A cytoplasmic activator of DNA replication is involved in signal transduction in antigen-specific T cell lines.

Cytoplasmic extracts prepared from T cell lines undergoing antigen-specific, interleukin-2 (IL-2)-dependent proliferation were tested for their ability to induce DNA synthesis in isolated, quiescent nuclei. A tetanus toxoid (TET)-specific T cell line, established from peripheral blood of a normal human volunteer, was stimulated in the presence of relevant antigen and 1 unit/ml IL-2. Cytoplasmic extracts prepared from these cells were capable of inducing DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated nuclei correlated positively with the degree of proliferation induced in these cells. In contrast, incubation of this T cell line in the absence of antigen failed to induce proliferation and cytoplasmic extracts prepared from these cells induced little to no DNA synthesis in isolated, quiescent nuclei. The factor present in the cytoplasm of T cells stimulated with relevant antigen in the presence of IL-2 is similar, if not identical, to a factor which we have previously demonstrated in cytoplasmic extracts prepared from transformed lymphoblastoid cell lines and from mitogenically stimulated normal human peripheral blood mononuclear cells. This factor, which we have called activator of DNA replication (ADR) is a heat-labile protein, and is inactivated by treatment with protease inhibitors, including aprotinin. The ability of cytoplasmic extracts from T cells undergoing antigen-specific, IL-2-dependent proliferation to induce DNA synthesis in isolated, quiescent nuclei was markedly inhibited in the presence of aprotinin, providing strong evidence that a cytoplasmic activator of DNA replication, ADR, is involved in the signal transduction process for antigen-specific, IL-2-dependent T cell proliferation. ADR may represent a common intracellular mediator of DNA synthesis in activated and transformed lymphocytes.     

Control of DNA replication in a transformed lymphoid cell line: coexistence of activator and inhibitor activities

Proliferating lymphocytes contain an intracellular factor, ADR (activator of DNA replication), which can initiate DNA synthesis in isolated quiescent nuclei. Resting lymphocytes lack ADR activity and contain an intracellular inhibitory factor that suppresses DNA synthesis in normal but not transformed nuclei. In this study we describe a MOLT-4 subline that produces both the activator and inhibitory activities which can be separated by ammonium sulfate fractionation. The inhibitor is heat stable and inhibits ADR-mediated DNA replication in a dose-dependent manner. It does not inhibit DNA polymerase alpha activity. The inhibitor must be present at the initiation of DNA replication to be effective, as it loses most of its effectiveness if it is added after replication has begun. The presence of inhibitory activity in proliferating MOLT-4 cells, taken with the previous observation that inhibitor derived from normal resting cells does not affect DNA synthesis by MOLT-4 nuclei, suggests that failure of a down-regulating signal may play an important role in proliferative disorder.     

Induction of proliferating cell nuclear antigen (PCNA) complex formation in quiescent fibroblasts from a xeroderma pigmentosum patient.

Accumulated evidence indicates that proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and forms tight association with DNA replication sites during DNA replication or DNA repair synthesis. In this study, such PCNA complex formation was investigated by the indirect immunofluorescence method, using both normal human fibroblasts and those derived from a xeroderma pigmentosum group A (XP-A) patient. XP-A fibroblasts in both proliferating and quiescent states did not show any differences from normal fibroblasts in the properties of PCNA-staining in the untreated conditions. The PCNA complex formation was induced in quiescent normal fibroblasts by both ultraviolet light (UV)- and X-irradiation, whereas in XP-A fibroblasts it was induced by X-irradiation, but not by UV-irradiation. However, PCNA complex was induced in quiescent XP-A fibroblasts by UV-irradiation when the cells had previously incorporated 5-bromodeoxyuridine (BrdU). These observations indicate a close correlation of PCNA complex formation and unscheduled DNA synthesis (UDS). Thus, it was concluded that PCNA complex formation was commonly induced in at least three conditions to produce UDS in spite of different types of DNA damages and DNA repair mechanisms.     

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.     

