The action of 5-amino uracil on log growth and division-synchronized Tetrahymena

The influence of 5-amino uracil (5-AU) was investigated on the cell cycle of log growth and division-synchronized Tetrahymena pyriformis GL. The division index of log growth phase Tetrahymena was suppressed by 50% after 40 min in 8 mM 5-AU. Cells division-synthronized by one heat shock per generation were also treated with 5-AU. Cells treated either prior to the first synchronous division (80 min EH) or up to 25 min prior to the second synchronous division (after 160 min EH) were not delayed in their progress through the cell cycle. Cells treated during the S phase of the first free running cell cycle, however, were delayed 5-30 min from reaching the second synchronous division. The effect of 5-AU on DNA and RNA synthesis was also examined. Incorporation of [3H]thymidine into acid-precipitable material was reduced in the presence of 5-AU; the rate of DNA synthesis was also reduced. The depression in the rate of DNA synthesis was greater at the beginning of S than at the end of S. The size of the thymidine pool (nucleosides + nucleotides) did not change during 5-AU treatment; however, an accumulation of thymidine tri-phosphate and a decrease in the amount of thymidine nucleoside was observed. A suppression of [14C]uridine incorporation resulting from 5-AU treatment was observed throughout the cell cycle. The rate of RNA synthesis as monitored by [14C]uridine incorporation into acid precipitable material was also reduced during 5-AU treatment. No change in either the size or the composition of the pool of uridine (nucleoside + nucleotide) was detected in 5-AU treated cells as compared to controls.     

DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures

Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 m. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4–6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4–6S) and large (20–30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm³. Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 m.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.Abbreviations BUdR 5-Bromodeoxyuridine - FUdR 5-Fluorodeoxyuridine     

Inhibition of Synchronized Cellular Deoxyribonucleic Acid Synthesis During Newcastle Disease Virus, Mengovirus, or Reovirus Infection

Cultures of L cells were synchronized with respect to deoxyribonucleic acid (DNA) synthesis with thymidine and 5-fluoro-2'-deoxyuridine (FUdR) and infected with Newcastle disease virus (NDV), mengovirus, or reovirus 3. Inhibition of incorporation of (3)H-cytidine into the DNA of synchronized cells is partially inhibited 2 hr after infection with NDV or mengovirus and nearly completely suppressed 4 hr after infection. With NDV and mengovirus, no evidence was obtained of differences in sensitivity of cells during early S phase as compared to later stages in DNA synthesis. When cells were infected with reovirus at the time of release from FUdR block, inhibition of cellular DNA synthesis was evident at 2 to 3 hr, and it was complete at 4 to 5 hr after infection. However, when cells were infected several hours prerelease, synthesis of DNA occurred in early S phase in spite of the fact that the cells had been infected for up to 6 hr. The results indicate that DNA synthesis in early S phase is relatively insensitive to the inhibitory function of reovirus. Colorimetric determinations (diphenylamine reaction) of the amounts of DNA produced in synchronized cells have substantiated the inhibition of DNA synthesis observed by isotope incorporation techniques.     

Lethal effects of fluorodeoxyuridine on cultured mammalian cells at various stages of the cell cycle

The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10^?5 M FUdR was very low (0.0001–0.0003) whether the drug was administered during early G₁, late G₁, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10^?7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.     

INHIBITION OF MYOBLAST FUSION AFTER ONE ROUND OF DNA SYNTHESIS IN 5-BROMODEOXYURIDINE

The thymidine analogue 5-bromodeoxyuridine (BUdR) has a differential effect on the synthesis of tissue-specific products and molecules required for growth and division. Proliferating myogenic cells cultured in BUdR fail to fuse and fail to initiate the synthesis of contractile protein filaments. Conversely, BUdR has but a minor effect on cell viability and reproductive integrity. Low concentrations of BUdR result in an enhancement of cell number relative to the controls; higher concentrations are cytotoxic. Suppression of myogenesis is reversible after at least 10 cell generations of growth in the analogue. Cells that do not synthesize DNA, such as postmitotic myoblasts and myotubes, are not affected by BUdR. Incorporation of BUdR for one round of DNA synthesis was accomplished by first incubating myogenic cells, prior to fusion, in 5-fluorodeoxyuridine (FUdR) to block DNA synthesis and collect cells in the presynthetic phase. The cells were then allowed to synthesize either normal DNA or BU-DNA for one S period by circumventing the FUdR block with BUdR or BUdR plus thymidine (TdR). The cultures were continued in FUdR to prevent dilution of the incorporated analogue by further division. After 3 days, the cultures from the FUdR-BUdR series showed the typical BUdR effect; the cells were excessively flattened and few multinucleated myotubes formed. Cells in the control cultures were of normal morphology, and multinucleated myotubes were present. These results were confirmed in another experiment in which BUdR-³H was added to 2-day cultures in which myotubes were forming. Fusion of thymidine-³H-labeled cells begins at 8 hr after the preceding S phase. In contrast, cells which incorporate BUdR-³H for one S period do not fuse with normal myotubes.     

