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1.
Recovery of the cell cycle in cells A 431 and in human embryo fibroblasts (EFH) differs much. Unlike EFH, A 431 cells have: 1) synchronized exit of cells from G1 into S phase after 5 Gr irradiation; 2) G2-block; 3) much less manifestation of these two phenomena in the presence of EGF; 4) a lesser effectiveness of the repair of DNA single-strand breaks. EGF stimulation of the repair of radiation-induced DNA lesions, SSB in particular, may be of great importance for the postirradiation cell cycle recovery.  相似文献   

2.
We have examined whether a human chromosome has distinct segments that can replicate autonomously as extrachromosomal elements. Human 293S cells were transfected with a set of human chromosomal DNA fragments of 8-15 kilobase pairs that were cloned on an Escherichia coli plasmid vector. The transfected cells were subsequently cultured in the presence of 5-bromodeoxyuridine during two cell generations, and several plasmid clones labeled in both of the daughter DNA strands were isolated. Efficiency of replication of these clones, as determined from the ratios of heavy-heavy and one-half of heavy-light molecules to total molecules recovered from density-labeled cells, was 9.4% per cell generation on the average. Replication efficiency of control clones excluded during the selection was about 2.2% and that of the vector plasmid alone was 0.3%. A representative clone p1W1 replicated in a semiconservative manner only one round during the S phase of the cell cycle. It replicated extrachromosomally without integration into chromosome. The human segment of the clone was composed of several subsegments that promoted autonomous replication at different efficiencies. Our results suggest that certain specific nucleotide sequences are involved in autonomous replication of human segments.  相似文献   

3.
The distribution of cells through the phases of the cell cycle by DNA flow cytofluorimetry was analysed to investigate the effects of UV irradiation on cell cycle progression in asynchronous Chinese hamster cells with different UV-sensitivity: cell line V79 (UV-resistant cells), and UV-sensitive clones: B6, CHS1, CHS2 and XII. The UV-irradiated cultures show a large accumulation of cells in S phase, the effect increasing with UV dose increase, which may point to an inhibition of the DNA chain elongation. UV-sensitive clones show a larger and more prolongated increase in the proportion of cells in S phase after irradiation with smaller dose than UV-resistant cells. Besides, the UV-sensitive clone XII shows an inhibition of movement of irradiated cells from G1 into S phase, that may testify to an inhibition of replicon initiation. These results suggest that there is a correlation in UV-irradiated Chinese hamster cells between alteration in cell cycle progression and UV-sensitivity of cells.  相似文献   

4.
The action of the increased intracellular content of adenosine monophosphate (cAMP) in CHO-K1 cells (clones 773 and ADr112eb), treated with isoproterenol, on gamma-induced DNA single-strand breaks repair has been investigated. The hormonal treatment stimulates gamma-induced (180 Gr) DNA single-strand repair during the post-irradiation incubation (45 min) by 75 +/- 16%. The results show the involvement of the cAMP system in radiosensitivity of cultured mammalian cells and in regulation of cellular mechanisms of DNA repair.  相似文献   

5.
Increased apoptosis in U937 cells over-expressing lipocortin 1 (annexin I)   总被引:2,自引:0,他引:2  
The potential involvement of endogenous lipocortin 1 in the process of cellular apoptosis, particularly in cells of the myelo-monocytic lineage, has been investigated. U937 cells were transfected either with an antisense or a sense DNA for lipocortin 1 and the stable clones 36.4AS clone (20-40% lower lipocortin 1 levels) and 15S (30% higher lipocortin 1 levels) were obtained. Cell apoptosis was induced by incubation with tumor necrosis factor-alpha: optimal responses were observed within a 24 h incubation period at a 5 ng/ml concentration. Apoptosis was assessed both morphologically, by annexin V binding and cell cycle analysis with propidium iodide. Whilst no consistent difference was seen between wild type cells and clone 36.4AS, a higher incidence of apoptosis (ranging from +30% to + 60%) was observed in the 15S clone. Release of arachidonic acid from loaded cells was promoted by 24 h incubation with the cytokine, and a higher degree of release was measured in the 15S clone. These data indicate that endogenous intracellular lipocortin 1 is involved in the promotion of apoptosis in cells of the myelo-monocytic derivation.  相似文献   

