DNA replication in mammalian cells after exposure to factors of a physical, chemical or biological nature. III. 5-Fluorodeoxyuridine induces DNA synthesis in cells not suppressed by gamma irradiation

The influence of preincubation of HeLa and Chinese hamster V79 cells with fluorodeoxyuridine (FUdR, 10(-6) M) on DNA replication and molecular weight of nascent DNA was studied after gamma-irradiation with a dose as much as 10 Gy. The 60Co-radiation inhibits DNA synthesis in both HeLa and V79 cells by 30-40 per cent. The incubation with FUdR before irradiation suppresses the inhibitory effect of irradiation on DNA synthesis. It is suggested that differences in gamma-radiation inhibition of DNA synthesis may result from the FUdR-induced changes in chromatin structure, rather than from synchronization of cell growth. This suggestion is based on the observation that the radioresistant mode of DNA synthesis occurred 18 hours following the short-term (6 hours) incubation with FUdR in cell cultures differing from each other in almost 2-fold their cell longevity.     

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.     

Effects of thymidine analogues on murine and human cells.

Three mouse tumour cell lines grew continuously in 3 micro M 5-bromodeoxyuridine (BUdR). One line (MC-2) produced a retrovirus and altered in morphology in the presence of BUdR or 5-iododeoxyuridine (IUdR). These effects, which could be reversed by growth in normal medium were similar to those reported for the B-16 mouse melanoma line. The B-16 line used in this study, however, as well as a variety of human cells (six melanoma lines and three fibroblast strains), were much more sensitive to BUdR, 0.03-0.1 micro M being the maximum tolerated levels for continuous growth. No virus production or changes in morphology were induced in these cells by BUdR, deoxyuridine (UdR), 5-fluorodeoxyuridine (FUdR) or thymidine (TdR). The results of cell labelling and growth studies showed a correlation of incorporation of BUdR into DNA with toxicity. Compared on a competitive basis with 1 micro M TdR, the order of incorporation of 1 micro M nucleosides by two human cell lines was TdR = BUdR = IUdR greater than UdR greater than FUdR. In contrast to previous reports that FUdR is incorporated into RNA but not into DNA, half of the FUdR label was found in alkalistable, DNase-sensitive material. Over 90% of the other compounds was incorporated into DNA. All of the UdR and 60% of the IUdR label was incorporated as thymidine; this conversion could be inhibited by labelling in the presence of FUdR.     

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 X 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.     

EVIDENCE FOR CYTOPLASMIC DNA IN ROOT CELLS OF NICOTIANA

Sterile root cultures from Nicotiana tabacum were grown with H³-thymidine added to the medium for various intervals. Incorporation of the labeled nucleoside into nuclear DNA occurred in a fraction of the nuclei which increased with time. In addition, the cytoplasm of all cells incorporated enough tritium to be readily detected by autoradiography. The tritium was not removed by hydrolysis in 1 N HCl at 60°C for 10 minutes, but was removed by digestion in a DNase solution which also removed nuclear DNA. The amount of tritium in the cytoplasm increased during the first 2 hours, but did not appear to increase significantly during the following 5 hours. If the roots were transferred to unlabeled medium after 2 hours, the label was diluted faster than expected by growth without turnover of the labeled component. If FUdR was added to the unlabeled medium, the depletion occurred faster during the first 6 hours, but later appeared to level off so that at 10 hours these cultures did not differ from those incubated without FUdR. However, the addition of an excess of unlabeled carrier had no effect on the rate of depletion of the cytoplasmic label. Actinomycin D, which inhibited the incorporation of H³-cytidine into RNA in the root tips, had no effect on the incorporation of H³-thymidine into the cytoplasmic component. However, Mitomycin C or a high concentration of deoxyadenosine inhibited the incorporation of H³-thymidine into the cytoplasmic component as well as into the nuclear DNA. It is concluded that H³-thymidine is incorporated into a cytoplasmic fraction which has the characteristics of DNA, with a measurable rate of turnover. This fraction is synthesized regardless of whether or not the nucleus is synthesizing DNA. Although the function of cytoplasmic fraction is not yet known, it does not appear to be that of supplying precursors for the synthesis of the nuclear DNA.     

Reorganization of DNA replicating system induced by 5-fluorodeoxyuridine and caffeine as the basis for radiosensitivity in human calls

Treatment of diploid human fibroblasts with 5-fluorodeoryuridine (FUdR) and caffeine results in the increase in cellular radiosensitivity in terms of survival and chromosomal aberrations, on the one hand, and in radioresistant DNA synthesis (RDS), on the other hand, i.e. rather mimics those in mutant cells from patients with AT, XPII, Down syndrome, and others. A study was made of the autoradiographic length of simultaneously active adjacent replicon clusters. After incubation of diploid human cells with FUdR (10(-6) M, 6 h), this parameter was shown to reduce by two-fold, remaining unchanged upon 5 Gy irradiation. In contrast, after incubation of the cells with caffeine (2 mM, 30 min), this parameter was longer, compared to that in intact cells; upon 5 Gy irradiation the values remained almost the same as in the control. A possible relation of the data to the cellular radiosensitivity and RDS in the cells incubated with FUdR and caffeine is discussed.     

