Bromodeoxyuridine/DNA analysis of replication in CHO cells after exposure to UV light

The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured asa function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction ( 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed.     

The recovery of normal DNA replication kinetics in ultraviolet-irradiated Chinese hamster cells

The kinetics of DNA replication were analyzed in the second S phase following UV irradiation of Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by treating cells selected in mitosis with hydroxyurea for 9 h. Following UV irradiation, the cells were allowed to progress until the next mitosis; at which time they were resynchronized at the beginning of the second S phase by the same procedure. The kinetics of DNA replication were determined by measuring the proportion of DNA which achieved hybrid buoyant density on CsCl density gradients as a function of the time of incubation in the presence of 5-bromodeoxyuridine.The results of these experiments showed that even though the rate of DNA replication is substantially depressed during the first S phase following UV irradiation with a fluence of 5 J/m², the rate has recovered to the extent that it is indistinguishable from the unirradiated control by the time the cells have entered their second S phase. It was concluded from these observations that the lesions in DNA which caused the rate of DNA replication to be initially depressed during the first S phase have been either removed or modified such that they no longer are able to cause a reduction in the rate of DNA replication in the second S phase following UV irradiation.     

Evaluation of S phase synchronization by analysis of DNA replication in 5-bromodeoxyuridine

The S phase kinetics have been evaluated in cells synchronized with either thymidine or hydroxyurea by direct analysis of the proportion of DNA semi-conservatively replicated as a function of time after release from the inhibitor. The proportion of DNA replicated was determined by growing the cells in medium containing 5-bromodeoxyuridine (BUdR) and subsequently measuring the amount of DNA that acquired increased buoyant density in CsCl gradients. The results confirm previous reports that substantial DNA synthesis occurs during TdR treatment. In contrast, HU provided a population of cells very nearly at the G 1-S interphase since 95 % of the DNA replicated synchronously after its removal. It is proposed that by measuring the rate and maximum extent of DNA replication with BUdR during S phase one can evaluate different synchrony methods for use in experiments designed to study aspects of semiconservative DNA replication.     

Flow cytometric estimation of cell cycle parameters using a monoclonal antibody to bromodeoxyuridine

An estimation of cell kinetic parameters was made by simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdUrd) contents of cells. The procedure described in this paper involves the incorporation of BrdUrd by S phase cells, labeling the BrdUrd with an indirect immunofluorescent technique using a monoclonal anti-BrdUrd antibody, and staining DNA with propidium iodide (PI). The amount of incorporated BrdUrd in HeLa cells was proportional to that of synthesized DNA through S phase. For all cell lines examined, the pattern of BrdUrd incorporation was essentially the same and the rate of DNA synthesis during S phase was not constant. The bivariate BrdUrd/DNA distributions showed a horse-shoe pattern, maximum in the mid S phase and minimum in the early and late S phases. Furthermore, the durations of cell cycle (Tc) and S phase (Ts) were estimated from a FLSm (fraction of labeled cells in mid S phase) curve that was generated by plotting the percentage of BrdUrd pulse-labeled cells in a narrow window defined in the mid S phase of the DNA histogram. The values of these parameters in NIH 3T3, HeLa S3, and HL-60 cells were in good accordance with the reported data. This FCM method using the monoclonal anti-BrdUrd antibody allows rapid determination of both cell cycle compartments and also Ts and Tc without the use of radioactive DNA precursors.     

Cell cycle analysis of synchronized chinese hamster cells using bromodeoxyuridine labeling and flow cytometry

Summary Chinese hamster ovary cells were synchronized into purified populations of viable G1-, S-, G2-, and M-phase cells by a combination of methods, including growth arrest, aphidicolin block, cell cycle progression, mitotic shake-off, and centrifugal elutriation. The DNA content and bromodeoxyuridine (BrdUrd) labeling index were measured in each purified fraction by dual-parameter flow cytometry. The cell cycle distributions determined from the DNA measurements alone (single parameter) were compared with those calculated from both DNA and BrdUrd data (dual parameter). The results show that highly purified cells can be obtained using these methods, but the assessed purity depends on the method of cell cycle analysis. Using the single versus dual parameter measurement to determine cell cycle distributions gave similar results for most phases of the cell cycle, except for cells near the transition from G1- to S-phase and S- to G2-phase. There the BrdUrd labeling index determined by flow cytometry was more sensitive for detecting small amounts of DNA synthesis. As an alternative to flow cytometry, a simple method of measuring BrdUrd labeling index on cell smears was used and gave the same result as flow cytometry. Measuring both DNA content and DNA synthesis improves characterization of synchronized cell populations, especially at the transitions in and out of S-phase, when cells are undergoing dramatic shifts in biochemical activity.     

