DNA synthesis in the nuclei of resting and proliferating cells of the NIH 3T3 line in monokaryons, homo- and heterodikaryons

Serum-deprived (0.5%) resting NIH 3T3 mouse fibroblasts were fused with stimulated cells taken at 2 hour intervals after changing the medium to the one containing 10% serum, and DNA synthesis was investigated in monokaryons, homodikaryons, and heterodikaryous using radioautography with double-labeling technique. The presence of the resting nucleus in the common cytoplasm has an inhibitory effect on the entry of the stimulated nucleus into the S period in the medium containing either 0.5 or 10% serum, but DNA synthesis continues. After a 24 hour stay in the 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 heterodikaryons still persists for at least 2 hours following stimulation. Preincubation of resting cells with cycloheximide for 4 hours abolishes their ability to suppress DNA synthesis in stimulated nuclei. The data suggest that the resting cells produce an endogenous inhibitor of cell proliferation whose formation depends upon the synthesis of protein(s). When stimulated, cell can proliferate only upon decreasing the level of this inhibitor. The obtained results are consistent with the idea of a negative control of cell proliferation.     

DNA synthesis in the nuclei of stimulated NIH 3T3 cells in heterodikaryons obtained by the fusion of these cells with resting cells treated with cycloheximide

Serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts preincubated with cycloheximide (7.5 micrograms/ml) were fused with stimulated cells taken 10 hours after changing the medium to the one containing 10% serum, and DNA synthesis was investigated in nuclei of heterodikaryons, homodikaryons, and monokaryons, using radioautography with double-labeling technique. Preincubation of resting cells with the inhibitor of protein synthesis cycloheximide for 4, 3, 2, but not for 1 or 0.5 hours abolishes their ability to suppress DNA synthesis in stimulated nuclei in heterodikaryons. Three hours after the removal of cycloheximide from the media, the resting cells acquire once again the inhibitory effect towards the entry of stimulated nuclei into the S-period. The data suggest that the resting cells may produce a labile endogenous inhibitor of cell proliferation, and support the idea on the active metabolic processes occurring in the resting cells.     

Hepatocytes in heterokaryons do not retard the nuclei of stimulated fibroblasts entering the S period]

Serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with hepatocytes from intact, regenerating and embryonic mouse livers to elucidate mechanisms of liver cell proliferation, DNA synthesis being investigated in nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes in heterokaryons were found to have no inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period, but on the contrary they were involved in DNA synthesis. In addition, the nuclei in heterokaryons mutually stimulated each other to enter the S-period. In their turn, the resting fibroblasts did not prevent the proliferating hepatocytes from the regenerating and embryonic livers to enter the S-period. Possible reasons of the absence of inhibitory effect of differentiated cells in heterokaryons are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in resting immortalized cells differs from that in differentiated cells where proliferation seems to be stopped without affecting the endogenous inhibitor postulated for the resting and ageing fibroblasts.     

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 effect of cycloheximide on the entry into the S period of the nuclei in heterodikaryons]

Serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts were fused with serum-stimulated (10%) proliferating cells to elucidate mechanisms of entering into S-period operating in the nuclei of the heterokaryons under the effect of cycloheximide--an inhibitor of protein synthesis. Using radioautography DNA synthesis was investigated in mono-, homo- and heterodikaryons. After short (0.5-3.0 h) depressing of protein synthesis, the nuclei of stimulated cells in heterokaryons were found to enter into S-period. Under these conditions no induction of DNA synthesis was found in the nuclei of resting cells in heterodikaryons. In other experiments, resting cells were under the effect of cycloheximide during 2-4 h before the fusion, that led to a great induction of DNA synthesis in the nuclei of these cells in heterodikaryons. The data obtained are consistent with the idea of fibroblast transition to the rest under the action of labile proteins-repressors.     

