DON抑制小麦种胚细胞周期启动的研究

小麦种子在０．５－４．０ｍｇ／Ｌ的脱氧雪腐镰刀菌烯醇（ＤＯＮ）中溶液中萌发,用流式细胞仪检测胚细胞的周期时相并观察黄化苗的生长。结果表明,对照组胚细胞在萌发１２ｈ开始启动细胞周期,此后Ｓ期细胞比率迅速增加并在２０ｈ达到最大值,较萌发１０ｈ的６．５％增加１．７８倍,约１４％的胚细胞在萌发的２２－２６ｈ间完成第一次分裂;ＤＯＮ抑制Ｇ１期胚细胞的启动,小麦胚中Ｓ期细胞比率随ＤＯＮ浓度增加呈指数下降,３．     

Radiosensitivity variation during the cell cycle in pronuclear mouse embryos in vitro

Abstract. The radiosensitivity of pronuclear mouse (B6D2 F1 x ICR) embryos has been measured in vitro as a function of time during the cell cycle. This was done by measuring the dose of X-rays (LD50) required to prevent development of 50% of the pronuclear embryos to the blastocyst stage in 5 days of culture. The LD50 was found to vary from 1 to 2 Gy during the period from G1 to the first cleavage. The cell cycle in the pronuclear embryo was analysed by [³H]thymidine autoradiography. Compared with earlier studies on two-cell mouse embryo radiosensitivity, the pronuclear embryos appear to be more sensitive to radiation than the two-cell embryos. If, however, one considers the radiation sensitivity on a blastomere basis, the pronuclear embryos are not different in their radiation sensitivity from the two-cell embryos. Thus, during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo.     

Interference of a synthetic C18 juvenile hormone with mammalian cells in vitro. II. Effects on cell cycle

Interference of a synthetic C18 juvenile (JH) with the cell cycle of mouse embryo cells (ME-cells) and mouse cells of established cell line (L-cells) was examined. After 3 hour in the medium with JH (20 mg/ml) the cells were transfered to the regular culture medium and labelled with H3-thymidine then incubated for 1 to 48 hours before processing them for autoradiography. The percentage of labelled mitosis was then calculated for all cells samples examined and the labelled mitosis curves were drown and analyzed. It was shown that in contrast to the solvent which had no effect on duration of any of the component phases of the cell cycle of ME-cells, the juvenile hormone under conditions of these experiments prolonged G1 and G2 intervals what resulted in prolongation of the total cell cycle of these cells. On the other hand it shortened G1 and prolonged G2 intervals of L-cells without changing duration of the total cell cycle. Thus, in the examined mouse cells, they were the G1 and G2 intervals which are affected by JH. This findings are considered as an argument for pleiotropic nature of the juvenile hormone interference with mouse cells, the more so as it interfered with both protein and DNA synthesis in these cells.     

Induction of differentiation and arrest of proliferation of mouse myeloid leukemia M1 cells

The relationship between differentiation and the cell cycle of mouse myeloid leukemia M1 cells was studied. The cells were induced to differentiate into macrophage-like cells by treatment with conditioned medium (CM) of hamster embryo cells. CM-treated cells traversed the S phase of the cell cycle at least once, then a fraction of the cells lost the ability to enter the S phase and accumulated in the G1 phase. Incorporation of [³H]thymidine in phagocytosis-induced cells decreased after 12–18 h of CM treatment. The morphology of the differentiated cells changed and the nucleus-cell ratio (NCR) of the individual cells decreased significantly between 12 h and 24 h of CM treatment. The decrease in NCR was well associated with arrest of proliferation in the G1 phase of the cells. The results suggest that G1 arrest of CM-treated M1 cells is an expression of cellular characteristics encoded in the differentiation program.     

Liquid-holding experiments with human lymphocytes. III. Experiments with G0 and G1 cells

Liquid holding (LH) experiments were performed with human peripheral lymphocytes treated in the G0 (G0-LH) or the G1 (G1-LH) phase of the cell cycle with diepoxybutane (DEB) or methylnitrosourea (MNU). In the G0-LH system, treatment with DEB but not with MNU led to a lowering of the frequencies of sister-chromatid exchanges (SCE). In the G1-LH system treatment with both chemicals led to a lowering of the SCE frequencies during the LH. These results are concluded to mean that lesions induced by DEB but not by MNU can be repaired in G0 cells and that G1 cells can repair both DEB and MNU induced lesions.     