6.
A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase alpha and delta (aphidicolin) and DNA polymerase beta (2', 3'-dideoxythymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gr) or by gamma-ray irradiation (150 Gr) is more sensitive to aphidicolin independently of cell proliferating status. Aphidicolin inhibits the recovery of single-strand DNA in quiescent and mitogen-stimulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase alpha. It is suggested that the regulation action of mitogens on the DNA SSB repair may be determined by qualitative changes of this enzyme or of some conditions in which it functions. The involvement of DNA polymerase delta in this process is not excluded.  相似文献   

7.
A cDNA library was screened for plant proliferating-cell nuclear antigen (PCNA) from Catharanthus roseus (periwinkle). A lambda gt11 cDNA library was constructed using poly(A)-rich RNA isolated from the cells in the S phase. A cDNA clone for PCNA was isolated by using a rice genomic clone, pCJ-1, which contains PCNA-related gene sequences. The cDNA contains an open reading frame of 804 nucleotides, encoding a protein of 268 amino acids with a molecular mass of 29,765 Da. When conservative substitutions were included, a high degree of similarity (about 85%) was observed between the predicted amino acid sequence of periwinkle PCNA and that of human PCNA. Expression of mRNA for periwinkle PCNA was undetectable or very weak in quiescent cells, such as phosphate-starved cells, auxin-starved cells and cells in the stationary phase. In the synchronous progression of the cell cycle induced by the addition of phosphate or auxin, the active accumulation of periwinkle PCNA mRNA was observed preferentially in the S phase. When an inhibitor of DNA synthesis, aphidicolin, was added to the cells at the G1 phase, an increase in the level of PCNA mRNA was observed. The partial inhibition of protein synthesis at the G1 phase by a protein inhibitor, anisomycin, caused the arrest of cells in the G1 phase. No increase of the level of periwinkle PCNA mRNA was observed in cells arrested at the G1 phase by the inhibition of protein synthesis. These results indicate that the induction of mRNA for periwinkle PCNA occurred independently of the initiation of DNA replication, but that synthesis of certain proteins at the G1 phase was required for the induction of periwinkle PCNA mRNA at the S phase.  相似文献   

8.
A comparison was made of the ability of either X-radiation or a DNA-specific ligand (the vital bis-benzimidazole dye; Hoechst 33342) to induce: cell killing, inhibition of de novo DNA synthesis, DNA strand breakage and the delay of cell division in human colon adenocarcinoma cells in vitro. Unlike radiation-induced cell killing, ligand-induced cytotoxicity appeared to be positively correlated with the extent of inhibition of de novo DNA synthesis--a feature consistent with the persistent binding of ligand molecules to nuclear DNA. Ligand-induced DNA strand-breaks disappeared slowly although ligand-treated cells retained apparently normal capacities to repair discrete radiogenic DNA strand-breaks. Pre-treatment of cells with Hoechst 33342 resulted in a dose-modifying enhancement of radiation resistance not associated with altered dosimetry for strand-break induction. However, radioresistance was accompanied by the protracted retention of cells in the G2 phase of the cell cycle. We suggest that the results provide direct evidence that the retention of cells in G2 phase is a sparing phenomenon and is triggered by the responses of chromatin domains to the presence of DNA damage. Our results have implications for the use of DNA-interactive agents in combined modalities for tumour therapy, and indicate a possible basis for the sparing of some tumour cells in dividing populations.  相似文献   

9.
The infection of permissive monkey kidney cells (CV-1) with simian virus 40 induces G1 growth-arrested cells into the cell cycle. After completion of the first S phase and movement into G2, mitosis was blocked and the cells entered another DNA synthesis cycle (second S phase). Growth-arrested CV-1 cells replicated significant amounts of viral DNA in the G2 phase with the majority of synthesis occurring during the second S phase. When mimosine-blocked (G1/S) infected cells were released into the cell cycle, a major portion of the viral DNA was detected in G2 with the largest accumulation in the second S phase. The total DNA produced per infected cell was 10-12C with approximately 0.5-2C of viral DNA replicated per cell. Therefore the majority of the DNA per cell was cellular, 4C from the first S phase and approximately 4-6C from the second cellular synthesis phase.  相似文献   

