Changes in H1 content, nucleosome repeat lengths and DNA elongation under conditions of hydroxyurea treatment that reportedly facilitate gene amplification

Depletion of histone H1, changes in nucleosome repeat lengths, and extents of DNA elongation were investigated in synchronized Chinese hamster (line CHO) cells using the general conditions of hydroxyurea treatment that appear to increase the frequency of gene amplification, i.e., synchronized cultures of G1 cells were allowed to begin to enter S phase before treatment with hydroxyurea was effected to retard DNA synthesis (Mariani, B.D. and Schimke, R.T. (1984) J. Biol. Chem. 259, 1901-1910). During the time that synchronized G1 cells begin to enter S phase, there occur considerable synchrony decay and accumulation of new DNA that increase with time before treatment with hydroxyurea is initiated. During exposure to hydroxyurea, there occur depletion of histone H1 and shortened repeat lengths for the DNA synthesized in the presence of hydroxyurea. In contrast, DNA synthesized in S phase before exposure to hydroxyurea has essentially the same repeat lengths as bulk chromatin at both the time that hydroxyurea treatment is effected and after 6 h in its presence. Sedimentation measurements indicate that the early replicating DNA undergoes considerable elongation both before and during 6 h of exposure to 0.3 mM hydroxyurea. Thus, nearly all of the early replicating DNA is elongated to greater than average replicon size under those conditions of hydroxyurea treatment that appear to favor gene amplification. Because the extents of DNA synthesis and cell cycle progression vary as functions of drug concentration, treatment times, and unknown factors (from experiment to experiment), it would appear that the parameters must be carefully monitored in each experiment if biochemical results are to be related to the position of cells in the growth cycle.     

Changes in histone H1 content and chromatin structure of cells blocked in early S phase by 5-fluorodeoxyuridine and aphidicolin

We have measured changes in histone H1 content and changes in chromatin structure of Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation and blockade with 5-fluorodeoxyuridine or aphidicolin. Both the H1:core histone ratio in isolated nuclei and the H1 content of the cell are reduced 20-60%, depending on the duration of the block. The new deoxyribonucleic acid (DNA) synthesized during S-phase block has a shorter nucleosome repeat length than that of bulk chromatin, but it is nearly equally resistant as bulk DNA to attack by micrococcal nuclease. During the time that H1 content is decreasing, bulk chromatin also undergoes structural changes so that its nucleosome cores appear to be more closely packed along the DNA chain. The losses in H1 content and changes in chromatin structure are similar to those reported for cells blocked in early S phase by hydroxyurea [D'Anna, J. A., & Prentice, D. A. (1983) Biochemistry 22, 5631-5640]. The results suggest that losses of H1 and changes in chromatin structure are general events which occur when the elongation of initiated replicons or the joining of intermediate-sized DNA fragments is retarded during replication. They are consistent with the notions that H1 is lost from initiated replicons and/or the loss of H1 is part of an alarm response in the cell which might facilitate events leading to gene amplification.     

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

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

Comparison of synchronized Chinese hamster ovary cells obtained by mitotic shake-off, hydroxyurea, aphidicolin, or methotrexate

Synchronized cell populations are necessary to study many aspects of cell biology. We have developed a method to obtain highly synchronized Chinese hamster ovary cell populations in S phase or G2 phase by utilizing mitotic selection followed by incubation with either hydroxyurea, aphidicolin, or methotrexate for 12 h. Flow cytometry analysis shows that the coefficient of variation in the spread of the cell population in S phase is as low as 6%. Drug toxicity studies compare the effects of the various drugs on G1 and S phase cells. The use of aphidicolin or hydroxyurea results in the most highly synchronized cell populations, but methotrexate yields inadequate synchronization. These results demonstrate that both aphidicolin and hydroxyurea are useful drugs for obtaining highly synchronized cell populations after an initial synchrony in mitosis. Aphidicolin is perhaps the best choice because of less toxicity to S phase cells when used in low concentrations.     

The cell cycle dependence of c-sis gene expression: artifactual conclusions in cells prepared by chemical but not physical techniques

The c-sis oncogene encoding the B-chain of platelet-derived growth factor (PDGF) may be involved in an autocrine growth stimulation of tumours expressing the PDGF receptor, such as glioblastomas and sarcomas. To investigate whether expression of c-sis RNA is regulated in a cell cycle dependent manner, human A172 glioblastoma cells were synchronized by either centrifugal elutriation or chemical blockage with the DNA synthesis inhibitors hydroxyurea or aphidicolin. In non-perturbed elutriated cells, c-sis RNA levels were lower in the S phase of the cell cycle than in the G1 phase. In contrast, the chemically synchronized cells revealed a transient rise in c-sis RNA shortly after drug release, in early S phase. The RNA changes occurring after release from drug inhibition represent cell recovery from drug induced metabolic disturbances rather than true cell cycle dependent effects.     