Hepatocytes in heterokaryons can suppress entry into the S-period of fibroblast nuclei from murine NIH 3T3 cells

Mouse liver regeneration after partial hepatectomy can be considered as a spectacular example of controlled tissue increase. In this study serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with primary hepatocytes isolated from normal (intact) and regenerating adult mouse liver at different times after partial hepatectomy (1-15 days) to elucidate mechanisms of liver cell proliferation cessation at the regeneration end. DNA synthesis was investigated in the nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes isolated from regenerating liver within 1-12 days following operation did not retard the entry of stimulated fibroblast nuclei into the S-period. In contrast, hepatocytes isolated within 15 days after hepatectomy were found to have inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period in heterokaryons. Preincubation of these hepatocytes with cyclocheximide for 2-4 h abolished their ability to suppress DNA synthesis in stimulated fibroblast nuclei in heterokaryons. Possible reasons of inhibitory effect of differentiated cells in heterokaryos are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in regenerating hepatocytes seems to be stopped being affected by the intracellular growth inhibitors, whose formation depends on protein synthesis.     

Inhibitors of Protein Biosynthesis Can Stimulate Proliferation of Mouse Hepatocytes in Vitro

Hepatocyte proliferation in the liver regenerating after partial hepatectomy ceases when the organ is restored, and the mechanism of this phenomenon is still unclear. In the experiments on fusing hepatocytes from the regenerated mouse liver (15 days after partial hepatectomy) with NIH 3T3 mouse fibroblasts, we revealed no DNA synthesis in the nuclei of stimulated fibroblasts in heterokaryons (in the presence of hepatocyte nuclei), whereas DNA synthesis in nonfused cells was undisturbed. In this work, our purpose was to find out whether the suppression of DNA synthesis in heterokaryons could be due to the appearance in hepatocytes of some endogenous factors having an inhibitory effect on proliferation. To this end, hepatocytes from the mouse liver regenerated after partial hepatectomy were treated with cycloheximide for 1–4 h and were then fused with stimulated fibroblasts. Such a short-term treatment of hepatocytes with cycloheximide proved to result in the loss of their ability to inhibit DNA synthesis in the nuclei of stimulated or quiescent fibroblasts in heterokaryons, but hepatocytes proper actively proliferated in the medium with a low serum content (0.2%). When the mice with the liver regenerated after partial hepatectomy were treated with a single sublethal dose of cycloheximide (3 mg/kg), their hepatocytes taken two days after this treatment had no inhibitory effect. Puromycin, another inhibitor of protein synthesis, had the same effect on hepatocytes. These results may be interpreted as evidence that the final stage of liver regeneration after damage is controlled by the factors having a negative effect on cell proliferation.     

DNA synthesis in heterokaryons obtained by the fusion of proliferating cells of the NIH 3T3 line with cells in transition to the resting state

NIH 3T3 mouse fibroblasts cultured in the medium containing 0.5% serum for 2, 4, 8, 24, 48 and 72 hours were fused to cells stimulated for proliferation by replacing the medium with a fresh one containing 10% serum; DNA synthesis was studied in monokaryons, homo- and heterodikaryons using radioautography and double-labelling technique. Cells that were cultured in the medium with a low serum content for 72 hours exerted their inhibitory effect on the entry of stimulated nuclei into the S period in heterodikaryons, whereas cells deprived of serum for shorter periods failed to exert this effect. It thus appears that in cell fusion studies with NIH 3T3 cells, the effects of endogenous growth inhibitor(s) produced by resting cells may be revealed not earlier than by the 3rd day of serum depletion.     

Dependence of the entry of NIH 3T3 cells into a period of DNA synthesis on the duration of their preliminary resting state

Using radioautography and cell fusion technique, we studied cell kinetics and functional properties of NIH 3T3 mouse fibroblasts stimulated to proliferate after being quiescent for 3, 7 and 14 days. The resting state was achieved by cultivating cells in the medium with 0.5% of serum, the stimulation being achieved by replacement of the depleted medium for a fresh one containing 10% of serum. It was found that the longer cells had been kept resting, the longer their prereplicative period lasted after the stimulation, the lesser was the fraction of cells that entered the S-period. Cell-fusion experiments revealed that the ability of the resting nuclei to suppress the onset of DNA synthesis in the nuclei of stimulated cells in heterodikaryons increased as the cells stayed in the resting state before fusion, and that the period of suppression was prolonged. The data are consistent with the idea of cells going into deeper resting states. It may be concluded that the resting cells undergo a gradual development resulting in the changes of their functional properties.     