10.
Senescent human diploid cells (HDC) were fused to T98G human glioblastoma cells and to RK13 rabbit kidney cells, and DNA synthesis was analyzed in the heterodikaryons. T98G and RK13 cells are “partially transformed” cell lines that have some characteristics of normal cells, yet are transformed to immortality, i.e., they do not senesce. Previous experiments have shown that “fully transformed” HeLa and SV80 cells induce DNA synthesis in senescent HDC nuclei, whereas normal young HDC do not. Our experiments show that T98G and RK13 cells do not induce DNA synthesis in senescent HDC nuclei. These results demonstrate that the ability to induce DNA synthesis in senescent HDC is not correlated with immortality per se. Our results show further that a T98G cell in S phase at the time of fusion to a senescent HDC will continue to make DNA. However, a T98G cell in G1 phase at the time of fusion is prevented from initiating DNA synthesis. RK13 cells behave similarly to T98G. These results are consistent with the hypothesis that the molecular basis for the senescent phenotype involves a block that prevents cells in G1 phase from entering S phase. Thus, we conclude that the senescent phenotype can be dominant in heterokaryons composed of senescent HDC fused with certain immortal cell lines. To explain the different results obtained with various immortal cell lines, we present a model that suggests that T98G and RK13 cells are immortal because they have lost a normal regulatory factor, whereas HeLa and SV80 are immortal because they have gained a dominant transformation factor.  相似文献   

11.
The synchronization effects of the plant amino acid mimosine on proliferating higher eukaryotic cells are still controversial. Here, I show that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks. The DNA content of nuclei isolated from mimosine-treated cells was determined by flow cytometry. The presence or absence of DNA replication forks in these isolated nuclei was then detected by DNA replication run-on assays in vitro. Treatment of asynchronously proliferating HeLa or EJ30 cells for 24 h with 0.5 mM mimosine resulted in a population synchronized in late G1 phase. S phase entry was inhibited by 0.5 mM mimosine in cells released from a block in mitosis or from quiescence. When added to early S phase cells, 0.5 mM mimosine did not prevent S phase transit, but delayed progression through late stages of S phase after a lag of 4 h, eventually resulting in a G1 phase population by preventing entry into the subsequent S phase. In contrast, lower concentrations of mimosine (0.1-0.2 mM) failed to prevent S phase entry, resulting in cells containing active DNA replication foci. The G1 phase arrest by 0.5 mM mimosine was reversible upon mimosine withdrawal. This synchronization protocol using 0.5 mM mimosine can be exploited for studying the initiation of human DNA replication in vitro.  相似文献   

12.
Treatment of a nontumorigenic clone of AKR mouse embryo cells in culture with a variety of polycyclic aromatic hydrocarbons has resulted in the development of derivative clones which are highly tumorigenic and exhibit other characteristics of the transformed phenotype. A 3-methylcholanthrene-transformed derivative clone (clone MCA) has been compared to the parent clone (clone 2B) with respect to the abundance and diversity of polysomal poly(A)-containing mRNA sequences. Hybridization kinetic experiments show that the poly(A)-containing sequences of both clones are organized into indistinguishable abundance classes, and that the vast majority of the sequences are common to both the parent and derivative clones. The levels of two specific messenger RNAs (α- and β-globin mRNA) which characterize highly differentiated mouse erythroid cells were much less than 1 molecule per cell in either cell type. Titration of a balanced complementary DNA probe to AKR murine leukemia virus (AKR-MuLV) 70S RNA with purified polysomal poly(A)-containing RNA from both parent and derivative clones shows that approximately 5000 and 1200 viral 35S RNA equivalents are present in the cytoplasm of growing and resting clone MCA cells, respectively. Rapidly growing clone 2B cells contain less than about 30 viral 35S RNA equivalents per cell. Viral specific sequences therefore correspond to members of the high abundance class of poly(A)-containing RNA sequences in clone MCA cells and to the low abundance class of sequences in clone 2B cells. Within the limits of detection, this large increase in abundance is characteristic only of viral specific RNA sequences.  相似文献   