The relationship of DNA synthesis to protein accumulation in the cell nucleus

The relationship between DNA synthesis and protein accumulation in cell nucleus and cytoplasm has been investigated by the use of a combination of ultramicrointerferometric and ultramicrospectrophotometric methods. 5-Fluoro-2'-deoxyuridine (FUdR) inhibited DNA synthesis, resulting in inhibition of cell proliferation in G-1 and early S-phase. However, synthesis and accumulation of protein continued in the presence of FUdR, as indicated by a 54% increase in the average dry mass value per individual cell during 18-hour exposure to FUdR; due primarily to protein accumulation in the cytoplasm, the average cytoplasmic dry mass increased by as much as 85%, while the dry mass of the nucleus increased by only 21%. The dry mass values of individual nuclei were well-correlated to the nuclear DNA content throughout the period of exposure to FUdR. In contrast to the continued accumulation of protein in the cytoplasm during inhibition of DNA synthesis, protein accumulation in the nucleus was inhibited. When cells were released from inhibition of DNA synthesis by the addition of 2'-deoxythymidine, the nuclear DNA content and nuclear dry mass increased in near-synchrony, there being some evidence that DNA synthesis was initiated somewhat prior to initiation of increase in nuclear dry mass. Thus, it appears that DNA synthesis (or an increase in nuclear DNA content) is intimately related to the regulation of protein accumulation in the nucleus.     

DNA synthesis in growth and encystment of Acanthamoeba castellanii.

Differentiation of Acanthamoeba castellanii into dormant cysts occurs spontaneously in stationary phase cultures, or can be induced experimentally by starvation. Although no further increase in cell density occurred after induction in either case, incorporation of [H]thymidine into DNA continued at a reduced rate through the period when differentiated products (cyst wall components) were formed. No net accumulation of DNA occurred during differentiation, indicating that the DNA synthesis occurring at this time was balanced by breakdown. When either 5-fluorodeoxyuridine (FUdR) or hydroxyurea was added to exponentially growing cultures, growth was terminated and the subsequent spontaneous encystment was delayed in comparison with untreated stationary phase cultures. A similar delay was observed for experimentally induced encystment of FUdR-pretreated cells. In all cases, delay of encystment was correlated with inhibition of 32PO4 incorporation into DNA, and unexpectedly also into RNA. Addition of FUdR at zero-time of experimental induction of cells not previously exposed to FUdR, on the other hand, had no effect on encystment or on 32PO4 incorporation. The delay of encystment produced by FUdR and hydroxyurea, therefore, appeared to reflect a requirement for normal synthesis of DNA and/or RNA not only during encystment, but also during the period of exponential growth just before encystment induction.     

Decreased Sensitivity to Hydroxyurea and to [3H]Thymidine Suicide In the Middle of the S Phase

Pluripotent haemopoietic stem cells (CFUs) move synchronously through the cell cycle in hydroxyurea-treated mice in a cohort 1–2 hr broad. Ten to fifteen hours after hydroxyurea they pass through S phase. DNA synthesis appears to be depressed 5–10 times when the cells are in the middle part of the S phase but does not seem to be completely interrupted. High concentrations of [³H]thymidine must be used for ‘suicide’ in order to achieve lethality for the cells with depressed DNA synthesis. At the time when DNA synthesis is depressed, the sensitivity of the cells to hydroxyurea also decreases. This may lead to a significant underestimation of the S phase fraction by the hydroxyurea method, because CFUs with low DNA synthesis rate are resistant to hydroxyurea although being in S phase.     