Comparison of the subunit organization of early and late replicating chromatin

A comparison was made of the subunit organization of chromatin from regions of the genome with different metaphase chromosome banding characteristics by analyzing the accessibility of early and late replicating DNA in synchronized Chinese hamster ovary cells to digestion with staphylococcal nuclease. Three measures of nuclease susceptibility were employed: (1) the release of acid-soluble material; (2) a digestion index, P, which corresponds to the proportion of internucleosome segments which experienced at least one cleavage event; and (3) the size distribution of DNA fragments isolated from digested chromatin. Little or no difference was observed in the initial rates with which nuclease converted early and late replicating chromatin to acid-soluble material, although the initial digestion rates varied with time of cell collection in the cycle (metaphase > G1 mid-S > late-S or G2). Measurements of the digestion indices of material isolated from interphase cells suggested that initial cleavage events were more rapid in early replicating chromatin than in late replicating chromatin. In contrast, electrophoretic analysis revealed that oligomer DNA fragments from early labelled metaphase chromatin were slightly larger than corresponding fragments from late labelled metaphase chromatin. The size distribution of DNA in submonomer fragments obtained from extensively digested chromatin appeared to be identical regardless of the timing of replication or cell collection. Those small differences in chromatin digestibility that were observed may reflect subtle variations in the accessibility of internucleosome regions or perhaps in the higher-order arrangement of nucleosomes. However, no gross variation in accessibility to staphylococcal nuclease digestion was observed in chromatin localized to metaphase chromosome regions with vastly different cytological staining properties.     

Variation through the cell cycle in the dose-response of DNA neutral filter elution in X-irradiated synchronous CHO-cells

Dose-response curves for DNA neutral (pH 9.6) filter elution were obtained with synchronized CHO cells exposed to X-rays at various phases of the cell cycle. The dose response was similar in synchronized and plateau-phase G1 cells, as well as in cells that were arrested at the G1/S border using aphidicolin; it flattened as cells progressed into S phase and reached a minimum in the middle of this phase. An increase in DNA elution dose response, to values only slightly lower than those obtained with G1 cells, was observed as cells entered G2 phase. Significant alterations in the sedimentation properties of the DNA during S phase were also observed in Ehrlich ascites tumor cells using the neutral sucrose gradient centrifugation technique. A significant proportion of the DNA from S cells irradiated with 10 Gy sedimented at speeds (350S-700S) well above the maximum sedimentation speed expected for free sedimenting DNA molecules (Smax = 350S), indicating the formation of a DNA complex. DNA from G1, G1/S, or G2 + M cells sedimented as expected for free sedimenting molecules. These results indicate significant alterations in the physicochemical properties of the DNA--probably caused by DNA replication-associated alterations in DNA structure and chromatin conformation--as cells enter S phase, and are invoked to explain the observed variation in DNA elution dose response throughout the cycle. It is proposed that the formation of a complex DNA structure, resistant to the proteolytic enzymes and detergents used, affected the elution characteristics of the DNA and gave rise to the observed curvilinear DNA elution dose-response curves, as well as to the fluctuations in elution characteristics observed throughout the cell cycle.     

Organization of 5S genes in chromatin of Xenopus laevis.

The chromatin organization of the genes coding for 5S RNA in Xenopus laevis has been investigated with restriction endonucleases and micrococcal nuclease. Digestion of nuclei from liver, kidney, blood and kidney cells maintained in culture with micrococcal nuclease reveals that these Xenopus cells and tissues have shorter nucleosome repeat lengths than the corresponding cells and tissues from other higher organisms. 5S genes are organized in nucleosomes with repeat lengths similar to those of the bulk chromatin in liver (178 bp) and cultured cells (165 bp); however, 5S gene chromatin in blood cells has a shorter nucleosome repeat (176 bp) than the bulk of the genome in these cells (184 bp). From an analysis of the 5S DNA fragments produced by extensive restriction endonuclease cleavage of chromatin in situ, no special arrangement of the nucleosomes with respect to the sequence of 5S DNA can be detected. The relative abundance of 5S gene multimers follows a Kuhn distribution, with about 57% of all HindIII sites cleaved. This suggests that HindIII sites can be cleaved both in the nucleosome core and linker regions.     

