Analysis of cellular DNA distributions

In the fluorescent-flow cytophotometric measurement of cellular DNA content the DNA distributions usually have two peaks. The second peak, which corresponds to the 4C DNA content of G2 and M cells, is often positioned at lower values of DNA content than twice that of the 2C DNA peak which contains G1 cells. Computerized numerical analyses were performed on artificial DNA distributions in which the proportion of S-phase cells was varied. It was demonstrated that the contribution of late S-phase cells to the 4C DNA peak in the histogram shifts the second peak to a position below twice the 2C DNA value. Also, increasing the coefficient of variation of the DNA measurement shifts the second peak position to lower values. A group of 33 DNA distribution histograms was found to have an average G2/G1 peak position ratio of 1.90, in keeping with typical values obtained from the numerical analysis of the artificial populations.     

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.     

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12-starved cells

Vitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12-deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.     

Two Aspects of DNA Base Composition: G+C Content and Translation-Coupled Deviation from Intra-Strand Rule of A=T and G=C

The relative contribution of mutation and selection to the G+C content of DNA was analyzed in bacterial species having widely different G+C contents. The analysis used two methods that were developed previously. The first method was to plot the average G+C content of a set of nucleotides against the G+C content of the third codon position for each gene. This method was used to present the G+C distribution of the third codon position and to assess the relative neutrality of a set of nucleotides to that of the G+C content of the third codon position. The second method was to plot the intrastrand bias of the third codon position from Parity Rule 2 (PR2), where A=T and G=C. It was found that whereas intragenomic distributions of the DNA G+C content of these bacteria are narrow in the majority of species, in some species the G+C content of the minor class of genes distributes over wider ranges than the major class of genes. On the other hand, ubiquitous PR2 biases are amino acid specific and independent of the G+C content of DNA, so that when averaged over the amino acids, the biases are small and not correlated with the DNA G+C content. Therefore, translation coupled PR2-biases are unlikely to explain the wide range of G+C contents among different species. Considering all data available, it was concluded that the amino acid-specific PR2 bias has only a minor effect, if any, on the average G+C content. In addition, PR2 bias patterns of different species show phylogenetic relationships, and the pattern can be as a taxal fingerprint. Received: 5 November 1998 / Accepted: 1 March 1999     

Computer-aided S-phase fraction determination in DNA static cytometry in breast cancer. A preliminary methodologic study on cytologic material.

This methodologic study was performed on a single-cell-cycle breast carcinoma to evaluate the feasibility of computer-aided S-phase fraction determination in DNA static cytometry. The investigation was performed on Feulgen-stained cytologic material in which the total optical density values of 1,000 consecutive, randomly selected nuclei were analyzed (MultiCycle software). A good correlation in the S-phase fraction value with flow cytometry was obtained when the G2/G1 ratio was fixed at 1.95, when the histogram data points were smoothed at least once and the coefficient of variation of the G2 peak was the same as that of G0-G1 or when a first-order S-phase polynomial model was used. The percentages of nuclei in G0-G1 and G2 were somewhat similar to those obtained with flow cytometry. The greatest discrepancy with flow cytometry was observed in the value of the coefficient of variation of the G0-G1 peak of the static cytometric data: it was at least twice as great. It always remained high despite the software options used. As for the influence of the sample size in the S-phase calculation, the software was also run on samples of 600 and 200 nuclei. When the G2/G1 ratio was fixed at 1.95, the data obtained from 600 nuclei did not differ from those obtained with 1,000 nuclei, whereas an analysis on 200 nuclei showed a substantial variation. The software also allowed calculation of the ratio of the G0-G1 peak of the neoplastic population against that of the diploid reference (DNA index), the value of which in flow cytometry was 1.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

The induction of DNA synthesis in the chick red cell nucleus in heterokaryons during the first cell cycle after fusion with HeLa cells.

Induction of DNA synthesis in embryonic chick red cells has been examined during the first and second cell cycles after fusion with HeLa cells synchronized in different parts of G1 and S-phase. The data indicate that: (i) the younger the embryonic blood the more rapidly the red cells are induced into DNA synthesis; (ii) the greater the ratio of HeLa to chick nuclei in the heterokaryon, the more rapidly the induction occurs; (iii) DNA synthesis in the chick nucleus can continue after the HeLa nucleus has left S-phase and entered either G2 or mitosis; (iv) the induction potential of late S-phase HeLa is somewhat lower than that of early or mid S-phase cells; (v) less than 10% of the chick DNA is replicated during the first cycle after fusion and only a small proportion (15%) of the chick nuclei approach the 4C value of DNA during the second cycle after fusion; (vi) the newly synthesized DNA is associated either with the condensed regions of the nucleus or with the boundaries between condensed and non-condensed regions; (vii) the chick chromosomes at the first and second mitosis after fusion are in the form of PCC prematurely condensed chromosomes); they are never fully replicated and are often fragmentary; (viii) DNA synthesis in the chick nuclei is accompanied by an influx of protein (both G1 and S-phase protein) from the HeLa component of the heterokaryon.     

