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.     

A possible involvement of polyamines in the initiation of DNA synthesis by human WI-38 and mouse BALB/3T3 cells

Changing the medium, or adding fresh serum, induces a large proportion of the proliferatively quiescent cells in confluent monolayers of human WI-38 and mouse BALB/3T3 cells to initiate a growth-division cycle. Exposure at the time of the medium change or serum addition to MGBG (methyl glyoxal bis [guanylhydrazone]), an inhibitor of spermidine and spermine synthesis and function, reduces or stops the subsequent flow of cells into the DNA-synthetic phase, without grossly affecting RNA synthesis. Mediation of MGBG action by an actual or functional shortage of spermidine or spermine (but not putrescine), and consequently an involvement of these polyamines in DNA synthesis, is strongly suggested by the reduction of the inhibitor's effectiveness by a brief (1-hour), early prereplicative exposure of the treated cells to exogenous spermidine and spermine (but not putrescine).     

Calcium-dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor-promoting phorbol ester (PMA).

During the first six passages after their arrival in this laboratory, BALB/c 3T3 mouse cells did not proliferate in serum containing-medium having an ionic calcium concentration of 0.05 mM or less, but by the ninth passage they had become able to multiply in the presence of these lower calcium levels. In low calcium (e.g., 0.02 mM) medium, passage 1-6, cells in sparse cultures were blocked at the Gl/S boundary of their cycle. These blocked cells could be induced to start making DNA within only one hour either by returning the ionic calcium level to a normal range of values (1.25 mM), or by adding 0.05 mug/ml of PMA (12-O-tetradecanoyl-phorbol-13-acetate). PMA probably acted by sensitizing the blocked cells to calcium rather than replacing the ion, because it was ineffective in ionic calcium-free medium. Finally, PMA did not by itself induce proliferation of cells (regardless of the number of passages) which had been proliferatively inactivated by density-dependent factors in confluent cultures. However, PMA did promote DNA synthesis by these cells during their brief transition to the "cycling" state caused by exposure to fresh serum-containing medium.     

An examination of the roles of cyclic nucleotides in the initiation of cell proliferation.

The initiation of proliferative activity in proliferatively quiescent confluent monolayers of BALB/3T3 cell cultures by a fresh medium-serum replacement is independent of early (within minutes) fluctuations in total cellular cyclic AMP or cyclic GMP levels. However, the possibility of a later prereplicative cyclic AMP surge being involved in the initiation of DNA synthesis will be discussed.     

Amine inhibition of serum-induced DNA synthesis in 3T3 and 3T6 cell cultures

Several amines were shown to inhibit growth stimulation in quiescent confluent and sparse cultures of Swiss 3T3 and 3T6 cells by changing for a fresh medium containing 10-20% serum. Proliferation was inhibited by dansylcadaverine (0.1 mM), chloroquine (0.05 mM), 5-methoxytryptamine (0.1 mM), cystamine (0.1 mM), dimethylurea (100 mM), methylurea (100 mM), and in some experiments--by urea (100 mM). The inhibitory activity was not associated with a direct influence of amines on DNA synthesis or thymidine phosphorylation. The findings suggest that amines may influence the process of clustering of growth factor-receptor complexes, or the fusion of plasma membrane and intracellular vesicles containing some components required for cell proliferation.     

Inhibition by 2-deoxy-D-ribose of DNA synthesis and growth in Raji cells

When Raji cells were cultured for 3 days in serum-free medium, addition of 2-deoxy-D-ribose at the start of culture inhibited incorporation of [3H]thymidine and cell division. At deoxyribose concentrations between 1 and 5 mM, viability was 80% or greater after 3 days of culture even though 5 mM deoxyribose inhibited thymidine incorporation 95-99%. Inhibition by deoxyribose could be completely reversed if the culture medium was replaced with fresh medium up to 8 hr after the start of culture. The inhibition was specific for deoxyribose since other monosaccharides had no effect. Inhibition of DNA synthesis did not appear to be due to depletion of essential nutrients in the medium since the percentage inhibition of thymidine incorporation by cells cultured either in suboptimal serum-free media or in media supplemented with 0.025-5% human AB serum was similar. When DNA repair synthesis was measured as hydroxyurea-resistant thymidine incorporation, addition of deoxyribose to Raji cultures caused increased thymidine incorporation. These results, together with data from others, suggest that deoxyribose damages DNA.     

