Altered growth factor requirements and cell cycle control in rat hepatoma cells versus adult rat hepatocytes in culture

Adult rat hepatocytes multiply in primary cultures when incubated in arginine-free MX-83 medium supplemented with dialyzed fetal calf serum, insulin, glucagon, hydrocortisone, epidermal growth factor, and transferrin. In the absence of mitogens, the fraction of the cells engaged in DNA synthesis dropped sharply. However, cells initiated DNA synthesis in response to the mitogenic mixture indicating that hepatocyte proliferation is controlled by G1----S transition rates. In contrast, rat hepatoma line DTH-3, derived from Morris 7777 "minimal deviation" hepatoma, required only insulin for proliferation in chemically defined MX-83 medium. The lengths of their cell cycle phases varied with the growth rate. The phases of the growth cycle were proportionately shortened (expanded) when the growth rate was increased (decreased). It is concluded that DTH-3 hepatoma cells, which display a decreased growth factor requirement as compared with adult rat hepatocytes differ from normal hepatocytes by fundamental alterations in the mechanisms controlling the progression of the cell cycle.     

STUDIES ON PRIMARY CULTURES OF DIFFERENTIATED FETAL LIVER CELLS

A method for culturing non- or slowly growing, differentiated fetal rat liver cells is described. It involves the use of collagenase as a digesting agent and of a selective medium deficient in arginine which suppresses the growth of nonparenchymal liver cells. Evidence is presented that surviving cells (a) retain liver-specific urea cycle functions measured by their capacity to transform ornithine into arginine, (b) synthesize DNA in glucose-deficient medium, and (c) synthesize and secrete albumin. This primary cell culture responds to partially hepatectomized rat serum and may be an appropriate assay system for the study of mechanisms which regulate liver regeneration.     

Factors regulating the induction of ornithine decarboxylase in fetal rat liver cells in culture.

Various hormonal and non-hormonal agents were tested for their ability to induce ornithine decarboxylase (EC 4.1.1.17) in primary cultures of fetal rat liver cells that retain many of the differentiated functions of hepatocytes. The only agents to induce ornithine decarboxylase in this cell type were fetal calf serum, prostaglandin E1 and cyclic AMP derivatives. Also, the amino acid arginine would induce ornithine decarboxylase in this cell type following arginine starvation for 24 h. These observations are in contrast to the wide range of hormones, e.g. insulin, hydrocortisone, glucagon and growth hormone, than can induce ornithine decarboxylase in vivo in the adult rat liver but which are all without effect on fetal rat liver cells.     

Non-neuronal cells of rat hypothalamus in dissociated cell culture

Summary The neurophysin that is biosynthesised in association with the neurohypophysial hormone vasopressin (vasopressin-neurophysin) affects the growth and DNA synthesis of rat hypothalamic non-neuronal cells in culture. Over a narrow range of concentrations vasopressin-neurophysin stimulated growth, as assessed by increase in cell numbers, about five-fold, in conditions where fetal calf serum concentration was limiting (0.2% fetal calf serum). Maximum stimulation occurred in the presence of 20 to 30 ng vasopressin-neurophysin per ml of medium. DNA synthesis was increased by a factor of three in the presence of 30 ng vasopressin-neurophysin per ml of medium. At least two populations of non-neuronal hypothalamic cells were present in the cultures, and these were both affected by vasopressin-neurophysin.This study allows the suggestion that neurophysin may be acting as a growth-regulating factor at its release site, playing a part in the interactions of neurones and glial cells in the hypothalamo-neurohypophysial system.     

Factors regulating the induction of ornithine decarboxylase in fetal rat liver cells in culture

Various hormonal and non-hormonal agents were tested for their ability to induce ornithine decarboxylase (EC 4.1.1.17) in primary cultures of fetal rat liver cells that retain many of the differentiated functions of hepatocytes. The only agents to induce ornithine decarboxylase in this cell type were fetal calf serum, prostaglandin E₁ and cyclic AMP derivatives. Also, the amino acid arginine would induce ornithine decarboxylase in this cell type following arginine starvation for 24 h. These observations are in contrast to the wide range of hormones, e.g. insulin, hydrocotisone, glucagon and growth hormone, that can induce ornithine decarboxylase in vivo in the adult rat liver but which are all without effect on fetal rat liver cells.     

