Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

Mechanisms of chromosomal aberration production II. Aberrations induced by 5-bromodeoxyuridine and visible light

We have allowed synchronized V79B Chinese hamster tissue culture cells to incorporate 5-bromodeoxyuridine (BUdR) during one DNA synthetic (S) period of the cell cycle and then determined chromosomal aberration yields induced by illumination of the cells with visible light during the succeeding pre- and post-DNA-synthetic (G₁and G₂) phases of the cell cycle. At the level used, BUdR by itself induces no aberrations. Illumination during the G₁ phase following incorporation induces aberrations of the chromatid type, but none of the chromosome type. All types of chromatid aberrations are induced, including isochromatid deletions and exchange types. In contrast, when cells are illuminated during the immediately following G₂ phase, large numbers of achromatic lesions and chromatic deletions are seen at the first post-illumination mitosis, but no isochromatid deletions and few exchange-type aberrations occur. When G₂-illuminated cells are examined in their second mitosis, however, chromatid aberrations of all types are again seen.

These results are interpreted within the “repair” model of chromosomal aberration production by UV light presented earlier³. The model assumes that the vertebrate chromosome is mononeme, consisting of but a single DNA double helix during the prereplication G₁ phase. The initial lesions induced by illumination of BUdR-containing DNA are believed to be single-chain breaks, and the observation that G₁ illumination produces only chromatid-type aberrations is taken as additional evidence for the mononeme chromosome. Conversion of single-chain breaks into double chain breaks through the action of a single-strand nuclease is postulated to account for the production of chromatid deletions at the first mitosis of G₂-illuminated cells. The action of this enzyme, plus a recombinational or post-replication repair mechanism, are postulated to account for the production of isochromatid deletions in G₁-illuminated cells. A rapid decline in achromatic lesion frequency with increasing time between G₂ illumination and fixation of the cells is considered evidence for rapid rejoining of most of the initial chain breaks.     

Cyclin gene expression and growth control in normal and neoplastic human breast epithelium

Recent advances in defining the molecular mechanisms of cell cycle control in eukaryotes provide a basis for beter understanding the hormonal control of cell proliferation in normal and neoplastic breast epithelium. It is now clear that a number of critical steps in cell cycle progression are controlled by families of serine/threonine kinases, the cdks. These kinases are activated by interactions with various cyclin gene products which form the regulatory subunits of the kinase complexes. Several families of cyclins control cell cycle progression in G₁ phase, cyclins C, D and E, or in S, G₂ and mitosis, cyclins A and B. Recent studies have defined the expression and regulation of cyclin genes in normal breast epithelial cells and in breast cancer cell lines. Following growth arrest of T-47D breast cancer cells by serum deprivation restimulation with insulin results in sequential induction of cyclin genes. Cyclin D1 mRNA increases within 1 h of mitogenic stimulation and is followed by increased expression of cyclins D3 and E in G₁ phase, cyclin A in late G₁/early S phase and cyclin B1 in G₂. Similar results were observed following epidermal growth factor stimulation of normal breast epithelial cells. Other hormones—oestrogens and progestins—and growth factors—insulin-like investigated for their effects on G₁ cyclin gene expression. In all cases there was an excellent correlation between the induction of cyclin D1 mRNA and subsequent entry into S phase. Furthermore, growth inhibition by antioestrogens and concurrent G₁ arrest were preceded by an acute decrease in cyclin D1 gene expression. These observations suggest a likely role for cyclin D1 in mediating many of the known hormonal effects on cell proliferation in breast epithelial cells.     

Circadian and Circannual Variations of Cell Cycle Distribution in the Mouse Bone Marrow

The cell cycle distribution of bone marrow cells from the femurs of female C3H mice has been investigated by flow cytometry according to the time of the day and month of the year. Both circadian and seasonal variations were found for the different cell cycle phases as well as the total cell numbers per femur. Both the mesor, the acrophase and the amplitude of the S, G₂ and (G₁ + G₀) phases varied significantly in some months, while in other months only insignificant rhythms were found. The relative cell cycle distribution only partly reflected variations in the total numbers of proliferating cells, since the total cell number per femur was also variable.

