Sulfhydryl groups during the S phase: comparison of cells from G 1 , plateau-phase G 1 , and G 0

The non-protein sulfhydryl (NPSH) content of cells moving into S from G₁, plateau phase G₁, and G₀ was measured. Chinese hamster ovary (CHO) cells accumulated in G₁ by growth into plateau phase contain only one-fourth the NPSH concentration of cycling C₁ cells or G₁ cells accumulated by brief growth in isoleucine-deficient medium. Upon dilution of plateau cultures with fresh medium, cellular NPSH content increases rapidly, reaching the same level as that in cycling cells within four hours. This increase is prevented by cycloheximide but not by actinomycin D or hydroxyurea. Neither CHO cells cycling in vitro nor salivary gland G₀ cells stimulated with isoproterenol in vivo show significant changes in intracellular NPSH concentrations during S phase. This suggests that the concentration of intracellular NPSH (glutathione) remains constant during the cell cycle except when cells are grown to plateau phase in exhausted or deficient medium, in which case normal degradation exceeds synthesis and the gross level falls until fresh medium is provided and synthesis, apparently on preexisting RNA templates, accelerates.     

Cell Synchrony Techniques. I. A Comparison of Methods

Abstract Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G₁ S, or G₂+ M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G₁ phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G₁ peak yielded a relatively pure but heterogeneous G₁ population (i.e. early to late G₁). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

Factors affecting the formation of phytol and its incorporation into chlorophyll by homogenates of the leaves of the french bean Phaseolus vulgaris

The biosynthesis and phosphorylation of histone fractions were measured in synchronized CHO Chinese hamster cells arrested in late G₁ by hydroxyurea treatment. Hydroxyurea was found to inhibit the initiation of both DNA and histone synthesis, thus confirming the conclusion that it arrests cells in G₁ slightly before the $\text{G}{}_1\text{S}$ boundary. However, hydroxyurea did not inhibit the phosphorylation of histone f1 or histone f2a2. The phosphorylation of histone f1, which normally is absent in early G₁, begins 2 hr prior to DNA synthesis. In the presence of hydroxyurea, f1 phosphorylation occurs on schedule at this same time in G₁, resulting in significant G₁-phase f1 phosphorylation. This offers strong evidence that (a) f1 phosphorylation is not restricted to S phase; (b) “old” f1 which was synthesized in previous cell cycles is phosphorylated in G₁ before “new” f1 which is synthesized in S phase; and (c) G₁-phase f1 phosphorylation does not require new histone or new DNA synthesis.Histone f1 phosphorylation was observed to occur at accelerated rates in S phase over phosphorylation rates observed in late G₁-arrest. Data support the proposal that three different levels of f1 phosphorylation occur during the cell cycle: (1) a G₁-related phosphorylation of “old” f1; (2) an S-related phosphorylation of both “old” and “new” f1; and (3) a superphosphorylation of f1 associated with chromosome condensation during the G₂ to M transition. It is also possible that a limited proportion of f1 may be phosphorylated in G₁, perhaps at the initial DNA synthesis sites, and that an increased proportion of f1 is phosphorylated in S as DNA is synthesized. Similarities between the kinetics of histone f1 phosphorylation and the association of DNA with lipoprotein in synchronized control and hydroxyurea-treated cells suggest an involvement of f1 phosphorylation in cell-cycle-dependent chromatin structural changes.     

Enhancement of DNA polymerase II activity in E. coli after treatment with N-methyl-N'-nitro-N-nitrosoguanidine.

The G₁(G₀) arrest induced in NRK cells by picolinic acid was preceded by marked changes in iron metabolism. In contrast, picolinic acid did not significantly prevent zinc uptake and changes in intracellular zinc were small and clearly preceded by changes in iron. A kinetic study revealed that iron uptake by NRK cells was rapidly halted by picolinic acid. Experiments with radioiron-labeled cells indicated that picolinic acid, in a dose dependent manner, effectively removed iron from the cells. The dose of picolinic acid that exactly removed iron from the cells was also the concentration that induced the G₁(G₀) arrest. Picolinic acid, therefore, may induce the growth inhibition by selectively withholding iron from the cells. These data strongly suggest that iron availability may be a controlling factor in the initiation of DNA synthesis in NRK cells.     

Hydroxyurea inhibits ODC induction, but not the G1 to S phase transition.

