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.     

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.     

Phenotype modulation in primary cultures of arterial smooth-muscle cells. Dual effect of prostaglandin E1

The effects of prostaglandin E1 (PGE1) on the phenotypic state of enzymatically isolated arterial smooth-muscle cells in primary culture were studied by transmission electron microscopy, thymidine autoradiography, and cell counting. Early in culture (day 0-2), PGE1 stimulated conversion of the cells from contractile (less euchromatic nucleus and cytoplasm dominated by myofilament bundles) to synthetic state (more euchromatic nucleus and cytoplasm dominated by cisternae of rough endoplasmic reticulum and a large Golgi complex). The rate of entrance of the cells into DNA synthesis and mitosis was also increased at this time. Later on (day 3-6), when the majority of the cells had entered synthetic state, PGE1 inhibited DNA synthesis and cellular proliferation. These observations indicate that the effect of prostaglandins on arterial smooth muscle is dual in nature and dependent on the state of differentiation of the cells.     

Epidermal growth factor-induced topoisomerase(s). Intracellular translocation and relation to DNA synthesis

We have used epidermal growth factor (EGF) to investigate the relationship between eukaryotic topoisomerases and DNA synthesis. We found that EGF stimulates topoisomerase activity in human fibroblasts and Swiss/3T3 mouse fibroblasts. The first increase is seen in the cytoplasm, followed by increased activity in the nucleus. The nuclear increases correspond to increases in DNA synthesis. A type II topoisomerase is stimulated as indicated by the ATP dependence of the relaxing reaction and by the formation of catenanes. We have also found that the topoisomerase activity in the cytoplasm is sedimentable indicating that it is either membrane-associated or in a supramolecular complex. The stimulation of topoisomerase activity by EGF may represent a key step in the process by which EGF induces DNA synthesis and cell division.     

The relationship of DNA synthesis to protein accumulation in the cell nucleus

The relationship between DNA synthesis and protein accumulation in cell nucleus and cytoplasm has been investigated by the use of a combination of ultramicrointerferometric and ultramicrospectrophotometric methods. 5-Fluoro-2'-deoxyuridine (FUdR) inhibited DNA synthesis, resulting in inhibition of cell proliferation in G-1 and early S-phase. However, synthesis and accumulation of protein continued in the presence of FUdR, as indicated by a 54% increase in the average dry mass value per individual cell during 18-hour exposure to FUdR; due primarily to protein accumulation in the cytoplasm, the average cytoplasmic dry mass increased by as much as 85%, while the dry mass of the nucleus increased by only 21%. The dry mass values of individual nuclei were well-correlated to the nuclear DNA content throughout the period of exposure to FUdR. In contrast to the continued accumulation of protein in the cytoplasm during inhibition of DNA synthesis, protein accumulation in the nucleus was inhibited. When cells were released from inhibition of DNA synthesis by the addition of 2'-deoxythymidine, the nuclear DNA content and nuclear dry mass increased in near-synchrony, there being some evidence that DNA synthesis was initiated somewhat prior to initiation of increase in nuclear dry mass. Thus, it appears that DNA synthesis (or an increase in nuclear DNA content) is intimately related to the regulation of protein accumulation in the nucleus.     

DNA polymerase activities in growing cells infected with simian virus 40.

Growing CV1 cells were infected with simian virus 40 (SV40), and the levels of DNA polymerases-alpha, -beta, and -gamma were analyzed in the cytoplasm, nuclear Triton wash, and nucleus. In the cytoplasmic fraction, the amount of alpha-, beta-, or gamma-polymerase remained unaltered after SV40 infection. The activity of DNA polymerase-alpha increased five- to sixfold in the nuclear Triton wash and threefold in the nuclei and then remained enhanced only inside the nuclei. That of DNA polymerases-beta and gamma increased mostly in the nuclei after infection. These results suggest that DNA polymerase-alpha could be the major enzyme involved in SV40 DNA replication.     