Differential sensitivity to 5-bromodeoxyuridine during the S phase of synchronized myogenic cells

Synchronized myogenic cell cultures have been used to demonstrate differential sensitivity to BUdR during segments of the S period. Synchronization of the cells was achieved by two methods. First, cells were initiated in medium containing FUdR, an inhibitor of DNA synthesis. Following FUdR blockade reversal with TdR after 19 hr in vitro, the synchronized cells were allowed to replicate their DNA with BUdR for periods corresponding to early and late S. Determinations of percentage labeled cells during synchronization with FUdR indicate that about 90% of the cycling population of cells accumulates at the G1/S interface of the cell-cycle and that the duration of the S period following blockade reversal with TdR is not altered. Since BUdR is pulsed to these cultures immediately after the point of synchronization, a high degree of synchrony is obtained. In the second method of synchrony, cohorts of cells which had been in G2, late S, or early S during a BUdR pulse were collected in metaphase arrest with Colcemid and selectively removed from the cultures. With the mitotic selection method the point of synchronization occurred several hours after the BUdR pulse. In both methods the cells were allowed to resume myogenesis and scored for percentage fused nuclei after approx 50 hr in vitro. With both methods of synchrony, BUdR incorporation into early replicating DNA results in a striking decline in myoblast fusion, whereas incorporation into late replicating DNA is without effect. The results cannot be attributed to a disproportionate uptake of nucleotide during early S. Further fractionation of the 4-hr S phase into 1-hr periods indicates that the BUdR sensitive target is replicated during the second hr of DNA synthesis.     

RATE AND TIME OF DNA SYNTHESIS OF INDIVIDUAL CHINESE HAMSTER CELLS

The duration of DNA synthesis of a diploid cell line of Chinese hamster fibroblasts was determined in a comparative study by the FLM technique, and also by a new technique for measuring the rate of DNA synthesis of individual cells. These methods produced comparable results when applied during exponential growth of the cells. The rate of DNA synthesis was measured by means of quantitative autoradiography following a short-term incubation of the cells with 5 × 10^-6 M FUdR and 10^-5 M ¹⁴C-TdR. The choice of the medium for this purpose did not seem to be critical. The autoradiographic silver grains over cells and ¹⁴C-standard sources are counted by microphotometry using incident light bright-field. The direct measurements of DNA synthesis rate are ‘compartment’ statistics which have been converted into ‘flux’ parameters for comparison with the FLM method and applicability in cell-kinetic calculations. Frequency distributions of the rate of DNA synthesis of individual cells thus obtained may resemble normal distributions quite closely. They result from several factors: differences in the rate of synthesis in different parts of the S-phase, the density distribution of cells within the S-phase, the variation in the time of DNA synthesis among individual cells, and the experimental error. In the case of a pronounced partial synchronization as probably has been present in one experiment performed in the lag phase, an incorrect time of DNA synthesis may result from the rate values. Due to the variation in DNA synthesis rate in different parts of the S-phase it is not possible to determine the duration of DNA synthesis of an individual cell. However, the mean values of DNA synthesis time are reliable. The new method will be preferentially applied for determining the duration of DNA synthesis of human cells in as far as difficulties are encountered with the classical methods. In addition, it may be used to advantage for studying cells which make up low percentages in mixed populations. It finally permits a safer morphological classification of the cells under study than is possible with the classical methods.     

Mechanism of mitotic synchronization induced by 5-aminouracil.

The mechanism of mitotic synchronization induced by 5-aminouracil (5-AU) in onion root meristems has been investigated. We studied the effect of the chemical upon asynchronous meristematic cells, as well as upon a synchronous fraction of them the position of which in the cell cycle was known. The kinetic studies have shown a fairly linear relationship between the treatment time and the induced mitotic wave. Therefore, cell accumulation in a particular zone of interphase appears to be the cause of mitotic synchronization. This zone seems to be located at the end of S period. 5-AU induces maximal mitotic delay on the cells located in the accumulation zone and severely depresses the S-G2 transit while the passage through G 2 remains relatively unaffected. The possibility that preferential inhibition affects the late replicating DNA synthesis is discussed.     

