Differential regulation of DNA synthesis in heterokaryons between chicken erythrocytes and culture cells with various proliferative potentials

The regulation of DNA synthesis in heterokaryons between chicken erythrocytes and culture cells of various proliferative potential was studied. The following regularities were found: 1) Both immortalized and non-immortalized cells can efficiently reactivate DNA synthesis in erythrocyte nuclei. 2) Erythrocytes drastically inhibit the entry of non-malignant culture cell nuclei into the S-period, not acting upon DNA synthesis. 3) The inhibitory action is characteristic of erythrocytes from different stages of chicken ontogenesis (from 5-day-old embryos to the adult bird). 4) Malignant cells are completely refractory to the inhibitory action of erythrocytes. The ability of erythrocytes to inhibit the onset of replication in heterokaryons may be connected with the mechanisms of maintaining these terminally differentiated cells in a non-proliferating state.     

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.     

Effect of cell density on the inhibition of tumor cell attachment and nucleic acid synthesis by selenite

The effect of cell density on the sensitivity of tumor celles to selenite has been examined. The inhibitory effect of selenite on cellular DNA and RNA synthesis was significantly greater in higher density cultures of HeLa cells and A2780 ovarian tumor cells. High-density cells were also more sensitive to the inhibitory effect of selenite on cell attachment. This difference could not be accounted for by a higher intracellular level of glutathione, since there was no significant difference between the cells at high or low density. The high-density cells were found to take up more selenium per cell during the exposure period; the resulting higher level of intracellular Se could explain their greater sensitivity to selenite. This hypothesis is supported by the observation that DNA synthesis in nuclei isolated from high-density cells did not exhibit higher sensitivity to inhibition by selenite than synthesis in nuclei isolated from low-density cells.     

Positive and negative regulation of replication in hybrid cells]

DNA replication blockage in various differentiated cells was investigated on the model of heterokaryons. Two distinct types of DNA synthesis regulation in heterokaryons "differentiated cell + proliferating cell" were revealed: I. Neutrophils and nucleated erythrocytes efficiently prevented the entry of non-malignant proliferating cells nuclei into the S-period but usually failed to substantially inhibit the replication in malignant cells nuclei. Both "mortal" and immortalized proliferating cells activated the DNA synthesis in neutrophil and chicken erythrocyte nuclei. II. Macrophages did not influence the DNA synthesis in the nuclei of non-malignant cells in heterokaryons but drastically inhibited that in the nuclei of malignant cells. Only immortalized cells reactivated DNA synthesis in the nuclei of macrophages. These data show that the mechanisms maintaining differentiated cells in non-proliferating state are not uniform. Nucleated erythrocytes were shown to suppress the duplication of centrioles in partner cells. The possibility of the blockage of DNA replication upon the fusion of two proliferating cells (fibroblast + leukemia cell) was demonstrated for the first time in the present work. The influence of various oncogenes upon the regulation of DNA synthesis in heterokaryons was investigated in detail. New modifications of the methods of cell fusion, enucleation and heterokaryon identification were proposed.     

Inhibition of DNA synthesis in permeabilized L cells by novobiocin

Novobiocin was equipotent in inhibiting DNA and RNA synthesis in cultured mouse L cells. It also suppressed in vitro DNA and RNA synthesis in permeabilized L cells and nuclei; 50 percent inhibition of DNA and RNA synthesis was obtained by 1 mM and 20 mM novobiocin, respectively. ATP antagonized the effect of novobiocin. Nalidixic acid had a weak inhibitory effect on in vitro DNA synthesis; 10 mM nalidixic acid showed 60 percent inhibition. ATP did not antagonize nalidixic acid. The inhibitory effect of novobiocin exceeded that of aphidicolin. These findings suggest a participation of a gyrase- and/or type II topoisomerase-like enzyme in the DNA replication machinery in L cells.     

Stimulation of DNA synthesis in senescent human cells following incubation with plasma membranes

DNA synthesis and mitosis were increased in mitogen-stimulated senescent WI-38 cells following incubation with plasma membranes prepared from young or senescent WI-38 cells, A431 cells, 3T3 cells, or NR6 cells. The percentage of [3H]thymidine-labeled nuclei in senescent cultures was two- to fivefold greater than that seen in controls in which cells were incubated in the absence of membranes or in the presence of boiled membranes. The effect was trypsin sensitive, suggesting that a protein moiety is necessary for stimulation of DNA synthesis. As the culture age increased, basal levels of DNA synthesis, as well as maximal stimulation of DNA synthesis following incubation with plasma membranes, decreased. These observations are consistent with the hypothesis that different subpopulations exist in senescing cultures and suggest a complex pattern of inhibitory and stimulatory regulation of cell proliferation.     

Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production.

Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Suppression of DNA synthesis in normal, but not neoplastic, nuclei by cytoplasmic extracts from resting cells

We have previously shown that a heat-stable protein in cytoplasmic extracts from human quiescent peripheral blood lymphocytes (PBL) is capable of inhibiting the induction of DNA synthesis in isolated resting nuclei. We now report that these cytoplasmic extracts are also capable of suppressing DNA synthetic activity in replicative nuclei isolated from mitogen-activated PBL. PBL extracts had little or no inhibitory effect, however, on replicative nuclei derived from several transformed lymphoblastoid cell lines. These results suggest that the growth of normal lymphocytes may be negatively controlled by cytoplasmic inhibitory factors. Furthermore, the relative resistance of tumor cell nuclei to these inhibitory signals provides a possible explanation for the loss of growth control in neoplastic cells.     

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.     

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.     

Oxidative Stress Induces a Form of Programmed Cell Death with Characteristics of Both Apoptosis and Necrosis in Neuronal Cells

Abstract: Oxidative stress is implicated in a number of neurological disorders including stroke, Parkinson's disease, and Alzheimer's disease. To study the effects of oxidative stress on neuronal cells, we have used an immortalized mouse hippocampal cell line (HT-22) that is particularly sensitive to glutamate. In these cells, glutamate competes for cystine uptake, leading to a reduction in glutathione and, ultimately, cell death. As it has been reported that protein kinase C activation inhibits glutamate toxicity in these cells and is also associated with the inhibition of apoptosis in other cell types, we asked if glutamate toxicity was via apoptosis. Morphologically, glutamate-treated cells underwent plasma membrane blebbing and cell shrinkage, but no DNA fragmentation was observed. At the ultrastructural level, there was damage to mitochondria and other organelles although the nuclei remained intact. Protein and RNA synthesis inhibitors as well as certain protease inhibitors protected the cells from glutamate toxicity. Both the macromolecular synthesis inhibitors and the protease inhibitors had to be added relatively soon after the addition of glutamate, suggesting that protein synthesis and protease activation are early and distinct steps in the cell death pathway. Thus, the oxidative stress brought about by treatment with glutamate initiates a series of events that lead to a form of cell death distinct from either necrosis or apoptosis.     

Telomere elongation in immortal human cells without detectable telomerase activity.

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.     

Relationship between contact inhibition and intranuclear S100C of normal human fibroblasts

Many lines of evidence indicate that neoplastic transformation of cells occurs by a multistep process. For neoplastic transformation of normal human cells, they must be first immortalized and then be converted into neoplastic cells. It is well known that the immortalization is a critical step for the neoplastic transformation of cells and that the immortal phenotype is recessive. Thus, we investigated proteins downregulated in immortalized cells by two-dimensional gel electrophoresis. As a result, S100C, a Ca(2+)-binding protein, was dramatically downregulated in immortalized human fibroblasts compared with their normal counterparts. When the cells reached confluence, S100C was phosphorylated on threonine 10. Then the phosphorylated S100C moved to and accumulated in the nuclei of normal cells, whereas in immortalized cells it was not phosphorylated and remained in the cytoplasm. Microinjection of the anti-S100C antibody into normal confluent quiescent cells induced DNA synthesis. Furthermore, when exogenous S100C was compelled to localize in the nuclei of HeLa cells, their DNA synthesis was remarkably inhibited with increase in cyclin-dependent kinase inhibitors such as p16(Ink4a) and p21(Waf1). These data indicate the possible involvement of nuclear S100C in the contact inhibition of cell growth.     

