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.     

Protein synthesis inhibitors, like growth factors, may render resting 3T3 cells competent for DNA synthesis: a radioautographic and cell fusion study

Serum-deprived (0.1-0.2%) resting NIH 3T3 mouse fibroblasts pre-incubated with cycloheximide (7.5 micrograms/ml), or puromycin (10 micrograms/ml), were fused with stimulated cells taken 10 h after changing the medium to one containing 10% serum, and DNA synthesis was investigated in the nuclei of monokaryons, homodikaryons and heterodikaryons using radioautography with the double-labelling technique. Pre-incubation of resting cells with inhibitors of protein synthesis for 1-4 h abolished their ability to suppress DNA synthesis in stimulated nuclei in heterokaryons. Three hours after the removal of cycloheximide from the medium, the resting cells acquired once again the inhibitory capacity for entry of stimulated nuclei into the S period. This inhibitory influence disappeared also in the case of post-fusion cycloheximide application as well as following an 8-12 h pre-treatment of resting cells with actinomycin D (1 microgram/ml) prior to fusion. Pre-incubation of resting cells for 12 h with PDGF (1 u/ml-1) followed by an 8-48 h incubation in serum-free medium stimulated the onset of DNA synthesis. A brief exposure (45 min) of resting cells to cycloheximide (7.5 micrograms/ml), or puromycin (7.5 micrograms/ml), exerted a similar effect, inducing by itself the entry of cells into the S period. The results support the assumption that acquirement, by resting cells, of competence for DNA replication includes as a necessary step the down-regulation of intracellular growth inhibitors whose formation depends on protein synthesis.     

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.     

Metabolic turnover of proliferation-related nuclear proteins in serum-stimulated Swiss mouse 3T3 cells

Stimulation of quiescent Swiss mouse 3T3 cells either by serum or by pure growth factors induces DNA synthesis after a lag period of about 15 h. Following restimulation by serum or by growth factors there is an overall increase of 2-4-fold in the rate of biosynthesis of nuclear proteins. Two nuclear polypeptides show specific temporal correlations with the transition from quiescence to proliferation. The synthesis of p30 (30 kDa, pI 5.2) is at a maximum within 5 h of restimulation, while the synthesis of p36 (36 kDa, pI 4.25) is first seen at 10-12 h after restimulation. The synthesis of p36 correlates well with the initiation of DNA biosynthesis. The metabolic turnover of both of these proteins has been estimated by pulse-chase and by cycloheximide inhibition experiments. They both have a half-life of 10-15 h and appear to be cell-cycle related.     

Dissociation of RNA synthesis from the calcium requirement for serum-increased ornithine decarboxylase activity in rat glioma cells

When C6-2B rat glioma cells were stimulated with calf serum in the presence of calcium, ornithine decarboxylase activity increased maximally in 6-8 h after an initial 2-3 h lag period wherein RNA synthesis occurred. The increase of ornithine decarboxylase activity in serum-stimulated C6-2B cells was prevented by the calcium chelator EGTA, but EGTA had no effect upon RNA synthesis as judged by [3H]uridine incorporation into RNA. In addition, the calcium requirement for increased ornithine decarboxylase activity was temporally distal to the lag period. EGTA appeared to inhibit the synthesis of ornithine decarboxylase, because the half-life values of ornithine decarboxylase activity were similar (37-47 min) in the presence of EGTA or protein synthesis inhibitors such as cycloheximide or emetine. Also, calcium readdition rapidly reversed EGTA inhibition of ornithine decarboxylase activity by a mechanism which could be blocked by cycloheximide.     

Cytosolic free calcium and DNA synthesis in BALB/c 3T3 cells: aequorin luminescence studies

In order to investigate how growth factors stimulate DNA synthesis, we studied the relationship between early increases in the intracellular concentration of free calcium (Cai) and early mitogenic events (competence) in quiescent BALB/c 3T3 cells. Cai was measured by aequorin luminescence, and DNA synthesis was quantitated by autoradiography. While both serum (2%) and platelet-derived growth factor (2 U/ml) stimulated large increases in Cai without inducing competence, fibroblast growth factor (100-400 ng/ml) produced a large number of competent cells without causing a detectable increase in Cai. Thus, there was no correlation between the magnitude of Cai increase and subsequent competence for DNA synthesis.     