Heterokaryons in the analysis of genes and gene regulation.

Cytological and chemical analysis of heterokaryons, the immediate product of cell fusion, offer new possibilities for studying the factors responsible for genetic regulation in eukaryotic cells. In comparison with proliferating cell hybrids the heterokaryon state offers the important advantage that a heterokaryon contains two complete genomes since chromosome loss does not occur, but since segregation and recombination are absent, heterokaryons cannot be used for gene mapping in the same way as proliferating cell hybrids. However, if two cell types carrying different genetic defects are fused the analysis can be used for studies of gene complementation. The biological information obtained with heterokaryons has emphasized the role of the cytoplasm in the control of nuclear activity. When a G1 nucleus is brought into contact with the cytoplasm of an S phase cell the G1 nucleus is stimulated to synthesize DNA. If the nucleus is brought into a mitotic cell, the chromatin of the G1 nucleus is forced to condense into prematurely condensed chromosomes. Inactive nuclei such as the dormant chick erythrocyte nucleus will be stimulated to initiate RNA and DNA synthesis when brought into contact with an active cytoplasm by cell fusion. Specific nuclear proteins have been shown to be responsible for this process of reactivation. Other inactive nuclei such as the nuclei of macrophages and spermatozoa have likewise been shown to be reactivated by fusion with active cells. The degree of activation in all of these cases appears to be determined by the state of the active cell. Inactive nuclei are activated to the same level as the active nucleus but seldom beyond this level. If differentiated cells are fused with undifferentiated cells, usually the differentiated character is lost rapidly after fusion. This observation is in agreement with several studies on proliferating cell hybrids indicating some type of negative control of differentiated properties. In heterokaryons obtained by fusion of cells of a similar type of histotypic differentiation usually coexpression of the differentiated markers is observed.     

Reactivation of chick erythrocyte nuclei in young and senescent WI38 cells

The chromatin of the dormant chick nucleus is dispersed in the heterokaryons made by Sendai virus fusion of phase II WI38 cells with chick erythrocyte nuclei. The erythrocyte nucleus resumes RNA synthesis and enters into DNA synthesis with the host nucleus. In the heterokaryons of phase III WI38 cells and chick erythrocytes, the nuclear chromatin is not dispersed and RNA synthesis occurs at a reduced rate. The differences in the physiological state of the young and senescent cells measured by [³H]uridine incorporation into nuclear RNA is reflected in the extent of reactivation of the chick erythrocyte nuclei in the cytoplasm of these cells. The reactivation of the chick nucleus in enucleated fibroblasts parallels the nucleated cells. The results of these studies are interpreted as evidence that there is a specific loss of nuclear function in the senescent cells.     

Hepatocytes from the liver of mice with experimental post-toxic cirrhosis stimulate in heterokaryons DNA synthesis in the nuclei of resting fibroblasts of the NIH 373 line]

Hepatocytes from mouse liver with experimental post-toxic cirrhosis (received by means of 10-12 inhalations with CCl4) were fused with serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts to elucidate mechanisms of liver stroma cells proliferation at cirrhosis. After fusion, nuclei of fibroblasts in such heterokaryons were found to enter into S-period without any exogenous stimulation of cell proliferation (in the medium with low content of serum). The obtained data allow us to suggest that hepatocytes from mouse liver with experimental post-toxic cirrhosis can produce and secrete into the medium (blood) factor (s) capable of stimulating the mesenchymal origin cell proliferation.     