Differentiation of mitotic melanoma cells from G2 cells and their isolation by use of 5-bromo-2'-deoxyuridine and propidium iodide

This report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5-bromo-2'-deoxyuridine (BrdUrd) and double-stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 micrograms/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti-BrdUrd and PI. PGE1-treated cells monitored at 3-hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti-BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd-negative "S phase" cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move "backwards" in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to over-estimation of S phase cells in kinetic studies because of contaminating mitotic cells.     

Analysis of the replication pattern of Chinese hamster chromosomes using 5-bromodeoxyuridine suppression of 33258 Hoechst fluorescence

Chinese hamster fibroblasts were synchronized and given 5-bromodeoxyuridine for DNA synthesis except during one hour of the S phase when thymidine was present in the medium. In the next mitosis, chromosomes stained with 33258 Hoechst were banded in appearance when photographed by fluorescence microscopy. The bright regions corresponded to the chromosome segments replicated during the thymidine exposure in the S phase. The segments replicated together during any one hour produced three distinct patterns which were characteristic of early, middle, and late S phase. Most of the fluorescent regions corresponded in size and position with G-bands of these chromosomes. There was no correlation between the staining behavior of a band in G-band procedure and its time-of-replication, i.e., both light and dark G-bands were replicated during early, middle, and late S phase. However, it appears that all of the DNA within a single band is replicated together within one third of the S phase.     

Detection of replication initiation by a replicon family in DNA of synchronized pea (Pisum sativum) root cells using benzoylated naphthoylated DEAE-cellulose chromatography

Fractionated replicating DNA from pea was obtained from both synchronized cells just starting replication and from carbohydrate-starved cells ending replication. Benzoylated naphthoylated DEAE-cellulose chromatography of pulse-labeled DNA digested with EcoR I gave evidence that a family of replicons initiated replication 45 to 60 min after synchronized cells were released from the G₁/S phase boundary. DNA from cells labeled in late S phase, on the other hand, showed no signs of additional replication initiations before entering G₂ phase. Results with DNA from both early and late S phase cells comply with a model based on the premise that with short pulses of [³H]-thymidine the isotope is localized at replication forks and that longer pulses label both replication forks and recently replicated segments of double-stranded DNA. The model applies only to DNA subjected to fragmentation before chromatography.The results also suggest that benzoylated naphthoylated DEAE-cellulose chromatography is a useful means to isolate origins and replication forks from synchronized plant cells.     

Evaluation of four methods of DNA distribution data analysis based on bromodeoxyuridine/DNA bivariate data

Four published methods of DNA-content histogram analysis (those of Fried, Dean and Jett, simplified Dean, and Fox) were compared using a double labeling of different cell populations. Partially synchronized and asynchronous cell populations were incubated with bromodeoxyuridine (BrdUrd) and then stained with an anti-BrdUrd monoclonal antibody and propidium iodide (PI). The fractions of cells in the G1, S, and G2 + M phases were calculated by each method and compared with those derived from G1, S, and G2 + M areas plotted on BrdUrd/DNA bivariate histograms, taken as the "true" values. This procedure enabled an optimal choice of method for a given cell population.     

Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions.

An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.     

Early replication signals in nuclei of Chinese hamster ovary cells

Summary DNA replication sites generally known as replicon domains were resolved as individual replication signals in interphase nuclei of permeabilized Chinese hamster ovary cells by immunofluorescent microscopy. Biotin-11-dUTP was utilized as a tool to label newly replicated DNA in permeable cells and to study the distribution of nascent DNA in pulselabel and in pulsechase experiments. Active sites of DNA replication were visualized in exponentially growing cells and in synchronized cultures throughout the S phase. Fluorescent images of replication sites were analyzed by standard fluorescense microscopy and in three dimensions by confocal laser scanning microscopy. The rapid increase in number of discrete foci of newly replicated DNA is an indication that DNA synthesis starts at limited number of sites in mammalian nuclei rather than at thousands of foci at the same time.     

DNA sequence of the Bryan high-titer strain of Rous sarcoma virus: extent of env deletion and possible genealogical relationship with other viral strains

Raji cells, collected at various times from a synchronized culture, were gently lysed, and the high-molecular-weight DNA was enriched ca. 10-fold for latent Epstein-Barr virus (EBV) genomes by equilibrium density gradient centrifugation in neutral CsCl. The heavy-density DNA pool, which included more than 90% of the total intracellular EBV DNA sequences, was further fractionated by velocity sedimentation on neutral glycerol gradients, and material from fractions containing potential EBV DNA replicative forms was examined in the electron microscope. Early in the cellular S phase, when the EBV DNA content was found to be doubling in parallel with host chromosome replication, half of the 50- to 55-micron circular EBV genomes were observed to have two or more DNA branch points or forks. Most molecules were in a relaxed theta configuration, indicative of the Cairns mode of DNA replication. In the supercoiled state, the two daughter strands of the partially replicated molecules were seen to be wrapped around each other. Two theta structures had more than two DNA forks, indicating that DNA replication can initiate more than once on the same DNA molecule. Late in the S phase, the EBV DNA sedimenting at positions where theta structures were found with early S phase samples was composed of catenated dimers rather than partially replicated genomes. It is concluded that the circular EBV genomes, which are the major intracellular form in latently infected cells, are maintained as independent replicons and are not synthesized from an integrated template.     