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.     

Repair replication in human cells. Simplified determination utilizing hydroxyurea.

A simplified and shortened procedure has been developed for the determination of repair replication of DNA in cultured mammalian cells. The procedure, using the bromodeoxyuridine density label and a radio-isotopic label has been applied to normal diploid human cells (WI38) and to their SV40 transformants (VA13). After incubation with the repair label the cells are lysed and digested for two hours at 50 degrees C with proteinase K. This digest can then be immediately subjected to alkaline cesium chloride density gradient centrifugation with no need for DNA extraction. Hydroxyurea is used to reduce the level of semi-conservative synthesis that a quantitative determination of repair replication can be accomplished by a single centrifugation. The method is not affected by variation in the effectiveness of the inhibitor although a small amount of semi-conservative synthesis normally occurs in the presence of the drug. The time course of repair replication in WI38 cells is unaffected by the drug. The apparent amount of repair synthesis in ultraviolet irradiated cells is increased 25 to 40% in the presence of hydroxyurea when thymidine is used as tracer. Under certain conditions in which the level of semiconservative synthesis is low (e.g., contact inhibited cells, high ultraviolet doses) the use of hydroxyurea is unnecessary.     

DNA replication in mammalian cells after the action of physical, chemical or biological factors. VI. The recovery of DNA synthesis in a culture of irradiated cells

The kinetics of DNA synthesis restoration in cultured HeLa cells and in L-929 mouse fibroblasts irradiated by gamma-rays of 60Co with a dose of 10 Gy was studied. Early after irradiation the rate of DNA synthesis in HeLa cells measured with 3H-thymidine incorporation was seen to decrease. Two hours later the incorporation starts to increase to reach the control level 4 hours after irradiation and then becomes even higher than this level. The distribution of cells among phases of the cell cycle measured with flow cytometry undergoes changes. 4-6 hours after irradiation part of S-phase cells increased contributing presumably to the elevating of 3H-thymidine incorporation observed at this time. The restoration of the incorporation was suppressed by inhibitors of protein and RNA synthesis--cycloheximide and actinomycin D. It is suggested that the processes of restoration of DNA synthesis in irradiated cells can be of inducible nature. In irradiated HeLa and L-929 cells the restoration of DNA synthesis is resistant to novobiocin, an inhibitor of DNA replication.     

Effects of inhibitors of DNA synthesis on the stimulation of mouse erythroid cells by erythropoietin

In cultures of foetal mouse liver cells the acceleration of haemoglobin synthesis provoked by erythropoietin is inhibited by FUdR, cytosine arabinoside, hydroxyurea and BUdR, even when these have little effect on either the basal rate of DNA synthesis or on the increase which occurs soon after the addition of erythropoietin to the medium. This seems to be correlated with an inhibition of the production of macro-erythroblasts and macrocytes which normally follows treatment of these cells with erythropoielin.     

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.     

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.     

Repair Replication and Photorepair of DNA in Larvae of DROSOPHILA MELANOGASTER

Repair replication of DNA has been studied in first instar larvae of Drosophila melanogaster with isopycnic centrifugation techniques. Larvae were fed BUdR, FUdR, streptomycin, penicillin, and Fungazone for two to four hours prior to exposure to UV, X-rays, MMS, or EMS. Feeding was continued for four hours in the presence of (3)HBUdR and DNA was isolated from whole larvae. Repair replication is stimulated by each of these agents. MMS is about 10 times as potent as EMS in stimulating repair synthesis. A dose of 200 ergs/mm(2) largely saturates the level of repair replication observed after UV irradiation. Repair replication rises between 0 and 80,000 R of X-rays before falling off. Semiconservative synthesis is seriously inhibited above a dose of 40,000 R of X-rays. Photorepair has been detected as a reduction in repair synthesis resulting from post-irradiation exposure to photoreactivating light. The same treatment has no detectable effect on X-ray-stimulated repair replication. Repair replication is insensitive to the presence of caffeine or hydroxyurea during the final incubation, although semiconservative synthesis is strongly inhibited by these agents. A mixture of BUdR and (3)HTdR can be used to replace (3)HBUdR in detecting repair replication.     

