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.     

Cell proliferation and cell cycle dependent changes in the methylthioadenosine phosphorylase activity in mammalian cells

The objective of this study is to investigate the activity of methylthioadenosine phosphorylase (MTA-Pase) in mammalian cells stimulated by serum to proliferate and during their cell cycle. A direct correlation between growth rate and MTA-Pase activity in chinese hamster ovary (CHO) cells was observed. High MTA-Pase activity was observed during the exponential growth phase followed by a low enzyme activity during plateau phase of growth. To understand whether the fluctuations in the enzyme activity was cell cycle dependent, initially the activity of MTA-Pase was studied in plateau phase (G0) CHO cells as they synchronously go into S phase upon plating in fresh medium. The MTA-Pase activity in G0 cells before initiation of growth was 10.3 n.mol/mg protein/30'. A peak activity of 16.0 n.mol/mg/30 min was found at 12 hr after stimulation of proliferation by serum. These results indicate a peak MTA-Pase activity between 10-12 hr after stimulation of proliferation coinciding with the initiation of DNA synthesis. The activity of the enzyme slowly decreased as the cells completed their DNA synthesis. To understand whether these fluctuations are cell cycle specific, HeLa cells were synchronized in different phases and MTA-Pase activity was studied. The specific activities of the enzyme were 2.76, 2.99, 3.97, 3.28 and 3.65 n.moles/mg/30 min. in mitosis, early G1, late G1, S and G2 phases of the cell cycle respectively. These results indicate that MTA-Pase activity peaks in late G1 phase before the initiation of DNA synthesis, similar to the polyamine biosynthetic enzymes and might play a role in the initiation of DNA synthesis by salvage of adenine into nucleotide pools.     

Human cyclin A is required for mitosis until mid prophase.

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21(Cip1/Waf1/Sdi1) (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.     

Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner

The synchronization effects of the plant amino acid mimosine on proliferating higher eukaryotic cells are still controversial. Here, I show that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks. The DNA content of nuclei isolated from mimosine-treated cells was determined by flow cytometry. The presence or absence of DNA replication forks in these isolated nuclei was then detected by DNA replication run-on assays in vitro. Treatment of asynchronously proliferating HeLa or EJ30 cells for 24 h with 0.5 mM mimosine resulted in a population synchronized in late G1 phase. S phase entry was inhibited by 0.5 mM mimosine in cells released from a block in mitosis or from quiescence. When added to early S phase cells, 0.5 mM mimosine did not prevent S phase transit, but delayed progression through late stages of S phase after a lag of 4 h, eventually resulting in a G1 phase population by preventing entry into the subsequent S phase. In contrast, lower concentrations of mimosine (0.1-0.2 mM) failed to prevent S phase entry, resulting in cells containing active DNA replication foci. The G1 phase arrest by 0.5 mM mimosine was reversible upon mimosine withdrawal. This synchronization protocol using 0.5 mM mimosine can be exploited for studying the initiation of human DNA replication in vitro.     

Correlation between cell cycle duration and RNA content.

The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen-stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high- and low-RNA, 25 percentile subpopulations, respectively. Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5-fluorodeoxyuridine showed extremely high intercellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA-, six to nine hours for cells with moderate RNA- and up to 27 hours for cells with minimal RNA-content. The data suggest that the rate of progression through the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.     

Towards a further understanding of the growth-inhibiting action of oxygen deficiency. Evaluation of the effect of antimycin on proliferating Ehrlich ascites tumour cells

The effect of 1 microM antimycin on the proliferative properties, metabolism and basic cell composition of Ehrlich ascites tumour cells cultured in the second in vitro passage was studied. Continuous drug exposure of asynchronous cells caused rapid cessation of cell growth, characterized by the cell number and DNA, RNA and protein content of cultures. Cells cease to consume oxygen and enhance their glycolytic activity. Uptake of labelled thymidine into acid-insoluble material was far below that of the controls, whereas incorporation of labelled uridine exceeded that of controls, as was also observed with other inhibitors of the respiratory chain (sodium cyanide, 2-thenoyltrifluoroacetone, or anaerobiosis). The influence of antimycin on cells at different stages of the cell cycle was tested using cells enriched in either G1, S or G2 phase by centrifugal elutriation. DNA histograms (flow cytometry) and pulse-labelling index curves gave detailed insight into cell-cycle progression of antimycin-treated cells: G1 and early S cells remained stationary; G2 cells still passed from G2 into mitosis to remain subsequently in a non-growing state in G1; S cells were either slowed or halted. Supplementation of antimycin-containing cultures with exogenous pyrimidine nucleosides stimulated reprogression of G1 cells without changing their ATP content. The results of the current experiments are interpreted as supporting the concept that growth cessation of G1 cells under respiratory insufficiency is not predominantly caused by impairment of respiratory phosphorylation but may be the consequence of a lack of precursors for DNA and RNA synthesis.     