An analysis of DNA replication in synchronized CHO cells treated with benzo[a]pyrene diol epoxide

Synchronized Chinese hamster ovary (CHO) cells treated with (+/-)7 beta,8 alpha- dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-dihydrobenzo[a]pyrene (BP diol epoxide I) were used to test the 'block-gap' model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 microM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the 'block-gap' model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the 'block-gap' model of replicative bypass repair in carcinogen damaged mammalian cells.     

Changes in nucleosome repeat lengths precede replication in the early replicating metallothionein II gene region of cells synchronized in early S phase

Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. Here we report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated [D'Anna, J. A., & Prentice, D. A. (1983) Biochemistry 22, 5631-5640]. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 micrograms mL-1 aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression.     

Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry.

Hairless mice were injected intraperitoneally with bromodeoxyuridine (Brd-Urd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate-buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cycle progression of a pulse-labelled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H-thymidine labelling studies. The percentage of cells in S phase was highest at night and lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase. Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation-keratin K10 was turned on only in G1 phase and after the last division.     

Excision and replication of extrachromosomal DNA of pea (Pisum sativum).

Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80% of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 X 10(6) to 140 X 10(6) daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 X 10(6) daltons and another of 7 X 10(6) daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 X 10(6) and 7 X 10(6) daltons.     

Gene replication in the presence of aphidicolin

DNA replication in the nucleus of eukaryotic cells is restricted to the S phase of the cell cycle, and different genes are duplicated at specific times, according to a well-defined temporal order. We have investigated whether activation of initiation sites, in proximity to genes that are replicated in different portions of the S phase, could be detected when synchronized 10T1/2 cells were maintained in aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta. Cells released from confluence arrest into medium containing 2 micrograms/mL APC progressed into the S phase, and nascent DNA accumulated during incubations of 24 and 32 h. Exposure to APC for 40 or 48 h resulted in growth of the radiolabeled DNA into larger molecules. Replicating DNA was isolated in CsCl gradients and probed with 32P-labeled gene probes for early-replicating genes (e.g., Ha-ras, mos, and myc) and a late-replicating gene (VH Ig). DNA replicated during the 24-h incubation in APC was enriched in Ha-ras gene sequences. The VH Ig gene did not replicate in cells incubated for as long as 56 h with APC. The myc and the mos genes were detected after 32 and 40 h in APC, respectively. The myc gene is replicated in 10T1/2 cells after Ha-ras but before mos. Therefore, the order of activation of these genes was conserved in the presence of APC. The delay in replication of myc and mos correlated well with the slowing of DNA replication by APC.     

DNA contents of replication without DNA density labeling

A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and "time of replication" for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of less than or equal to +/- 10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5'-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.     

Three-way differential staining of sister chromatids in M3 chromosomes : Evidence for spontaneous sister chromatid exchanges in vitro

Three types of Giemsa differential staining of sister chromatids were observed in HeLa cells when they were exposed continuously to 5-bromodeoxyuridine (BrdUrd) for three replication cycles. In type-1, about a half set of chromosome complements were composed of pairs of darkly-stained and intermediately-stained chromatids; the other half consisted of pairs of intermediately-stained and lightly-stained chromatids. In type-2, one fourth of chromatids was stained darkly and the remaining ones were stained lightly. In type-3, about a half set of chromosomes consisted of the pairs of darkly-stained and lightly-stained chromatids and the rest of pairs of intermediately-stained and lightly-stained chromatids. Cells showing each differentiation pattern at the third mitotic phase were dependent on the stages of the first DNA synthetic (S) phase at which BrdUrd treatments were initiated. Type-1 cells were observed, when BrdUrd treatment was initiated anywhere from G1 to early S phase, type-2 when treatments were begun in middle S stage, and type-3 when treatments were initiated in the late stages of the first S phase. The appearance of the three types seems to be caused by a different amount of BrdUrd incorporated into DNA between the first (S1) and the second S period (S2). The amount of BrdUrd incorporated is as follows: in type-1 S1>S2, in type-2 S1 S2 and in type-3 S2>S1.By analysing type-1 cells, all of the sister chromatid exchanges (SCEs) occurring during each replication cycle can be accurately counted and distinguished from one another. In cells exposed to BrdUrd above 5 μg/ml, the frequencies of SCEs occurring during S1, S2, and S3 are higher than those detected at lower BrdUrd concentrations. On the other hand, at lower concentrations (0.1–1.0 μg/ml) they occurred at the same frequency during S1, S2, and S3. Thus, SCEs detected at low concentrations are free from the incremental effect of BrdUrd incorporated, and enable us to estimate the spontaneous level of SCE frequency.