13.
Serum stimulation of stationary cultures of Chinese hamster ovary cells CHO-K1 (clone 773) is accompanied by sustained increase in ouabain-sensitive rubidium (potassium) influx which results in the elevation of intracellular potassium content from 0.5-0.6 to 0.7-0.8 mmole per gram of protein. Cytofluorometric studies of serum-stimulated CHO-773 cultures have shown that the intracellular potassium increase is necessary for successful G1----S progression. The elevation of intracellular potassium was found to occur simultaneously with the cellular protein growth. Cycloheximide (10 micrograms/ml) does not influence the early Na,K-ATPase activation induced by serum; however, it abolishes the sustained increase of both rubidium influx and intracellular potassium content. In serum stimulated cells ouabain increases the potassium efflux; this ouabain effect is not observed after S phase, when rubidium (potassium) influx decreases and intracellular potassium content stops growing.  相似文献   

14.
Studies suggest that Gr1(+)CD11b(+) cells have immunoregulatory function, and these cells may play an important role in autoimmune diseases. In this study, we investigated the regulatory role of Gr1(+)CD11b(+) cells in protecting against type 1 diabetes in NOD mice. In this study, we showed that temporary B cell depletion induced the expansion of Gr1(+)CD11b(+) cells. Gr1(+)CD11b(+) cells not only directly suppress diabetogenic T cell function but also can induce regulatory T cell differentiation in a TGF-β-dependent manner. Furthermore, we found that Gr1(+)CD11b(+) cells could suppress diabetogenic CD4 and CD8 T cell function in an IL-10-, NO-, and cell contact-dependent manner. Interestingly, single anti-Gr1 mAb treatment can also induce a transient expansion of Gr1(+)CD11b(+) cells that delayed diabetes development in NOD mice. Our data suggest that Gr1(+)CD11b(+) cells contribute to the establishment of immune tolerance to pancreatic islet autoimmunity. Manipulation of Gr1(+)CD11b(+) cells could be considered as a novel immunotherapy for the prevention of type 1 diabetes.  相似文献   

15.
Helper T cell clone 52.3 supernatant (52.3 SN) was previously shown to be able to stimulate gradient-purified murine resting B cells in the absence of any additional stimulus. However, the proportion of cells that were accounting for the thymidine uptake and the Ig production was unknown. In this paper, we have studied induced changes that can be measured at the single cell level, and have thus determined the frequency of resting B cells that respond to 52.3 SN. Results indicate that 52.3 SN induces an increased I-A expression and a cell size enlargement on virtually all resting B cells. A significant proportion (30%) of these cells later becomes large blasts. Acridine orange staining revealed that in the presence of 52.3 SN a large fraction of the resting B cells undergoes the G0 to G1 transition. Furthermore, 52.3 SN is able to induce at least 20% of the cells to continue through the cell cycle into S phase as indicated by propidium iodide staining of DNA. Finally, a fraction of the 52.3 SN-stimulated cells differentiate to Ig-producing cells. Our present results suggest that resting B cells express functional receptors for some lymphokines and that these lymphokines can act in the absence of membrane Ig occupancy. Our findings further support the existence of a B cell-activating factor acting in a MHC-unrestricted manner and responsible for the entry of resting B cells into cell cycle. The relationship between this factor and other lymphokines is discussed.  相似文献   