Ascorbate free radical and its role in growth control

Summary Ascorbate and its free radical potentiates proliferation of HL-60 cells in serum-limiting media. Dehydroascorbate does not affect growth. This stimulation of growth is due to a general shortening of the cell cycle. The incubation of HL-60 cells with ascorbate free radical produces a significant change of the redox potential of cells. The presence of cells in culture media avoids the total oxidation of ascorbate, and also HL-60 cells induce the short-term stabilization of ascorbate. Ascorbate free radical potentiates also the onset of DNA synthesis in CCL39 cells induced by fetal calf serum, although itself does not affect quiescense to proliferation transition. This transition induced by fetal calf serum also potentiates the capacity of CCL39 cells to stabilize ascorbate. We discuss here the role of ascorbate free radical on growth control by its reduction by the plasma membrane redox system and its meaning for cell physioslogy.     

Effects of withdrawal of a mitogenic stimulus on progression of fibroblasts into S phase: differences between serum and purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA) for chicken embryo fibroblasts was purified from serum-free medium conditioned by the growth of a rat liver cell line. A comparison between calf serum and purified MSA was made regarding the regulation of the fibroblast cell cycle. Addition of serum or MSA to stationary, quiescent cells stimulates them to enter the DNA synthetic phase after a characteristic lag period. Exposure to serum for shorter periods of time will irreverisbly commit cells to continue through the cell cycle and initiate DNA replication in the absence of serum. In contrast, the withdrawal of purified MSA from the medium results in an abrupt halt in the progression of cells towards S phase. The results of labeled thymidine incorporation and autoradiographic experiments clearly indicate that the point at which cells become irreversibly committed to enter the DNA synthetic period is at or near the G1-S boundary. The abrupt decay of the stimulation upon withdrawal of purified MSA provides a unique opportunity to investigate the biochemistry of this discrete phase of the cell cycle.     

Soluble insulin-like growth factor II/mannose 6-phosphate receptor inhibits DNA synthesis in insulin-like growth factor II sensitive cells

The soluble form of the insulin-like growth factor II (IGF-II)/mannose 6-P (IGF-II/M6P) receptor is released by cells in culture and circulates in the serum. It retains its ability to bind IGF-II and blocks IGF-II-stimulated DNA synthesis in isolated rat hepatocytes. Because these cells are not normally stimulated to divide by IGF-II in vivo, the effect of soluble IGF-II/M6P receptor on DNA synthesis has been further investigated in two cell lines sensitive to IGF-II; mouse 3T3(A31) fibroblasts, stimulated by low levels of IGF-II following priming by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and Buffalo rat liver (BRL) cells, which secrete IGF-II and proliferate in the absence of exogenous growth factors. Soluble IGF-II/M6P receptor (0.2-2.0 microgram/ml) purified from a rat hepatoma cell line inhibited DNA synthesis (determined by dThd incorporation) in both cell lines. Basal DNA synthesis was very low in serum-free 3T3 cells, but high in serum-free BRL cells, possibly as a result of autocrine IGF-II production. The inhibitory effect was reversible in cells preincubated with soluble receptor prior to incubation with growth factors and could also be overcome by excess IGF-II. Soluble receptor was more potent in IGF-II-stimulated 3T3 cells and serum-free BRL cells than in BRL cells incubated with serum. Mean inhibition by four preparations of soluble receptor (1 microgram/ml) was 34.7% +/- 4.4% in BRL cells stimulated with fetal calf serum (FCS) (5%) compared to 54.8% +/- 4.2% in serum-free BRL cells (P = 0.05) and 60.6% +/- 6.5% (P = 0.02) in 3T3 cells stimulated by PDGF, EGF, and IGF-II. Soluble receptor had no effect on DNA synthesis in 3T3 cells stimulated with IGF-I. These results demonstrate that soluble receptor, at physiological concentrations, can block proliferation of cells by IGF-II and could therefore play a role in blocking tumor growth mediated by IGF-II.     