Analysis of cellular DNA distributions

An analytical formula for calculating peak channel ratios in fluorescent cytophotometric determinations of DNA content per cell was derived to assess the effects of inaccuracies in the model-dependent derivation of S-phase cell populations and of systematic instrumental errors. The DNA distribution histograms usually have two peaks, corresponding to the 2C DNA content of G₁ cells and to the 4C DNA content of G₂ and M cells. In the presence of S-phase cells, the ratio of peak channels G₂/G₁ becomes less than 2. The calculation uses the model-dependent number of S-phase cells per channel and instrumental resolution to obtain G₂/G₁. The peak channel ratio calculated in this way decreases with increasing coefficient of variation and increasing proportion of S-phase cells. The calculated G₂/G₁ peak channel ratios were compared with 17 experimental values ranging from 1.68 to 2.08. Significant differences were found for two experiments, and the calculated G₂/G₁ ratios were systematically low by ≈4% for the other experiments. When this systematic difference in predicted peak channel ratios is taken into account, the formula predicts the observed ratios with an accuracy of 1% showing the dominant effect of S-phase cells in modifying the observed spectrum. The possible experimental effects leading to the observed systematic discrepancy are discussed A programmable pocket calculator program to perform these calculations is also described in detail.     

Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

Nuclear non-histone protein content of G0/G1 cells as related to growth fraction in mouse mammary tumors.

In eight mouse mammary tumors with varying growth fractions DNA and non-histone nuclear protein (NHNP) were determined by absorption cytophotometry of Feulgen-Naphthol Yellow S stained, isolated cells. It was found that: 1. The mean NHNP content of cells with postmitotic DNA content (G0 + G1) increased with increasing growth fraction. 2. The mean NHNP content of S and G2 cells in the eight tumors did not vary significantly with growth fraction. 3. The frequency distributions of NHNP in G0/G1 cells were unimodal and right-skewed. The results are interpreted as follows: A) G0 cells differ from G1 cells by their lower content of NHNP. B). If it is assumed that the G0 and G1 compartments are arranged in series, the cells in the transition from G0 to late G1 may account for the unimodality and skewedness of the NHNP frequency distributions of postmitotic cells.     

DNA synthesis in the second and third cell cycles of mouse preimplantation development. A cytophotometric study

Female Swiss mice were sacrificed at 2 h intervals between 16–30 and 40–56 h after insemination. One-, 2- and 4-cell embryos were stained by the Feulgen method and cytophotometric measurement of their nuclear DNA content was carried out. The cells with 2C and 4C DNA content were assumed to be in G1 and G2 phase and those with intermediate DNA content in S phase of the cell cycle. The fractions of cells which had passed a given phase of the cell cycle were calculated for various times after insemination and utilized for measurements of the second and third cell cycle timing. Results of measurements for the second cell cycle: G1 phase 1.3 h, S phase 6.1 h, G2 phase 15.4 h, whereas for the third cell cycle: G1 phase 1.6 h, S phase 7.4 h, G2 phase 0.5 h. The first cleavage division was calculated as 1.6 h, the second as 1.3 h and the third as 1.2 h. Complete intra-embryonic synchronization of the DNA-synthesizing nuclei was preserved during the entire synthesis phase of 2-cell embryos, while in 4-cell embryos they were slightly asynchronized. Among mitotic cells of the first cleavage division and G1 cells of 2-cell embryos a slight interembryonic asynchronization was found which deepened during subsequent cell cycle phases.     

Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells

Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (gammaH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in gammaH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced gammaH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of gammaH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of gammaH2AX. With the exception of xrs5 cells, gammaH2AX level was approximately 3 times lower in unirradiated G(1)-phase cells than S- and G(2)-phase cells, and the slope of the G(1)-phase dose-response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G(1)-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5-10-fold higher levels of gammaH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of gammaH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G(2)-phase cells.     

