Effect of amino acid deprivation on DNA synthesis in BHK-21/C13 cells

Removal of serum from BHK-21/C13 cells in culture results in a decline in thymidine incorporation extending over five days. Additional removal of any of several amino acids results in a rapid decrease in incorporation of thymidine to negligible levels by 24 hours. Replacement by complete medium then provokes a synchronous wave of DNA synthesis after only ten hours with DNA synthesis first increased at six hours. Starvation for glutamine results in a rapid decline in protein synthesis over the 24 hour period when DNA synthesis is falling. However, there is considerable degradation of total protein during this period, and RNA degradation is also greatly increased. Concurrently, synthesis of RNA falls to less than 10% of that in control cells.     

The mechanism of regulation of fibroblastic cell replication. I. Properties of the system

Sixty to eighty per cent of the cells in a culture of human diploid fibroblasts may be stimulated from the state of density dependent inhibition of replication to active DNA synthesis and division. The maximum response is effected by 50% serum within the pH range 7.2–8.0. The proportion of cells responding depends on the concentration of serum protein in the medium which may be effectively substituted by crystalling serum albumin. There is a differential sensitivity to the stimulus of cells in the densely packed centers of whorls and in the less dense areas between the whorls. The cell response is parasynchronous and the median durations of the various phases of the cell cycle are: G₁I 6 β ?æ^® ¿ ∞ 8 hours, G₂ = 6 hours and doubling time = 30 hours. The stimulatory effect of fresh medium is lost during contact with dense cultures so that it has only 50% of its initial capacity after 14 hours. It can be restored by dialysis against serum-free medium. The stimulus must be applied for at least ten hours to be effective in inducing DNA synthesis. During the latter half of ten hour induction period subsequent DNA synthesis becomes exquisitely sensitive to actinomycin D. After this time an increasing number of cells become irreversibly committed to replicate. The data are interpreted to indicate that during contact with serum proteins (including albumin) changes in the cell surface, if continued long enough, trigger a mechanism which involves the synthesis of a unique RNA species during the fifth to tenth hours. After this RNA has been synthesized the cells are then committed to DNA synthesis.     

Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production.

Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.     

Dolichol-Linked Glycoprotein Synthesis in G1 Is Necessary for DNA Synthesis in Synchronized Primary Cultures of Cerebral Glia

Primary cultures of newborn rat cerebrum, which are composed of glial cells (principally astroglia), were used for examining the relationship between dolichol-linked glycoprotein synthesis and DNA synthesis in developing cerebral glia. The cells were synchronized by reducing the content of fetal calf serum in the culture medium from 10 to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the quiescent state by return of the cultures to 10% serum causes a marked increase in DNA synthesis 12-24 h later. A sharp increase in glycoprotein synthesis (incorporation of [3H]mannose) occurred in the first 12 h after serum repletion, preceding the increase in DNA synthesis. Tunicamycin, an inhibitor of the dolichol-linked pathway to glycoprotein synthesis at the first committed step in oligosaccharide formation, promptly and completely prevented the increase in glycoprotein synthesis and, in addition, the subsequent increase in DNA synthesis. The effects of tunicamycin on glycoprotein and DNA syntheses were reversible, and no comparable effect on total protein synthesis was observed. When tunicamycin was added only during a temporally circumscribed period in G1, i.e., from 3 to 9 h after serum repletion, the increase in DNA synthesis between 12 and 24 h after repletion was still markedly inhibited, i.e., to approximately 45% of the value in untreated cultures. The data thus show that there is a requirement for dolichol-linked glycoprotein synthesis for the subsequent occurrence of DNA synthesis and that this requirement is expressed late in the G1 phase of the cell cycle.     

