NUCLEOCYTOPLASMIC RATIO REQUIREMENTS FOR THE INITIATION OF DNA REPLICATION AND FISSION IN TETRAHYMENA

Hydroxyurea (10 mM) arrests the exponential growth of Tetrahymena by blocking DNA replication during S-phase. After removal of the hydroxyurea (HU), they have a long recovery period during which they are active in DNA synthesis. ³H-TdR uptake showed that on completion of the recovery period, the cells divide (recovery division) and enter a cell cycle which lacks G₁. The frequency, size and DNA content of the extranuclear chromatin bodies (ECB) formed at this division are all markedly increased (2–4) over the corresponding values obtained from exponential growth phase controls. Microspectrophotometric analysis of macronuclear DNA content (N) coupled with the cytoplasmic dry mass (C) values suggest that specific N to C ratios (N/C) are required for the initiation of DNA replication and fission: during a normal (exponential growth) cell cycle, both N and C double, but asynchronously, so that the N/C of both post-fission-daughter cells and pre-fission cells is identical (standardized to N/C = 1) but late G₁ cells have a low N/C. During a 10 hr exposure to HU, the N remains essentially the same whereas the C increases. When the HU is removed, the N increases by 4× and the C continues to increase until just prior to recovery division when it also reaches a value 4× that of the original daughter cells. Thus, the N/C = 1 is re-established. The enlarged ECB formed during recovery division may function to lower the N/C in the daughter cells, which in turn may in some way stimulate immediate DNA replication, thus eliminating G₁. The elimination of G₁ (and shortening in a few subsequent cell cycles) allows less time for cytoplasmic growth and results in the return of the cells to the generation time and the N and C values observed prior to the HU treatment.     

Fish cell line (ULF-23HU) derived from the fin of the central mudminnow (Umbra limi): Suitable characteristics for clastogenicity assay

Summary A cell line (ULF-23HU) from the fin of the central mudminnow (Umbra limi) was characterized and tested for its suitability to assess cytogenetic damages induced by chemicals in fish. Cells of this line exhibit a fibroblastlike appearance and grew optimal at 25°C in, TC-199 medium containing 10% fetal bovine serum, but slower growth continued down to 4°C, where they could be stored for prolonged periods. Seeding efficiency of ULF-23HU cells on the plastic substratum was approximately 85% in the above culture medium at 25°C. They had a 32-h cell cycle time taken up by a 20-h S period as determined by the autoradiographic analysis of the fraction of labeled mitosis. Cultures showed relatively high mitotic index (0.84 to 2.35%) during exponential growth phase lasting about 7 d. Karyological analysis of the cells at the different subculture passages revealed constant chromosome modal number of 23 consisting of metacentric or submetacentric chromosomes, which were primarily similar to those of in vivo cells, with one additional chromosome. The spontaneous sister chromatid exchange rate was 5.3 per metaphse. When ULF-23HU cells were exposed toN-nitroso-N-methylurea, a clastogen in the mammalian cells, dose-dependent increases both in sister chromatid exchanges and chromosome aberrations were clearly detected. These results on the growth kinetics and cytogenetic characteristics offered the high possibility of the use of this cell line as a suitable in vitro model for clastogenicity studies in fish. This work was supported by grants from Korea Sciences and Engineering Foundation and Japanese Government Research Awards for Foreign Specialists to E.-H. Park.     

Time-dependent changes in H1 content, H1 turnover, DNA elongation, and the survival of cells blocked in early S phase by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine

Cells were synchronized in G1 by isoleucine deprivation and then released into medium containing 1 mM hydroxyurea (HU), 5 micrograms mL-1 aphidicolin (APC), or 1 microgram mL-1 5-fluorodeoxyuridine (fl5dU). Coulter volume, content of histone H1 per unit DNA, turnover of histone H1, the extent of DNA elongation, and the survival of cells were measured as functions of time after release into the presence of the drugs. At the concentrations used in the experiments, the drug differ in their toxicity (fl5dU greater than HU greater than APC), induction of unbalanced cell growth, and the distribution of new DNA fragment sizes allowed during block, but they all (1) allow cells to enter S phase, (2) cause similar time-dependent losses of histone H1 per unit DNA, which begin as synchronized G1 cells begin to enter S phase, (3) retard DNA elongation beyond replicon size, and (4) retard the turnover of histone H1. The results indicate that loss of histone H1, inhibition of histone turnover, the retarded ligation of newly replicated DNA into bulk chromatin, and chromatin structural changes may be part of the cell's general response to inhibition of DNA replication. Since transient S phase block increases the frequencies of gene amplification [Mariani, B. D., & Schimke, R. T. (1984) J. Biol. Chem. 259, 1901-1910] and sister chromatid exchanges (SCE) [Rainaldi, G., Sessa, M. R., & Mariani, T. (1984) Chromosoma 90, 46-49], the observed changes in H1 content and chromatin organization may also be essential features of gene amplification and SCE.     