The total numbers of cells in DNA synthesis seem to be higher in the first part of the year, indicating increased cell proliferation during winter and spring. In this period the acrophases of DNA synthesis and G₂ were in the morning, while the second half of the year showed the peak later in the day.

In general, hemopoietic cell proliferation seems to constitute a labile equilibrium with rapidly changing activities.     

Nitric oxide blocks the cell cycle of mouse macrophage-like cells in the early G2+M phase

The effects of nitric oxide produced by macrophage-like cells (Mml) on the cell cycle were investigated. Mml cells lost proliferative activity in the presence of interleukin-6 (IL-6) and a subpopulation accumulated in the G₂+ M phase. This level increased in proportion to the incubation time. The DNA content of the cells was slightly lower than that of Mml cells treated with vinbrastine or demecolcine, drugs which block the cell cycle in the M phase. The peak of the early G₂+M phase was reduced by treatment with N^G-mono-methyl- -arginine. However, after treatment with exogenous nitric oxide or sodium nitroprusside, the G₀/G₁ phase increased, but the early-G₂+M and the S phase decreased. The flow cytometry pattern in IL-6-treated Mml was the same as that of cytochalasin B-treated Mml. These data suggest that endogenous nitric oxide affects the microfilament system of IL-6-treated Mml cells and blocks the cell cycle in the early G₂+M phase.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

Abstract. The kinetics of isthmal cells in mouse antrum were examined in three ways: (a) the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; (b) the duration of interphase and mitotic phases was determined from how frequently they occurred; and (c) mice were killed at various intervals after an intravenous injection of ³H-thymidine to time the acquisition of label by the various phases of mitosis.
The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G₁ and G₂ stages were 6.8 and 1.0 hr, respectively.
From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr.
Ten minutes after an intravenous injection of ³H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase.
We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G₂. Also, nuclei are in telophase during the early half of G₁ but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G₁ into early and late periods is practical.     

Differentiation is coupled to changes in the cell cycle regulatory apparatus of human embryonic stem cells

Mouse embryonic stem cells (mESC) exhibit cell cycle properties entirely distinct from those of somatic cells. Here we investigated the cell cycle characteristics of human embryonic stem cells (hESC). HESC could be sorted into populations based on the expression level of the cell surface stem cell marker GCTM-2. Compared to mESC, a significantly higher proportion of hESC (GCTM-2⁺ Oct-4⁺ cells) resided in G₁ and retained G₁-phase-specific hypophosphorylated retinoblastoma protein (pRb). We showed that suppression of traverse through G₁ is sufficient to promote hESC differentiation. Like mESC, hESC expressed cyclin E constitutively, were negative for D-type cyclins, and did not respond to CDK-4 inhibition. By contrast, cyclin A expression was periodic in hESC and coincided with S and G₂/M phase progression. FGF-2 acted solely to sustain hESC pluripotency rather than to promote cell cycle progression or inhibit apoptosis. Differentiation increased G₁-phase content, reinstated cyclin D activity, and restored the proliferative response to FGF-2. Treatment with CDK-2 inhibitor delayed hESC in G₁ and S phase, resulting in accumulation of cells with hypophosphorylated pRb, GCTM-2, and Oct-4 and, interestingly, a second pRb⁺ GCTM-2⁺ subpopulation lacking Oct-4. We discuss evidence for a G₁-specific, pRb-dependent restriction checkpoint in hESC closely associated with the regulation of pluripotency.     