Liquid-holding experiments with human lymphocytes: III. Experiments with G0 and G1 cells

Liquid holding (LH) experiments were performed with human peripheral lymphocytes treated in the GO (G0-LH) or the G1 (G1-LH) phase of the cell cycle with diepoxybutane (DEB) or methylnitrosourea (MNU). In the G0-LH system, treatment with DEB but not with MNU led to a lowering of the frequencies of sisterchromatid exchanges (SCE). In the G1-LH system treatment with both chemicals led to a lowering of the SCE frequencies during the LH. These results are concluded to mean that lesions induced by DEB but not by MNU can be repaired in GO cells and that G1 cells can repair both DEB and MNU induced lesions.     

Flow cytometric analysis of unbalanced control of protein accumulation and DNA synthesis in differentiating mouse myeloid leukemia cells

The protein and DNA contents of mouse myeloid leukemia M1 (clone B24) cells were determined by flow cytometry (FCM) after double fluorescent staining of the cells with fluorescein isothiocyanate and propidium iodide. FCM analysis showed that there was a linear relationship between the DNA and protein contents in logarithmically growing cells, although the protein content showed some variation. B24 cells can be induced to differentiate into macrophage-like cells by treatment with a protein inducer(s) in conditioned medium (CM) of hamster embryo cells. When the cells were treated with various concentrations of CM, cells with a 2C DNA content, G1/0 cells, increased and protein accumulated in these G1/0 cells. The increases in the number of G1/0 cells and in their protein content per cell were proportional to the concentration of CM. Serial analysis of changes in the contents of DNA and protein in differentiating B24 cells showed that DNA synthesis was suppressed by differentiation-induced block of the cell cycle at the G1/0 phase, whereas increase in the protein content was not completely suppressed by block of the cell cycle. These results suggest that unbalanced control of the DNA and protein contents of B24 cells is involved in the mechanisms of the morphological changes during differentiation into macrophages.     

Circadian variation in the susceptibility of mouse epidermis to tumour induction by methylnitrosourea

To study possible circadian differences in the sensitivity of hairless mouse epidermis to a small dose of a short-acting alkylating carcinogen, groups of animals were painted once with 0.2 mg methylnitrosourea (MNU) at 08.00, 12.00, 20.00 and 24.00 h. Other animals were painted three times at weekly intervals at 08.00 and at 20.00 h, respectively. Significantly higher tumour yields were found in animals painted at 20.00 h (when the cell cycle progression and DNA synthesis rate are lowest, and when relatively large numbers of late G1 cells may accumulate) than at any other time point investigated. Hence a circadian variation in sensitivity to MNU in mouse epidermis is confirmed. This may be due to the variations in flux of cells through the S phase. The formation of DNA/carcinogen adducts may be facilitated at times of low flux with many cells in late G1, and fixation of these errors in DNA may take place by the subsequent increased flux through S, before repair is possible.     

RB-dependent S-phase response to DNA damage

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.     

Characteristics of the cell cycle of matrix cells in the mouse embryo during histogenesis of telencephalon

The cell cycle of matrix cells in the telencephalon of the mouse embryo at different stages at day 10, 13, and 17 of gestation was investigated by means of ³H-thymidine autoradiography.The cell cycle time of matrix cells in the day 10 group was found to be 7.0 h, and lengthened linearly with embryonic age. The cell cycle times of day 13 and 17 groups were 15.5 and 26.0 h, respectively.The duration of G1 and S phases also lengthened linearly with embryonic age. The durations of G1 phase were 0.1, 6.8, and 13.8 h, for day 10, 13, and 17 groups, respectively, and those of S phase were 5.1, 6.9, and 10.4 h, for day 10, 13, and 17 groups, respectively. On the other hand, the durations of both G2 and M phases remained unchanged and these were 1.0 and 0.8 h, respectively, throughout the embryonic stages.It was a characteristic of the alteration of the cell cycle of the telencephalon during mouse embryonic life that not only G1 but also S phases lengthened linearly with embryonic age and both G2 and M phases remained constant.     

