Inter- and intra-chromosomal distribution of chromatid breaks induced by X-rays during G2 in human lymphocytes

Cultures of blood from healthy adults were irradiated 48 h after stimulation with 240 R of X-rays and fixed after various time intervals (0–2 h, 2–4 h, 4–6 h). ³HTdR was added to several cultures after irradiation. Mitotic and labelling indices were used to distinguish between two cell samples inside the irradiated G₂ population: D − cells reaching mitosis without mitotic delay and a high frequency of chromatic breaks and D + cells with mitotic delay and which, during the delay, repair most of the damage produced. After R banding 450 chromatid deletions were located in each of the two cell samples. The D + cells showed a higher frequency of breaks than the D − cells with decreasing chromosome size, in the telomeric and centromeric region and in the junction between the R + and R − bands. These results can be interpreted as indicative of a non-random distribution of repair processes both between and within chromosomes.     

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.     

G0 chromosomal radiosensitivity in ataxia telangiectasia lymphocytes

Contrary to an earlier report, peripheral lymphocytes from 4 AT patients were not found to exhibit higher yields of unequivocal chromosome type aberrations following irradiation in the G₀ phase of the cell cycle, providing that only first post-irradiation metaphases were included in the samples (ensured by 5-bromodeoxyuridine (BrdU) incorporation and differential fluorescence or Giemsa staining). We were able, however, to confirm the earlier-reported increase in chromatid-type aberrations in the G₀-irradiated cells. AT lymphocytes were found to experience more cell-cycle delay following G₀ irradiation than normal cells. These observations appear consistent with the damaged base excision DNA-repair defect reported for AT cells.     

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.     

Formation of chromatid-type aberrations in G2 stage of the cell cycle

CHO cells were treated in G₁ stage of the cell cycle with chromosome-breaking agents that act in an S-dependent manner. The cells were challenged in G₂ stage, before fixation, with various inhibitors of DNA synthesis or repair. Short-wave UV, mitomycin C, decarbomyl mitomycin and 4-nitroquinoline oxide (4NQO) were used as chromosome-breaking agents. The inhibitors of DNA repair or synthesis used were hydroxyurea, aphidicolin and caffeine. Permeabilization of cells followed by a treatment with Neurospora endonuclease (a treatment to convert DNA single-strand breaks into double-strand breaks) did not have any influence on the frequencies of chromatid aberrations induced by the chemicals used, whereas with the inhibitors the extent of potentiation varied depending on the mutagen and the inhibitor used.     

Synergistic enhancement of the frequency of chromatid aberrations in cultured human lymphocytes by combinations of inhibitors of DNA repair

Whole-blood cultures of human lymphocytes were exposed in the G₂-phase to caffeine and to 4 inhibitors of DNA synthesis, hydroxyurea (HU), 2′-deoxyadenosine (dAdo), 1-β- -arabinofuranosylcytosine (araC) and aphidicolin (Aph), either individually or in pairs resulting in 10 different possible combinations. Since dAdo is rapidly deaminated in whole-blood cultures, all treatments involving dAdo were carried out in the presence of an inhibitor of the enzyme adenosine deaminase (ADA). The G₂-treatments were carried out on 3 different types of culture, (1) cultures that had not previously been exposed to any mutagenic treatment, (2) cultures that had been irradiated with 0.4 Gy of X-rays 3.5 h before harvesting, and (3) cultures that had been exposed for 2 h to 4 × 10⁻⁵ M thiotepa (TT) in G₀ immediately before stimulation with phytohaemagglutinin. The aim of the study was to find out in which combinations the inhibitors enhanced synergistically the frequencies of spontaneous and induced chromatid aberrations. In all 3 types of experiment, synergistic effects were observed with most of the 10 combinations, those involving HU being particularly effective. A very strong synergistic enhancement was also obtained when dAdo was combined with Aph.     

Effect of pH shifts on radiosensitivity of human lymphocytes irradiated in the G2 stage

Experiments were carried out using human lymphocytes in order to test the effect of pH shifts on radiosensitivity of cells irradiated in the G₂ stage. In our culture conditions constant variations in medium pH over the range of 7.4–6.8 were observed as a function of incubation time after PHA stimulation; in addition the pH of the medium was adjusted in parallel cultures over the range of 5.9–8.2. Then we exposed the cultures with different pH to a treatment of X-rays delivered 2 h before fixation.