Pteridines are produced during interleukin 2-induced T-cell proliferation and modulate transmission of this signal

Pteridine levels of interleukin 2 (IL-2) receptor+ T-cell populations have been determined by HPLC after iodine oxidation; neopterin was monitored in the culture supernatants by radio-immunoassay. Upon addition of IL-2, cellular levels of biopterin and 6-hydroxymethylpterin rise transiently from 0.02 to 0.9 pmol/10(6) cells, cellular levels of neopterin from 1.5 to 4.1 pmol/10(6) cells. They peak at 8 and 13 h, respectively, after exposure to IL-2. Neopterin is not accumulated in the culture supernatant. DNA synthesis in T cells begins 10-12 h after adding the lymphokine and the portion of cells that undergo S-phase transition gradually increases during the subsequent 10 h. Entry into DNA synthesis phase is markedly accelerated if IL-2 is supplied together with tetrahydrobiopterin (0.8-1.6 X 10(-6) M) and the kinetics of entry into the S-phase transition during the period of 6-20 h become linear. This indicates that tetrahydrobiopterin modulation of IL-2 activity (Ziegler, I. et al. Naturwiss 72 (1985) 330) is an early event occurring during IL-2 signal transmission.     

Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m2) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m2) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair.     

Ionic changes associated with lead stimulation of DNA synthesis in Balb/c3T3 cells

Lead at slightly subtoxic concentrations markedly stimulated the rate of DNA synthesis in cultured animal cells. This stimulation was closely correlated with formation of a precipitate that was adsorbed and taken up by the cells under certain medium conditions. Data suggest that a precipitate-induced perturbation of the surface membrane leads to intracellular changes responsible for stimulation of DNA synthesis. Maximum stimulation of³H-thymidine incorporation by optimum concentrations of lead is delayed about 8 h compared to that in serum stimulation. In cells stimulated significantly by lead, but not in unstimu-lated cells, a reproducible rise of about 13% in intracellular magnesium occurred over a 24 h period, with an 8 h lag in the increase compared to that observed in serum stimulation. In view of the increases in intracellular magnesium consistently associated with and preceding stimulation of DNA synthesis by several different mitogens including serum and insulin, the present time-coordinated positive correlation between magnesium and DNA synthesis provides evidence for the primary involvement of this divalent cation in growth stimulation produced by lead.     

Induction of the nuclear protein ''cyclin'' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis

The effect of serum and growth factors [platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)] on the synthesis of the nuclear protein cyclin and its correlation with DNA synthesis has been studied in quiescent mouse 3T3 cells by means of quantitative two-dimensional gel electrophoresis. Serum must be present in the medium for at least 8-12 h to induce maximal synthesis of cyclin (6- to 7-fold increase compared with quiescent cells). The stimulation of cyclin synthesis is dose-dependent and correlates directly with DNA synthesis. In addition, partially purified PDGF and FGF also induce cyclin and DNA synthesis in a coordinate way. Both growth factors, like serum, exhibit a similar lag phase to induce maximal cyclin (6- to 7-fold) and DNA synthesis (90% of the cells). Pure PDGF at a concentration as low as 10 ng/ml has the same effect as 10% serum. The coordinate induction of cyclin and DNA synthesis can only be observed with growth factors that induce DNA synthesis. These results strengthen the notion that cyclin is an essential component of the events leading to DNA replication.     

Nuclear DNA synthesis is blocked by UV irradiation in Dictyostelium discoideum

We have used a thymidine auxotroph of the simple eukaryote, Dictyostelium discoideum and alkaline sucrose gradients of isolated nuclei to study alterations in DNA synthesis following irradiation of replicating haploid cells with 254 nm UV light. Three responses were characterized using pulse-chase protocols: (1) Lags in DNA synthesis as measured by the amount of label incorporated were 4, 9, and 20 h after 10, 50, and 200 J/m2. (2) The DNA synthesized during a 15-min pulse immediately after irradiation was of lower single strand molecular weight: 7, 3.5, and 3 x 10(6) dalton after 0, 50, and 200 J/m2. (3) The time required for maturation of the nascent DNA to full-sized single strands of about 2 x 10(8) dalton was 45-50 min for unirradiated cells, 3 h after 10 J/m2, and 20 h after 200 J/m2. The DNA of the irradiated cells did not mature uniformly during these delays; instead, a period of no increase in size was followed by a rapid, nearly control rate of maturation. We conclude: (a) at least some UV lesions block elongation of replicons; (b) the elongation of the replicons and their subsequent joining to yield mature high molecular weight DNA occurs after most of the lesions are repaired; (c) the timing of the different aspects of recovery suggest that initiation of replication is also inhibited.     