Arginase as one of the inhibitory principles in the density-dependent as well as plasma membrane-mediated inhibition of liver cell growth in vitro

Liver cells isolated from the adult rat livers under mild conditions were preincubated for 1 day with Williams medium E (WE) containing serum, dexamethasone and insulin, and then the cells (monolayered) were incubated for 2-3 days with WE (1 ml) containing only insulin to measure DNA synthesis and/or mitosis. DNA synthesis of cultured liver cells was dependent on cell densities within a region from 0.1 X 10(6) to 1.0 X 10(6) nuclei/dish (Falcon, diameter 35 mm). The addition of EGF from the beginning of preincubation stimulated DNA synthesis (or replication) as well as cell proliferation in vitro, but the density-dependent inhibition of DNA synthesis was observed similarly in the presence of EGF. In contrast to the low and high density cultures, DNA synthesis in the intermediary density cultures was enhanced by enlarging the medium volume or by adding ornithine (arginase inhibitor). DNA synthesis in low density cultures was inhibited by liver plasma membranes in a concentration-dependent fashion. The inhibition of DNA synthesis by liver plasma membranes in low concentrations (less than 30 micrograms protein/ml) was reduced by adding either extra arginine or ornithine. DNA synthesis of cultured liver cells (low density) was inhibited by replacing arginine in WE with equimolar ornithine and urea or by adding a commercial arginase (bovine liver). These, together with earlier findings indicating the presence of arginase in liver plasma membranes (outer leaflet), seem to support the idea that arginase may be involved in density-dependent as well as plasma membrane-mediated inhibition of DNA synthesis of cultured liver cells. However, this does not exclude possible involvement of other inhibitory principle(s), such as direct cell-to-cell or cell-to-plasma membrane interactions, especially in higher cell densities or larger plasma membrane concentrations.     

Spontaneous immortalisation of Schwann cells in culture: short-term cultured Schwann cells secrete growth inhibitory activity.

In the developing peripheral nerve, Schwann cells proliferate rapidly and then become quiescent, an essential step in control of Schwann cell differentiation. Cell proliferation is controlled by growth factors that can exert positive or inhibitory influences on DNA synthesis. It has been well established that neonatal Schwann cells divide very slowly in culture when separated from neurons but here we show that when culture was continued for several months some cells began to proliferate rapidly and non-clonal lines of immortalised Schwann cells were established which could be passaged for over two years. These cells had a similar molecular phenotype to short-term cultured Schwann cells, except that they expressed intracellular and cell surface fibronectin. The difference in proliferation rates between short- and long-term cultured Schwann cells appeared to be due in part to the secretion by short-term cultured Schwann cells of growth inhibitory activity since DNA synthesis of long-term, immortalised Schwann cells was inhibited by conditioned medium from short-term cultures. This conditioned medium also inhibited DNA synthesis in short-term Schwann cells stimulated to divide by glial growth factor or elevation of intracellular cAMP. The growth inhibitory activity was not detected in the medium of long-term immortalised Schwann cells, epineurial fibroblasts, a Schwannoma (33B), astrocytes or a fibroblast-like cell-line (3T3) and it did not inhibit serum-induced DNA synthesis in epineurial fibroblasts, 33B cells or 3T3 cells. The activity was apparently distinct from transforming growth factor-beta, activin, IL6, epidermal growth factor, atrial natriuretic peptide and gamma-interferon and was heat and acid stable, resistant to collagenase and destroyed by trypsin treatment. We raise the possibility that loss of an inhibitory autocrine loop may contribute to the rapid proliferation of long-term cultured Schwann cells and that an autocrine growth inhibitor may have a role in the cessation of Schwann cell division that precedes differentiation in peripheral nerve development.     

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.     

The nature of a proliferation block in differentiated cells with heterokaryons as a model: various types of absence of proliferation in cells in terminal differentiation

Heterokaryons obtained by fusion of proliferating and terminally differentiated cells were studied. The data obtained suggest that mechanisms of proliferation arrest are different in macrophages on one hand and nucleate erythrocytes and polymorph leukocytes on the other. Macrophages appeared to be devoid of factors preventing replication in nontransformed and spontaneously immortalized cells. Inhibition of proliferation was probably due to certain modifications of macrophage genome which arise during differentiation and can be compensated by the effect of "immortalizing" oncogenes. On the contrary, nucleate erythrocytes and polymorphs evidently contain some factors mediating negative control of proliferation. For reactivation of DNA synthesis in these cell types after fusion with other cells the latter did not have to be immortalized. After cell fusion macrophages specifically inhibit DNA synthesis in cells containing active oncogenes.