The pH of the medium was found to greatly affect the yield of induced chromatid aberrations.     

DNA polymerase delta mediates increase in exchange production by X-radiation in human lymphocytes moving from G0 to G1

Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH₁ some hours after phytohemagglutinin stimulation than if they are irradiated in G₀ before stimulation. Post-treatment of lymphocytes irradiated in G₀ with the DNA polymerase inhibitor aphidicolin, which is effective against both pol and pol δ, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G₁ four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol ineffective in producing increased yields in either G₀ or G₁ lymphocytes, we conclude that much of the G₀ to G₁ increase in yields is mediated by pol δ.     

Induction of Cell Death by Cytokines in Cell Cycle-Synchronous Tumor Cell Populations Restricted to G1and G2

In the present work, cytokine-mediated induction of cell death was investigated by flow cytometry in cell cycle-synchronous human tumor cell populations gained by centrifugal elutriation or by cell cycle blockade with mimosine and aphidicolin. Attention was payed to the question of whether the effector phase of cell death takes place in the same phase of the cell cycle in which the death signal is received. Another point of interest was the question whether synchronization of cell populations with respect to the cell cycle leads to increased synchronicity of the death phase. The results demonstrate that supernatants from monocyte/tumor cell interaction cultures containing tumor necrosis factor-α, interferons, and interleukins-1 and -6 or appropriate combinations of pure cytokines cause cell cycle arrest predominantly in G₁and to a lesser extent in G₂. Cell death is initiated from both arrest points. Cytokine-treated G₁cells do not enter S phase. They die within the same G₁phase in which they receive the death signal. In contrast, a high proportion of cytokine-treated G₂cells pass through mitosis and are arrested and die in the subsequent G₁phase, whereas only a smaller proportion of cells are arrested and die in G₂. The synchronicity of the death phase cannot be increased by the diverse methods of cell cycle synchronization applied. Interestingly, aurin-tricarboxylic acid, an agent known for inhibitory effects on nucleolytic activities and other protein/nucleic acid interactions, not only prevents cell death, but also cell cycle arrest.     

Cell-cycle arrest and apoptosis induction in human breast carcinoma MCF-7 cells by carboxymethylated β-glucan from the mushroom sclerotia of Pleurotus tuber-regium

The mechanism for the anti-tumor activity of a water-soluble carboxymethylated β-glucan (CMPTR), partially synthesized from an insoluble native glucan isolated from the sclerotia of Pleurotus tuber-regium, was studied using human breast carcinoma MCF-7 breast cancer cells in vitro. CMPTR-induced anti-proliferative activity dose-dependently, with an IC₅₀ of 204 μg/ml. CMPTR inhibited the cell proliferation of MCF-7 by arresting the G₁ phase of its cell cycle after 48 h of incubation as shown by flow cytometry. Such G₁ phase arrest was associated with the down-regulation of cyclin D1 and cyclin E expressions in the breast cancer cells. In addition, the CMPTR-treated MCF-7 cancer cells were associated with decreased expression of anti-apoptotic Bcl-2 protein and increased expression of Bax/Bcl-2 ratio. This study shows that CMPTR can inhibit the proliferation of MCF-7 by cell-cycle arrest and apoptosis induction. The potential development of this mushroom polysaccharide as a water-soluble anti-tumor agent requires further investigation.     

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.     

Method For Probing Cells In Radiation-Induced G2Arrest: Demonstration of Potentially Lethal Damage Repair

Abstract. Chinese hamster ovary cells were arrested in the G₂ phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G₂, for the investigation of G₂ arrest. Comparisons were made of the number of cells retrieved from G₂ arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. the retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. the repair curves (surviving fraction us time) were each described by a single exponential. G₂ cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

Thermal sensitivity of chinese hamster V79 cells related to time after plating and cell-cycle redistribution

1. 1.|Cultured Chinese hamster V79 cells were trypsinized plated and grown attached or inoculated into spinner flasks and grown in suspension from exponnential growth to plateau-phase growth and their thermal sensitivity was measured after various growth times.