Effect of cell physiological state on infection by rat virus

Infection by rat virus has been studied in cultures of rat embryo cells to evaluate the Margolis-Kilham hypothesis that the virus preferentially infects tissues with actively dividing cells. An enhancement of infection was seen in cultures infected 10 hr after fresh medium was added as compared to infection of stationary cultures (infected before addition of fresh medium). Since addition of fresh medium stimulates deoxyribonucleic acid (DNA) synthesis, the number of cells per culture synthesizing DNA at the time of infection was compared with the proportion of cells which synthesized viral protein. Cells were infected before the medium change and 10 or 24 hr after the medium change and were pulse-labeled with ³H-thymidine at the time virus was added. The cells were allowed to initiate viral protein synthesis before they were fixed and stained with fluorescein-conjugated anti-rat virus serum. Fluorescence microscopy permitted both labels to be counted simultaneouly and showed that the greatest proportion of cells synthesizing viral protein were those which had incorporated ³H-thymidine at the time of infection.     

The mitogenic activity of pp60v-src, the oncogenic protein product of the src gene of avian sarcoma virus, is independent of external serum growth factors

The oncogenic pp60v-src product of ASV (avian sarcoma virus) is shown to be a potent endogenous mitogen, which, unlike mitogens such as PDGF (platelet derived growth factor), is able to stimulate host cell proliferation without the help of other growth factors. Thus, NRK rat cells, infected with a temperature-sensitive ASV mutant which produces an abnormally thermolabile pp60v-src, became proliferatively quiescent at a pp60v-src-inactivating 40 degrees C in medium containing either 0.2% calf serum or no serum at all. Adding PDGF stimulated the quiescent tsASV-NRK cells at 40 degrees C to initiate DNA replication in medium containing 0.2% serum, but not in serum-free medium. By contrast, activating internal pp60v-src by dropping the temperature to a permissive 36 degrees C stimulated these quiescent cells to transit G1, initiate DNA replication and to enter mitosis even in serum-free medium. Thus, relative to PDGF, endogenous pp60v-src behaves as a complete mitogen.     

Time dependency of a critical effect of EGTA on DNA synthesis in cultures of Swiss 3T3 cells.

In a temporal analysis of the mitogenic response to serum, a critical period has been demonstrated just prior to the onset of replicative DNA synthesis during which transient calcium depletion blocks the subsequent entry of the cells into the S phase of the mitotic cycle. Transient washington of monolayer cultures of 3T3 cells with 2.5 mM EGTA between 6 and 8 h after serum-stimulated initiation of DNA synthesis was found to reduce cell-associated calcium levels and to inhibit thymidine incorporation, whereas similar treatment before (1-5 h) and after (8-9 h) had no detectable effect on either of these parameters when estimated after 21 h incubation. The effects during the chelation-sensitive period were reversed by the subsequent addition of fresh serum.     