Induced changes in the rates of uridine- 3 H uptake and incorporation during the G 1 and S periods of synchronized Chinese hamster cells

The rates of uridine-5-³H incorporation into RNA and the rates of uridine uptake into the acid-soluble pool during the cell cycle of V79 Chinese hamster cells were examined. Cells cultured on Eagle''s minimal essential medium supplemented with fetal calf serum, lactalbumin hydrolysate, glutamine, and trypsin displayed rates of incorporation and uptake which increased only slightly during G₁ and accelerated sharply as DNA synthesis commenced. In contrast, cells cultured on minimal essential medium supplemented only with calf serum exhibited rates of incorporation and uptake which increased linearly through both G₁ and S. The transition from one pattern to the other can be induced within 24 hr and is completely reversible. The nonlinear pattern exhibited by cells grown on the supplemented fetal calf serum medium can also be overcome with high exogenous uridine concentrations. In the presence of 200 µM uridine, these cells display a linear pattern of increase in rates of uridine incorporation and uptake. It is concluded that at lower uridine concentrations the pattern of increase in the rate of uridine incorporation into RNA during the cell cycle for a given population of cells is dependent upon the rate of uridine entry into the cell, and that this pattern is not rigidly determined but can be modified by culture conditions.     

Stimulation of bumetanide-sensitive K+ transport in Swiss 3T3 fibroblasts by serum and mitogenic hormones

Rapidly growing Swiss 3T3 fibroblasts possess a bumetanide-sensitive K+ transport system that is dependent on both Na+ and Cl- ions; a smaller bumetanide-insensitive component of K+ transport is also present. In cells brought to the quiescent state by 8-11 days of incubation without a medium change, the bumetanide-sensitive rate of transport was reduced by 63%; the bumetanide-insensitive rate did not change. Removal of dialyzed fetal calf serum from the uptake medium resulted in a substantial reduction in bumetanide-sensitive uptake in both rapidly growing cells (33% reduction) and quiescent cells (68% reduction) but had no effect on bumetanide-insensitive uptake. Insulin was almost as effective as dialyzed fetal calf serum in stimulating bumetanide-sensitive uptake; insulin was maximally stimulatory at 2.5 micrograms/ml. The combination of insulin, epidermal growth factor, and arginine-vasopressin was maximally effective in stimulating both bumetanide-sensitive K+ uptake and 3H-thymidine incorporation in quiescent cells; bumetanide, however, did not interfere with the hormonal stimulation of DNA synthesis. Thus, the bumetanide-sensitive K+ transport system is not necessary for such stimulation to occur. Furthermore, concentrations of hormones which stimulated significant levels of DNA synthesis produced no elevation in the intracellular concentration of K+. We conclude that the bumetanide-sensitive pathway of K+ transport is modulated by serum and by mitogenic hormones, but does not play a role in the stimulation of DNA synthesis by these factors.     

Basic fibroblast growth factor and transforming growth factor-alpha are hepatotrophic mitogens in vitro

Basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF alpha) have been identified as potent hepatotrophic mitogens. bFGF and TGF alpha induce DNA synthesis in fetal and adult rat hepatocytes in primary culture and support fetal rat hepatocyte multiplication in chemically defined medium. No additional exogenous growth or progression factors are required by the cells for traversing the cell cycle or for cell division. These mitogenic polypeptides, previously identified in various cell types including liver and endothelial cells, platelets, and macrophages may act locally in a paracrine mode in controlling hepatocyte multiplication in the liver during development and regeneration.     