Cytoplasmic regulation of two G1-specific temperature-sensitive functions

tsAF8 and ts13 cells are temperature-sensitive (ts) mutants of BHK cells that specifically arrest, at nonpermissive temperature, in the G1 phase of the cell cycle. These two mutants can complement each other. Both cell lines can be made quiescent by serum deprivation (G0). When subsequently stimulated by serum, they can enter S phase at 34 degrees C but not at 39.5 degrees-40.6 degrees C. We have used these mutants to determine whether the nucleus is needed during the G0 leads to S transition for the expression of the G1 ts functions. For this purpose, we fused cytoplasts of G0-tsAF8 with whole ts13 cells in G0, and cytoplasts of G0-ts13 with whole tsAF8 cells in G0. Serum stimulation at the nonpermissive temperature induced DNA synthesis in both types of such fusion products. No DNA synthesis was induced by serum stimulation at the nonpermissive temperature in fusion products constructed between either G0-tsAF8 cytoplasts and whole G0-tsAF8 cells or G0-ts13 cytoplasts and whole G0-ts13 cells. These results demonstrate that the information for these two ts functions, which are required for entry of serum-stimulated cells into the S phase, are already present in the cytoplasm of G0 cells--that is, before serum stimulation commits them to the transition from the nonproliferating to the proliferating state.     

The effect of heat and radiation on the initiation and elongation processes of DNA synthesis

The pH step alkaline elution and alkaline sucrose gradient techniques were utilized to evaluate alterations in DNA replication (initiation and elongation) induced by heat and low dose X-irradiation is synchronized Chinese hamster ovary cells. The initiation and elongation process of DNA synthesis were radioresistant at the G1/S boundary (4 hours after mitosis) while in mid S phase (9 hours after mitosis) DNA initiation and elongation were sensitive to X-irradiation. The initiation and elongation processes of DNA synthesis which were radiation resistant at the G1/S boundary could be inhibited by a hyperthermia treatment (43 degrees C for 1 hour beginning at 4 hours after mitosis). The impairment of initiation in the heated cells was maintained through late S phase while that of elongation was reversible as judged by full recovery at 15 hours after mitosis. These data suggest that the known synergistic lethality of heat and radiation may be mediated by an impairment of initiation of DNA synthesis.     

Migration of newly synthesized RNA during mitosis : III. Nuclear RNA in the cytoplasm of metaphase cells

It was shown by autoradiography in previous papers that RNA which is synthesized before mitosis and located in the nuclei, enters the cytoplasm at the onset of mitosis and returns to the nuclei of the daughter cells after mitosis. In order to study thenature of this migrating RNA we performed a sedimentation analysis of RNA isolated from the cytoplasm and chromosomes (nuclei) of metaphase and interphase cells in the synchronized culture of the Chinese hamster. Whereas the cytoplasm of interphase cells is found to contain RNA with sedimentation constants not higher than 28S, the cytoplasm of metaphase cells includes precursors of ribosomal and messenger RNA with sedimentation constants 32S, 45S and even higher. This means that RNA migrating from nuclei to cytoplasm during cell division retains its nuclear character. It is suggested that this property provides for the return of RNA synthesized before mitosis to the nuclei of the daughter cells.     

Acceleration of CHO cells into mitosis and reduction of X-ray-induced G2 delay by cordycepin

The mitotic cell selection technique was used to monitor the effect of cordycepin and/or 100 rad of X-rays on the entry of asynchronous or synchronous Chinese hamster ovary cells into mitosis. Continuous exposure of asynchronous cells to 5–50 μg/ml of cordycepin caused a rapid increase in the relative numbers of cells entering mitosis. In irradiated cells, cordycepin also reduced a 120-min mitotic delay by about 80 min and shifted the X-ray transition point about 10 min farther away from mitosis. Further studies showed that synchronous cells, treated continuously with 15 μg/ml of cordycepin starting at mid-to-late S phase, proceeded into mitosis approx. 40 min ahead of controls. This acceleration was associated with a 30-min lengthening of S phase and a reduction in the length of G2 from 80 to about 10 min. Furthermore, cordycepin reduced the 70-min mitotic delay observed for cells irradiated in S phase by 20 min. In contrast to the results for treatment at mid-S phase, continuous treatment during G2 of unirradiated synchronous cells with 15 μg/ml of cordycepin had little effect on accelerating cells into mitosis, yet did reduce by about 60 min the 170-min mitotic delay observed for cells irradiated in G2. Unirradiated synchronous cells treated with cordycepin starting before mid-S did not reach mitosis. Thus, there are the following transition points or intervals for cordycepin: for treatment prior to mid-S phase, cell cycle progression through S is blocked; for treatment between mid-S and late S, progression through S continues but progression through G2 is accelerated; and for treatment during G2, the rate of progression in accelerated only if the cells have been irradiated. These results are discussed in relation to the synthesis during late S and G2 of critical protein molecules essential for mitosis.     

