Early alterations in amino acid pools and protein synthesis of diploid fibroblasts stimulated to synthesize DNA by addition of serum

Diploid human fibroblasts in culture (WI 38) were allowed to reach a stationary phase and were then stimulated to reenter DNA synthesis and cell division by addition of serum to the culture medium. The rate of protein synthesis increased during the first hours after addition of serum reaching at three hours a plateau value that continued for at least 24 hours after serum addition. Inhibition of protein synthesis during the early hours after serum addition abolished the stimulation of DNA synthesis occurring 20 to 28 hours later. Increased protein synthesis was preceded by a rapid decrease in the intracellular pool size of most amino acids. These changes were independent of concomitant protein synthesis. They suggest that serum exerts an immediate effect on the function of the cell membrane.     

On the regulation of DNA synthesis in a line of adrenocortical tumor cells: effect of serum, adrenocorticotropin and pituitary factors.

Y-1 adrenal cells responded to serum step down by a several fold decrease in DNA synthesis. Serum starved cells resumed DNA synthesis upon serum step up. ACTH and cAMP inhibited DNA synthesis both at low and high serum concentrations, a fact previously known. Pituitary, brain and liver crude extracts stimulated DNA synthesis in serum starved cells. Purified pituitary factors preparations contained two activities: one specific for Y-1 cells and another active with both fibroblasts and Y-1 cells. The kinetics of restimulation of DNA synthesis by serum and pituitary factors was studied. DNA synthesis restimulation occurred after a lag of 11 hours. This lag did not vary irrespective of the type of stimulator or its concentration. Cells entered S phase continuously at a rate which increased with increasing concentrations of the stimulator. Cells became refractory to the inhibitory action of ACTH five hours before entering S phase. The implications of these data to the understanding of cell growth control are considered.     

Stimulation by serum of multiplication of stationary chicken cells

When serum was added to cultures of stationary chicken fibroblasts, there was an increase in the percentage of cells synthesizing DNA after a delay of about five hours. If the serum was removed before DNA synthesis began, some cells were still found to start DNA synthesis and to enter mitosis. The delay between the time of addition of serum and the start of DNA synthesis was not affected by the type of serum, the concentration of serum, nor the means of preparing the stationary cells. However, all of these factors affected the proportion of cells stimulated by serum. If two pulses of serum were given immediately following each other, their effects were synergistic. If there were twelve hours between them, there was no effect of the first pulse. These results were summarized in a new model of the cell cycle which subdivided the G1 phase into Gla, b, and c. The effects of serum were seen in a balanced salt solution, and were sensitive to several metabolic inhibitors.     

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.     

Histone acetylation and histone synthesis in mouse fibroblasts during quiescence and restimulation into S-phase

Mouse NIH 3T3 fibroblasts were starved by serum depletion and subsequently restimulated by addition of serum. Histone acetylation and histone synthesis were studied from the beginning of starvation to the point where most of the cells were in S-phase utilizing electrophoretic and fluorographic techniques. We found that the major part of histone acetylation and histone synthesis occurs during S-phase but that also in the absence of DNA synthesis there are significant changes in the acetylation and synthesis rates of the core histones which occur during the first 6 hours of serum stimulation of quiescent cells, and between 24 and 48 hours of serum starvation.     

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.     

Synthesis of serum proteins by cultures of chick embryo yolk sac endodermal cells

Endodermal cells were isolated from yolk sacs of 3-day chick embryos and cultured for 6 days in Eagle's minimal essential media plus 10% fetal calf serum. During this period cells rapidly lost their ability to synthesize DNA as judged by [3H]thymidine incorporation into DNA. In spite of this loss of DNA synthesis serum protein synthesis and secretion remained at a constant 45% of total protein synthesis and secretion. This was determined by immunoprecipitation of culture media using antibodies directed against embryonic chick serum proteins. Media were also analyzed for the synthesis and secretion of specific serum proteins using polyacrylamide gel electrophoresis. The relative synthesis and secretion of the individual serum proteins followed that previously observed in ovo with the exception of alpha-globulin-a which became undetectable. When culture media were supplemented with ovalbumin or insulin the relative synthesis and secretion of certin specific serum proteins were altered. However, analysis of these same media samples showed that the total amounts of serum protein synthesis and secretion were unaffected.     

The different roles of serum and calcium in the control of proliferation of BALB/c 3T3 mouse cells.

Proliferatively inactive BALB/c 3T3 mouse cells in dense cultures initiate a growth-division cycle upon exposure to fresh calf serum in a low-calcium (0.01 mM) medium. If these calcium-deprived cells are not supplied with calcium sometime during the first 10 hours after serum stimulation, they will rapidly return to a proliferatively inactive state without initiating DNA synthesis. The prereplicative development of such stimulated calcium-deprived cells appears to stop at an advanced stage, because addition of calcium as late as 10 hours after serum exposure rapidly initiates DNA synthesis, and enables the culture's DNA-synthetic activity subsequently to reach its peak value at the same time as in control cultures.     

Autoantibodies from patients with systemic lupus erythematosus can affect DNA and RNA synthesis in cultured cells

Sera reacting positively for anti-DNA antibodies from systemic lupus erythematosus (SLE) patients were tested for their effect on DNA and RNA synthesis in permeabilized cultured cells and isolated nuclei. The immunoglobulin fraction obtained by ammonium sulfate precipitation of serum was shown to exert considerable influence on DNA and RNA synthesis in cultured cells and nuclei. A component of this antibody population is anti-DNA. These antibodies exert different effects on DNA template activity which is a function of their conformational specificity. Intracellular penetration of autoantibodies as noted in SLE may be one of the reasons for clinical manifestations of disease in these patients.     