Chinese hamster ovary cells, selected in mitosis and plated into medium containing hydroxyurea, can progress through G₁ and enter S phase although bulk DNA synthesis is prevented. As the cells progress through G₁ in the presence of hydroxyurea, ornithine decarboxylase activity remains low while general protein synthesis appears unaffected. After hydroxyurea is removed, ornithine decarboxylase activity increases, but only after approximately 20% of the DNA has been replicated. These results suggest that ornithine decarboxylase induction is not essential for cellular progression into S phase but is required for the completion of DNA synthesis.     

Replication and G2 checkpoints: their response to caffeine

Under long hydroxyurea treatments, evidence was obtained for the sequential activation of four checkpoints located between the onset of S phase and mitosis in Allium cepa L. root meristems. Bi-parametric flow cytometry (Br-DNA/total DNA) showed that cells initially accumulated at early S phase but, after a delay, they resumed replication and paused again at mid S phase. Cells not only overrode this second replication block but also any G₂ checkpoint they encountered. Thus, a late mitotic wave was produced in the presence of hydroxyurea. The wave was formed by cells that had apparently completed their replication (normal mitoses), while others displayed anaphases/telophases with less than the expected DNA content and with chromosomal breaks (aberrant mitoses). The presence of aberrant mitoses is direct evidence for the undue override of the two G₂ checkpoints responsible for surveillance of completion of DNA synthesis and repair, respectively. Caffeine selectively abrogated the G₂ block produced by the checkpoint that controls post-replication DNA repair, as it advanced the entry of cells into an aberrant mitosis. However, caffeine proved not to be the universal checkpoint-evading agent as postulated. Caffeine did not modify the spontaneous override of the replication checkpoints. Moreover, it seems to enforce the checkpoint that controls the completion of DNA synthesis, as the appearance of the late wave of normal mitoses produced in the presence of hydroxyurea was prevented by the use of caffeine. Received: 21 February 2000 / Accepted: 31 July 2000     

Enhanced GTP-dependent activities of the adenylate cyclase system: basis for increased hormonal responsiveness.

Normal rat kidney (NRK) cells growth arrested by picolinic acid and isoleucine deprivation exhibit an increased response to certain agents (i.e., prostaglandin E₁, (?)-isoproterenol, and cholera toxin) which elevate intracellular cyclic AMP levels. The enhanced hormonal response is apparently due, at least in part, to increased adenylate cyclase activity. Adenylate cyclase activities measured in the presence of GTP, GTP plus prostaglandin E₁, and GTP plus (?)-isoproterenol are increased two- to threefold in membranes prepared from treated cells. In contrast, basal activity is potentiated only 20 to 50% and activity determined in the presence of fluoride is only marginally altered. Also of interest is the increase in cholera toxin activation of cyclase activity in the treated cells. Lower concentrations of cholera toxin (5 ng/ml) are required to achieve maximal stimulation of cyclase activity from picolinic acid-treated and isoleucine-deprived cells; maximal stimulation of control cell adenylate cyclase is attained with 25 to 50 ng/ml cholera toxin. Picolinic acid treatment and isoleucine deficiency both have been shown to arrest NRK cell growth in the G₁ phase of the cell cycle. However, results with cells arrested in G₁ by serum starvation and by growth to high cell population density indicate that G₁ specific growth arrest does not appear to account for the increase in hormonal responsiveness. Chelation of inhibitory metals and proteolytic activation also do not appear to be involved in the mechanism by which picolinic acid enhances cyclic AMP formation. Rather, the results suggest that the treated cells have an increased amount of an active GTP-dependent function required for hormone and cholera toxin stimulation of adenylate cyclase. Thus, picolinic acid treatment and isoleucine deprivation may provide a useful means of modulating the GTP-dependent step required to potentiate hormonal responsiveness.     

Cytological effects on Chinese hamster cells of synchronizing concentrations of hydroxyurea

The cytological effects of 2 mM hydroxyurea upon Chinese hamster cells at various phases of the cell cycle were examined. Cells in the G₁, G₂, or M phases of the generation cycle treated with hydroxyurea showed no chromosomal aberrations. Cell treated in S phase became moribund and eventually lysed. Some of these moribund S cells reached mitosis much later and were found to have chromatid aberrations. Cells in the log phase of growth, surviving exposure to 2 mM hydroxyurea for six hours, also showed no aberrations. Thus, viable (colony-forming) cells, resulting from synchrony procedures with hydroxyurea are free of chromosomal aberrations.     