Leucine transport in relation to the activities of Na+-H+ antiporter and Na+/K+ pump stimulated by serum and a tumor promoter

Fetal calf serum and 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the rate of leucine uptake by Chang liver cells in Na+-containing medium. Addition of monensin to the incubation medium also increased the leucine uptake. All these agents were capable of raising the cytoplasmic pH, which was blocked by a prior addition of amiloride or removing Na+ from assay medium, suggesting activation of Na+-H+ exchange across the cell membrane by fetal calf serum and TPA. The stimulation of leucine uptake by monensin and fetal calf serum was blocked completely or incompletely by addition of ouabain or amiloride. The basal and fetal-calf-serum- or TPA-stimulated leucine uptake was extensively depressed by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid in the incubation medium. Based on these results it is proposed that the transport of leucine by the system L is stimulated by fetal calf serum and TPA with a high concentration of Na+ outside the cells as a result of alkalinization of the cytoplasm and coordinated activation of (Na+ + K+)-ATPase by these stimulators to maintain the transmembrane Na+ gradient and also hyperpolarize the cell membrane.     

Primary culture of normal adult rat liver cells which maintain stable urea cycle enzymes.

Normal adult rat liver cells have been successfully cultured as monolayers without subjecting donor animals to a partial hepatectomy before cell isolation. Coating plastic tissue culture dishes with acid soluble calf skin collagen increases the efficiency of cell attachment. Hepatocytes form a monolayer in 24 hr in serum-free L-15 medium although 10% fetal calf serum for 24 hr increases efficiency of attachment. In serum-free medium the mono-layer remains viable for at least one week without added insulin. Cultured cells maintain tyrosine transaminase and four of the five urea cycle enzymes at levels above or equal to freshly isolated cells for 72 hr. Adenylyl cyclase activities are maintained for at least 72 hr, and are stimulated by epinephrine, glucagon and fluoride. Tyrosine transaminase activity is increased in cultured cells by glucagon and dexamethasone, but urea cycle enzymes are not.     

Bi-functional action of transforming growth factor-beta on DNA synthesis in early passage human fetal fibroblasts

We investigated the influence of transforming growth factor-beta (TGF-beta) on DNA synthesis in human fetal fibroblasts, as measured by the incorporation of [3H]thymidine and cell replication. In serum-free medium, without additional peptide growth factors, TGF-beta had no action on thymidine incorporation. However, in the presence of 0.1% v/v fetal calf serum, TGF-beta exhibited a bi-functional action on the cells. A dose-dependent stimulation of [3H]thymidine incorporation, and an increase in cell number, occurred with fibroblasts established from fetuses under 50 g body weight, with a maximum stimulation seen at 1.25 ng/ml. For fibroblasts from fetuses of 100 g or greater body weight, TGF-beta caused a dose-related decrease in thymidine uptake with a maximal inhibition at 2.5 ng/ml, and a small decrease in cell number. When DNA synthesis was stimulated by the addition of somatomedin-C/insulin-like growth factor I, epidermal growth factor, or platelet-derived growth factor, their actions were potentiated by the presence of TGF-beta on cells derived from fetuses under 50 g body weight, but inhibited on cells obtained from the larger fetuses weighing more than 100 g. Similar results were found for changes in cell number in response to TGF-beta when stimulated by SM-C/IGF I. The ability of TGF-beta to modulate [3H] thymidine incorporation did not involve a change in the time required for growth-restricted cells to enter the S phase of the replication cycle. These data suggest that TGF-beta may exert either a growth-promoting or growth-inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.     

Lack of a correlation between polyamine synthesis and DNA synthesis by cultured rat liver cells and fibroblasts

Growth stimulation of either fetal rat liver cells or rat embryo fibroblasts in culture results in considerable increases in intracellular polyamine levels as cells proceed through the cell cycle. Treatment of such cell cultures with appropriate levels of two inhibitors of polyamine synthesis, namely α-hydrazino ornithine and methylglyoxal bis(guanylhydrazone), can essentially completely block these increases in cellular polyamine content. Under such conditions, where the elevation in intracellular polyamine content is prevented, cell cultures are nevertheless able to initiate DNA synthesis and subsequently synthesize DNA at rates comparable to untreated control cultures that have been growth-stimulated. These two cell types therefore contain sufficient polyamines when in a resting state (G₁) to enable them to enter from G₁ into S phase and traverse S phase at normal rates in the absence of further polyamine synthesis. The recruitment of cells into the first cell cycle, through serum stimulation of growth, therefore appears not to be mediated or regulated by the increases in intracellular levels of polyamines that occurs under these conditions. Conversely, the arrest of growth of these cell types resulting from serum deprivation is not mediated by a limitation of intracellular polyamine content.     