Variations in some molecular events during the early phases of the Reuber H 35 cell cycle. II.-Chromatin protein phosphorylation and protein kinases

Reuber H 35 hepatoma cells were synchronized by transfer in a serum free medium. Growth was re-initiated by addition of serum. Under these conditions DNA synthesis exhibited a maximum after 24 hours. Chromatin non-histone proteins prepared from cells at various phases of the cell cycle were incubated with [gamma-32P] ATP and the radioactive pattern of protein bound 32P was analysed by electrophoresis on polyacrylamide gels. No radioactive peak was observed in G0. Several peaks appeared 3 hours after the addition of serum. The radioactivity progressively increased until the cells reached the S phase. When most of the cells were in the S phase the radioactivity strongly decreased. Chromatin protein kinase activities were found to increase in late G1 and continued to increase in the S phase. The increase was 65% when phosvitin was the substrate, 100% with casein and histone H1. It is suggested that chromatin phosphorylated proteins could be involved in the mechanism which initiates DNA synthesis in G1 phase cells.     

Responses of protein synthesis and degradation in growth control of WI-38 cells

The overall rates of protein synthesis and degradation in perfusion-grown WI-38 cells were followed in the three days after a stepdown in the serum concentration of the culture medium, from 10% to 0.3%. Within three hours after the stepdown, the rate of protein synthesis had decreased and the rate of protein degradation had increased, the combined result being the cessation of protein accumulation. The degradation rate returned over the next three days to its original value, but a zero rate of accumulation was retained because the synthesis rate continued to decline. The rate of DNA synthesis remained constant for six hours after the stepdown. It then declined steadily until reaching a minimum about eight hours later. The results show that extracellular control of protein accumulation depends on adjustments in both protein synthesis and protein degradation, and that the adjustments take place rapidly. This behavior suggests that the cell cycle is arrested after a stepdown because post-mitotic cells are unable to accumulate additional protein. However, an alternative interpretation of the data is that at least part of the changed accumulation is the result, rather than the cause, of the cycle arrest, and that the arrest is caused by other, more specific, reactions than those of general protein metabolism.     

Oligonucleotides antisense to catalytic subunit of cyclic AMP-dependent protein kinase inhibit mouse mammary epithelial cell DNA synthesis

Mouse mammary epithelial cells were plated onto 24-well culture plates (50,000 per well), allowed to attach and serum starved for 24 h. Following serum starvation, DNA synthesis was induced by the addition of 10% fetal calf serum and determined by a 1-h pulse with [3H]thymidine from 17 to 18 h after serum addition. Addition of oligonucleotides antisense to the translation start region of cyclic AMP-dependent protein kinase (kinase A) mRNA inhibited thymidine incorporation into DNA (total or percentage of cells incorporating thymidine, as measured by autoradiography). This inhibition was apparent whether compared to controls with no oligonucleotide addition, sense oligonucleotides, or mismatch oligonucleotides. Enzymatic assays indicated that the antisense oligonucleotides lowered kinase A activity in cells. Time course studies indicated that the inhibition in DNA synthesis was not an artifact of the time at which DNA synthesis was estimated. Long-term (4 day) cultures indicated that effects on induction of DNA synthesis were reflected in long-term cell proliferation.     

Inhibition of rat fibroblast cell proliferation at specific cell cycle stages by cocaine

We have analyzed the role of cocaine in the control of the rat fibroblast (EL2) cell proliferation. Our data show a dose-related effect on the inhibition of DNA synthesis and cell growth when cocaine is added with serum or with a pure growth factor [Epidermal Growth Factor (EGF)]. Pretreatment by drug did not appreciably enhance the inhibition of S-phase entry above that obtained when cocaine and mitogen were added simultaneously. On the contrary, exposure of quiescent EL2 cells to cocaine has little or no effect on DNA synthesis, when drug is removed before the mitogenic stimulus. Moreover, even when cocaine is added after EGF, an exposure only within 1–8 hours is required in order to inhibit stimulation of DNA synthesis. Cocaine also suppressed the general increase in protein synthesis that occurs during the first hour after EGF addition. The combined data suggest that cocaine inhibits the traverse of mitogen-stimulated quiescent EL2 cells from Go to S phase by acting on processes that take place during the initial phase of the cell cycle.     