DNA replication in mammalian cells exposed to the action of physical, chemical or biological factors. IV. 5-fluorodeoxyuridine modifies the effect of hyperthermia on DNA synthesis and the survival of HeLa cells

Preliminary incubation of logarithmically growing HeLa cells with FUdR decreases an inhibitory effect of hyperthermia (43 degrees C, 1 hour) on DNA synthesis. The hyperthermia alone inhibits DNA synthesis considerably: the label in acid-precipitable material accounts for 30% of control level. Preliminary incubation of the cells with FUdR (10(-6)) for 24 or 6 hours (plus 18 hours in fresh medium) decreases the effect: the label yields account for 50 or 90% of the respective control levels. A molecular weight of nascent DNA synthetized in the cells after hyperthermia or incubation with FUdR is lower than the control one but it increases rapidly during postincubation. Nucleoid of cells treated with FUdR has a sedimentation velocity which exceeds that of the control cells by more than 25%. Preliminary incubation with FUdR sensitizes the cells to hyperthermia. The effect is not believed to be associated with cells synchronization since the treatment of the cells with FUdR for 2 or 6 hours, when FUdR itself does not exert its toxic effect, brings about sensibilization of cells to hyperthermia. It is suggested that modification of the cell viability and DNA replication are related to some changes of chromatine structure induced by FUdR.     

DNA replication in mammalian cells after the action of factors of a physical, chemical or biological nature. V. The structural reorganization of replicating DNA in HeLa cells after incubation with 5-fluorodeoxyuridine

A study was made of sedimentation properties of the nucleoid (chromatin) of HeLa cells with radio- and thermostable mode of DNA synthesis induced by 5-fluorodeoxyuridine (FUdR). After the incubation of HeLa cells with FUdR (10(-6) M, 6 h or 24 h) the rate of nucleoid sedimentation was shown to rise by 40 and 25%, respectively. Maximum relaxation of the nucleoid was observed under 5 mg/ml ethidium bromide concentration in sucrose gradients. After the incubation with FUdR the nucleoid relaxes to a lesser extent, and after irradiation its response to ethidium bromide in various concentrations was similar to that of intact nucleoid, and by this property the "FUdR nucleoid" differs essentially from the irradiated "normal nucleoid". A model of chromatin structure of cells exposed to FUdR is proposed, based on the transformation of large domains in small ones, for the explanation of radioresistant DNA synthesis.     

An artifact in measurement of S phase initiation and its implication for the kinetics of S phase-specific enzyme activities

It is shown that the different onset of S phase as measured by autoradiography vs cumulative thymidine uptake is an artifact. We consequently propose that S phase-specific enzyme activities may accumulate a few hours prior to the actual initiation of DNA synthesis. A “pre-S” DNA synthesis that can be readily detected only by autoradiography has been proposed. Published data show that DNA synthesis in cultured animal cells is initiated approx. 2 h later when measured by cumulative incorporation of [³H]thymidine ([³H]TdR) as compared with autoradiography. We show here that the difference is in reality an artifact, owing to not taking into account both gradual, asynchronous entry of cells into S phase, as well as time-dependent accumulation of radioactivity into each cell after it has entered S phase. Combination of these two factors leads to the conclusion that [³H]TdR should be incorporated approximately as the square of time following entry of the first cell into S. Taking this into account, the two methods then are in agreement, as predicted. This argument also applies to the enzyme activities shown to increase with DNA synthesis in synchronized cultures. Such an enzyme accumulation really could begin some time earlier than indicated by conventional plots of cumulative enzyme activity vs time and may, in fact, precede the onset of S by a few hours.     

Relationship between Tracheary Element Differentiation and DNA Synthesis in Single Cells Isolated from the Mesophyll of Zinnia elegans--Analysis by Inhibitors of DNA Synthesis

Effects of inhibitors of DNA synthesis on tracheary element(TE) differentiation were investigated in a culture of singlecells isolated from the mesophyll of Zinnia elegans L. cv. Canarybird. In this system, neither mitosis nor replication of thewhole genome during the S phase in the cell cycle is a prerequisitefor TE differentiation [Fukuda and Komamine (1980) Plant Physiol.65: 61, unpublished data]. Fluorouracil (FU), fluorodeoxyuridine(FUdR), mitomycin G (MC), arabinosyl cytosine (ara-C) and aphidicolin,inhibitors of DNA synthesis, prevented the incorporation of[³H]-thymidine into nucleic acid, cell division and cytodifferentiationto TE. However, neither FUdR nor aphidicolin prevented the incorporationof [14C]-leucine into protein. Thymidine reversed the inhibitoryeffect of FUdR when given simultaneously with FUdR. These resultsshow that the inhibitors of DNA synthesis prevent TE differentiationvia blockage of the synthesis of some DNA, although replicationof the whole genome during the S phase is not a prerequisitefor cytodifferentiation. The role of DNA synthesis in TE differentiationis discussed. (Received October 13, 1980; Accepted November 17, 1980)     