Autophagy is activated,but is not required for the G0 function of BCL-2 or BCL-xL

Cell cycle arrest in G0 and autophagy have features in common, but the inter-relationship between the two processes is not well defined. The anti-apoptosis molecules BCL-2 and BCL-xL promote G0 arrest through upregulation of p27 protein, which can also induce autophagy. We tested the hypothesis that autophagy was involved in the cell cycle arrest function of BCL-2 and BCL-xL. We found that in IL-3-dependent FL5.12 cells, NIH3T3 cells, and mouse embryo fibroblasts induced to arrest, treatment with 3-methyladenine did not affect the expected decrease in cell size and ribosomal RNA synthesis, or upregulation of p27 levels. Using the m5-7 ATG5-/- MEF cell line with doxycycline-regulated ATG5 expression, we demonstrated that autophagy was activated during serum withdrawal and contact inhibition, but inhibition of autophagy had no measurable effect on G0 arrest in parental or BCL-xL-expressing cells. Thus, our data indicate that, in cell culture models, autophagy occurs but is not required for entrance into quiescence or for the G0 function of BCL-2 or BCL-xL.     

High resolution analysis of cell cycle-correlated vimentin expression in asynchronously grown,TPA-treated MPC-11 cells by the novel flow cytometric multiparameter BrdU-hoechst/PI and immunolabeling technique

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC-11 mouse plasmacytoma cell cultures treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA-treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time-dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replication. © 1994 Wiley-Liss, Inc.     

Malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by malachite green: transformation is associated with abrogation of G2/M checkpoint control.

Malachite green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells by MG. In this study, we have studied the effects of MG on cell cycle phase distribution of normal and MG transformed Syrian hamster embryo cells in asynchronous and synchronous cell population. DNA flow cytometric analysis indicated that culturing cells for 48 h in medium containing MG at different concentrations induced dose-dependent G2/M arrest in normal cells. Malignantly transformed cells showed no such dose-responsive accumulation of cells at the G2/M phase of the cell cycle in response to MG. Synchronization studies indicated that in the control, both in the presence and absence of MG, cells followed a normal cell cycle pattern up to 16 h. After 16 h in the absence of MG, cells continued a normal cell cycle, whereas in the presence of MG they accumulated at G2/M phase of the cell cycle. This pattern of accumulation of cells at the G2/M checkpoint control was not observed in either untreated or MG-treated transformed cells. The present study indicates efficient operation of G2/M checkpoint control in control SHE cells and its abrogation in transformed SHE cells.     

Digital Holographic Microscopy for Non-Invasive Monitoring of Cell Cycle Arrest in L929 Cells

Digital holographic microscopy (DHM) has emerged as a powerful non-invasive tool for cell analysis. It has the capacity to analyse multiple parameters simultaneously, such as cell- number, confluence and phase volume. This is done while cells are still adhered and growing in their culture flask. The aim of this study was to investigate whether DHM was able to monitor drug-induced cell cycle arrest in cultured cells and thus provide a non-disruptive alternative to flow cytometry. DHM parameters from G1 and G2/M cell cycle arrested L929 mouse fibroblast cells were collected. Cell cycle arrest was verified with flow cytometry. This study shows that DHM is able to monitor phase volume changes corresponding to either a G1 or G2/M cell cycle arrest. G1-phase arrest with staurosporine correlated with a decrease in the average cell phase volume and G2/M-phase arrest with colcemid and etoposide correlated with an increase in the average cell phase volume. Importantly, DHM analysis of average cell phase volume was of comparable accuracy to flow cytometric measurement of cell cycle phase distribution as recorded following dose-dependent treatment with etoposide. Average cell phase volume changes in response to treatment with cell cycle arresting compounds could therefore be used as a DHM marker for monitoring cell cycle arrest in cultured mammalian cells.     

Clastogenic effects of methylnitrosourea and ethylnitrosourea on chromosomes from human fibroblast cell lines.