Effect of hydroxyurea on procyclic Trypanosoma brucei: an unconventional mechanism for achieving synchronous growth

Procyclic Trypanosoma brucei cells were synchronized with 0.2 mM hydroxyurea. The cells did not arrest at the G₁/S boundary but proceeded through one round of replication and arrested near the end of S phase. The mitochondrial genome (kinetoplast DNA network) replicated, forming two progeny networks, but the repair of minicircle gaps was inhibited.     

The role of the G1 period in the life cycle of eukaryotic cells

The objective of this study was to test the concept that the G₁ period lacks any specific function in the life cycle of mammalian cells and hence could be drastically reduced without any effect on the generation time. HeLa cells were grown in medium containing an optimum dose (60 μM) of hydroxyurea at which the duration of S period was prolonged with little or no increase in generation time. At this concentration of hydroxyurea, we observed a maximum of 3 h (or 28.5%) reduction in the G₁ period. We also studied the effects of synchronization in S phase by single and double thymidine blocks on cell size and its relationship to the duration of G₁ in the subsequent cycle. By these treatments, we could reduce the G₁ period by not more than 2 to 3 h. The reduction in G₁ period was not directly proportional to the size (volume) of the G₁ cells. These results suggest that G₁ period has certain specific functions and cannot be eliminated by alterations in culture conditions.     

Dynamics of cell cycle phase perturbations by trabectedin (ET-743) in nucleotide excision repair (NER)-deficient and NER-proficient cells, unravelled by a novel mathematical simulation approach

Abstract. Objectives: Trabectedin (ET‐743, Yondelis^®) is a natural marine product, with antitumour activity, currently in phase II/III clinical trials. Previous studies have shown that cells hypersensitive to ultraviolet (UV)‐rays because of nucleotide excision repair (NER) deficiency, were resistant to trabectedin. The purpose of this study was to investigate whether this resistance was associated with different drug‐induced cell cycle perturbations. Materials and Methods: An isogenic NER‐proficient cellular system (CHO‐AA8) and a NER‐deficient one (CHO‐UV‐96), lacking functional ERCC‐1, were studied. Flow cytometric assays showed progressive accumulation of cells in G₂ + M phase in NER‐proficient but not in NER‐deficient cells. Applying a computer simulation method, we realized that the dynamics of the cell cycle perturbations in all phases were complex. Results: Cells exposed to trabectedin during G₁ and G₂ + M first experienced a G₁ block, while those exposed in S phase were delayed in S and G₂ + M phases but eventually divided. In the presence of functional NER, exit from the G₁ block was faster; then, cells progressed slowly through S phase and were subsequently blocked in G₂ + M phase. This G₂ + M processing of trabectedin‐induced damage in NER‐proficient cells was unable to restore cell cycling, suggesting a difficulty in repairing the damage. Conclusions: This might be due either to important damage left unrepaired by previous G₁ repair, or that NER activity itself caused DNA damage, or both. We speculate that in UV‐96 cells repair mechanisms other than NER are activated both in G₁ and G₂ + M phases.     

Effect of cell cycle on the regulation of the cell surface and secreted forms of type I and type II human tumor necrosis factor receptors

The cell cycle has been shown to regulate the biological effects of human tumor necrosis factor (TNF), but to what extent that regulation is due to the modulation of TNF receptors is not clear. In the present report we investigated the effect of the cell cycle on the expression of surface and soluble TNF receptors in human histiocytic lymphoma U-937. Exposure to hydroxyurea, thymidine, etoposide, bisbensimide, and democolcine lead to accumulation of cells primarily in G₁/S, S, S/G₂/M, G₂/M, and M stages of the cell cycle, respectively. Whilie no significant change in TNF receptors occurred in cells arrested in G₁/S or S/G₂ stages, about a 50% decrease was observed in cells at M phase of the cycle. Scatchard analysis showed a reduction in receptor number rather than affinity. In contrast, cells arrested at S phase (thymidine) showed an 80% increase in receptor number. The decrease in the TNF receptors was not due to changes in cell size or protein synthesis. The increase in receptors, however, correlated with an increase in total protein synthesis (to 3.8-fold of the control levels). A proportional change was observed in the p60 and p80 forms of the TNF receptors. A decrease in the surface receptors in cells arrested in M phase correlated with an increase in the amount of soluble receptors. The cellular response to TNF increased to 8- and 2-fold in cells arrested in G₁ and S phase, respectively; but cells at G2/M phase showed about 6-fold decrease in response. In conclusion, our results demonstrate that the cell cycle plays an important role in regulation of cell-surface and soluble TNF receptors and also in the modulation of cellular response. © 1995 Wiley-Liss, Inc.     