Phenotype modulation in primary cultures of arterial smooth-muscle cells

Abstract. The effects of prostaglandin E₁ (PGE₁) on the phenotypic state of enzymatically isolated arterial smooth-muscle cells in primary culture were studied by transmission electron microscopy, thymidine autoradiography, and cell counting. Early in culture (day 0–2), PGE₁, stimulated conversion of the cells from contractile (less euchromatic nucleus and cytoplasm dominated by myofilament bundles) to synthetic state (more euchromatic nucleus and cytoplasm dominated by cisternae of rough endoplasmic reticulum and a large Golgi complex). The rate of entrance of the cells into DNA synthesis and mitosis was also increased at this time. Later on (day 3–6), when the majority of the cells had entered synthetic state, PGE₁ inhibited DNA synthesis and cellular proliferation. These observations indicate that the effect of prostaglandins on arterial smooth muscle is dual in nature and dependent on the state of differentiation of the cells.     

Cell-cycle dependent biosynthesis and localization of p53 protein in untransformed human cells

Summary— Localization of p53 in human cultured lymphocytes and in cultured skin fibroblasts was studied by immuno-fluorescent microscopy and post-embedded immunoelectron microscopy using Lowicryl K4M. In quiescent lymphocytes, p53 was found in small amounts in both the cytoplasm and the nucleus. p53 in the nucleus was found associated with the non-chromatin structure. At 24 h or 72 h of PHA stimulation, p53 increased markedly just beneath the plasma membrane and in the nucleus, which stained diffusely with anti-p53. In resting fibroblasts, small amounts of p53 were present in both the cytoplasm and the nucleus. After 16 h of stimulation of confluent-resting fibroblasts by trypsinization and replating, a phase just prior to the initiation of DNA synthesis, p53 slightly increased in both the cytoplasm and the nucleus. Afterwards, p53 was present predominantly in the cytoplasm, closely associated with the cytoskeletal actin filaments. In mitotic cells, p53 was distributed throughout the cytoplasm. When fibroblasts were extracted with saponin, p53 was still associated with the actin filaments, as well as mitochondrial membranes and granular structures of the nuclear matrix. Our data suggest that the initial increase of p53 in cells that enter the cell cycle through G1 first bind to the actin cytoskeleton, and that some of the p53 then move into the nucleus to initiate gene activation and DNA synthesis for cell proliferation. This implies that there is some functionally significant interaction between p53 and actin in the cells.     

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.     

AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.     

Simultaneous photometry of the DNA and silver-grain count in liver cells labelled with 3H-thymidine or 3H-leucine

A modified method was proposed for reflected light simultaneous measurement of DNA content and of the silver grain number in the nucleus or cytoplasm of the same cell. Specimens-smears of isolated liver cells incorporated 3H-thymidine and 3H-leucine were prepared on coverslips and after processing were mounted on the slide glasses with smeared side facing downwards to avoid the influence of grains on DNA content measurements. To decrease the background, label measurements were carried out in polarized light. It was shown that the intensity of 3H-leucine incorporation in hepatocytes increases proportionally with cell ploidy degree.     

IQGAP1 translocates to the nucleus in early S-phase and contributes to cell cycle progression after DNA replication arrest

IQGAP1 is a plasma membrane-associated protein and an important regulator of the actin cytoskeleton, contributing to cell migration, polarity and adhesion. In this study, we demonstrate the nuclear translocation of IQGAP1 using confocal microscopy and cell fractionation. Moreover, we identify a specific pool of IQGAP1 that accumulates in the nucleus during late G1-early S phase of the cell cycle. The nuclear targeting of IQGAP1 was facilitated by N- and C-terminal sequences, and its ability to slowly shuttle between nucleus and cytoplasm/membrane was partly regulated by the CRM1 export receptor. The inhibition of GSK-3β also stimulated nuclear localization of IQGAP1. The dramatic nuclear accumulation of IQGAP1 observed when cells were arrested in G1/S phase suggested a possible role in cell cycle regulation. In support of this, we used immunoprecipitation assays to show that the nuclear pool of IQGAP1 in G1/S-arrested cells associates with DNA replication complex factors RPA32 and PCNA. More important, the siRNA-mediated silencing of IQGAP1 significantly delayed cell cycle progression through S phase and G2/M in NIH 3T3 cells released from thymidine block. Our findings reveal an unexpected regulatory pathway for IQGAP1, and show that a pool of this cytoskeletal regulator translocates into the nucleus in late G1/early S phase to stimulate DNA replication and progression of the cell cycle.     