Requirements for competence and commitment of quiescent 3T3-cells to initiate DNA synthesis after growth stimulation in low serum concentration

Quiescent serum-starved 3T3 cells in sparse cultures can be stimulated to initiate DNA synthesis and undergo one cell division in low serum concentration after brief exposure to alkaline pH [19]. This method of mitogenic stimulation was used to investigate the requirements of low molecular weight components during the first cell cycle after onset of stimulation. It was shown that each of the low molecular constituents in Dulbecco's Modified Eagle Medium (DMEM) could be excluded (one at a time) without influencing the stimulatory response to brief alkaline treatment, with the exception of glutamine, calcium and phosphate ions. The temporal requirements of these factors were studied from onset of stimulation to initiation of DNA synthesis. It was found that phosphate ions were required immediately after the alkaline treatment, but glutamine was not until 6 h after onset of alkaline stimulation.     

Onset of DNA replication in nuclei of proliferating and resting NIH 3T3 fibroblasts following fusion

NIH 3T3 mouse fibroblasts arrested in medium containing 0.5% serum were fused with stimulated cells taken at 2-h intervals after replacing the medium with one containing 10% serum, and DNA synthesis was studied in mono-, homo- and heterokaryons using radioautography with double-labelling technique. The presence of a resting nucleus in a common cytoplasm with a stimulated nucleus from the prereplicative period has an inhibitory effect on the entry of the stimulated nucleus into the S period in medium containing either 0.5 or 10% serum, but ongoing DNA synthesis continues. After a 24-h stay in a common cytoplasm with resting nuclei the stimulated nuclei return into the state of rest. When resting cells are stimulated by 10% serum, their inhibitory effect on stimulated nuclei in heterokaryons still persists, at least for 2 h following stimulation. Preincubation of resting cells with cycloheximide for 4 h abolishes their ability to suppress DNA synthesis in stimulated nuclei.The data suggest that resting cells produce an endogenous inhibitor of cell proliferation, whose formation depends upon the synthesis of protein. When stimulated, the cells can proliferate only after decreasing the level of this inhibitor. The results obtained are consistent with the idea of a negative control of cell proliferation.     

Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells

Addition of the cAMP derivatives butcAMP or 8BrcAMP to quiescent cultures of Swiss 3T3 causes synergistic stimulation of DNAk synthesis with insulin, phorbol esters, vasopressin, epidermal growth factor, or fetal bovine serum (2-5%). In the presence of insulin, 8BrcAMP, and butcAMP stimulate [3H]-thymidine incorporation into acid-precipitable material in a dose-dependent manner. The effect of these agents is specific since 8Br5'AMP, 5'AMP, butyrate, or 8BrcGMP fail to stimulate DNA synthesis under identical experimental conditions. Furthermore, the mitogenic effects of the cAMP derivatives were markedly potentiated by 1-methyl-3-isobutyl xanthine and 4-(3-butoxy-4-methoxy benzyl)-2-imidazolidine, both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. The growth-promoting effects of the cAMP derivatives were demonstrated by [3H]-thymidine incorporation (either by scintillation counting or by autoradiography), by flow cytofluorometric analysis, and by increase in cell number. When quiescent Swiss 3T3 cells were exposed to butcAMP and insulin, DNA synthesis began after a lag of 17h. The result of sequential additions of cAMP derivatives and insulin to quiescent 3T3 cells suggest that these agents must act simultaneously in G0/G1 to stimulate entry into DNA synthesis in these cells. The findings support the proposition that an increase in cellular levels of cAMP (but not cGMP) act sas a mitogenic stimulus for confluent and quiescent Swiss 3T3 cells.     