An in vivo study on the synchronizing effect of hydroxyurea

The effect of hydroxyurea (HU; 0.5 mg/g body wt) on L 1210 ascites tumor cells has been studied using various cell kinetic methods. In contrast to the general assumption that HU blocks cells at the G1/S boundary [J. Brachet (1985) Molecular Cytology, Vol. I, p. 266, Academic Press, New York], the present results show that the cells are not held at G1/S but enter S at about the normal rate and are accumulated in early S phase due to a dose-dependent inhibiting effect of HU on DNA synthesis. Partial synchronization of the cells demonstrated by a distinct mitotic peak 10 h after HU application is not due to a G1/S block of the cells and their subsequent synchronous passage through the cycle after release from the block but is due to rather complex mechanisms of action of HU: a differential cytocidal effect and an effect on the passage of the cells through the cycle, both depending on the position of the cells throughout the cycle. HU kills S-phase cells, mainly cells in early S phase; i.e., a great portion of the cells "accumulated" in early S phase is killed by the drug, while G1-phase cells are almost not affected by the lethal effect of HU. These G1-phase cells pass through the cycle more rapidly after cessation of the HU effect. The same is true for the surviving cells accumulated in early S phase, while part of the cells in the remaining S phase are delayed in their passage through the cycle. This causes partial synchronization, since a great portion of all cells that survive HU treatment reach mitosis at the same time.     

Hydroxyurea-induced mitotic synchronization in Allium sativum root meristems

The synchronized divisions following a treatment with hydroxyurea (HU) — an inhibitor of DNA synthesis — were studied in root meristems of Allium sativum using two methods: autoradiography of median sections and morphological labeling with a cytokinesis inhibitor. It is shown that the second wave of mitoses is heterogeneous: it is composed mostly of cells which have been synchronized in the S phase by the HU treatment, of cells coming from the quiescent center stimulated to enter DNA synthesis and of cells which were not blocked by the 23 h HU treatment (slow cycling cells). It is also shown that the cell cycle following the first synchronized division is considerably shortened by the synchronization procedure.Abbreviations QC quiescent center - HU hydroxyurea - MHQD methyl-3 hydroxy-6 quinazoline dione 2–4     

A TECHNIQUE FOR DETERMINING THE PROPORTION OF THE CLONOGENIC CELLS IN S PHASE IN EMT6 CELL CULTURES AND TUMORS

The survival of cultured EMT6 cells was examined after treatment with hydroxyurea (HU) or high specific activity tritiated thymidine (³H-TdR). The concentrations of the agents, duration of exposure to the agents, and post-exposure treatment of the cultures were found to influence the cell survival; the effects of these factors are reported. Conditions were defined under which the proportions of cells killed by HU and by ³H-TdR were the same and were also the same as the proportion of labeled cells seen on autoradiographs of cultures labeled with small doses of ³H-TdR. Under these conditions, either ³H-TdR or HU could be used to determine the proportion of the clonogenic cells in S phase. Single cell suspensions prepared from solid EMT6 tumors were treated in vitro with HU or ³H-TdR, using the conditions found optimal for each agent with cultured cells. The proportion of the tumor cells killed by treatment with HU in vitro was the same as the proportion killed by HU in vivo and as the proportion labeled by ³H-TdR in vivo, and incubation of tumor cell suspensions with HU in vitro appeared to provide a valid measurement of the proportion of clonogenic tumor cells in S phase. Incubation of tumor cell suspensions with ³H-TdR in vitro proved difficult to perform and the results were relatively unreliable because of severe problems with reutilization of ³H-TdR during the incubation for colony formation.     

Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis

Cultures of Euglena gracilis (strain Z from French CNRS collection) can be made cadmium resistant if grown in a medium with 5x10^-4M cadmium chloride. This resistance is reflected by the appearance of a second exponential growth phase. The development of this resistance was studied at the cellular level by determining the relative content of DNA at different stages of the cell cycle in an asynchronously grown culture. The culture was followed until the second, cadmium resistant, growth phase had reached its stationary state. During the first exponential growth phase, cells were mostly in the late period of DNA synthesis (stage S of the cell cycle), or in the gap preceding mitosis (stage G₂ of the cell cycle). In addition, some cells contained high multiples of the normal amount of DNA. In the beginning of the second exponential growth phase, a few cells were again in G₁ (the post mitotic stage of the cell cycle preceding DNA synthesis). These G₁ cells were predominant at the end of the second growth period. During the second stationary phase the DNA content of the cadmium treated cells was similar to the stationary phase of the control culture. Cells had stopped growing in G₁ with an unreplicated genome. The implications of these data are discussed.     

Unbalanced growth and cell death in HeLa S3 cultures treated with DNA synthesis inhibitors

Flow cytometry indicated that significant amounts of dsRNA were accumulated in HeLa S3 cells blocked at or near G₁/S boundary by hydroxyurea (HU) or excess thymidine (TdR). The dsRNA/DNA ratio increased in these cells in a manner characteristic of unbalanced cell growth. In HU-treated cells, dsRNA content was maximal 16 hours after addition of the drug and did not change significantly during the next 24 hours. The DNA content in blocked cells increased by 10%. Cell viability assessed by colony formation in soft agar decreased exponentially in HU-treated cultures after 16 hours of incubation. Correlation between loss of cell viability and rate of cell proliferation after removal of HU was observed, as determined by cell count and analysis of cell cycle progression. In TdR-treated cultures cells slowly progressed into mid S-phase during 40 hours and dsRNA accumulation continued during this period. Cell viability was not significantly affected by treatment with excess TdR, indicating that unbalanced growth per se, as measured by dsRNA accumulation, is not lethal for the cells. After reversal of DNA synthesis inhibition by removal of the drug, cells treated with HU for 16 hours or TdR for 16–24 hours promptly progressed through the cell cycle. This progression was accompanied by accumulation of significant amounts of dsRNA. As a result, cells in G₂ phase had a very high dsRNA content leading to retention of the unbalanced condition (increased dsRNA/DNA ratio) in the daughter cells. It is suggested that dsRNA accumulation in the cell is controlled to a certain degree by cell progression through the S phase. This type of control, evidently, was reflected in limited dsRNA accumulation in the cells blocked at or near G₁/S border, in continuous dsRNA accumulation in the cells slowly progressing through S phase, and in accumulation of large amounts of dsRNA after renewal of progression through the S phase.     

DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa

Background and Aims

Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis.

Methods

Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H₂O₂ production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation).

Key Results

Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H₂O₂, γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants.

Conclusions

The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus.     

Chromatin‐level regulation of the fragmented dothistromin gene cluster in the forest pathogen Dothistroma septosporum

Genes required for fungal secondary metabolite production are usually clustered, co‐regulated and expressed in stationary growth phase. Chromatin modification has an important role in co‐regulation of secondary metabolite genes. The virulence factor dothistromin, a relative of aflatoxin, provided a unique opportunity to study chromatin level regulation in a highly fragmented gene cluster that is switched on during early exponential growth phase. We analysed three histone modification marks by ChIP‐qPCR and gene deletion in the pine pathogen Dothistroma septosporum to determine their effects on dothistromin gene expression across a time course and at different loci of the dispersed gene cluster. Changes in gene expression and dothistromin production were associated with changes in histone marks, with higher acetylation (H3K9ac) and lower methylation (H3K9me3, H3K27me3) during early exponential phase at the onset of dothistromin production. But while H3K27me3 directly influenced dothistromin genes dispersed across chromosome 12, effects of H3K9 acetylation and methylation were orchestrated mainly through a centrally located pathway regulator gene DsAflR. These results revealed that secondary metabolite production can be controlled at the chromatin‐level despite the genes being dispersed. They also suggest that patterns of chromatin modification are important in adaptation of a virulence factor for a specific role in planta.     