Synchronization in the cell cycle by inhibitors of DNA replication induces histone H2AX phosphorylation: an indication of DNA damage

Several methods to synchronize cultured cells in the cell cycle are based on temporary inhibition of DNA replication. Previously it has been reported that cells synchronized this way exhibited significant growth imbalance and unscheduled expression of cyclins A and B1. We have now observed that HL-60 cells exposed to inhibitors of DNA replication (thymidine, aphidicolin and hydroxyurea), at concentrations commonly used to synchronize cell populations, had histone H2AX phosphorylated on Ser-139. This modification of H2AX, a marker of DNA damage (induction of DNA double-strand breaks; DSBs), was most pronounced in S-phase cells, and led to their apoptosis. Thus, to a large extent, synchronization was caused by selective kill of DNA replicating cells through induction of replication stress. In fact, similar synchronization has been achieved by exposure of cells to the DNA topoisomerase I inhibitor camptothecin, a cytotoxic drug known to target S-phase cells. A large proportion of the surviving cells 'synchronized' by DNA replication inhibitors at the G1/S boundary had phosphorylated histone H2AX. Inhibitors of DNA replication, thus, not only selectively kill DNA replicating cells, induce growth imbalance and alter the machinery regulating progression through the cycle, but they also cause DNA damage involving formation of DSBs in the surviving ('synchronized') cells. The above effects should be taken into account when interpreting data obtained with the use of cells synchronized by inhibitors of DNA replication.     

Action of heparin on mammalian nuclei. II. Cell-cycle-specific changes in chromatin organization correlate temporally with histone H1 phosphorylation

The interaction of the polyanion heparin with the inner histones of chromatin has been used to detect changes in chromatin organization associated with cell-cycle traverse. Synchronized populations of Chinese hamster cells were obtained either in early G1 or near the G1/S boundary. The rate of interaction of heparin with chromatin-associated inner histones was measured using nuclei isolated from synchronized cell populations in different phases of the cell cycle. A G1-specific decrease in rate of interaction of heparin with inner histones was observed and found to be independent of the presence of hydroxyurea during traverse of G1. A further decrease in heparin-inner histone interaction occurred in late S and G2. These changes correlate temporally with the interphase phosphorylation(s) of histone H1. This correlation is discussed within the framework of current models of higher order chromatin structure (i.e. organization above the nucleosome level). Analysis of the cooperativity of interaction of heparin with inner histones was performed using the kinetic analog of the Hill equation. This analysis suggests that the organization of inner histones on chromatin does not undergo large variations during the cell cycle.     

Sizes and rates of elongation of replicons in the frog embryo

Summary DNA fiber autoradiography has shown an increase in size of replicons during early development of the frog embryo. Replicons of endoderm cells were considerably larger than those of dorsal ectoderm and mesoderm cells in tailbud embryos. Late replicating DNA in partially synchronized tailbuds has a more rapid rate of replicon elongation than does early replicating DNA.     

Altered chromatin structure associated with a partial trisomy of chromosome 7 in the mouse

The chromatin of a mouse that is trisomic for part of chromosome 7 was investigated. Chromatin from trisomic tissue has a smaller average nucleosome DNA repeat length than chromatin from tissue taken from normal diploid littermates. DNA of the nucleosome cores is the same size in both normal and trisomic tissues. Not all of the nucleosome monomers have different repeat lengths. Normal and trisomic mouse kidney cells in tissue culture maintained their nucleosome repeat-length differences.     

Screening of five specific cell cycle inhibitors using a T Cell lymphoma cell line synchrony/release assay

To obtain different cell populations at specific cell cycle stages, we used a cell culture synchronization protocol. Effects of five different cell cycle inhibitors acting throughout the cell cycle were examined by DNA flow cytometric analysis of a synchrony/release lymphoma cell line (CEM). The screening synchronized protocol showed that staurosporine, mimosine and aphidicolin are reversible G1 phase inhibitors that act at different times. Staurosporine acted in early G1, exhibited the strongest cytotoxic effect, and induced apoptosis. Mimosine and aphidicolin acted in late G1 and at the G1/S boundary, respectively. Hydroxyurea arrested CEM cells in early S phase, but later than the aphidicolin arrest point. Nocodazole synchronized CEM cells in M phase. All the inhibitors examined in this study can be used to synchronize cells at different phases of the cell cycle and were reversible with little toxicity except for staurosporine which is highly toxic. Because the regulatory mechanism of the cell cycle is disrupted by their effects on protein synthesis, however, these drugs must be used with caution.     