Cell Cycle Regulation of DNA Replication Initiator Factor Dbf4p

The precise duplication of eukaryotic genetic material takes place once and only once per cell cycle and is dependent on the completion of the previous mitosis. Two evolutionarily conserved kinases, the cyclin B (Clb)/cyclin-dependent kinase (Cdk/Cdc28p) and Cdc7p along with its interacting factor Dbf4p, are required late in G1 to initiate DNA replication. We have determined that the levels of Dbf4p are cell cycle regulated. Dbf4p levels increase as cells begin S phase and remain high through late mitosis, after which they decline dramatically as cells begin the next cell cycle. We report that Dbf4p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). In addition, Dbf4p is modified in response to DNA damage, and this modification is dependent upon the DNA damage response pathway. We had previously shown that Dbf4p interacts with the M phase polo-like kinase Cdc5p, a key regulator of the APC late in mitosis. These results further link the actions of the initiator protein, Dbf4p, to the completion of mitosis and suggest possible roles for Dbf4p during progression through mitosis.     

Tissue-specific G1-phase cell-cycle arrest prior to terminal differentiation in Dictyostelium

The cell cycle status of developing Dictyostelium cells remains unresolved because previous studies have led to conflicting interpretations. We propose a new model of cell cycle events during development. We observe mitosis of about 50% of the cells between 12 and 18 hours of development. Cellular DNA content profiles obtained by flow cytometry and quantification of extra-chromosomal and chromosomal DNA suggest that the daughter cells have half the chromosomal DNA of vegetative cells. Furthermore, little chromosomal DNA synthesis occurs during development, indicating that no S phase occurs. The DNA content in cells sorted by fluorescent tissue-specific reporters indicates that prespore cells divide before prestalk cells and later encapsulate as G1-arrested spores. Consistent with this, germinating spores have one copy of their chromosomes, as judged by fluorescence in situ hybridization and they replicate their chromosomes before mitosis of the emergent amoebae. The DNA content of mature stalk cells suggests that they also attain a G1 state prior to terminal differentiation. As prestalk cells appear to be in G2 up to 22 hours of development, our data suggest that they divide just prior to stalk formation. Our results suggest tissue-specific regulation of G1 phase cell cycle arrest prior to terminal differentiation in Dictyostelium.     

EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells

The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5-10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4-day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF-treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF-treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF-induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.     

Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.     

Early replicating DNA involved in the G2 to M transition in Allium cepa L meristematic cells

Summary— The involvement of genome portions replicating at different times of the S phase in the control of cell cycle events was analysed in Allium cepa L meristematic cells. 5-Azacytidine (5-azaC) was incorporated into discrete replicating DNA portions in synchronous cells. Cells treated with 5-azaC during the late S phase went through normal cell cycles while cells treated with 5-azaC during the early S phase were able to go through the immediately succeeding mitosis, as well as to begin the subsequent S phase. However, they were unable to enter a second mitosis. Thus, sequences replicating in the early S phase appear to code for a mitotic inhibitor which represses the emergence of a positive mitotic signal. This inhibition took place in the first half of the interphase (ie during G1) and resulted in a cell cycle blockage in G2.     

Arrest of 3T3 cells in G1 phase in suspension culture.

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.     

Circadian rhythms in mouse epidermal basal cell proliferation. Variations in compartment size, flux and phase duration.

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G2 phase DNA content were measured by flow cytometry of isolated basal cells, and the [3H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G2 phase and traversed the G2 phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G2 phase and mitosis. Circadian variations in the duration of the S phase, the G2 phase and mitosis were clearly demonstrated.     

Arrest of 3T3 cells in G1 phase in suspension culture

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.     

Non-histone protein synthesis during G1 phase and its relation to DNA replication.

The kinetics of non-histone chromosomal protein (NHCP) synthesis were studied in Chinese hamster ovary (CHO) plateau phase cells stimulated to proliferate and were compared to NHCP synthesis kinetics in two populations of synchronous G1 traversing cells. In all cases, NHCP synthesis rates increase 3- to 5-fold as cells traversed G1 and attained maximum values one hour before semi-conservative DNA replication began. Similar to results in synchronous G1 cells, the molecular weight distributions of the NHCP fraction from stimulated plateau phase cells underwent only minor changes, measured by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, as these cells moved toward S phase. Yet, during this progression after plateau phase and in the transition from early G1 to late G1 in synchronous cells, the total NHCP fraction increased significantly (1.5-2-fold) in amount per cell. These data indicate that plateau phase cells are similar to early G1 cells both in terms of their amounts of non-histone per cell and in their subsequent NHCP synthesis kinetics as they move toward S phase. These results extend previous findings which suggested that NHCP synthesis was coupled to DNA replication and demonstrate that the increased NHCP synthesis and accumulation in chromatin may be a biochemical marker for G1 progression.