THE ACTION OF HYDROXYUREA ON MOUSE L-CELLS*

The toxic and inhibitory properties of hydroxyurea (HU) have been studied in asynchronous and synchronized populations of mouse L-cells. Hydroxyurea is a potent growth inhibitor and appears to be specifically lethal for cells which are in the early part of S phase at the time the compound is introduced. Cells in late S phase, G₂, mitosis and G₁ appear to progress normally around the cycle in the presence of the compound until they reach the G₁/S boundary. There are indications that at least some G₁ cells are able to enter the S phase even in the presence of the drug; however their flow into S is much slower than that of control cells and therefore they are killed at a slow rate. Upon prolonged exposure to the drug a second phase of more rapid killing is observed, beginning at about the time division would occur in uninhibited cells. Hydroxyurea exhibits a rapid and marked inhibition on DNA synthesis but its effect on RNA synthesis is much less pronounced and may be a consequence of the inhibition of DNA synthesis. The effects of hydroxyurea on cell viability and DNA synthesis can be partially prevented by the addition of deoxyribonucleosides which in sufficient concentration appear to compete temporarily with the drug. The fact that the protection is only temporary would appear to rule out the hypothesis that the primary mode of action of the drug is the inhibition of the reduction which converts ribonucleotides to deoxyribonucleotides. The data presented in this communication taken together with observations of other workers would appear to suggest that the effect of the drug may be directly on the DNA molecule.     

Effects of inhibition of RNA or protein synthesis on CHO cell cycle progression

Chinese hamster ovary (CHO) cells, synchronized by selective detachment at mitosis, were treated with various concentrations of actinomycin D (AMD) or cycloheximide (CHX) either immediately, or 1, 2, or 3 hr after mitosis. Since the minimum duration of G₁ phase in these cultures was 3.4 hr, the addition of RNA or protein synthesis inhibitors took place at the beginning, first third, second third, or end (G₁–S boundary) of G₁ phase. The kinetics of exit from G₁ phase, the rate and extent of traverse of S phase, and the reaccumulation of RNA were estimated under each set of growth conditions by flow cytometry of acridine orange-stained cells. A mathematical model was constructed to describe the trajectories of the cell populations with respect to their increase in RNA and DNA content in the absence or presence of the inhibitor. The chronologic synchrony imposed on the CHO cell population began to decay within 3 hr, resulting in stochastic entrance of cells into S phase in the absence of inhibitor. Addition of AMD or CHX at 0, 1, 2, or 3 hr after mitosis, regardless of the inhibitor concentration, did not provide evidence of a critical restriction point in G₁ beyond which cells were committed to enter S phase and were no longer sensitive to moderate suppression of RNA or protein synthesis. The observed kinetics of cell entrance into and traverse of S phase were consistent with an inherently heterogenous response to serum stimulation occurring at or just after cell division.     

DNAase I and cellular factors that affect chromatin structure

The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134–144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G₁ phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G₁ phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G₁- and S-phase cells, respectively. The G₁ cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G₁ nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.     

Histone phosphorylation in late interphase and mitosis

Histone phosphorylation in late interphase has been investigated employing cells synchronized by the isoleucine-deprivation method, followed by resynchronization at the $\text{G}{}_1\text{S}$ boundary using hydroxyurea. Phosphorylation occurred in both f1 and f2a2 as cells synchronously entered S phase following removal of hydroxyurea. The relative rates of phosphorylation of both species of histone increased in G₂-rich and metaphase-rich cultures. A small amount of histone f3 phosphorylation was also observed in M-rich cultures which was not seen in G₁, S, or G₂-rich cultures. It is concluded that f1 phosphorylation is not dependent on continous DNA replication. These experiments suggest consideration of the concept that f1 phosphorylation is initiated as a preparation for impending cell division.     