Catecholamine modulation of embryonic palate mesenchymal cell DNA synthesis

Development of the mammalian embryonic palate depends on the precise temporal and spatial regulation of growth. The factors and mechanisms underlying differential growth patterns in the palate remain elusive. Utilizing quiescent populations of murine embryonic palate mesenchymal (MEPM) cells in vitro, we have begun to investigate hormonal regulation of palatal cell proliferation. MEPM cells in culture were rendered quiescent by 48 hr serum deprivation and were subsequently released from growth arrest by readdition of medium containing 10% (v/v) serum. The progression of cells into S-phase of the cell cycle was monitored by autoradiographic analysis of tritiated thymidine incorporation. Palate mesenchymal cell entry into S-phase was preceded by a 6- to 8-hr prereplicative lag period, after which time DNA synthesis increased and cells reached a maximum labeling index by 22 hr. Addition of 10 microM isoproterenol to cell cultures at the time of release from growth arrest lengthened the prereplicative lag period and delayed cellular entry into S-phase by an additional 2 to 4 hr. The rate of cellular progression through S-phase remained unaltered. The inhibitory effect of isoproterenol on the initiation of MEPM cell DNA synthesis was abolished by pretreatment of cells with propranolol at a concentration (100 microM) that prevented isoproterenol-induced elevations of cAMP. Addition of PGE2 to cell cultures, at a concentration that markedly stimulates cAMP formation, mimicked the inhibitory effect of isoproterenol on cellular progression into S-phase. These findings demonstrate the ability of the beta-adrenergic catecholamine isoproterenol to modulate MEPM cell proliferation in vitro via a receptor-mediated mechanism and raise the possibility that the delayed initiation of DNA synthesis in these cells is a cAMP-dependent phenomenon.     

Calmodulin stimulates DNA synthesis by rat liver cells

Incubation in low (0.02 mM)-calcium medium prevented T51B rat liver cells from initiating DNA synthesis. Raising the calcium concentration in the medium from 0.02 to 1.25 mM caused these arrested cells to initiate DNA synthesis 1–2 hours later. The possibility of this rapid DNA-synthetic response to calcium addition being mediated through Ca-calmodulin complexes was suggested by the following observations: It was blocked by the putative Ca-calmodulin blockers chlorpromazine and trifluoperazine; the trifluoperazine-inhibited cells were stimulated by purified rat calmodulin; and purified rat calmodulin itself (10^?7 to 10^?6 moles/l) mimicked calcium action, unless the already low ionic calcium concentration in the calcium-deficient medium was reduced further by adding the specific calcium chelator EGTA.     

Shaking 3T3 cells: further studies on diffusion boundary effects

Confluent quiescent cultures of 3T3 cells can be stimulated to grow—with or without a medium change—simply by shaking (20 mm excursions, 3 per sec). Shaking without medium change, or with low serum concentration in fresh medium (4%), has the greatest effect on thymidine labeling and mitotic index compared with unshaken cultures, but high serum concentration (20%) stimulates both shaken and unshaken cultures equally. Even without medium change, the majority of cells can be stimulated to incorporate thymidine and to divide over 3 days, though initial shaking for 7 hr is sufficient to initiate growth. We conclude that growth in confluent 3T3 cell cultures is limited by a diffusion barrier in the microenvironment, probably affecting uptake of a serum component. The limitation can be overcome by increased fluid velocity as an alternative to raising the serum concentration.Shaking also causes increased cell movement and a change in cell morphology (which may be partly due to alterations in serum caused by shaking). Despite increased movement in the cell sheet, emigration from the border of a wound is diminished by shaking, suggesting that chemotaxis may be involved.To test whether loss of contact between the migrating cells is essential for growth stimulation at the border of a confluent layer, wounds were made in unshaken cultures in the presence of cytochalasin B, which prevents locomotion of cells and maintains the original topography. Nevertheless, the thymidine labeling index was still increased along the wound boundary, and included cells which were near but not on the edge. This supports the view that short range effects on cell growth at discontinuities in cell populations can be due to modification of a diffusion barrier and not simply alteration in cell contacts.     