Circadian rhythms of DNA synthesis in nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) occurs frequently in southern China. The circadian rhythm of DNA synthesis of a poorly differentiated NPC human cell line (CNE2) was investigated as an experimental prerequisite for designing chrono-chemotherapy schedules for patients with this disease. Twenty-two nude mice with BALB/c background were synchronized alternatively in 12 h of light and 12 h of darkness (LD12:12) for at least 3 wk prior to the transplantation of a CNE2 tumor fragment into each flank (area of ∼2×2 mm²). Ten days later, a tumor sample (area of ∼5 mm²) was obtained at 3, 9, 15, and 21 h after light onset (HALO) alternatively from different sites in each mouse. Single-cell suspensions were prepared and stained with propidium iodide. Cellular DNA content was measured with flow cytometry. Data were analyzed by ANOVA and cosinor methods. The average proportion of tumor cells in G₁, S or G₂-M phase varied according to circadian time with statistical significance. The maximum occurred at 9 HALO for G1, 2 HALO for S and 21 HALO for G₂-M phase cells. The approximate average distribution patterns of G₁ and G₂-M phases of cosine curve was 24 h. This was not the case for S-phase cells, which displayed a bimodal temporal pattern. Inter-individual variability in peak time was large, possibly due to relatively sparse sampling time. Nevertheless, no more than 6% of the time series displayed a maximum at 3 HALO for G₁, 21 HALO for S and 15 HALO for G₂-M. The cell cycle distribution of this human NPC cell line displayed circadian regulation following implantation into nude mice. The mechanisms involved in this rhythm and its relevance to the chrono-chemotherapy of patients deserve further investigation.     

Mechanisms of chromosomal aberration production I. Aberration induction by ultraviolet light

We have studied chromosomal aberration production in V-79 Chinese hamster tissue culture cells by UV light administered during the post-DNA-synthetic G₂ phase of the cell cycle. The treatment produced achromatic lesions and some chromatid deletions in the first post-irradiation mitosis, but no isochromatid deletions or chromatid exchange aberrations. In contrast, when G₂ UV-irradiated cells were examined in their second post-irradiation mitosis, there were significant yields of chromatid-type aberrations of all types, including isochromatid deletions and chromatid exchanges.

We have earlier reported²¹ that UV-irradiation during the pre-DNA-synthetic G₁ phase of the cell cycle induces only chromatid aberrations and also that most chromosomal aberration production by UV in G₁ can be photoreactivated in cells possessing the photoreactivating enzyme. We present here a model for chromosomal aberration production by UV. In the model all aberration production is enzymatically mediated, a consequence of the functioning of known molecular repair mechanisms. The important elements in the model are the following:

1. (1) The vertebrate chromosome is mononeme; i.e., contains but a single DNA double helix during the prereplication G₁ phase of the cell cycle.

2. (2) The UV-induced DNA lesion leading to the production of most aberrations is the cyclobutane dimer between adjacent pyrimidines in one polynucleotide strand.

3. (3) Single chain breaks appear at metaphase as achromatic lesions.

4. (4) Dimer removal sometimes leaves unrepaired single chain gaps, possibly as a result of incomplete excision repair.

5. (5) The single-stranded DNA opposite a single chain gap can be cleaved by a single-strand DNAase.

6. (6) Gaps are left in newly synthesized DNA polynucleotide chains opposite defective template chains (i.e., opposite dimers and chain breaks).

7. (7) Double-strand breaks present following local DNA replication may “spread” to the other chromatid by a recombinational process between template and new polynucleotide chains, one from each of the homologous double helices.

The model predicts the occurrence of isoachromatic lesions and of chromatid deletions paired (isolocus) with achromatic lesions. Though often not reported, both do, in fact, occur. In addition, the model accounts for the phenomenon of sister-chromatid exchange as a manifestation of a recombinational, or post-replication, repair mechanism. Finally, the model offers a simple interpretation of chromosomal aberration production by a variety of chemical agents.     