Reversible nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase upon serum depletion

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E.C. 1.2.1.12) functions as a glycolytic enzyme within the cytoplasm, but beside its metabolic function it is involved in early steps of apoptosis, which trigger the translocation of GAPDH into the nucleus. As apoptosis can be induced by serum withdrawal, which otherwise causes cell cycle arrest, the linkage between serum deprivation, cell cycle and nuclear transport of GAPDH has been investigated. The intracellular distribution of GAPDH was monitored by confocal laser scanning microscopy of either immuno-stained NIH 3T3 fibroblasts or of cells overexpressing GFP-tagged GAPDH. Serum withdrawal led to an accumulation of GAPDH in the nucleus. In contrast to investigations published so far, this nuclear translocation was a reversible process: cytoplasmic location of endogenous GAPDH or of GFP-GAPDH could be recovered upon serum addition to arrested cells and was not inhibited by cycloheximide treatment. In addition, the nuclear import upon serum depletion had no influence neither on the catalytic activity nor on the expression level of GAPDH. The nuclear export of GFP-GAPDH in serum-deprived cells could be stimulated by serum or directly by the growth factors EGF or PDGE The transport process is not regulated via an initiation of cell cycle arrest, as olomoucine, which causes G1-arrest neither stimulated nuclear accumulation nor prevented nuclear export after serum addition to serum-depleted cultures. Moreover, SV40-transformed 3T3 cells transported GAPDH into the nucleus upon serum deprivation, though the expression of the viral large T-antigen enabled growth factor-independent cell proliferation in this cell line. The recruitment of GAPDH to the cytoplasm upon serum stimulation of arrested cells was not impaired by the inhibition of the MAPK signalling pathway with PD 098059. However, further analysis of the growth factor signalling pathway with specific inhibitors revealed that nuclear export was prevented by LY 294002, an inhibitor of the PI-3 kinase. PI3K links the growth factor signalling pathway with cell death via the repression of an apoptotic inducer. Thus, the nuclear accumulation of GAPDH upon growth factor depletion is a reversible process not related directly to cell cycle and likely triggered by survival signals.     

Immunocytochemical evaluation of protein kinase C translocation to the inner nuclear matrix in 3T3 mouse fibroblasts after IGF-I treatment

The complex pathway which links the agonist-cell membrane receptor binding to the response at the genome level involves, among other elements, protein kinase C (PKC). Agonists acting at the cell membrane can affect an autonomous nuclear polyphosphoinositide signaling system inducing an activation of nuclear phosphoinositidase activity and a subsequent translocation of PKC to the nuclear region. The fine localization of PKC has been investigated by means of electron microscopy quantitative immunogold labeling in 3T3 mouse fibroblasts, mitogenically stimulated by IGF-I. The enzyme, which in untreated cells is present in the cytoplasm, except for the organelles, and in the nucleoplasm, after IGF-I treatment is reduced in the cytoplasm and almost doubled in the nucleus. The PKC isoform translocated to the nucleus is the isozyme, which is found not only associated with the nuclear envelope but mainly with the interchromatin domains. By using in situ matrix preparations, PKC appears to be retained at the nuclear matrix level, both at the nuclear lamina and at the inner nuclear matrix, suggesting a direct involvement in the phosphorylation of nuclear proteins which are responsible for the regulation of DNA replication.     

Electrofocusing analysis of DNA polymerase beta in hypophysectomized rat testis

Isoelectric focusing studies indicated that three peaks of DNA polymerase beta (beta-enzyme) activity were focused at isoelectric point of 7.1, 8.2 and 9.1 in testes of sham-operated rats, whereas in testes of hypophysectomized rats only two peaks of the beta-enzyme activity appeared at pH 7.1 and 9.1 and the peak at pH 8.2 was reconstituted by the treatment of gonadotropins and testosterone. In the brain hypophysectomy did not affect the activities of isoelectrically different beta-enzymes. These facts imply that under in vivo conditions the beta-enzymes in testes focused at different isoelectric points are modified with unknown factors and the beta-enzyme focused at pH 8.2 is affected by pituitary hormones.     