Induction of myogenic differentiation in serum-free medium does not require DNA synthesis

Cells of the myogenic rat cell line L6 can be obtained as a confluent, quiescent population of undifferentiated myoblasts after growth in F12 medium supplemented with fetal calf serum. Myogenic differentiation can be induced in these cells by changing to Dulbecco's modified Eagle's (DME) medium containing insulin as the only protein component. Labeling of the cells with [3H]thymidine demonstrates that this induction of fusion occurs in the absence of DNA synthesis in about 85% of the cells. This result was confirmed using cytosine arabinoside: fusion of quiescent L6 cells was induced in the presence of this inhibitor of DNA synthesis. The myotubes formed in DME + insulin medium, with or without cytosine arabinoside, synthesize or accumulate proteins characteristic of differentiated muscle cells including myosin heavy and light chains, alpha-actin, alpha- and beta-tropomyosins, and the acetylcholine receptor. These experiments represent a direct demonstration that DNA synthesis is not required for the induction of myogenic differentiation in undifferentiated quiescent cells.     

Purified multiplication-stimulating activity from rat liver cell conditioned medium: comparison of biological activities with calf serum, insulin, and somatomedin

Multiplication-stimulating activity (MSA) for chicken embryo fibroblasts was purified from serum-free medium conditioned by the growth of a line of rat liver cells (CRL), The biological activities of purified CRL MSA for chicken embryo fibroblasts were compared with those of calf serum to determine which activities are important for the stimulation of DNA synthesis and mitosis. In a balanced salt solution, only glucose and amino acids were needed in addition to purified CRL MSA to stimulate DNA synthesis maximally. Purified CRL MSA stimulated the rates of uptake of glucose and α-aminoisobutyric acid. Only the stimulation of the rate of glucose uptake appeared to be a primary response to purified CRL MSA since the stimulation was not inhibited by actinomycin D or cycloheximide. The stimulation of the rate of uptake of α-aminoisobutyric acid was inhibited by actinomycin D. CRL MSA differed from calf serum in its inability to commit cells irreversibly to synthesize DNA after the removal of CRL MSA and in its lack of the ability to stimulate the migration or prolong the survival of chicken embryo fibroblasts. Comparative studies indicated that purified CRL MSA had functional similarities to insulin and somatomedin. CRL MSA may be representative of a family of small polypeptide hormones having insulin-like activity which are involved in the control of cell multiplication.     

Obligatory Relationship Between the Sterol Biosynthetic Pathway and DNA Synthesis and Cellular Proliferation in Glial Primary Cultures

Primary cultures of newborn rat brain, which are composed predominantly of astroglia, were used to examine the relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation. Reduction of the fetal calf serum content of the culture medium from 10 to 0.1% (vol/vol) for an interval of 48 h between days 4 and 6 in culture resulted in a quiescent state characterized by inhibition of DNA synthesis and cellular proliferation. When 10% fetal calf serum was returned to the medium for these quiescent cells, within 24 h DNA synthesis increased markedly. Preceding the rise in DNA synthesis was an increase in sterol synthesis, which occurred within 12 h of the return of the quiescent cells to the 10% fetal calf serum. Exposure of the quiescent cells to mevinolin, a specific inhibitor of sterol synthesis at the 3-hydroxy-3-methylglutaryl-CoA reductase step, completely inhibited the increase in DNA synthesis that followed serum repletion. The increase in total protein synthesis that followed serum repletion was not similarly inhibited by mevinolin. When mevinolin was removed after causing the 24-h inhibition of DNA synthesis, the cultured cells underwent active DNA synthesis and proliferation. Thus, inhibition of the sterol biosynthetic pathway resulted in a specific and reversible inhibition of DNA synthesis and glial proliferation in developing glial cells. These findings establish a valuable system for the examination of glial proliferation, i.e., primary glial cultures subjected to serum depletion and subsequent repletion. Moreover, the data establish an obligatory relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation in developing glia.     