Serum-stimulated phosphate uptake and initiation of fibroblast proliferation

Previous studies have shown that initiation of proliferation of density-inhibited fibroblasts by fresh serum is accompanied by a rapid increase in phosphate uptake. This increase might be a key event in the initiation of DNA synthesis. The present studies examined this possibility. Mouse 3T3, secondary chick embryo, or human diploid foreskin cultures were grown to quiescence in medium containing varying levels of serum. When proliferation of the cultures was initiated by addition of fresh serum, the changes in phosphate uptake were inversely related to the final increases in cell number. Additional experiments showed that the change in phosphate uptake following serum addition was determined by the level of phosphate uptake prior to serum addition. Addition of dexamethasone to quiescent 3T3 cultures caused them to proliferate but did not increase phosphate uptake. Similarly, trypsin or insulin stimulated proliferation of quiescent secondary chick embryo cultures, but caused little or no change in phosphate uptake. Quiescent 3T3 cultures switched to medium containing fresh serum and reduced levels of phosphate showed a decrease in both phosphate uptake and intracellular phosphate pool size. Cell proliferation in these cultures, however, was stimulated to the same degree as cultures switched to medium containing fresh serum and the normal amount of phosphate. In addition, quiescent secondary chick embryo cultures switched to medium containing fresh serum and no phosphate showed a decrease in the intracellular phosphate pool size. Thymidine incorporation and final cell number in these cultures, however, was stimulated to the same or higher degree than in cultures switched to medium containing fresh serum and the normal amount of phosphate. These results demonstrate that the rapid increase in phosphate uptake following addition of fresh serum to quiescent fibroblasts is not a necessary event for the initiation of proliferation.     

INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G₁ phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N⁶, O²-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.     

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.     

Regulation by calcium of the proliferation of heart cells from young adult rats

Summary Cells of primary and secondary cultures of rat heart ventricle were grown in a medium supplemented with homologous plasma to avoid the non-specific proliferogenic effects of foreign sera. Specific chelation of calcium from the medium arrested the cells' progression through their cycle at the G1 phase. A permanent or brief restoration of the extracellular calcium level (48 hr later) was followed, after a 5 to 6 hr delay, by a burst of DNA synthesis, and the resumption of mitotic activity. NRCC No. 14976.     

Cycloheximide or puromycin can substitute for PDGF in inducing cellular DNA synthesis in quiescent 3T3 cells

A brief exposure of quiescent (Go) Swiss 3T3 mouse fibroblasts to inhibitors of protein synthesis can replace platelet-derived growth factor in the stimulation of cellular DNA synthesis. When 3T3 cells, after a 6 hr exposure to either cycloheximide or puromycin, are incubated with platelet-poor plasma, a significant percentage of cells enters DNA synthesis. Either inhibition of protein synthesis, or platelet poor plasma by themselves are totally ineffective. A possible mechanism by which inhibitors of protein synthesis may initiate cell cycle progression is through the activation of the c-myc gene.     

Serum stimulation of stationary human glia and glioma cells in culture

Stationary cell cultures can be stimulated to re-enter the cell cycle by giving medium containing fresh serum. The mechanism for this initiation was studied in normal human glia and neoplastic glioma cells in order to demonstrate any differences between normal and neoplastic cells regarding the kinetics of their march through G 1 into the S phase of the cell cycle. Glia cells were found to demand at least 12 h of serum stimulation to enter the cell cycle and were committed to DNA synthesis about 4 h prior to the actual onset of this synthesis. On the other hand, as little as l h of serum stimulation induced DNA synthesis in a fraction of glioma cells and these malignant cells were committed during the largest part of the prereplicative phase. This difference in commitment kinetics could not be explained by differences in cell cycle parameters or medium deficiencies and is considered to reflect a part of the fundamental difference between normal and neoplastic cells as regards their growth control.     

Effect of cultivation conditions on the growth properties of Swiss 3T3 cells

A possibility of using Swiss 3T3 cells, adapted to the growth in the Eagle basal medium and bovine serum, in studies of cell proliferation and quiescent state was shown on the basis of their growth characteristics. Proliferative activity of cultures was estimated by measuring the intensity of DNA synthesis (incorporation of labeled thymidine and flow cytofluorometric analysis), mitotic index and cell number counts. Growth rate and saturation density of the culture were analyzed depending on serum concentration, substrate quality and medium changes both in growth and quiescent states. In spite of repeated medium changes such adapted cells had saturation density within 4.10(4)--7.10(4) cells/cm2, standard for this line. Besides, a distinct inhibition of cell proliferation at confluence or after incubation with low serum (0.5%) and a possibility of the following stimulation of cell divisions by adding a fresh medium containing different concentrations of serum were demonstrated. The increased rate of adipose conversion was detected in resting confluent 3T3 cells cultivated in closed vessels, as compared to cells growing in tissue culture dishes in the CO2 incubator.