Induction of the replicative synthesis of DNA by SV40 virus T antigen introduced into cells using liposomes

The role of virus SV40 T-antigen in the induction of cell DNA synthesis during its incorporation into cell liposomes was studied, using monolamellar liposomes obtained by phase reversal with incorporated highly purified T-antigen. Immunofluorescence studies revealed that T-antigen effectively penetrates inside the cells and after 10 hours is accumulated in the nuclei, where its level remains unchanged for 24 hours. Injections of purified T-antigen into the renal cells of serum-starved CV1 monkeys resulted in an almost 10-fold increase in the number of DNA-synthesizing cells 18 hours after the exposure. The same effect was observed during stimulation of a 10% serum culture. Removal of T-antigen from the preparation by specific immunoadsorption eliminated this effect. Centrifugation of cells grown in the presence of bromodeoxyuridine in a CsCl gradient was used to demonstrate the replicative type of cell DNA synthesis during T-antigen induction.     

The relationship of serum fibronectin and cell shape to thrombin-induced inhibition of dna synthesis in human fibroblasts

In a previous report it was shown that inhibited DNA synthesis and altered morphology resulted when human fibroblasts (HF) were plated in 3% fetal calf serum (FCS) medium preincubated with thrombin (Hall and Ganguly, 1980a). This was in contrast to the stimulatory effects of this enzyme when added to cells several hours after subculture. Those observations suggested that thrombin may act upon serum components of the growth medium necessary for initial culture establishment following cell plating. In this report, the relationship of serum fibronectin (FN) to this thrombin-mediated inhibitory phenomena was investigated. It was found that the development of altered morphology and inhibited DNA synthesis could be completely prevented by the addition of this glycoprotein to medium preincubated with thrombin. Cell shape and DNA synthesis appeared to be closely related and both parameters showed a dose-dependent sensitivity to added fibronectin. To further investigate this, a technique was developed in which cell shape could be selectively varied and DNA synthesis measured in the absence of serum or thrombin. These studies indicated that cell shape was closely related to DNA synthesis and morphologies identical to that seen in thrombin-treated medium were produced. As observed in the thrombin system, normal cellular appearance and DNA synthesis could be restored by the addition of fibronectin. The results of this work suggest that thrombin acts upon medium components necessary for normal morphological development, possibly fibronectin, in cells following subculture. Inhibited DNA synthesis and growth seem to arise as a direct consequence of this effect.     

X-irradiation of equine peripheral blood lymphocytes stimulated with phytohaemagglutinin in vitro.

Small lymphocytes were isolated from the peripheral blood of horses and incubated at 37 degrees C in Eagle's medium supplemented with 20 per cent foetal calf serum. The addition of phytohaemagglutinin (PHA) to the cultures resulted in: increased RNA and protein synthesis; the enlargement of the small lymphocyte into a lymphoblast-like cell; the initiation of DNA synthesis, and cell division. When survival was measured 24 hours after X-irradiation by means of phase-contrast microscopy, the lymphoblast-like cell was much more radio-resistant (D0 = 250 rad) than the small lymphocyte (D0 = 20 rad). This increase in radioresistance, however, was not observed until 12-24 hours after PHA treatment. To investigate which of the changes occurring during the transformation of the small lymphocyte was responsible for the increased resistance to irradiation, the percentage of cells surviving irradiation was compared with the percentage of cells incorporating significant amounts of 3HTdR, 3H-UR, or 3H-leucine at the time of irradiation. For this comparison, a dose of 100 rad was used because 100 rad killed essentially all of the small lymphocytes, but less than 35 percent of the cells which had become radioresistant from the PHA treatment. The results indicated that the increase in radioresistance was not associated with DNA synthesis, but instead correlated with the increase in RNA and protein synthesis which the cells had attained at the time of irradiation.     