Initiation of lymphocyte DNA synthesis

The initiation of DNA replication in T lymphocytes appears to be regulated by two distinct activities: one associated with proliferation which mediates initiation, and another associated with quiescence which blocks initiation. Activated lymphocytes and proliferating lymphoid cell lines produce an activity, termed ADR, which can initiate DNA replication in isolated, quiescent nuclei. ADR is heat-labile, has protease activity or interacts closely with a protease, and is distinct from the DNA polymerases. ADR activity is absent in quiescent lymphocytes and appears in mitogen-stimulated lymphocytes after IL-2 binding. The generation of active ADR appears to be mediated by phosphorylation of a precursor which is present in resting cells. Nuclei from mitogen-unresponsive lymphocytes fail to initiate DNA replication in response to ADR, of potential importance in the age-related decline of immunity. Quiescent lymphocytes lack ADR and synthesize an ADR-inhibitory activity. The ADR inhibitor is a heat-stable protein which suppresses the initiation of DNA synthesis, but is ineffective at suppressing elongation once DNA strand replication has begun. Nuclei from several neoplastic cell lines fail to respond to the ADR inhibitor, which may play a role in the continuous proliferation of these cells. At least one of these neoplastic cell lines produces both ADR and an inhibitory factor. These findings suggest that the regulation of proliferation is dependent on the balance between activating and inhibitory pathways.     

Changes in the nuclear distribution of cyclin (PCNA) but not its synthesis depend on DNA replication.

Synthesis of cyclin in serum-stimulated quiescent 3T3 cells increases shortly before DNA synthesis after 10 h of stimulation, reaching a maximum after 16 h. Inhibition of DNA synthesis by hydroxyurea does not affect the increase of cyclin following stimulation, as determined by quantitative two-dimensional gel electrophoresis. The levels of cyclin decrease dramatically at the end of the S-phase. Cells kept in the presence of hydroxyurea (G1/S boundary) do not show this decrease in cyclin, indicating that its amounts are regulated by events occurring during the S-phase. Immunofluorescence studies of serum-stimulated quiescent cells in the presence of hydroxyurea, using proliferating cell nuclear antigen (PCNA) autoantibodies, confirm the results obtained by protein analysis. They also reveal that there are dramatic changes in the nuclear distribution of cyclin and that these depend on DNA synthesis or events occurring during the S-phase. Cyclin (PCNA) is no longer detectable at the end of the S-phase. However, pulse-chase experiments indicate that this protein is very stable, suggesting that it possibly interacts with other macromolecules rendering it inaccessible to the antibody. These results strengthen the notion that cyclin is an important component of the events leading to DNA replication and cell division.     

A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.

Geminiviruses are plant DNA viruses that replicate through DNA intermediates in plant nuclei. The viral components required for replication are known, but no host factors have yet been identified. We used immunolocalization to show that the replication proteins of the geminivirus tomato golden mosaic virus (TGMV) are located in nuclei of terminally differentiated cells that have left the cell cycle. In addition, TGMV infection resulted in a significant accumulation of the host DNA synthesis protein proliferating cell nuclear antigen (PCNA). PCNA, an accessory factor for DNA polymerase delta, was not present at detectable levels in healthy differentiated cells. The TGMV replication protein AL1 was sufficient to induce accumulation of PCNA in terminally differentiated cells of transgenic plants. Analysis of the mechanism(s) whereby AL1 induces the accumulation of host replication machinery in quiescent plant cells will provide a unique opportunity to study plant DNA synthesis.     

On the mechanism of erythropoietin-induced differentiation. XII. A cytoplasmic protein mediating induced nuclear RNA synthesis

Erythropoietin, the primary inducer of red blood cell differentiation, has no effect on RNA synthesis by isolated bone marrow nuclei. A cytoplasmic fraction from marrow cells exposed to erythropoietin does, however, stimulate RNA synthesis by such nuclei. This marrow cell cytoplasmic factor (MCF) also stimulates RNA synthesis by liver and kidney nuclei, whereas erythropoietin has no effect on intact kidney or lung cells. MCF appears rapidly in cells after addition of erythropoietin, and its formation does not require protein synthesis. MCF is inactivated by trypsin, but not by ribonuclease. The data suggest that erythropoietin acts on the responsive cells to generate a cytoplasmic protein that mediates the effect of the hormone on nuclear RNA synthesis.     

Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei

Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery.     

Proliferating cell nuclear antigen (PCNA) is expressed in activated rat skeletal muscle satellite cells

Skeletal muscle satellite cells from uninjured muscle of adult animals are generally found to be in a quiescent state, and when cultured, they remain quiescent in vitro for a period of time which is directly related to the age of the donor animal. A technique for studying the activation of satellite cells in primary cultures has been developed and employs proliferating cell nuclear antigen (PCNA) as a marker for entrance into the S phase of the cell cycle. PCNA is a protein involved in DNA replication and is maximally expressed in S phase of the cell cycle. We monitored PCNA expression in satellite cells isolated from young (3 week) and adult (9 month) rats, and our results indicate that satellite cells begin to accumulate PCNA prior to changes in cell number in both age groups. Using ELISA techniques, we demonstrated that addition of an extract of crushed muscle (CME) activated satellite cells and significantly reduced the length of the lag phase in cells from both age groups. Addition of bFGF shortened the lag phase of PCNA synthesis in satellite cells from 3-week-old rats but had no effect on the kinetics of PCNA expression in cells from 9-month-old rats. Based on our experiments, PCNA expression can be used as a marker to follow the entry of satellite cells into the cell cycle in primary mass cultures. © 1993 Wiley-Liss, Inc.     

Molecular cloning of the gene for plant proliferating-cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells

A cDNA library was screened for plant proliferating-cell nuclear antigen (PCNA) from Catharanthus roseus (periwinkle). A lambda gt11 cDNA library was constructed using poly(A)-rich RNA isolated from the cells in the S phase. A cDNA clone for PCNA was isolated by using a rice genomic clone, pCJ-1, which contains PCNA-related gene sequences. The cDNA contains an open reading frame of 804 nucleotides, encoding a protein of 268 amino acids with a molecular mass of 29,765 Da. When conservative substitutions were included, a high degree of similarity (about 85%) was observed between the predicted amino acid sequence of periwinkle PCNA and that of human PCNA. Expression of mRNA for periwinkle PCNA was undetectable or very weak in quiescent cells, such as phosphate-starved cells, auxin-starved cells and cells in the stationary phase. In the synchronous progression of the cell cycle induced by the addition of phosphate or auxin, the active accumulation of periwinkle PCNA mRNA was observed preferentially in the S phase. When an inhibitor of DNA synthesis, aphidicolin, was added to the cells at the G1 phase, an increase in the level of PCNA mRNA was observed. The partial inhibition of protein synthesis at the G1 phase by a protein inhibitor, anisomycin, caused the arrest of cells in the G1 phase. No increase of the level of periwinkle PCNA mRNA was observed in cells arrested at the G1 phase by the inhibition of protein synthesis. These results indicate that the induction of mRNA for periwinkle PCNA occurred independently of the initiation of DNA replication, but that synthesis of certain proteins at the G1 phase was required for the induction of periwinkle PCNA mRNA at the S phase.     

Restoration of proliferating cell nuclear antigen (PCNA) complex formation in xeroderma pigmentosum group A cells following cis-diamminedichloroplatinum (II)-treatment by cell fusion with normal cells.