Flow cytometry of the differentiation of Physarum polycephalum myxamoebae to cysts

Myxamoebae of Physarum polycephalum, strain Cld, were grown on agar lawns on live bacteria. Myxamoebae were harvested, fixed and stained with propidium iodide. Flow cytometry showed that, as in the case of Physarum plasmodia, there is no G1 phase during rapid exponential growth. However, an apparent G1 phase was observed at the end of exponential growth when the culture arrested with the G1 DNA content for about a day between growth and differentiation. Most myxamoebae differentiated into cysts, but some formed microplasmodia and others appeared to lose DNA. The cysts possessed the G2 phase DNA content and there was an S phase connecting the G1-arrested state with the encysted state. Encystment was blocked by hydroxyurea (HU) suggesting that DNA synthesis is essential for encystment. The natural temporary synchronization in G1 phase may provide the basis of a method for selecting mutants with a conditional block in G2 or M phases.     

Hydroxyurea synchronization of bovine fetal spleen cells

Hydroxyurea (HU) was shown to be an effective synchronization agent for bovine fetal spleen (BFS) cells. Following exposure of cells to 2 mM HU for 32 h, DNA synthesis above background levels was not observed. BFS cells released from the HU block by washing began to synthesize DNA immediately. Within 2 h, 80–85% of the cells were in S phase, as determined by autoradiography, and the maximum rate of DNA synthesis occurred 2–4 h following removal of HU. The rapid induction of DNA synthesis in BFS cells and the high percentage of cells synthesizing DNA immediately after removal of HU demonstrate that HU produces a highly synchronized population of S phase BFS cells. Although RNA and protein synthesis were maintained at near normal rates early after cells were exposed to HU, the rates decreased to 40–50% of those observed in cells seeded in medium without HU by the time of release. These reduced rates of synthesis of RNA and protein in the absence of DNA synthesis may account for the low toxicity of HU for BFS cells.     

Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae.

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.     

An improved method for the cell cycle synchronization of <Emphasis Type="Italic">Vicia faba</Emphasis> root meristem cells

Plant root meristem cells divide asynchronously which makes biochemical analysis of cell cycle regulation particularly difficult. In the present article a high level of cell cycle synchronization in Vicia faba root meristems was obtained by using a rich medium (HNS), special culture conditions and a double-block method with replication inhibitor—hydroxyurea (HU). Two HU concentrations were tested and different periods of the first and the second synchronization, and of cycle recommencement between the first and the second blockage. The level of synchronization was estimated on the basis of ³H-thymidine labeling indices, mitotic, and phase indices and indices determining the percentage of G1 and G2 cells, which were identified by cytophotometric measurements of DNA content in individual nuclei. The highest level of cell cycle synchronization was obtained after double treatment of meristems with 1.25 mM HU (18 and 12 h) separated by 6-h incubation in HNS without HU. During the second postincubation in HNS in subsequent hours: 4, 7, 10, 11, over 90% of cells in the S phase, nearly 70% in G2 phase, 86% in mitosis, and nearly 70% in G1 phase were received, respectively. The use of 2.5 mM HU in a similar experimental procedure caused disturbed divisions.     

The Effect of Nonanal on Dipodascus aggregatus II. Influence of the Prehistory of the Inoculum and of the Presence of Ethanol

Nonanal, added in ethannlic solution, in concentrations lower than 40 to 80 μM did not affect the growth of Dipodascus aggregatus, provided the inoculum had been harvested from the exponential phase of growth. Growth could even be inhibited by 80 μM. If the inoculum had been grown to the exponential phase and then for another period, to the acceleration phase, in fresh liquid medium, growth was strongly promoted by 80 μM nonanal. If cells from the exponential phase were grown for another period in the supernatant fluid of centrifuged cultures from the exponential phase, 80 μM affected growth in the following way: in five different experiments growth was not stimulated, in one experiment undoubtedly promoted, and weakly stimulated in another one. The growth of cultures inoculated with cells grown only on malt agar was not affected by 80 μM nonanal. Pretreatment of cells, harvested from the acceleration phase, with nonanal (80 μM) in the presence of ethanol did not diminish the growth-promoting action of nonanal on the cultures inoculated with these cells. Nonanal, in the absence of ethanol, in a concentration of 10 μM did not affect the growth of cells, harvested from the acceleration phase, whereas 100 μM nonanal strongly inhibited growth. An attempt is made to explain the results starting from the endogenous metabolism.     