The uncoupling of macromolecular synthesis from cell division in SV3T3 cells by glucocorticoids: The imposition of a G2 block

Through a receptor-mediated process glucocorticosteroids block cell division by 20–45 hours in SV40-transformed 3T3 (SV3T3) mouse fibroblasts growing in a low calf serum (0.30% v/v) medium containing biotin. However, the rate of DNA synthesis, determined at various times after dexamethasone addition by the incorporation of radioactive thymidine into acid-insoluble material, is not inhibited by this steroid as late as 66 hours. A modest decrease is observable by 91 hours. There is also no reduction in the uptake of exogenous thymidine into acid-soluble cellular pools. Similarly, RNA synthesis and the uptake of radioactive uridine are not affected by the glucocorticoid up to 69 hours. Measurements of the amounts of cellular DNA (by the fluorescent dye, 4′,6-diamidino-2-phenylindole) and protein revealed that both macromolecules are present in elevated quantities in steroid-treated cells. (The constancy of the protein content in the nonproliferative stage suggests that protein synthesis and degradation are occurring at equal rates.) If the steroid is removed and fresh 10% calf serum medium added, cell division commences (even if nearly 90% of protein synthesis is inhibited by cycloheximide) as early as 45 minutes later such that by 2 hours the viable cell count increases by as much as 70%. Since the growth curve after recovery resembles a step function, it appears that the cells are partially synchronized by the glucocorticoid. These results demonstrate that the glucocorticoid cytostatic effect in SV3T3 cells is the result of a block not in G₁, as previously thought, but in G₂.     

Induction of hyaluronic acid synthetase activity in rat fibroblasts by medium change of confluent cultures

Hyaluronic acid synthesis in cultured cells usually occurs during the growth phase. The relation between hyaluronic acid synthetase activity and cell proliferation is studied. The synthetase activity in rat fibroblasts is high during the growth phase, but low in the stationary phase. When the old medium of stationary cultures is renewed with fresh medium containing 20% calf serum, DNA synthesis occurs synchronously between 12 and 20 hours, followed by cell division. Under these conditions, the hyaluronic acid synthetase activity is significantly induced within two hours, reaching a maximum level at 5–8 hours, and then decreases gradually. This induction of the synthetase, which shows a high turnover rate, requires continued synthesis of both RNA and protein. Furthermore, the induction of both DNA and hyaluronic acid synthesis is found to be caused by calf serum added in the medium. However, dialysis and ultrafiltration of the serum permit us to concentrate an active fraction with a high molecular weight, which induces the synthetase activity, but not DNA synthesis.     

Replication kinetics of three DNA sequence families in synchronized mouse cells

Balb/C3T3 cells entered the quiescent Go state following serum deprivation. On addition of fresh serum, more than 95% of the culture resumed growth, but with asynchronous kinetics. If hydroxyurea were added just before the first cells reached S phase, at at least 90% of the cells accumulated at the Gl/S border over the next ten hours. When the block was removed, the culture moved synchronously into S phase. As the cells traversed S, the replication kinetics of three classes of rapidly renaturing DNA were analyzed. Main band highly repeated DNA and foldback DNA replicated continuously, In contrast, satellite DNA replication did not commence until three hours into S, whereupon its rate of synthesis increased ver rapidly, reaching a maximum with the next two hours. These results are discussed in the light of earlier work utilizing other methods of cell synchronization.     

The capacity of chondrocytes to respond to serum is enhanced by organ culture in the absence of serum,stimulated by serum,and modified by ascorbate

Cartilage slices maintained in organ culture have been shown to develop an enhanced capacity to respond to serum. The response was measured at the initiation of culture and after 3 and 7 days of culture in medium containing an inhibitor of DNA synthesis and 0, 1, or 16% serum. At these times, cartilage slices were washed to remove serum and inhibitor, and then exposed to various concentrations of serum for evaluation of DNA and proteoglycan synthesis. The range of the derived dose-response curves and the indicated sensitivity to low serum concentrations were the parameters used to evaluate the response capacity. Response capacity increased gradually, reaching a maximum after 8 days of culture. Considerable enhancement was obtained after maintenance in the absence of serum using both DNA and proteoglycan synthesis as markers. Additional, graded enhancement of response capacity was obtained when the cartilage slices were maintained in 1 or 16% serum. The effects of maintenance in serum were much greater when DNA synthesis rather than proteoglycan synthesis was used to measure the response. However, this serum-dependent enhancement was only prominent when ascorbate was present during the dose-response assay. Ascorbate caused a similar but less-marked increase in sensitivity to serum when proteoglycan synthesis was measured. The possibility that ascorbate may function as a cofactor during the progression phase of cell proliferation is discussed.     