X-ray induction of 6-thioguanine-resistant mutants in division arrested, G0/G1 phase Chinese hamster ovary cells

The cytotoxic and mutagenic effect of X-irradiation was determined with Chinese hamster ovary cells arrested in the G₀/G₁ phase of the cell cycle through 9 days incubation in serum-free medium. In comparison with exponential phase cultures, the arrested cells showed increased cytotoxicity and mutation induction over the dose range of 50–800 rad. Exponential cultures showed a linear mutant frequency-survival relationship while the arrested cells showed a biphasic linear relationship. A post irradiation holding period of 24 h does not result in any change in the mutant frequency. The increased sensitivity of the arrested cells to the mutagenic effects of X-rays appears to be a cell-cycle phase phenomenon. Upon readdition of serum, the arrested cells re-enter the cell cycle in a synchronous manner, reaching S phase at 10–12 h. Cells irradiated at 5 h after serum addition, i.e. in G₁, show a similar does response for mutant frequency, while those irradiated at 10 h or later, i.e. in late G₁, S or G₂, show lower mutation induction. These observations are consistent with a chromosome interchange mechanism of mutation induction by X-rays, possibly through interactions between repairing regions of the DNA. Irradiation of cells in the G₀/G₁ phase allow more time for such interactions in the absence of semiconservative DNA replication.     

Targeted deletion of mouse Rad1 leads to deficient cellular DNA damage responses

The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombeRad1 ortholog promotes cell survival against DNA damage and is required for G₂/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1^-/- ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G₂/M as well as S/M checkpoints. These data indicated that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G₂/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.     

cAMP IN THE CELL CYCLE OF THE DINOFLAGELLATE CRYPTHECODINIUM COHNII (DINOPHYTA)

The second messenger cAMP is a key regulator of growth in many cells. Previous studies showed that cAMP could reverse the growth inhibition of indoleamines in the dinoflagellate Crypthecodinium cohnii Biecheler. In the present study, we measured the level of intracellular cAMP during the cell cycle of C. cohnii . cAMP peaked during the G₁ phase and decreased to a minimum during S phase. Similarly, cAMP-dependent protein kinase activities peaked at both G₁ and G₂+M phases of the cell cycle, decreasing to a minimum at S phase. Addition of N6, O₂'-dibutyryl (Bt₂)-cAMP directly stimulated the growth of C. cohnii . Flow cytometric analysis of synchronized C. cohnii cells suggested that 1 mM cAMP shortened the cell cycle, probably at the exit from mitosis. The size of Bt₂-cAMP treated cells at G₁ was also larger than the control cells. The present study demonstrated a regulatory role of cAMP in the cell cycle progression in dinoflagellates.     

The symmetry of radiation-induced chromatid exchanges in relation to the cell cycle in Chinese hamster cells in vivo and in vitro

The symmetry of radiation-induced chromatid exchanges was studied in relation to the cell cycle in Chinese hamster cells in vivo and in vitro. Both in vivo and in vitro, the ratio between symmetrical and asymmetrical chromatid exchanges was about 1 to 1 during G₂ and S phase of the cell cycle.     

Human herpesvirus 6 infection arrests cord blood mononuclear cells in G(2) phase of the cell cycle

We here report that after infection with human herpesvirus 6A, human cord blood mononuclear cells accumulate in G₂/M phase of the cell cycle. Experiments with foscarnet or ultraviolet (UV)-irradiated virus stocks pointed at an (immediate-)early, newly formed viral protein to be responsible for the arrest. At the molecular level, p53, cyclin B₁, cyclin A and tyrosine¹⁵-phosphorylated cdk1 accumulated after HHV-6A infection, indicating an arrest in G₂. However, no change was observed in the levels of downstream effectors of p53 in establishing a G₂ arrest, i.e. p21 and 14-3-3σ. We thus conclude that the HHV-6A-induced G₂ arrest occurs independently of p53 accumulation.     