DNA Damage and Replication Checkpoints in Fission Yeast Require Nuclear Exclusion of the Cdc25 Phosphatase via 14-3-3 Binding

In fission yeast as well as in higher eukaryotic organisms, entry into mitosis is delayed in cells containing damaged or unreplicated DNA. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. In this study, we generated a mutant of fission yeast Cdc25 that is severely impaired in its ability to bind 14-3-3 proteins. Loss of both the DNA damage and replication checkpoints was observed in fission yeast cells expressing the 14-3-3 binding mutant. These findings indicate that 14-3-3 binding to Cdc25 is required for fission yeast cells to arrest their cell cycle in response to DNA damage and replication blocks. Furthermore, the 14-3-3 binding mutant localized almost exclusively to the nucleus, unlike wild-type Cdc25, which localized to both the cytoplasm and the nucleus. Nuclear accumulation of wild-type Cdc25 was observed when fission yeast cells were treated with leptomycin B, indicating that Cdc25 is actively exported from the nucleus. Nuclear exclusion of wild-type Cdc25 was observed upon overproduction of Rad 24, one of the two fission yeast 14-3-3 proteins, indicating that one function of Rad 24 is to keep Cdc25 out of the nucleus. In support of this conclusion, Rad 24 overproduction did not alter the nuclear location of the 14-3-3 binding mutant. These results indicate that 14-3-3 binding contributes to the nuclear exclusion of Cdc25 and that the nuclear exclusion of Cdc25 is required for a normal checkpoint response to both damaged and unreplicated DNA.     

Stimulated and Non-stimulated Rat Spleen Cells Release Different DNA-Complexes

Isolated rat spleen cells released into the culture medium a complex containing newly-synthesized DNA. Upward sucrose density gradient centrifugation yielded material from non-stimulated cells at a major peak of density 1.03, with minor peaks at 1.04 and 1.055. However, the complex from cells stimulated with phytohaemagglutinin showed a major peak at 1.052, with little material at 1.03. The low densities indicated that the complexes contained a high proportion of lipid, confirmed by the incorporation of ³H-ethanolamine. Data indicate that newly synthesized DNA leaves the nucleus, crosses the cytoplasm, and associates with the cell membrane prior to leaving the cell as a complex with the DNA protected against nuclease activity.     

The relative effects of different types of growth factors on DNA replication, mitosis, and cellular enlargement

It was recently demonstrated that growth in cell size can be dissociated from DNA synthesis and mitosis. 3T3 cells starved to quiescence in low serum concentration can be stimulated to undergo DNA synthesis and one cell division without growing in size (unbalanced growth) (42-44). We report here that in cells stimulated to undergo unbalanced growth, the cell nucleus undergoes balanced growth, i.e., nearly doubles in size prior to mitosis. The reduced ability to grow in cell size under unbalanced growth conditions is thus mainly ascribable to the cytoplasm. Furthermore, the extent to which cells grow in size prior to mitosis is dependent on the serum concentration in the tissue culture medium (44). This data suggests that some macromolecular factor or factors in serum are required for growth in cell size prior to mitosis. We report in this study that epidermal growth factor (EGF) alone exerts a small but significant stimulatory influence on DNA synthesis and mitosis but does not affect cellular enlargement. In contrast, insulin added at supraphysiological concentrations does not stimulate quiescent cells to enter S phase but instead stimulates growth in cell size in the small fraction of dividing cells. Furthermore, cells stimulated to proliferate by EGF could be induced to undergo balanced growth when insulin was added concomitantly. Finally, platelet-derived growth factor (PDGF) stimulates quiescent sparse 3T3 cells to undergo DNA synthesis and mitosis. PDGF also exerts a limited but significant effect on cellular enlargement. However, PDGF alone could not induce a complete balanced growth, i.e., a doubling in cell size prior to mitosis.