoma KB cells exhibit rapid induction of membrane ruffling in response to epidermal growth factor (EGF), insulin, and insulin-like growth factor-I (IGF-I) (Kadowaki, T., Koyasu, S., Nishida, E., Sakai, H., Takaku, F., Yahara, I., and Kasuga, M. (1986) J. Biol. Chem. 261, 16141-16147). We have analyzed the role of protein kinase C (PKC) in this response. Treatment of KB cells with 4 beta-phorbol 12,13-dibutyrate (PDBu) (100 ng/ml) for 30 min caused translocation of PKC to the membrane. This treatment completely inhibited the induction of membrane ruffling by EGF, insulin, and IGF-I. Prolonged treatment with PDBu (200 ng/ml for 15 h) induced complete depletion of the PKC activity in the cells. Under these conditions, EGF binding to cells and autophosphorylation of the EGF receptor occurred normally, while EGF could not induce membrane ruffling. In contrast, insulin- or IGF-I-induced membrane ruffling occurred normally in the PKC-depleted cells. Moreover, H-7 (PKC inhibitor) inhibited only EGF-induced membrane ruffling in a dose-dependent manner. We further found that EGF, but not insulin/IGF-I, caused transient translocation of PKC to the membrane. All these results suggest that PKC is required for the membrane ruffling induced by EGF but not for that induced by insulin/IGF-I. Therefore, there are PKC-dependent and independent pathways in the growth factor-induced membrane ruffling. Furthermore, we propose dual roles of PKC in the EGF signaling, a signal transmitting role and a negative feedback role.     

Purified plasma membranes inhibit polypeptide growth factor-induced DNA synthesis in subconfluent 3T3 cells

Plasma membranes derived from NR-6 cells, a variant line of Swiss mouse 3T3 cells that does not have cell surface receptors for epidermal growth factor (EGF), inhibited EGF-induced stimulation of DNA synthesis by 50% in serum-starved, subconfluent 3T3 cells. Membranes derived from SV3T3 cells were much less effective in inhibiting EGF-induced DNA synthesis. This inhibition on DNA synthesis by NR-6 membranes was not a direct effect of membranes on EGF, nor could it be overcome by high concentrations of EGF. NR-6 membranes were most effective when added 3 h before EGF addition and had little effect when added 2 h or more after EGF. NR-6 membranes also reduced the stimulation of DNA synthesis induced by platelet-derived growth factor or fibroblast growth factor in serum-starved 3T3 cells. These findings indicate that membrane- membrane interactions between nontransformed cells may diminish their ability to proliferate in response to serum polypeptide growth factors.     

Enhancement in rats by the liver tumor promoter ethinyl estradiol of a serum factor(s) which is stimulatory for hepatocyte DNA synthesis

Fractionation of female rat serum or plasma on Sephadex G-200 revealed the presence of an activity stimulatory for hepatocyte DNA synthesis. Treatment of female rats with the liver tumor promoter ethinyl estradiol (EE) at 2.5 micrograms/day caused a 1.6 fold increase in the level of this activity at 24 hr in both serum and plasma. The stimulatory activity had a molecular weight of 135 kD, was sensitive to trypsin and heating and was not inhibited by the antiestrogen tamoxifen or antibody to epidermal growth factor (EGF). However, the pooled active fractions from EE-treated rats competed to a greater extent than comparable fractions from control rats for specific [125I]-EGF binding to rat liver membranes. These results demonstrate that treatment of female rats with EE, under conditions known to stimulate liver growth, caused an increase in level of a factor(s) stimulatory for hepatocyte DNA synthesis and whose activity may be mediated through the EGF receptor.     

Biological effects of biosynthetic human EGF on the growth of mammalian cells in vitro

The effects were examined of biosynthetic human epidermal growth factor (Bh-EGF) produced from cloned E. coli on DNA synthesis and all divisions of 13 different kinds of primary and established cell lines. Primary cultures of mammary epithelia, hepatocytes and stomach cells were strongly stimulated by EGF to undergo DNA synthesis in serum-free culture medium with concentrations of Bh-EGF as low as 0.1-10 ng/ml. In sharp contrast, 0.1-100 ng/ml of Bh-EGF failed to enhance thymidine incorporation into DNA when applied to established cell lines using the serum-free medium. Higher concentrations of Bh-EGF (30-100 ng/ml) promoted morphological changes only in hepatocytes, e.g., elongation, enlargement and projection of their cytoplasm. The above results were also obtained in mouse EGF (m-EGF). In our binding assay, Bh-EGF competed against [125I]-m-EGF with a one-fourth to one-fifth efficacy when compared with m-EGF. It was concluded that the in vitro biological activity of Bh-EGF was similar to that of m-EGF.     

Behavior of transforming growth factors in serum-free media: An improved assay for transforming growth factors

Summary Transforming growth factors (TGFs) are a relatively new category of factors that induce the anchorage-independent growth of non-transformed cells. These factors are usually detected by their ability to induce normal rat kidney (NRK) fibroblasts to grow in soft agar. Until now, this assay has been performed in serum-containing medium (SCM). Unfortunately, the background activity of this assay is variable and dependent on several factors, including passage number of the cells and the serum lot used. Furthermore, the addition of either EGF or TGF-β alone results in the appearance of additional colonies, which decreases the sensitivity of the assay. To circumvent these problems, serum-free media have been developed that support the growth of the NRK cells at low density in both monolayer culture and soft agar. Long-term growth in monolayer cultures occurs in serum-free medium supplemented with laminin, insulin, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and high density lipoprotein (HDL). Growth in soft agar occurs when TGFs are added to a serum-free medium, AIG medium, that contains insulin, transferrin, FGF and HDL. In contrast to the background activity observed when the assay is performed in SCM, no colonies form in the AIG medium unless TGFs are added and few, if any, colonies form if EGF or TGF-β are added alone. Thus, the AIG medium provides an improved assay for TGFs. In addition, the AIG medium should prove useful for examining other factors, including serum factors, for TGF activity. Editor's Statement This communication describes a modification of the standard assay for transforming growth factors. The techniques employed make use of advantages provided by recent advances in serum-free cell culture to provide a well-defined detection system that is more sensitive than conventional procedures. Experimental approaches described in this article also should be helpful in unraveling differences in cellular behavior encountered under anchorage-dependent vs. anchorage-independent conditions. D. W. Barnes     

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.