Detailed Studies on the Application of Three Fluorescent Antibiotics for Dna Staining in Flow Cytometry

The effects of pH, ionic strength, stain concentration, magnesium concentration, and various fixative agents on DNA staining with the fluorescent antibiotics olivomycin, chromomycin A3, and mithramycin were examined with DNA in solution and in mammalian cells. Ethanol-fixed Chinese hamster cell populations (line CHO) stained with mithramycin and analyzed by flow cytometry provided DNA distribution patterns with a high degree of resolution. Glutaraldehyde-fixed cells exhibited about one-half the fluorescence intensity of ethanol-fixed cells; however, the percentages of cells in G₁, S, and G₂ + M were comparable. DNA distributions obtained for formalin-fixed cells were unacceptable for computer analysis. Cell staining over a pH range of 5-9 in solutions containing 0.15-1 M NaCl and 15-200 mM MgCl₂ provided optimal results based on the DNA profiles obtained by flow cytometry. The intensity of cells stained in 1 M NaCl was one and one-half times greater than cells stained in the absence of NaCl; however, spectrophotofluorometric analysis of mithramycin-magnesium-DNA complexes in solution revealed no significant changes in fluorescence intensity over a range of 0-1.75 M NaCl. These results and those obtained by flow cytometry analysis indicate that the increase in fluorescence of stained cells as a function of increasing ionic strength is due to changes in chromatin structure, providing a larger number of binding sites for the dye-magnesium complex.     

Sensitivity to cell killing and the induction of cytogenetic damage following gamma irradiation in wild-type and thymidine kinase-deficient Friend mouse erythroleukaemia cells

Wild-type Friend erythroleukaemia (clone 707) cells and 2 thymidine kinase-deficient subclones, 707BUE and 707BUF, having thymidine kinase activities of 1.4% and 0.7% that of clone 707, were compared for sensitivity to killing and the induction of cytogenetic damage following irradiation. Three doses of gamma irradiation were used (150, 300 and 450 cGy), and cells were harvested for metaphase spreads after 4, 8, 12, 15, 29 and 43 h. G₂ delay was evident at 4 h following gamma irradiation in the 3 cell clones examined, and recovery of mitosis was observed to be dose-dependent. G₂ delay was found to be most prolonged in subclone 707BUE and most prompt in clone 707. Increased sensitivity to the induction of cytogenetic aberrations at all three doses was apparent in the 2 thymidine kinase-deficient subclones (as compared to wild-type cells) at 15, 29 and 43 h. Th thymidine kinase-deficient subclones also showed increased sensitivity to gamma radiation-induced cell killing. Furthermore, subclone 707BUE consistently exhibited greater to gamma irradiation than did the subclone with lower thymidine kinase activity, 707BUF. The importance of thymidine kinase levels and extended G₂ delay for DNA repair processes is discussed.     

Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose

Sal1p, a novel Ca²⁺-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K _m of 0.20 ± 0.03 mM and 0.28 ± 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca²⁺ dependent. Ca²⁺ increases the V _max with a S _0.5 of 15 μM, and no changes in the K _m for ATP-Mg. Glucose sensing in yeast generates Ca²⁺ transients involving Ca²⁺ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H⁺-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca²⁺-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.     

Nucleostemin siRNA对肝癌细胞HepG2增殖的影响

Nucleostemin(NS)作为核仁蛋白,在神经干细胞、胚胎干细胞以及某些肿瘤细胞中均高表达,在多种肿瘤细胞增殖和凋亡调控中具有重要作用.本文通过瞬时转染NS siRNA降低NS的表达,以探究NS对HepG2细胞增殖和凋亡的影响.结果显示,下调NS表达使HepG2细胞增殖加快,G₁期细胞减少,S期及G₂/M期细胞增加,凋亡减少. 激光共聚焦实验表明,NS与S期激酶相关蛋白2 (S-phase kinase associated protein 2,Skp2)在HepG2细胞中存在共定位现象; Co-IP实验证明,NS与Skp2能相互作用|NS下调后,Skp2出核仁的数量增加,p27和p53表达降低. 总之,下调NS可促进HepG2细胞中Skp2从核仁逸出,p27降解增强,同时p53表达下降,或由此促进HepG2细胞增殖,抑制其凋亡.