Control of muscle differentiation in BC3H1 cells by fibroblast growth factor and vanadate

We have examined the control of actin isoform synthesis by pituitary-derived fibroblast growth factor and serum in BC3H1 cells, a tumor-derived nonfusing muscle cell line. Under differentiating conditions in BC3H1 cells, the synthesis of beta- and gamma-actin ceases, and the rate of alpha-actin synthesis is increased concomitant with cessation of cell growth. Addition of fetal calf serum to differentiated cells reverses the process, whereas the addition of pituitary-derived fibroblast growth factor inhibits synthesis of alpha-actin but fails to induce the synthesis of beta- and gamma-actin. Analysis of RNA from differentiated BC3H1 cells after the addition of fetal calf serum indicated that the serum-induced increase in beta- and gamma-actin synthesis reflected an increase in their mRNA levels. In contrast, the repression of alpha-actin synthesis by fetal calf serum or fibroblast growth factor appears to reflect the translation efficiency of alpha-actin mRNA. Fibroblast growth factor is a competence factor for BC3H1 cells which allows them to progress from G0 4 h into the G1 phase of the cell cycle. In order to understand the nature of the intracellular signals responsible for the effect of fibroblast growth factor, we treated cells with vanadate, a known inhibitor of tyrosine-specific protein phosphatases. Vanadate fully mimics the action of fibroblast growth on actin synthesis and creatine phosphokinase synthesis and causes BC3H1 cells to exit the G0 portion of the cell cycle, as demonstrated by the induction of the c-fos proto-oncogene following addition of serum, vanadate, or bovine pituitary-derived fibroblast growth factor to these cells. We conclude that repression of alpha-actin synthesis and induction of the synthesis of beta- and gamma-actin are under independent control and that the induction of beta- and gamma-nonmuscle actin synthesis following serum addition is independent from movement into the cell cycle, and dependent on as yet unidentified serum components. The rate of synthesis of alpha-actin can be controlled by a defined mitogenic polypeptide fibroblast growth factor, which in short term experiments primarily affects the rate of translation of alpha-actin mRNA. The repression by fibroblast growth factor is most likely due to activation of a tyrosine specific protein kinase(s).     

Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts.

Human fibroblasts, cultured in medium containing 10% fetal calf serum, responded dramatically to choleragen with an increase in cyclic adenosine monophosphate content to greater than 48 times basal levels. Analysis of these cells for gangliosides indicated that the major ganglioside was N-acetylneuraminylgalactosylglucosylceramide (GM3) with trace amounts (less than or equal to 100 pmol/mg of protein) of other gangliosides including GM1, the putative choleragen receptor. Although the cells contained three glycosyltransferases required for ganglioside synthesis, the N-acetylgalactosaminyltransferase activity necessary for the conversion of GM3 to more complex gangliosides was not detected. When the cells were grown in medium containing [14C]galactose or N-acety[3H]mannosamine, however, all of the gangliosides became labeled, indicating that the cells can synthesize complex gangliosides. Although fetal calf serum contains gangliosides including GM1, [3H]GM1 was taken up poorly from the growth medium and uptake at the rate observed could have accounted for less than 2% of the GM1 content of the cells. When the cells were incubated in chemically defined medium containing [3H]GM1 at the concentrations present in fetal calf serum, rapid uptake of the ganglioside occurred and the total GM1 content of the cells increased threefold in less than 3 h. Thus, although the cells are capable of binding exogenous gangliosides, the gangliosides in fetal calf serum are in a form not readily available to the cells.