Changes in ribo- and deoxyribonucleoside triphosphate pools within the cell cycle of a synchronized mouse fibroblast cell line

Intracellular pool levels of ribo- and deoxyribonucleoside triphosphates were monitored throughout the cell cycle of C3H10T1/2 mouse embryo fibroblast cells synchronized by isoleucine deprivation. Absolute pool sizes of ribonucleoside triphosphates were approximately 30 fold greater than those of the corresponding deoxyribonucleoside triphosphates. Of the ribonucleoside triphosphates, pool sizes of ATP exhibited the greatest change, increasing from a low of 32.7 nmol/10(7) cells during G1 to a high of 81.6 nmol/10(7) cells 2 h prior to mid S-phase. Levels of ATP subsequently declined to 40.2 nmol/10(7) cells during late S-phase, followed by a second peak of 65.8 nmol/10(7) with the onset of cell division. No significant changes in the pool sizes of UTP and GTP were found throughout the cell cycle. Of the deoxyribonucleoside triphosphates, pool sizes of pyrimidine deoxyribonucleoside triphosphates were approx. 5-10 fold greater than those of purine deoxyribonucleoside triphosphates. Low levels of deoxyribonucldoside triphosphates during G1 (0.3-1.3 pmol/10(7) cells) increased coordinately with the initiation of DNA synthesis to an initial peak during mid S-phase (0.5-6.4 pmol/10(7) cells). Declining levels of deoxyribonucleoside triphosphates during late S-phase were followed by a subsequent larger second peak (1.7-10.7 pmol/10(7) cells) during G2-M.     

Cytophotometry of granulocytes in chronic granulocytic leukemia patients. Part I. Cell cycle distribution, S-phase transition and quantitative cytochemistry

In the chronic phase of CGL the proportion of granulocytes in S + G2 was lower (18.7 +/- 1.3% in marrow and 16.7 +/- 2.4% in blood) than in normal bone marrow (42.4 +/- 2.9%) as studied by Feulgen-DNA cytophotometry. During the blast crisis the percentage of S + G2 blasts was 39.3 +/- 8.4 in marrow and 38.7 +/- 7.8 in blood which was much higher than in acute myeloblastic leukemia patients (10.8 +/- 1.4 and 5.1 +/- 1.0). Thymidine labelling index values were lower than the percentage of cytophotometrically detected S-phase cells: up to 28% of cells with Feulgen-DNA content corresponding to S-phase did not incorporate 3H-thymidine. The rate of DNA synthesis remained constant during the S-phase but 3H-thymidine uptake increases towards the end of the S-phase. Morphometric parameters and quantitative cytochemical (PAS, Sudan, myeloperoxidase activity) characteristics of polymorphonuclear neutrophils were altered during the chronic phase of the disease but remained in the normal range during the blast crisis. Mature neutrophils in the blast crisis are assumed to originate from normal granulocyte progenitors.     

Polyamines and HeLa-cell DNA replication.

HeLa cells were synchronized for S-phase DNA synthesis by the double thymidine-block procedure. A comparison was made of the polyamine content and S-phase DNA synthesis in cells from control cultures and cultures to which an inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine, was added to the synchronization medium. Control cells showed a peak of synchronous DNA synthesis at 3 h and a maximum concentration of polyamines at 6-9 h after release of the second thymidine block. Cells from cultures containing the inhibitor were severely inhibited in the synthesis of DNA and contained no putrescine and only traces of spermidine while the spermine content was lowered by as much as 80%. Supplementation of cultures containing alpha-difluoromethylornithine with a polyamine, at the time of release of the second thymidine block, replenished the intracellular pool of the administered polyamine and partially restored S-phase DNA synthesis, with a lag of 3-6 h. Almost complete restoration of DNA synthesis in cells depleted of polyamines was achieved by the addition of a polyamine to cultures at least 10 h before release of the second thymidine block. The lag in initiation of synchronous S-phase DNA synthesis was eliminated in these cells. It is concluded that reversal by polyamines of the deficiency in S-phase DNA synthesis, in polyamine-depleted HeLa cells, is a time-dependent process indicative of the necessity for the replenishment of replication factors or their organization into an active replication complex.     

Amiloride added together with bumetanide completely blocks mouse 3T3-cell exit from G0/G1-phase and entry into S-phase.

