NUCLEIC ACID CONTENT OF HeLa S3 CELLS DURING THE CELL CYCLE: VARIATIONS BETWEEN CYCLES

The process of continuous resynchronization with excess thymidine provides sufficient cell material for accurate chemical determination of DNA and RNA in HeLa S3 cells at hourly intervals during the cell cycle. Total DNA is constant during the non-S phase portion of the cell cycle but varies widely among cycles of synchronous growth. Total cellular RNA content increases linearly in the G₁ phase and accelerates to a higher linear rate of accumulation, which remains constant during most of the S and G₂ phases. The ratios of early and late cycle rates of RNA accumulation are not constant among cycles.     

The synthesis and distribution of DNA in the macronucleus of Euplotes eurystomus

In an attempt to recognize any ordering of DNA synthesis which might occur in ciliates, the distribution of labelled DNA in the G₁ and S periods of the first, second, and third generations of a synchronized population of Euplotes eurystomus was studied by means of autoradiography. The results presented here show that the replicating DNA which at the time of label incorporation is restricted to a morphologically identifiable narrow region of the nucleus (the replication band), becomes dispersed and is evenly distributed throughout the nucleus. This dispersal of labelled DNA occurs previous to division and is observable throughout the G₁ period of the following generation. During the S phase of this second generation, this previously labelled DNA once again becomes restricted to a small portion of the nucleus. Now, however, it is present at the tips of the macronucleus independently of the position of the replication band. Again the labelled DNA is found to be dispersed during G₁ of the third generation. In the S period of this third generation however the radioactive DNA again appears localized in the replication bands which are found at the same position in the nucleus where they were when the pulse was given two generations earlier. The observations derived from the autoradiographic analysis suggest a non-permanent organization for at least those DNA molecules which are replicated during the first third of the S period. This DNA can be associated in either of two specific locations, replication band or tip, and these two patterns of organization alternate from generation to generation.     

Microspectrophotometric measurements of DNA by automated computerized scanning in cycling cells of theChrysanthemum segetum shoot apex

Summary A new technique of exploitation of the data was proposed after DNA scanning microdensitometry. By using all of the measurements obtained from the seriated sections of a single nucleus, this method made it possible to estimate six characteristic parameters during the different phases of the cell cycle in the various shoot apical cells. The cells whose rate of proliferation was the highest showed the biggest variations of their nuclear and nucleolar volumes during the cell cycle. In the axial zone, where the cells have a slow cell cycle and display the longest duration of the G₁ phase, the volume occupied by dispersed DNA was greater than in the cells of the lateral zone and of the rib meristem, where the cell cycle and the G₁ phase were short. No matter what the cell type, the proportion of the dispersed and condensed DNA varied little when the G₁ and G₂ phases were compared. In the Z phase, characterized by a decondensation of the DNA, the mean DNA amount was 3.4 C. The evolution of the nuclear density during the interphase was also estimated. It is demonstrated that the main feature of the shoot apex zonation was the decondensation of the condensed DNA in the axial zone in both the G₁ and G₂ phases.     

Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells

DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G₂ and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G₁/G₀ (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G₁/G₀ cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G₁/G₀ and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected.     

SET8 plays a role in controlling G1/S transition by blocking lysine acetylation in histone through binding to H4 N-terminal tail

We report evidence suggesting that methyltransferase SET8 plays a novel role in regulating cell cycle by suppressing DNA replication through histone binding. First, the distribution of SET8 is strongly cell cycle-dependent. SET8 was concentrated in the nucleus during G₁ and G₂ phases, and was excluded from the nucleus during S phase. Second, at G₁/S transition, SET8 was degraded through ubiquitination via SCF/Skp2. Third, it was evident that the SET8 binds to the H4 N-terminal tail (H4NT) and blocks the acetylation of lysine residues K5, K8 and K12 of histone H4 during G₁. Such a blockage can hinder DNA replication. Fourth, SET8 binds to hypoacetylated but not hyperacetylated H4NT. Finally, overexpressing the histone-binding domain of SET8 appeared to suppress DNA replication and arrest the cell cycle before the G₁/S transition. Taken together, these findings suggest that SET8 can be a negative regulator of DNA replication and the destruction of SET8 is required for the onset of S phase.     

Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2–7 complex onto chromatin during G₁ phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G₁ phase. The immobile fraction of MCM2 and MCM4 increases during G₁ phase, suggestive of reiterative licensing. In late G₁ phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G₁ phase progresses and do not colocalize with sites of DNA synthesis in S phase.     

Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

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

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

Change of chromatin morphology during the cell cycle detected by means of automated image analysis

Mouse embryo fibroblasts growing asynchronously in vitro stained with Feulgen method and their nuclear chromatin was analysed by means of the image analysing computer Quantimet 720D. Cells with 2C, 3C and 4C content of DNA were considered as being in G₁, middle S and G₂ phase of cell cycle, respectively. It was found that the projected area of nuclei increases during the cell cycle and that the mean optical density of chromatin increases from G₁ through S to G₂ phase. The curves showing the areas of chromatin at different optical density thresholds are different for cells in G₁, S and G₂ phase. The results demonstrate cyclic changes in chromatin morphology in the interphase nuclei during the cell cycle.     

BRCA1-Ku80 Protein Interaction Enhances End-joining Fidelity of Chromosomal Double-strand Breaks in the G1 Phase of the Cell Cycle

Quality control of DNA double-strand break (DSB) repair is vital in preventing mutagenesis. Non-homologous end-joining (NHEJ), a repair process predominant in the G₁ phase of the cell cycle, rejoins DSBs either accurately or with errors, but the mechanisms controlling its fidelity are poorly understood. Here we show that BRCA1, a tumor suppressor, enhances the fidelity of NHEJ-mediated DSB repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G₁. BRCA1 binds and stabilizes Ku80 at DSBs through its N-terminal region, promotes precise DSB rejoining, and increases cellular resistance to radiation-induced DNA damage in a G₁ phase-specific manner. These results suggest that BRCA1, as a central player in genome integrity maintenance, ensures high fidelity repair of DSBs by not only promoting homologous recombination repair in G₂/M phase but also facilitating fidelity of Ku80-dependent NHEJ repair, thus preventing deletional end-joining of chromosomal DSBs during G₁.     

Conjugation Between G1 and G2 Phase Cells in Paramecium tetraurelia1

Cells of Paramecium tetraurelia, stock hr^d, cultured in a micro-capillary containing 1 μl fresh culture medium, expressed mating activity through the whole cell cycle. Mating-reactive G₂ phase cells can conjugate with cells of other phases. The G₂ phase cells, which have double (4C) the normal micronuclear DNA content, undergo pre-meiotic DNA synthesis when conjugated with G₁ phase cells. The micronucleus of the progeny from the cross between a G₁ and a G₂ cell becomes triploid.     

Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G₁ and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-β and the β-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G₁ phase was enriched in origin sequences in comparison with matrix-attached DNA from early G₁ phase cells. The concentration of the early firing ori-β in DNA attached to the matrix decreased in early S phase, while the late firing β-globin origin remained attached until late S phase. We conclude that replication origins associate with the nuclear matrix in late G₁ phase and dissociate after initiation of DNA replication in S phase.     

Effects of ganglioside GM1 on DNA synthesis in isolated nuclei and on the activity of DNA polymerase α derived from S-phase HeLa cells

Ganglioside G_M1 inhibited either DNA synthesis in isolated nuclei or the activity of DNA polymerase α fractionated from S-phase HeLa cells. The concentrations of G_M1 necessary for 50% inhibition were about 5 μM and 10 μM for nuclei and DNA polymerase α, respectively. The G_M1 inhibition of the enzyme activity was suppressed by the addition of 0.05% Triton X-100. Neither gangliotetraosylceramide (asialo-G_M1) nor free N-acetylneuraminic acid inhibited the enzyme activity. These facts suggest that G_M1, probably in the form of micelles, could influence the enzyme activity by behaving as a polyanionic macromolecule. The kinetic studies indicate that the G_M1 inhibition of the enzyme activity was not competitive with the substrate, deoxythymidine triphosphate, but rather with the template DNA. Binding of G_M1 and DNA polymerase α was suggested by the cocentrifugation of G_M1 and the enzyme fraction after their preincubation. It was also observed that other acidic glycolipids, i.e., brain sulphatide and seminolipid, also inhibited the enzyme activity, whilst neutral galactosylceramide did not. The inhibitory influences of these sulphate esters of glycolipids were, similarly to G_M1, suppressed by the addition of 0.05% Triton X-100.     

Progressive Increase In Polyamine Levels In 9l Cells in vitro During the Cell Cycle: Comparison Between Cells Isolated By Centrifugal Elutriation and Cells Grown In Synchrony

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G₁, S, or G₂/M phases of the cell cycle. Cells enriched in early G₁, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G₂/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G₁, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G₁, S, or G₂/M. Because we did not obtain pure S or G₂/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G₁, S, and G₂/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.     

Analysis of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogen during the cell cycle. II. Ultraviolet light

UV-induction of thymine dimers in cellular DNA and their excision during different phases of the cell cycle of HeLa S3 cells were studied. Induction of thymine dimers was higher in the mitotic phase and the middle of the S phase than in the G₁ phase and from the late S phase to the early G₂ phase which are rather insensitive to UV. However, there is no significant difference in excision rate of UV-induced thymine dimers from the irradiated cells through the cell cycle. These findings indicate that the cyclic variation of UV-survivals during the cell cycle may be due to differences in the amount of thymine dimers in cellular DNA induced by UV-irradiation.     