Subdivision of the G1 phase of human glia cells in culture

Human glia cells blocked post-mitotically by serum deprivation require about 8–12 h of continuous stimulation by growth factors to become committed to DNA synthesis. DNA synthesis begins about 5 h after growth factor withdrawal. The length of time until the S phase began and the length of the apparent commitment period, i.e. the time when cells progressed towards the G1/S transition point even in the absence of growth factors were independent of the nature of the growth factors studied (calf serum, platelet-rich human serum, epidermal growth factor). Epidermal growth factor and calf serum were mutually interchangeable during the pre-commitment period. Increasing cell density reduced the number of cells which entered DNA synthesis, but had no effect on the length of the apparent commitment period or the latent time until DNA synthesis commenced. The requirement for a long exposure to a growth factor may be an important safeguard in normal cells against “accidental” entry into the cell cycle, since malignant glia cells do not show the same requirement.     

Regulation of adherens junction protein expression in growth-activated 3T3 cells and in regenerating liver

The expression of the adherens junction proteins vinculin, alpha-actinin, and talin was compared in serum-stimulated 3T3 cells and in regenerating rat liver following partial hepatectomy. The levels of vinculin RNA and protein synthesis were rapidly and transiently elevated in growth-activated fibroblasts (peaking at 2-3 h) and in regenerating liver (at 4-8 h), preceding the replicative stage. alpha-Actinin expression was also induced, but more slowly (peaking at 6-8 h in 3T3 cells and at 28 h in regenerating liver), and remained elevated when DNA synthesis was proceeding in both systems. The expression of talin RNA was only slightly elevated in 3T3 cells following serum stimulation, and it remained largely unchanged in regenerating liver. The levels of RNA coding for fibronectin and for the beta 1-integrin subunit were transiently and extensively induced during liver regeneration (fibronectin with a peak at 8 h and beta 1-integrin at 12 h). The uvomorulin RNA level, and the expression of the liver-specific genes albumin and transthyretin, decreased in regenerating liver. The results suggest a physiologically significant regulation in the expression of structural components which link the extracellular matrix to the microfilament system in growth-activated fibroblasts and in regenerating liver.     

Uncoupling of RNA and DNA synthesis after plasma stimulation of G0-arrested BALB/C-3T3 cells.

The addition of whole serum to G0-arrested, confluent Balb/c-3T3 cells induces them to progress through G1 and synthesize DNA after a 12-h lag period. Prior to the onset of DNA synthesis, RNA is synthesized and RNA content increases. Serum has been fractionated into two sets of growth factors: a platelet-derived growth factor present in heat-treated (100 degrees C) platelet extracts and platelet-poor plasma. Addition of whole serum, platelet-derived growth factor or platelet-poor plasma induces quiescent cells to increase their cytoplasmic RNA content, but the cells treated with platelet-poor plasma do not synthesize DNA. Messenger RNA content increases within 2 h after stimulation with whole serum or platelet-poor plasma, and after 18 h, mRNA has accumulated to a greater degree than rRNA.     

Intracellular free Ca2+ in the cell cycle in human fibroblasts: transitions between G1 and G0 and progression into S phase.

Intracellular free calcium ([Ca2+]i) has been proposed to play an important part in the regulation of the cell cycle. Although a number of studies have shown that stimulation of quiescent cells with growth factors causes an immediate rise in [Ca2+]i (Rabinovitch et al., 1986; Vincentini and Villereal, 1986; Hesketh et al., 1988; Tucker et al., 1989, Wahl et al., 1990), a causal relationship between the [Ca2+]i transient and the ability of the cells to reenter the cell cycle has not been firmly established. We have found that blocking the mitogen-induced elevation of [Ca2+]i with the cytoplasmic [Ca2+]i buffer dimethyl BAPTA (dmBAPTA) also blocks subsequent entry of cells into S phase. The dose response curves for inhibition of serum stimulation of [Ca2+]i and DNA synthesis by dmBAPTA are virtually identical including an anomalous stimulation observed at low levels of dmBAPTA. Reversal of the [Ca2+]i buffering effect of dmBAPTA by transient exposure of the cells to the Ca2+ ionophore ionomycin also reverses the inhibition of DNA synthesis 20-24 h later. Ionomycin by itself does not stimulate DNA synthesis. These data are consistent with the conclusion that a transient increase in [Ca2+]i occurring shortly after serum stimulation of quiescent fibroblasts is necessary but not sufficient for subsequent entry of the cells into S phase. This study is the first to show a direct relationship between early serum stimulated Cai2+ increase and subsequent DNA synthesis in human cells. It also goes beyond recent studies on BALB/3T3 cells by providing dose response data and demonstrating reversibility, which are strong indications of a cause and effect relationship.