Effect of damage to early, middle, and late-replicating DNA on progress through the S period in Chinese hamster ovary cells

In order to explain sequential replication of DNA in eukaryotic cells, the duplication of a given bank of replicons is proposed to be initiated by specific events coupled to synthesis of the preceding replicons in the sequence. This model predicts that DNA synthesis in mid or late S should depend upon the synthesis and/or integrity of previously replicating DNA, but should not depend upon the integrity of DNA replicating later in the sequence. By incorporating BUdR into DNA during a given short interval of one S period in synchronous Chinese hamster ovary cells, we are able to selectively damage this DNA by irradiation at selected times before or during the next S period. Utilizing this technique, we find that damage to early replicating DNA before entry into the second S phase markedly suppresses DNA synthesis in the entire S period. Damage to mid or late replicating DNA prior to entry into the second S period has no effect on early S, but markedly reduces DNA synthesis commencing in mid or late S, respectively. Furthermore, if early replicating DNA is damaged with light in mid-S, no effect on subsequent DNA synthesis is observed. These results can be fitted to a model in which sequential triggering of replicon synthesis promotes orderly progression through S.     

The S-phase cytotoxicity of camptothecin

The DNA topoisomerase I inhibitor camptothecin (CAM) is selectively cytotoxic to S-phase cells of HL-60, and some other myelogenous leukemic lines. The early effects of cell exposure to 0.05-0.2 micrograms/ml CAM are seen after 2 h; at that time a progressive degradation of DNA in the chromatin of S-phase cells is initiated. The degradation manifests by "pulverization" of chromatin followed by coalescence of the fine granules and nuclear disintegration. Between 2 and 6 h of treatment, a loss of about 30-70% of DNA from S-phase nuclei is detected by flow cytometry. A 10-min pulse of CAM is adequate to trigger subsequent DNA degradation. Agarose gel electrophoresis of DNA from CAM-treated cells reveals a typical nucleosome core particles "ladder," suggestive of preferential degradation of spacer DNA. Despite extensive loss of DNA and nuclear disintegration, the cell membrane of CAM-treated S-phase cells remains intact for several hours, excluding trypan blue or propidium iodide. Mitochondria, assayed for their ability to maintain a transmembrane potential (rhodamine 123 retention), as well as the lysosomal proton pump (probed by supravital uptake of acridine orange) also remain unchanged in these cells. G1 cells are refractory to CAM under these conditions. Synchronization of cells in S phase by aphidicolin increases the sensitivity of the whole cell population to CAM. The data suggest that CAM or other topoisomerase I inhibitors may be effective in some myelogenous leukemias, especially in combination with treatments synchronizing cells in 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.     

Nucleosome rearrangement in human cells following short patch repair of DNA damaged by bleomycin.

We have examined the structure of newly repaired regions of chromatin in intact and permeabilized human cells following exposure to bleomycin (BLM). The average repair patch size (in permeabilized cells) was six to nine bases, following doses of 1-25 micrograms/mL BLM, and greater than 80% of the total repair synthesis was resistant to aphidicolin. In both intact and permeabilized cells, nascent repair patches were initially very sensitive to staphylococcal nuclease, analogous to repair induced by "long patch" agents, and are nearly absent from isolated nucleosome core DNA. Unlike long patch repair, however, the loss of nuclease sensitivity during subsequent chase periods was very slow in intact cells, or in permeabilized cells treated with a low dose of BLM (1 microgram/mL), and was abolished by treatment with hydroxyurea (HU) or aphidicolin (APC). The rate of repair patch ligation did not correlate with this slow rate of chromatin rearrangement since greater than 95% of the patches were ligated within 6 h after incorporation (even in the presence of HU or APC). In permeabilized cells, repair patches induced by either 5 or 25 micrograms/mL BLM, where significant levels of strand breaks occur in compact regions of chromatin, lost the enhanced nuclease sensitivity at a rate similar to that observed following long patch repair. This rapid rate of rearrangement was not affected by APC. These results indicate that short patch repair in linker regions of nucleosomes, and/or "open" regions of chromatin, involves much less nucleosome rearrangement than long patch repair or short patch repair in condensed chromatin domains.