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

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

The Circadian Variations In the Eithelial Growth of the Hamster Cheek Pouch: Quantitative Analysis of Dna Distributions*

The pronounced diurnal rhythm in DNA distribution of the hamster check pouch epithelium both in the S fraction and in the (G₂+ M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. the DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. the DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. the S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. the DNA distributions showed a diurnal release of G₁ cells into S phase proceeding through (G₂+ M) phase and returning to G₁ phase within a 24 hr period.     

The specificity of epidermal chalone action: the results of in vivo experimentation with two purified skin extracts.

To date two inhibitors of epidermal cell proliferation have been characterized: (1) a factor which depresses DNA synthesis, and (2) a factor which depresses mitotic rate. In the absence of experimental proof it has been assumed that the respective targets for these purified inhibitory factors are in G₁ and G₂ phases of the cell cycle. In the experiments reported here both these fractions were subjected to cell cycle phase specificity tests in order to verify these assumptions. In addition, an epidermally derived “cell line” (the sebaceous gland) and two nonectodermal tissues were examined for a response. The results suggest that the response induced by the inhibitor of DNA synthesis is cell cycle phase-specific, that the target cells are at the G₁-S phase boundary, and that only epidermal cells respond. Similarly the factor which depresses the flow of cells from G₂ into mitosis had no measurable effect on DNA synthesis by any of the tissues tested. The G₂ inhibitor lacks an inhibitory effect on mitosis in the sebaceous gland.The physiological roles which epidermal chalones may play are briefly discussed. It is suggested that a G₁–G₂ chalone system may have been effective in isolating kinetically cell populations with modified function during the evolutionary development in the vertebrates.     

Threshold expression of cyclin E but not D type cyclins characterizes normal and tumour cells entering S phase

Complexes of cyclin-dependent kinases (cdk) and their partner cyclins drive the cell through the cell cycle, each such complex phosphorylating a distinct set of proteins at a particular check-point or phase of the cycle. Immunocytochemical detection of cyclins combined with measurement of cellular DNA content by flow cytometry makes it possible to relate expression of each of these proteins with the actual cell cycle position, without the necessity of cell synchronization. In the present study, we have investigated expression of E and D type cyclins in G₁ cells and in cells entering S phase, in eight different human hematopoietic and solid tumour cell lines (two leukaemias, a lymphoma, three breast carcinomas, a colon carcinoma and a bladder transitional cell carcinoma) during their exponential phase of growth, as well as in normal mitogen stimulated lymphocytes. In all the cell types studied, the average level of D type cyclin expression was invariable throughout the cell cycle. A great intercellular variability, in particular of the G₁ cell subpopulations, and the presence of a large fraction of G₁, S and G₂+ M cells that were cyclin D negative (20–40% in tumour cell lines and about 80% among lymphocytes), were other characteristic features of D type cyclin expression. In contrast to D type cyclins, the expression of cyclin E was discontinuous during the cycle, peaking at the time of cell entrance to S. Also, a well defined threshold in expression of cyclin E characterized cells that were entering S phase, and virtually no cyclin E negative cells were seen during the early portion of S phase. The data indicate that while cell entrance to S phase is unrelated to expression of D type cyclins (at the time of entrance), accumulation of cyclin E up to critical level is a prerequisite for initiation of DNA replication. The great intercellular variability in expression of D type cyclins and their invariant average level across the cell cycle suggest that these cyclins, in addition to their acknowledged function in promoting cell progression through mid- to late-G₁ may have other role(s), related or unrelated to the cell cycle progression. The presence of a large number of D type cyclin negative cells in all phases of the cycle suggests that during exponential growth the cells may not express this protein and yet may traverse the cycle, including G₁ phase.     

Effects of 1000 R whole-body X-irradiation on DNA synthesis and mitosis in the duodenal crypts of the BCF1 mouse

Summary Effects of 1000 R, whole-body X-irradiation on the proliferative cells of the mouse duodenal crypts, in the four phases of the generation cycle; namely, the DNA synthesis phase, S; the pre-mitotic gap, G ₂; the division phase or mitosis, M; and the pre-synthesis gap, G ₁. As pointed out by Whitmore and Till (1964) G₁ and G₂ are characterized only by the fact that no DNA synthesis is taking place in these phases.In the intestinal crypts of BCF₁ mice, a 1000 R whole-body X-ray exposure blocks cells in G₂ for approximately 18 hours, and reduces the number of cells in S to less than 1/2 that observed in control animals during the first 12 hours after exposure. Cells synthesizing DNA, and undergoing division, remain few in number for more than 48 hours. Between 48 and 72 hours a compensatory reaction begins, and the number of cells in M and S increases from 28 at 48 hours to 150 at 72 hours and reaches a mean value of 482 at 96 hours.Work supported under the auspices of the US Atomic Energy Commission.