eIF2alpha phosphorylation, stress perception, and the shutdown of global protein synthesis in cultured CHO cells

The perception of environmental stress in animal cells engineered to produce heterologous protein leads to the induction of stress signaling pathways and ultimately apoptosis and cell death. Protein synthesis is regulated in response to various environmental stresses by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have utilized a model system of Chinese hamster ovary cells engineered to secrete recombinant TIMP-1 protein to investigate the relationship between the cellular rate of protein synthesis, eIF2alpha phosphorylation, cellular stress perception, and the rate of cell specific recombinant protein synthesis. The rate of total protein synthesis was maximal after 48 hours of culture, remaining relatively high until 96 hours of culture, after which a decline was observed. Towards the end of culture a marked increase in labeled secreted protein was observed. Total eIF2alpha expression levels were high during the exponential growth phase and decreased slightly towards the end of culture. On the other hand, the relative expression of phosphorylated eIF2alpha showed a bi-phasic response with a small increase in phosphorylated eIF2alpha observed at 48 hours of culture, and a significant increase at 120 hours post-inoculation. The large increase in phosphorylated eIF2alpha coincided with the observed increase in labeled secreted protein and the decline in total cellular protein synthesis. A marked increase in ubiquitination was also observed at 120 hours post-inoculation that coincided with reduced rates of cellular protein synthesis and mRNA translation attenuation. We suggest that eIF2alpha phosphorylation is an indicator of cellular stress perception, which could be exploited in recombinant protein manufacturing to commence feeding and engineering strategies.     

Uterine epithelial cells in primary culture: a model system to study cell proliferation

Guinea-pig uterine glandular epithelial cells were grown in primary culture. During the 4-day initial culture period, a 6.7 fold increase in DNA synthesis and a doubling time of approximately 30 hours were observed. Then the cells were submitted to serum depletion (60 hours) and the quiescent cells were stimulated with 15% fetal calf serum (FCS). The control cells were submitted to 1% heated and dextran-coated charcoal stripped FCS. In stimulated cells, the DNA synthesis increased and peaked between the 12th and 24th hour. In these cells, c-fos mRNAs increased rapidly, within 30 min., peaked at 75 min. (ratio to the control = 2.5), and returned to basal level within 90 min. These results prove that uterine epithelial cells in primary culture are able to respond to unspecific mitogen by both rapid expression of c-fos gene and DNA synthesis, suggesting that this cell culture system will be useful in studying the growth regulation in endometrium.     

Polyamine biosynthesis and DNA synthesis in cultured mammary gland explants from virgin mice

The mammary cells in virgin mice are essentially non-proliferative, but they can be induced to undergo DNA synthesis in vitro in the presence of insulin. Time course studies on polyamine biosynthesis and DNA synthesis showed that insulin elicits sequential stimulation of the activity of the polyamine biosynthetic enzymes, ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase (SAMDC) and spermidine synthase, and an increase in the concentration of spermidine prior to the augmentation of DNA synthesis. At 48 to 72 hours of culture when DNA synthesis is maximal, the concentration of spermidine increased 2? to 3-fold, whereas the level of spermine remained unchanged. Addition of methyl glyoxal bis(guanylhydrazone) (5—10 μM), a potent inhibitor of SAMDC, to the medium at the onset of culture resulted in inhibition of spermidine formation and DNA synthesis, but when added at 24 hours or 48 hours of culture, the inhibitory effect on DNA synthesis was greatly reduced. The drug, however, produced little inhibition of RNA and protein synthesis. Inhibition of DNA synthesis by the drug can be reversed by addition of spermidine or other polyamines such as putrescine, cadaverine and spermine to the culture. Spermidine is, however, the only polyamine that is effective at physiological concentrations (100～150 pmoles/mg tissue). These results suggest a possibility that spermidine may play a key role in the regulation of mammary cell proliferation.