We examined the role of the factor deficient in xeroderma pigmentosum group A (XP-A) cells in the formation of proliferating cell nuclear antigen (PCNA) complex with DNA in the DNA repair process in human fibroblasts following cis-diamminedichloroplatinum (CDDP)-treatment. Immunofluorescence staining after methanol fixation was used to detect the PCNA complex formation. When quiescent normal cells were PCNA-stained at 3 h after 100 microM CDDP treatment for 1 h, almost all nuclei of the cells showed a punctuated staining pattern. On the other hand, nuclei of XP-A cells were not stained. These results were the same with the findings following 10J/m2 of ultraviolet light (UV)-irradiation. The quantitative analysis of the PCNA immunofluorescence intensity of normal cells revealed that the mean intensity was increased by 4.8 times by the CDDP-treatment and 6.1 times by the UV-irradiation, compared with that of untreated cells. The intensities among nuclei ranged widely in both treatments. In contrast, the mean intensity was not increased in XP-A cells by the same treatments. However, when XP-A cells were fused with normal cells with polyethylene glycol (PEG) treatment, the nuclei of the XP-A cells showed positive PCNA-staining following CDDP-treatment or UV-irradiation in almost all cases. These results suggest that the PCNA complex formation may play a role in the DNA repair process after the step where the factor deficient in XP-A cells is involved following CDDP-treatment as well as following UV-irradiation.     

The relationship between DNA strand-scission and DNA synthesis inhibition in HeLa cells treated with neocarzinostatin.

Neocarzinostatin inhibits DNA synthesis in HeLa S3 cells and induces the rapid limited breakage of cellular DNA. The fragmentation of cellular DNA appears to precede the inhibition of DNA synthesis. Cells treated with drug at 37 degrees C for 10 min and then washed free of drug show similar levels of inhibition of DNA synthesis or cell growth, or of strand-scission of DNA as when cells were not washed. If cells are preincubated with neocarzinostatin at 0 degrees C before washing, the subsequent incubation of 37 degrees C results in no inhibition of DNA synthesis or cell growth, or cutting of DNA. Isolated nuclei or cell lysates derived from neocarzinostatin-treated HeLa S3 cells are inhibited in DNA synthesis but this can be overcome in cell lysates by adding activated DNA. A cytoplasmic fraction from drug-treated cells can stimulate DNA synthesis by nuclei isolated from untreated cells, whereas nuclei from drug-treated cells are not stimulated by the cytoplasmic fraction from untreated cells. By contrast, neocarzinostatin does not inhibit DNA synthesis when incubated with isolated nuclei, but it can be shown that under these conditions the DNA is already degraded and is not further fragmented by the drug. These data suggest that the drug's ability to induce breakage of cellular DNA in HeLa S3 cells is an essential aspect of its inhibition of DNA replication and may be responsible for the cytotoxic and growth-inhibiting actions of neocarzinostatin.     

Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis

UV irradiation of quiescent human fibroblasts immediately triggers the appearance of the nuclear protein cyclin/proliferating cell nuclear antigen (PCNA) as detected by indirect immunofluorescent staining after methanol fixation. This was found to be independent of new synthesis of cyclin/PCNA by two-dimensional gel analysis and cycloheximide treatment. The intensity of the immunofluorescent staining of cyclin/PCNA observed in UV-irradiated cells corresponded with the UV dose used and with the DNA repair synthesis detected by autoradiography. The nuclear staining remains as long as DNA repair activity is detected in the cells. By extracting the UV-irradiated quiescent cells with Triton X-100 and fixing with formaldehyde, it was possible to demonstrate by indirect immunofluorescence rapid changes in the cyclin/PCNA population after irradiation, a small proportion (5-10%) of which is tightly associated to the nucleus as determined by high salt extraction. By incubating at low temperature and depleting the ATP pools of the cells before UV irradiation, we have demonstrated that the changes in cyclin/PCNA distribution observed involve at least two different nuclear associations.     