HYDROXYUREA EFFECTS IN THE C3H MOUSE II. MAMMARY TUMOR CELL KINETICS

The cellular kinetics of C3H mouse mammary tumors were studied following a single dose (3 mg/g body weight) of hydroxyurea (HU). This dose was large enough to cause a significant perturbation in the growth curves of these tumours. This was accomplished by labeling the cells with tritiated 5-iodo-2'-deoxyuridine and performing detailed autoradiographic analysis. This dose of HU caused a temporary inhibition in growth and completely inhibited DNA synthesis for 4–5 hr. The HU-killed cells (pyknotic and karyorrhectic) reach a maximum around 10–12 hr and are apparently all removed in about 1 day. Tumors from a fast-growing line (S102F) showed some evidence for cell synchrony upon recovery from HU inhibition but desynchronization occurred within one cell cycle. The cell generation time was not decreased during the acute recovery phase, but the growth fraction shifted from 0·6 to 1·0, and the data suggested that the normal flow of cells from the proliferating pool to the degenerate pool was temporarily interrupted. The cellular kinetic parameters have probably returned to normal by 48 hr after the HU injection.     

Hydroxyurea exposure alters mouse testicular kinetics and sperm chromatin structure

Abstract. The effects of hydroxyurea (HU) on testicular cell kinetics and sperm chromatin differentiation were investigated in mice. Whole testis, minced testicular cell suspensions and caudal epididymal sperm cells were obtained at 8 and 29 days after i.p. injections containing 0, 25, 50, 100, 200, 400 and 500 mg/kg HU X 5 days. Testis weights were unaffected by 25 mg/kg HU while 500 mg/kg caused up to a 50% loss of testicular weight by 29 days. Flow cytometrically measured acridine-orange (AO) stained testicular cells revealed altered population ratios at the highest dosages at 8 days and for all dosages except 25 mg/kg HU at 29 days. At 8 days, 400–500 mg/kg HU caused a near depletion of tetraploid cells. Flow cytometry of AO stained sperm, previously treated with acid to potentially induce DNA denaturation, was used to follow the shift from normal chromatin structure to an abnormal form with increased sensitivity to DNA denaturation in situ. The extent of DNA denaturation was quantitated for each cell by the computer-derived value alpha t, α_t= [red/(red+green) fluorescence]. The flow cytometry measures, standard deviation of α_t (SDα_t), mean of α_t (Xα_t) and cells outside the main peak of α_t (COMPα_t), gave similar dose response curves to the sperm head morphology assay. SDα_t was more sensitive than the Xα_t as a measure of HU-induced alteration of chromatin structure. The major conclusions reached are that HU inhibits DNA synthesis, probably by inhibiting ribonucleotide reductase, causing maturation depletion of pachytene spermatocytes and, subsequently, depletion of meiotic daughter cells and differentiated cell types leading to mature sperm. This inhibition of DNA synthesis is related to an alteration of sperm chromatin structure and abnormal sperm head morphology.     

Conversion of Mitotic to Meiotic Cells in Saccharomyces cerevisiae II. Relation between Premeiotic DNA Replication and Revertibility to Mitosis

Meiotic differentiation was investigated using a synchronousmeiotic system of the yeast, Saccharomyces cerevisiae, withrespect to revertibility to mitotic division. When cells weretransferred from a sporulation (SPM) to a growth (YHA) medium,reversion to mitosis took place at all stages up to and includingthe post synthetic phase of DNA. Continuous treatment with 4mM hydroxyurea (HU) during sporulation resulted in the extensionof the the premeiotic S phase. Revertibility to mitosis alsowas extended after the lengthened S phase. Pulse treatment with50 min HU for 2 h during the premeiotic S phase caused a 2-hdelay in the revertibility to mitosis. Similar results wereobtained by treatment with elevated temperatures. The resultsdemonstrate an irreversible commitment of cells to meiosis afterthe completion of premeiotic DNA replication. When cells were transferred from SPM to poor nutrient media,the timing of their reversion to mitosis varied. If transferredto glucose-based media, cells in the premeiotic S phase underwentmitosis; whereas, if transferred to acetate-based media, theycontinued meiotic development. Thus, conversion to meiosis dependson the nutritional environment to which cells are transferred,which implies that a sequence of intracellular change duringpremeiosis leads to meiotic differentiation. (Received February 10, 1983; Accepted June 1, 1983)