2. 2.|For attached cells, cultures were trypsinized and replated either 2 h before or after heating and the results were qualitatively the same: the thermal sensitivity remained approximately the same for the first 20 h and then increased and reached a maximum at 40–70 h. During this time, cells were in an exponential growth phase, and little or no change was observed in the cell-cycle age distribution as measured by flow cytometry (FMF) or [³H]thymidine pulse ([³H]Tdr) labelling.

3. 3.|At longer times after plating, cells grew into plateau phase, and thermal sensitivity decreased and became less than for the cells in exponential growth phase at the beginning of the experiment.

4. 4.|FMF and [³H]Tdr labelling showed that cells were accumulating in G₁ phase as the population density increased and that this accumulation was maximum at about 120–140 h as cells grew into plateau phase. This would account for the decrease in heat sensitivity and the increase in radiosensivity observed in plateau-phase cells.

5. 5.|For cells cultured in suspension there was no change in thermal sensitivity while cells were in exponential growth phase, As cell entered plateau phase, thermal resistance increased and most of the cell population had accumulated in G₁ as measured by FMF.

Monocytic differentiation of HL60 cells induced by potent analogs of vitamin D3 precedes the G1/G0 phase cell cycle block

Abstract. Differentiation of mammalian cells is accompanied by reduced rates of proliferation and an exit from the cell cycle. Human leukemic cells HL60 present a widely used model of neoplastic cell differentiation, and acquire the monocytic phenotype when exposed to analogs of vitamin D₃ (VD₃). The maturation process is accompanied by two blocks in the cell cycle: an arrest in the G₁/G₀ phase, and a recently described G₂+ M block. In this study we have analyzed the traverse of the cell cycle phases of the well-differentiating HL60-G cells exposed to one of ten analogs of VD₃, and compared the cell cycle effects of each compound with its potency as a differentiation-inducing agent. We found that in general there was a good correlation between the effects of these compounds on the cell cycle and on differentiation, but the best cell cycle predictor of differentiation potency was the extent of accumulation of the cells in the G₂ compartment. All analogs induced a marked decrease in the mitotic index, and polynucleation of HL60 cells was produced, especially by compounds which were effective as inducers of differentiation. Time course studies showed that induction of differentiation was accompanied by a transient increase of the proportion of cells in the G₂+ M compartment, but preceded the G₁ to S, and the G₂ compartment blocks. These studies indicate that complex changes in the cell cycle traverse accompany, but do not precede, the acquisition of the monocytic phenotype by HL60 cells.     

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.     

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.     

Influence of confluent holding time on UV light mutagenesis in human diploid fibroblasts

We have investigated the induction of mutants resistant to 6-thioguanine (6TG) following 254 nm ultraviolet light exposure of density-inhibited cultures of human diploid fibroblasts. Phenotypic expression of 6TG resistance was maximal within 9 days and remained stable through 19 days after irradiation. In reconstruction studies, complete recovery of 6TG-resistant mutants occurred at cell densities of up to 35 000 cells per 100-mm petri dish. The induced mutation frequency increased linearly with dose over the range of 3–9 J/m²; the D₀ of the survival curve was 4.2 J/m². Delaying subculture to low density for 1.5–24 h after irradiation produced unexpected alterations in induced mutation frequencies. An increase in UV-induced mutations of approximately 3-fold was observed in cultures maintained in confluence for 3 h. This trend was reversed with longer holding times: the mutation frequency declined sharply in cultures held for 6 h compared to the 3-h value, and thereafter showed a steady and gradual diminution to background levels.

These data suggest that the repair of potentíally mutagenic damage is a complex phenomenon which can lead to an increase or decrease in mutation frequency as a function of holding time. Although the decline in mutation frequency observed following longer holding intervals is consistent with the notion of an error-free process, we hypothesize that the increased mutation frequency produced by a short holding period reflects the existence of a cell-mediated process which enhances the mutagenic potential of at least some UV-induced DNA photoproducts.     

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.