Sensitivity of excision repair in normal human,xeroderma pigmentosum variant and Cockayne's syndrome fibroblasts to inhibition by cytosine arabinoside

Inhibition of the gap-filling, polymerizing step of excision repair by 1-β-D-arabinofuranosylcytosine (ara-C) after irradiation with ultraviolet light in human diploid fibroblasts resulted in the formation of persistent DNA strand breaks in G₁, G₂, and plateau phase cells, but not in S phase cells. Addition of hydroxyurea to ara-C resulted in partial inhibition of repair in S phase cells. These observations can be explained either in terms of changing roles in repair for different DNA polymerases throughout the cell cycle or by the presence of a pool of deoxycytidine nucleotides during S phase equivalent to an external source of deoxycytidine at 50 μM concentration. A similar concentration dependence on ara-C was observed for inhibition of repair in normal human, xeroderma pigmentosum (XP) variant, and Cockayne's syndrome cells. Ara-C produced a similar number of breaks in normal and Cockayne's syndrome cells but slightly more in XP variant cells. Exonuclease III and S1 nuclease independently both degraded about 50% of the ₃H-thymidine incorporated into repaired regions in the presence of ara-C. Sequential digestion with both enzymes degraded nearly 90% of the repaired regions. These observations can be explained if excision repair proceeds by displacing the damaged strand so that both the ₃H-labeled patch and the damaged region are still ligated to high molecular weight DNA and compete for the same complementary strand during in vitro incubation with the nucleases. The amount of ₃H-thymidine incorporated in DNA by repair decreased with increasing concentrations of ara-C and hydroxyurea, suggesting that the incomplete patches became shorter under these conditions. Extrapolation of the digestion kinetics with exonuclease III permits an estimate of the normal patch size of about 100 nucleotides, consistent with previous estimates.     

CELL CYCLE-SPECIFIC CHANGES IN HISTONE PHOSPHORYLATION ASSOCIATED WITH CELL PROLIFERATION AND CHROMOSOME CONDENSATION

Preparative polyacrylamide gel electrophoresis was used to examine histone phosphorylation in synchronized Chinese hamster cells (line CHO). Results showed that histone f1 phosphorylation, absent in G₁-arrested and early G₁-traversing cells, commences 2 h before entry of traversing cells into the S phase. It is concluded that f1 phosphorylation is one of the earliest biochemical events associated with conversion of nonproliferating cells to proliferating cells occurring on old f1 before synthesis of new f1 during the S phase. Results also showed that f3 and a subfraction of f1 were rapidly phosphorylated only during the time when cells were crossing the G₂/M boundary and traversing prophase. Since these phosphorylation events do not occur in G₁, S, or G₂ and are reduced greatly in metaphase, it is concluded that these two specific phosphorylation events are involved with condensation of interphase chromatin into mitotic chromosomes. This conclusion is supported by loss of prelabeled ³²PO₄ from those specific histone fractions during transition of metaphase cells into interphase G₁ cells. A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests involvement of f1 phosphorylation in chromatin structural changes associated with a continuous interphase "chromosome cycle" which culminates at mitosis with an f3 and f1 phosphorylation-mediated chromosome condensation.     

Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis

Summary Microspectrophotometry following Feulgen staining and autoradiography following (³H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G₂ phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G₁ (=G₀). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G₁ arrest in the cell cycle of the vegetative cell.     

S phase synchrony in monolayer CHO cultures

Parameters are described for reproducible S phase synchrony of Chinese hamster ovary cells growing in monolayer, adapting a method described by Tobey & Crissman [1] for CHO cells growing in suspension culture. Cells are collected at the G1/S boundary in hydroxyurea after reversal of an early G1 block induced by isoleucine deprivation. The entire population enters the S period within 60 min after removal of hydroxyurea and proceeds through the S period with minimal decay of synchrony, as evidenced by autoradiographic and rate studies on [³H]TdR uptake. In addition, a method is described for obtaining cells synchronized during two successive S periods. The presence of hydroxyurea during G1 does not measurably affect the rate of uptake of [³H]uridine or [³H]leucine into TCA-insoluble material; however, cultures released from the hydroxyurea block at 10 h incorporate slightly more [³H]uridine (but not [³H]leucine) in the next 6 h than cultures maintained in hydroxyurea over this interval. Delaying entry into S with hydroxyurea for as long as 15 h does not significantly change the initial rate or duration of DNA synthesis upon removal of hydroxyurea, arguing against the build-up of substances responsible for initiation of replicons. Furthermore, if DNA synthesis is delayed with hydroxyurea in one cell cycle, a constant minimal interval of 15 h elapses before the population enters into the next S phase, suggesting that the timing of the S period is coupled to the timing of the previous S.