Effects of the tumor promoter TPA on the induction of DNA synthesis in normal and RSV-transformed rat fibroblasts

Induction of DNA synthesis by the tumor promoter tetradecanoyl phorbol acetate (TPA) was studied in a line of cultured rat fibroblasts (Rat-1) and their ffRous sarcoma virus-transformed derivative (Rat-1(RSV)). Following serum deprivation for 54 h to achieve quiescene, semiconservative DNA replication was measured by incubation of cells in BrdUrd and FdUrd after serum stimulation in the presence or absence of TPA. Optimal concentrations of TPA (0.1–0.5 μg/ ml) in serum-free medium induced a small increase (10–15%) in the amount of DNA made over a 30-h period in both Rat-1 and Rat-1 (RSV) cells. When Rat-1 cells were stimulated by a 4-h serum pulse, 30% of the DNA was replicated by 30 h. If the serum pulse was follwed by TPA addition, 702% DNA replication wass observed. If the serum pulse was preceded by TPA addition, the onset of DNA synthesis waas delayed by several hous, but stimulation of DNA synthesis occurred. In contrast, the Rat-1 (RSV) cells did not show an increase in DNA synthesis induced by TPA in similar protocols, but the serum-induced onset on DNA synthesis was delayed by several hours in the presence of TPA. Therefore, TPA acts as a co-inducer of DNA synthesis in the Rat-1 but not in the Rat-(RSV) cells. The parent alcohol, phorbol, was inactive in Rat-1 cells, but delayed the onset of DNA synthesis in the Rat(RSV) cells. We conclude that the co-inducing and delaying activities of TPA on DNA synthesis appear to be distinct and to act at different points in the G₁ phase of the cell cycle.     

Reversible alterations in the mitotic cycle of chick embryo cells in various states of growth regulation

Chick embryo cells which have been kept overnight at pH 6.8 in the absence of serum multiply very slowly. Only a small fraction of cells is in the S period at any given time, and the rate of uptake of 2-deoxy-D-glucose is very low. Upon raising the pH to 7.4 and adding serum (“turn-on”) the uptake of 2-deoxy-D-glucose increases immediately; the rate of DNA synthesis increases after a lag of about 4 hours, and represents an increase in the fraction of cells synthesizing DNA. The uptake of 2-deoxy-D-glucose is rapidly returned to its original low rate at any time by again lowering the pH and removing serum (“turn-off”). The synthesis of DNA in the culture remains constant or continues to rise at a markedly reduced rate following the same treatment. Lowering pH or removing serum independently of each other is less efficient at inhibiting the increase in DNA synthesis than the combined treatment but each accomplishes a similar result. Cultures which have been “turnedoff” during the early stages of the rapid increase in DNA synthesis, resume their prior rate of increase immediately if “turned-on” again within 2.5 hours. If the cultures have been “turned-off” for 5.5 hours before restoring the “turn-on,” there is a 2 hour delay before they resume an increased rate of DNA synthesis. The results indicate that chick embryo cells do not become committed to the initiation of DNA synthesis until shortly before, or at the time of the onset of the S period. Up to 96% of the cells in post-confluent cultures growing in conventional medium become labeled upon continuous, prolonged exposure to ³H-thymidine. Seventy-eight percent of the cells in serum-deprived cultures growing at a very low rate become labeled. These and other considerations suggest that the inhibition of cell multiplication by high population density or serum deprivation is caused by a lengthening of the time cells remain in the prereplicative G₁ period rather than by shifting cells into a qualitatively distinct G₀ period. There may, however, be a period common to all cells regardless of growth rate, in which cells are not progressing toward the S period. The length of this variable period would then determine the growth rate of a population of cells.     

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.     

Induction of T-suppressor cells by immunization with allogenic spleen cells in the H-2 system of mice]

The nature of suppressor cells induced by immunization with the allogenic spleen cells and inhibiting the DNA synthesis activation in the mixed lymphocyte culture was studied. Suppressor cells are resistant to mitomycin C and carrageenan. They are not inactivated by the treatment with rabbit anti-B- and anti-Ig- as well as with mouse antibodies (anti-Mls serum) against B lymphocytes in the presence of complement but eliminated by rabbit anti-lymphocyte and anti-T globulins and by mouse anti-theta serum. These T suppressor cells are concentrated in the large lymphocyte fraction in the ficoll gradient. Their blocking of the DNA synthesis activation is evidently non-specific.     

Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60 degrees C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases alpha, beta, gamma and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.     

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.     

Responsiveness of the increase in C-fos mRNA levels depends on the inducer and the cell's past

In this paper we show that in C3H10T1/2 mouse fibroblasts, the inducibility of c-fos mRNA by heat shock or serum addition is strongly dependent on the cell's past. Four hours after a heat shock, a time point where the induced c-fos mRNA has disappeared, c-fos mRNA could not be induced again by a second heat shock. Four hours after serum addition, by which c-fos was induced, a second serum addition also failed to induce c-fos mRNA again. When, however, serum was added 4 hours after heat shock or heat shock was given 4 hours after serum addition, levels of c-fos mRNA could be enhanced again. The induction by serum of c-fos mRNA levels in thermotolerant cells might be related to their increased stimulation of DNA synthesis as compared to control cells.