In this study we tested the hypothesis that stimulation of univalent-cation fluxes which follow the addition of growth factors are required for cell transition through the G1-phase of the cell cycle. The effect of two drugs, amiloride and bumetanide, were tested on exit of BALB/c 3T3 cells from G0/G1-phase and entry into S-phase (DNA synthesis). Amiloride, an inhibitor of the Na+/H+ antiport, only partially inhibited DNA synthesis induced by serum. Bumetanide, an inhibitor of the Na+/K+ co-transport, only slightly suppressed DNA synthesis by itself, but when added together with amiloride completely blocked cell transition through G1 and entry into S-phase. Similar inhibitory effects of the two drugs were found on the induction of ornithine decarboxylase (ODC) (a marker of mid-G1-phase) in synchronized cells stimulated by either partially purified fibroblast growth factor (FGF) or serum. To test this hypothesis further, cells arrested in G0/G1 were stimulated by serum, insulin or FGF. All induced similar elevations of cellular K+ content during the early G1-phase of the cell cycle. However, serum and FGF, but not insulin, released the cells from the G0/G1 arrest, as measured by ODC enzyme induction. This result implies that the increase in cellular K+ content may be necessary but not sufficient for induction of early events during the G1-phase. The synergistic inhibitory effects of amiloride and bumetanide on the two activities stimulated by serum growth factors, namely ODC induction (mid-G1) and thymidine incorporation into DNA (S-phase), suggested that the amiloride-sensitive Na+/H+ antiport system together with the bumetanide-sensitive Na+/K+ transporter play a role in the mitogenic signal.     

A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content

Cultures of ts BN75, a temperature-sensitive mutant of BHK 21 cells, show a gradual biphasic drop in [3H]thymidine incorporation together with an accumulation of cells having a G2 DNA content when incubated at 39.5 degrees. However, when higher (41 degrees - 42 degrees) nonpermissive temperatures were used, the major block was in S-phase DNA synthesis. The cultures of ts BN75 shifted to 42 degrees at the start of the S phase, cell-cycle progress was arrested in the middle of S, while under these conditions wild-type BHK cells underwent at least one cycle of DNA synthesis. When ts BN75 cells growth-arrested at high temperature with a G2 DNA content were shifted to the permissive temperature (33.5 degrees C), the restart of DNA synthesis preceded the appearance of mitotic cells. These data suggest that the ts defect of ts BN75 cells might affect primarily the S phase of the cycle rather than the G2 phase.     

Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.     

Discrepancies among the metaphase, telophase, and the G0/G1 and G2 DNA peaks of heteroploid cell lines

Heteroploid cell populations often show narrow peaks of G0/G1 and G2/M DNA content and broadly distributed chromosome numbers. This was originally explained by the selective metaphase arrest of the cells that have non-modal chromosome numbers. To test whether this explanation applies, we have measured the chromosome number distributions, as well as the G0/G1, G2, metaphase (M), and telophase (T) DNA distributions, of the cell lines WCHE-5, MCa-11, and HL-60. The WCHE-5 cells had narrowly distributed chromosome numbers and G0/G1 G2, M, and T DNA peaks. The MCa-11 and HL-60 cells also had narrowly distributed G0/G1 and G2 DNA peaks, but broadly distributed chromosome numbers and M and T DNA peaks. The widths of the MCa-11 and HL-60 M- and T-cell DNA peaks were similar to those of their chromosome number peaks, suggesting that all cells were completing mitosis, regardless of chromosome number or DNA content. Thus, selective metaphase arrest does not seem to be the cause of the narrow G0/G1 and G2 DNA peaks of heteroploid cell populations.     

Rad53 is essential for a mitochondrial DNA inheritance checkpoint regulating G1 to S progression

The Chk2-mediated deoxyribonucleic acid (DNA) damage checkpoint pathway is important for mitochondrial DNA (mtDNA) maintenance. We show in this paper that mtDNA itself affects cell cycle progression. Saccharomyces cerevisiae rho(0) cells, which lack mtDNA, were defective in G1- to S-phase progression. Deletion of subunit Va of cytochrome c oxidase, inhibition of F(1)F(0) adenosine triphosphatase, or replacement of all mtDNA-encoded genes with noncoding DNA did not affect G1- to S-phase progression. Thus, the cell cycle progression defect in rho(0) cells is caused by loss of DNA within mitochondria and not loss of respiratory activity or mtDNA-encoded genes. Rad53p, the yeast Chk2 homologue, was required for inhibition of G1- to S-phase progression in rho(0) cells. Pif1p, a DNA helicase and Rad53p target, underwent Rad53p-dependent phosphorylation in rho(0) cells. Thus, loss of mtDNA activated an established checkpoint kinase that inhibited G1- to S-phase progression. These findings support the existence of a Rad53p-regulated checkpoint that regulates G1- to S-phase progression in response to loss of mtDNA.