Brief exposures of resting fibroblasts to okadaic acid stimulate DNA synthesis

To study further the factors providing for cellular quiescence, we used okadaic acid (OA) at concentrations (0.1, 1, 10 or 100 nM) inhibiting type 1 and/or type 2A protein phosphatases in mammalian cell cultures. Brief (2 h) exposure of resting (0.2% serum for 72 h) NIH 3T3 mouse fibroblasts to OA with subsequent incubation of cells in a medium with 0.2% serum, stimulated DNA synthesis at all concentrations studied. Maximal stimulation was observed following pre-incubation of resting cells with 10 nM OA. Treatment of cycling cells (10% serum) with OA (2 h pulses at 12 h intervals for 72 h) prevented their exit to the resting state on transfer to a medium with 0.2% serum. Brief exposures of resting cells to OA did not affect the rate of protein synthesis. OA pulses in the late pre-replicative period had no effect on the entry of serum-stimulated cells into the S phase. Cell fusion experiments with resting (serum-deprived) and proliferating (serum-stimulated) NIH 3T3 cells, using radioautography with a double-labelling technique, revealed that pre-incubation of resting cells with OA for 2 h before and after fusion abrogates their ability to suppress the onset of DNA synthesis in the nuclei of proliferating cells in heterodikaryons. The results indicate that protein phosphatases of type 1 and/or 2A may be involved in the growth-arrest machinery that provides for cellular quiescence.     

Regulation of cell proliferation inhibitory and stimulatory factors diffused by 3T3 cultured cells

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density-dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of alpha globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by alpha globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.     

Reactivation of chick erythrocyte nuclei in heterokaryons with temperature-sensitive Chinese hamster cells.

Chinese hamster cell line K12 is temperature-sensitive for the initiation of DNA synthesis. K12 cells synchronized by serum deprivation were collected in early G1(G0). Heterokaryons were formed by fusing chick erythrocytes with serum-starved K12 cells through the use of UV-irradiated Sendai virus. At the permissive temperature (36.5 degrees C), erythrocyte nuclei in heterokaryons enlarged, the chromatin dispersed, and erythrocyte nuclei synthesized DNA at about the same time as the K12 nuclei. At the restrictive temperature (41 degrees C), erythrocyte nuclei enlarged, but neither erythrocyte nor K12 nuclei initiated DNA synthesis. When erythrocyte nuclei were fused with Wg-1A cells, the wild-type parent for ts K12 cells, both kinds of nuclei synthesized DNA at 36.5 degrees C and 41 degrees C. Activation of erythrocyte nuclei was inefficient in heterokaryons incubated in low-serum medium. The results indicate that serum factors and a cellular function defined by the K12 mutation are required for activation of chick erythrocyte nuclear DNA synthesis.     

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.     

DNA synthesis in the heterokaryons of non-dividing differentiated cells and culture cells with various proliferative potentials

Resident peritoneal mouse macrophages (non-dividing differentiated cells) were fused with mouse embryo fibroblasts (cells with a limited lifespan), NIH 3T3 and C3H 10T 1/2 cells ('immortal' cell lines) and SV 3T3 cells (a malignant cell line). DNA synthesis was investigated in the resultant heterokaryons. No inhibitory effect upon the transition of NIH 3T3 and mouse embryo fibroblasts nuclei to the S-phase was observed. C3H 10T 1/2, NIH 3T3 and SV 3T3 cells induced the reactivation of DNA synthesis in the macrophage nuclei in the heterokaryons. At the same time, no replication was detected in the macrophage nuclei after fusion with mouse embryo fibroblasts.     

Effect of azidothymidine on reactivation of DNA synthesis in macrophage nuclei contained in heterokaryons

EIn heterokaryons, DNA synthesis is reactivated in macrophage nuclei only in the case of fusion with immortal cells. Assuming that telomerase is responsible for reactivation, the effect of its inhibitor azidothymidine (AZT) was studied in heterokaryons of mouse resident peritoneal macrophages and immortal 3T3 Swiss cells. AZT suppressed reactivation of DNA synthesis in macrophage nuclei and had no effect on DNA synthesis in 3T3 Swiss cell nuclei, suggesting an altered telomere structure in normal mouse macrophages.     

The Effect of Azidothymidine on Reactivation of DNA Synthesis in Macrophage Nuclei Contained in Heterokaryons

In heterokaryons, DNA synthesis is reactivated in macrophage nuclei only in the case of fusion with immortal cells. Assuming that telomerase is responsible for reactivation, the effect of its inhibitor azidothymidine (AZT) was studied in heterokaryons of mouse resident peritoneal macrophages and immortal 3T3 Swiss cells. AZT suppressed reactivation of DNA synthesis in macrophage nuclei and had no effect on DNA synthesis in 3T3 Swiss cell nuclei, suggesting that telomere structure is impaired in normal mouse macrophages.