Cytological demonstration of DNA synthesis inhibition at late S by 5-amino-uracil

Meristematic cells of Allium cepa were treated with 5-amino-uracil (5-AU) while incorporating 5-bromodeoxyuridine (BrdU) into DNA until complete inhibition of mitosis was obtained. The pattern of BrdU substitution in interphase nuclei detected by FPG technique in the cells so treated was that known as corresponding to BrdU incorporation in only early and middle replicating DNA. In addition, when the cells were allowed to reach mitosis in the absence of BrdU after the synchronization induced by 5-AU all the metaphase chromosomes showed, likewise, the typical late-replicating pattern. These results are a cytological demonstration of a preferential inhibition of late-replicating DNA synthesis by 5-AU proposed by several authors.     

Premature chromosome condensation in a ts DNA-mutant of BHK cells

A temperature-sensitive mutant of BHK, designated is BN-2, shows a rapid drop in ³H-thymidine incorporation along with accumulation of the cells in the G1 phase of the cycle when asynchronous cultures are shifted from 33.5°C to the nonpermissive temperature of 39.5°C. Synchronized cultures of ts BN-2 cells did not enter DNA synthesis when shifted up in G1. Shift-up of cultures at the beginning of the S phase resulted in an approximately normal rate of DNA synthesis for about 2 hr. The rate of DNA synthesis then quickly declined, and the cells became arrested in mid-S after completion of approximately 0.5 rounds of DNA replication. At the same time, the majority of the cells were observed to lose the nuclear membrane and displayed premature chromosome condensation. These events were followed by the appearance of cells containing several micronuclei and eventual cell disruption and death. The nonpermissive temperature appeared to have no effect on either the elongation of short fragments of DNA or the execution of mitosis after the completion of the S phase under permissive conditions. The ts defect in this mutant may directly limit the initiation of DNA synthesis or alter the regulation of chromatin condensation.     

Strukturverändernde Wirkung von FUdR auf polytäne Chromosomen und Beziehungen zwischen Replikationsdauer und Bruchhäufigkeit von Querscheiben

Larvae of Chironomus th. thummi at the age of 10 hours after hatching were treated for 20 hours with 10^–4 M FUdR. The salivary gland chromosomes were studied at fourth instar. FUdR induces chromosomal constrictions and partial breakage of various diameters ranging from 1/2 to less than 1/16 of the total cross section of the polytene chromosomes. Breaks were predominantly found in chromosome regions containing bands of high DNA content. By H³-thymidine-autoradiography it is demonstrated that bands which are frequently broken are late replicating. This is shown by histograms correlating the distribution of breaks over the chromosomes with labeling patterns obtained in late S.—As bands with a great amount of DNA do not only replicate late but also spend the longest time in DNA synthesis, it is assumed that they also represent the largest replicons. It is discussed if this is the reason why FUdR induces breaks preferentialy in bands of high DNA content.     

The initiation of melanogenesis in the chick retinal pigment epithelium.

Observations have been made on chick pigment retinal epithelium between 2 and 5 days of development. 2-Thiouracil has been demonstrated to be an effective agent for measuring the rate of melanin synthesis.Using [³H]thymidine and colcimid, we have found that the cells undergo a marked withdrawal from the cell cycle between 3 and 3.5 days of incubation in ovo, indicating that a majority of the population is synchronized. This withdrawal is followed, approximately 24 hr later, by a rapid rise in melanin synthesis from the basal level which first appears at approximately 3 days.5-Bromodeoxyuridine (BUdR) has been used to determine the time at which melanin synthesis is initiated. When BUdR is administered as early as 2 days in ovo, it is incapable of blocking the appearance of basal levels of melanin even though the cells divide at least three times in the presence of this thymidine analog. However, BUdR is capable of delaying the rapid rise in the rate of melanin synthesis first observed at 4.5 days. This delay has been found to correlate, using [³H]BUdR, with a delay in the withdrawal of the cells from the division cycle.In pursuing the idea of a correlation between withdrawal and the rapid increase of melanin formation, 5-fluorodeoxyuridine (FUdR) was used. Histological and biochemical evidence suggests that those cells which have been prevented from dividing by FUdR increase their rate of melanin synthesis to the high level of the postmitotic control cells described above.Therefore, it seems that (1), in light of work done by others, the initial decision to make melanin is made prior to 2 days in ovo, and (2) the mechanism by which cells shift their synthetic capabilities to high levels of melanin production is withdrawal mediated.