Effect of cytochalasin D on DNA synthesis in cultured cells

The effect of cytochalasin D (CD), an agent specifically destroying actin cytoskeleton, on DNA replication of cultured mouse embryonic fibroblasts (MEF) and BALB/3T3 strain cells was studied. Incubation of normal cells with CD resulted in progressive inhibition of DNA synthesis: in the first 16-20 h the percentage of cells pulse-labelled with 3H-thymidine was similar to that in control cultures, on day 8 the percentage of labelled cells was 7-8 times lower than in the control. The transfer of cells into fresh medium upon 8-day incubation in the presence of CD resulted in the recovery of DNA synthesis. Similar curves of DNA synthesis inhibition in the presence of CD and of DNA synthesis recovery in fresh medium were observed both in mononuclear and binuclear cells. Thus, CD-induced reorganization of actin cytoskeleton can have an abrupt but reversible disturbing effect on normal cell cycle.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli DNA

Postreplication DNA repair (PRR) in UV-irradiated Escherichia coli WP2 uvrA (tryptophan-dependent strain) and K12 AB1886 uvrA6 pre-irradiated by gamma-rays in low doses (radioadaptation, the first stress effect) has been investigated. PRR was found to be more effective after incubation in the growth medium (for 45-60 min) than in non-radioadapted cells: the repair of postreplication gaps increased by 6-15%. If cells of WP2 uvrA strain were incubated after UV-irradiation in media lacking tryptophan or casamin acids (the second stress effect), PRR was seen to increase as early as within 15 min of incubation and it is more effective than at the first stress. After a 30-60 min incubation the double stress effect leads to an increase in postreplication gap repair by 23-45%. In this case almost all the gaps prove to be repaired. The second stress alone exerts no influence on PPR efficiency. It is supposed that a preliminary radioadaptation may stimulate synthesis of a protein (proteins) of the SOS-response (presumably DNA polymerase V). The second stress effect apparently induces synthesis of an unknown factor (or depreesses synthesis of a MmrA-like protein), and this in cooperation with a protein newly synthesized during radioadaptation significantly increases the efficiency of PPR.     

Stimulation of DNA synthesis in senescent human cells following incubation with plasma membranes

DNA synthesis and mitosis were increased in mitogen-stimulated senescent WI-38 cells following incubation with plasma membranes prepared from young or senescent WI-38 cells, A431 cells, 3T3 cells, or NR6 cells. The percentage of [3H]thymidine-labeled nuclei in senescent cultures was two- to fivefold greater than that seen in controls in which cells were incubated in the absence of membranes or in the presence of boiled membranes. The effect was trypsin sensitive, suggesting that a protein moiety is necessary for stimulation of DNA synthesis. As the culture age increased, basal levels of DNA synthesis, as well as maximal stimulation of DNA synthesis following incubation with plasma membranes, decreased. These observations are consistent with the hypothesis that different subpopulations exist in senescing cultures and suggest a complex pattern of inhibitory and stimulatory regulation of cell proliferation.     

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.     

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     

DNA photobinding of 7-methylpyrido[3,4-c]psoralen and 8-methoxypsoralen. Effects on macromolecular synthesis, repair and survival in cultured human cells

The photobinding to DNA of tritiated 7-methylpyrido[3,4-c]psoralen (MPP), a recently synthesized monofunctional compound of therapeutical interest, and of 8-methoxypsoralen (8-MOP) was determined in cultured normal human fibroblasts. Employing compounds at 10(-6) M, MPP photobinds approximately 11 times more efficiently than 8-MOP: one molecule is fixed respectively per 7.5 X 10(4) or 8.1 X 10(5) base pairs/kJ . m-2 of 365-nm radiation (UVA). Removal of bound material from DNA is slow and limited in 48-72 h of post-treatment incubation to 30-40% of initial adducts formed by MPP and to 50-60% of those of 8-MOP. For equivalent photobinding MPP and 8-MOP induce similar inhibitions of DNA synthesis. However, the recovery of DNA synthesis during post-treatment incubation is lower after photoaddition of MPP than after that of 8-MOP. MPP also exerts a much higher lethal effect than 8-MOP: one lethal hit corresponds to about 4400 and to 19,900 adducts per cell respectively. Alkaline elution experiments confirmed the monofunctional nature of MPP and indicated that in MPP-damaged cells DNA breaks accumulate with time of post-treatment incubation. In contrast, after photoaddition of 3-carbethoxypsoralen (3-CPs), another monofunctional furocoumarin, or irradiation with 254-nm UV, DNA breaks are induced only transiently. In 8-MOP-treated cells, DNA cross-links appear to be partially repaired. In conclusion, MPP monoadducts turn out to constitute more cytotoxic lesions than 8-MOP mono- and bi-adducts.     

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.