Human fibroblast cell lines were pulse-treated for 1 h with either methylnitrosourea (MNU) or ethylnitrosourea (ENU) at various time intervals before harvesting for chromosome analysis. Cells treated with 1 X 10(-3) M, 5 X 10(-4) M, and 1 X 10(-4) M final concentrations of MNU and ENU during the G2 or M phases of the cell cycle showed a significant increase in chromatid-type abnormalities over controls. Cells exposed to MNU or ENU 23 h before harvest showed some chromosome-type abnormalities, reflecting probable damage induced during the G1 phase of the cell cycle or derived from chromatid damage induced during the previous cell cycle. The mitotic indices and incidences of abnormalities suggested a dose response effect when cells were treated with the two higher concentrations and the three concentrations, respectively, of MNU or ENU. Chromatid abnormalities were observed in MUN and ENU-treated cells from each of four cell lines. From this investigation, it was concluded that MNU and ENU treatment of human diploid cell lines in vitro induced both chromatid and chromosome aberrations. MNU and ENU, both of which had previously been shown to be mutagenic in experimental animals, are, therefore, also considered to be mutagenic at the chromosome level in human fibroblasts grown and treated in cell culture.     

Correlation of reduced chilling injury with Increased spermine and spermidine levels in zucchini squash

By means of a biparametric cytofluorimetric analysis it is possible to distribute meristematic plant cells in a variety of cell cycle sub-compartments, unidentifiable by DNA measurements alone. In this work, an asynchronous proliferating cell population of pea root meristems was divided into different sub-compartments of the cell cycle phases, i.e. G1A, G1B, S. G2A and G2B on the basis of their DNA-nuclear protein content. By means of the same biparametric analysis, differentiated mesophyll cells and quiescent cells of embryo roots, indicated as G0 and G2Q, were distinguished from cycling cells by their low nuclear protein content. These results conform to those of some analyses performed on animal cells in culture and show that it is possible to get a major insight into cell cycle kinetics and its control in a natural system such as root meristem.     

Embryonal stem cells do not undergo cell cycle arrest upon exposure to damaging factors

As shown recently (Malashicheva et al., 2000), embryonic teratocarcinoma F9 mouse cells do not stop on the G1/S border after the treatment with agents causing G1 arrest in normal fibroblast cells. Since after a prolonged cultivation in vitro F9 cells could lose some properties characteristic of the stem cells, we studied here the capability of mouse ES cells to undergo cell cycle blocks following gamma-irradiation, adriamycin and PALA treatment as well as upon cultivation in the presence of nocodazol, an inhibitor of spindle assembly. The results obtained show that ES cells, similarly as their tumorigenic derivative F9 cells, do not demonstrate any delay on the G1/S boundary of the cell cycle. Moreover, nocodazol treatment for 48 h leads to accumulation of polyploid cells. Immunoblot experiments reveal a low level of p21/Waf1 expression both in F9 and in ES cells. Interestingly, the content of p21/Waf1 has been found to increase after cell treatment with proteasome inhibitor lactacystin, implying that p21/Waf1 level is regulated by proteasomal degradation. Thus, the p21/Waf1--dependent mechanisms of cell cycle control (checkpoint control) do not function properly in embryonic stem cells.     

Relieving Effects of Oligoglucosamine on the Inhibition Induced by Deoxynivalenol in Wheat Embryo Cells

The growth of etiolated wheat ( Triticum aestivum L. cv. Lumai No.22) seedlings and the activation of the cell cycle in embryo cells were estimated by flow cytometric analyses in wheat after the seeds being treated with oligoglucosamine and deoxynivalenol (DON). The results indicated that both the root number in etiolated wheat seedlings and the activation of the cells which had been arrested at G1 phase of the cell cycle in wheat embryos were enhanced by oligoglucosamine, suggesting that the mitosis in wheat embryo cells could be promoted by oligoglucosamine. The inhibition of DON on the growth of etiolated wheat seedlings and on the activation of the cell cycle in wheat embryo cells were relieved when the seeds were immersed in oligoglucosamine solution for 12 h before DON treatment. The results indicated that oligoglucosamine increased the hardiness to the poisoning of DON in wheat embryo cells. This might be the reason why such oligosaccharide elicits the resistance of plants to pathogen infection.