Contact-inhibition-induced quiescent state is marked by intense nuclear expression of statin

Statin, a nuclear protein of 57,000 daltons, is found in in vitro aged, nonproliferating human fibroblasts but not in their young, replicating counterparts or transformed derivatives; it is also found in the nuclei of young fibroblasts when their growth is arrested but rapidly disappears from the cells once the restriction to growth is removed. We reported earlier that as cells leave the quiescent state, the loss of statin from the nucleus precedes the initiation of DNA synthesis; here we report that in a confluent culture, as cells leave the traverse of the replicative cycle and assume the quiescent phenotype, statin is not expressed maximally until total contact inhibition of growth is achieved. This full manifestation of statin occurs in monolayer culture with cells forming the typical swirling pattern and fibronectin organized into large intercellular cables. The late expression of statin in cells approaching the quiescent state is also verified biochemically by immunoblotting assays. The present results, taken together with those reported earlier, indicate that the nuclear appearance of statin occurs only after the complete cessation of DNA synthesis and that the full manifestation of this protein can be used as a marker for the G0 quiescent state.     

Individual Nuclei Differ in Their Sensitivity to the Cytoplasmic Inducers of DNA Synthesis: Implications for the Origin of Cell Cycle Variability

Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesisasynchronouslyin cell-free extracts ofXenopuseggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they doin vivo,with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G₁variability in living cells is presented.     

Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle

Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.     

Nuclear accumulation of a heat-shock 70-like protein during herpes simplex virus replication

A monoclonal antibody defines an antigen, p68, related to hsp70, which is located in nuclei of uninfected exponential cells. Nuclear p68 is released by DNase but not RNase treatment suggesting an association with DNA. Lytic productive infection of confluent quiescent BHK 21 cells with herpes simplex virus type-2 causes p68 to accumulate in nuclei. The effect is specific for HSV-2, and does not occur in HSV-1 infected cells. Maximum nuclear accumulation of p68 requires virus DNA synthesis although a significant accumulation occurs in the absence of such synthesis. It is suggested that the nuclear accumulation of p68 is an aspect of a cellular stress response to lytic infection with HSV-2.Imperial Cancer Research Fund, Tumour Immunology Unit.     

Intracellular localization and metabolism of DNA polymerase α in human cells visualized with monoclonal antibody

Indirect immunofluorescence microscopy with monoclonal antibody against DNA polymerase α revealed the intranuclear localization of DNA polymerase α in G1, S, and G2 phases of transformed human cells, and dispersed cytoplasmic distribution during mitosis. In the quiescent, G0 phase of normal human skin fibroblasts or lymphocytes, the α-enzyme was barely detectable by either immunofluorescence or enzyme activity. By exposing cells to proliferation stimuli, however, DNA polymerase a appeared in the nuclei just prior to onset of DNA synthesis, increased rapidly during S phase, reached the maximum level at late S and G2 phases, and was then redistributed to the daughter cells through mitosis. It was also found that the increase in the amount of DNA polymerase a by proliferation stimuli was not affected by inhibition of DNA synthesis with aphidicolin or hydroxyurea.     

p21(CIP1) Controls proliferating cell nuclear antigen level in adult cardiomyocytes

Cell cycle withdrawal associated with terminal differentiation is responsible for the incapability of many organs to regenerate after injury. Here, we employed a cell-free system to analyze the molecular mechanisms underlying cell cycle arrest in cardiomyocytes. In this assay, incubation of S phase nuclei mixed with cytoplasmic extract of S phase cells and adult primary cardiomyocytes results in a dramatic reduction of proliferating cell nuclear antigen (PCNA) protein levels. This effect was blocked by the proteasome inhibitors MG132 and lactacystin, whereas actinomycin D and cycloheximide had no effect. Immunodepletion and addback experiments revealed that the effect of cardiomyocyte extract on PCNA protein levels is maintained by p21 but not p27. In serum-stimulated cardiomyocytes PCNA expression was reconstituted, whereas the protein level of p21 but not that of p27 was reduced. Cytoplasmic extract of serum-stimulated cardiomyocytes did not influence the PCNA protein level in S phase nuclei. Moreover, the hypertrophic effect of serum stimulation was blocked by ectopic expression of p21 and the PCNA protein level was found to be upregulated in adult cardiomyocytes derived from p21 knockout mice. Our data provide evidence that p21 regulates the PCNA protein level in adult cardiomyocytes, which has implications for cardiomyocyte growth control.