16.
The reduced efficiency of the mammalian immune system with aging increases host susceptibility to infectious and autoimmune diseases. However, the mechanisms responsible for these pathologic changes are not well understood. In this study, we demonstrate that the bone marrow, blood, and secondary lymphoid organs of healthy aged mice possess increased numbers of immature myeloid cells that are phenotypically similar to myeloid-derived suppressor cells found in lymphoid organs of mice with progressive tumors and other pathologic conditions associated with chronic inflammation. These cells are characterized by the presence of Gr1 and CD11b markers on their surfaces. Gr1(+)CD11b(+) cells isolated from aged mice possess an ability to suppress T cell proliferation/activation and produce heightened levels of proinflammatory cytokines, both constitutively and upon activation, including IL-12, which promotes an excessive production of IFN-γ. IFN-γ priming is essential for excessive proinflammatory cytokine production and the suppressive activities by Gr1(+)CD11b(+) cells from aged mice. These cells suppress T cell proliferation through an NO-dependent mechanism, as depletion of splenic Gr1(+) cells reduces NO levels and restores T cell proliferation. Insights into mechanisms responsible for the proinflammatory and immune suppressive activities of Gr1(+)CD11b(+) cells from aged mice have uncovered a defective PI3K-Akt signaling pathway, leading to a reduced Akt-dependent inactivation of GSK3β. Our data demonstrate that abnormal activities of the Gr1(+)CD11b(+) myeloid cell population from aged mice could play a significant role in the mechanisms responsible for immune senescence.  相似文献   

17.
The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.  相似文献   

18.
O P Clausen 《Cytometry》1987,8(6):612-617
DNA synthesis kinetics of P388 leukemic cells growing in ascites form in BDF1 hybrid mice were investigated during the periods of exponential growth and growth restriction. Incorporation of tritiated thymidine, and in some instances tritiated uridine, was studied by autoradiography in cells sorted from S-phase fractions during DNA flow cytometry. During exponential growth continuous labeling with tritiated thymidine indicated a growth fraction of unity, whereas the growth fraction was about 30% during growth restriction. At this growth phase the majority of cells with S phase DNA content remained unlabeled after pulse labeling with tritiated thymidine or uridine, indicating that both the "salvage" and the "de novo" DNA synthesis pathways were blocked in most S-phase cells. After pulse labeling with tritiated thymidine the DNA synthesis rate pattern was investigated by sorting of consecutive fractions of cells throughout the S phase followed by quantitative autoradiography. With exception of a reduced rate in the middle of S phase, the DNA synthesis rate increased as the cells progressed through S phase during exponential growth. In contrast, the DNA synthesis rate pattern had a relative peak in the middle of S phase during growth restriction, which is otherwise characterized by a low mean DNA synthesis rate.  相似文献   

19.
Hydroxyurea treatment affects the G1 phase in next generation CHO cells   总被引:1,自引:0,他引:1  
DNA replication kinetics were studied in populations of synchronized CHO cells treated in the previous generation with hydroxyurea. These CHO cells were re-synchronized by selective detachment of mitotic cells after previously synchronized G1 traversing cultures were treated with 0.1 mM and 2 mM hydroxyurea for 9 and 13 h. Our results show that these cells exhibit a shortening of G1 of at least 1 h relative to cells selected in mitosis from untreated exponentially growing cultures. Survival studies indicated that the hydroxyurea treatments did not affect plating efficiencies. Cell viability was reduced when the initially synchronized populations were blocked with 2 mM, but not 0.1 mM hydroxyurea for greater than 13 h. DNA replication measurements after these blocks showed that all cultures treated with 2 mM hydroxyurea for either 9, 13 or 15 h were blocked at the same point near the G1/S boundary, and then progressed through S phase with similar kinetics. The observed shortening of G1 in the next generation of these cells was independent of both the concentration (0.1 or 2.0 mM) and the time (9 or 13 h) of the hydroxyurea block. These results suggest that specific events relating to the next cell generation can be uncoupled from DNA synthesis and can occur when hydroxyurea inhibits normal cell cycle traverse of G1 cells into and through S phase.  相似文献   

20.
Human embryonic stem (ES) cells have an expedited cell cycle ( approximately 15 h) due to an abbreviated G1 phase ( approximately 2.5 h) relative to somatic cells. One principal regulatory event during cell cycle progression is the G1/S phase induction of histone biosynthesis to package newly replicated DNA. In somatic cells, histone H4 gene expression is controlled by CDK2 phosphorylation of p220(NPAT) and localization of HiNF-P/p220(NPAT) complexes with histone genes at Cajal body related subnuclear foci. Here we show that this 'S point' pathway is operative in situ in human ES cells (H9 cells; NIH-designated WA09). Immunofluorescence microscopy shows an increase in p220(NPAT) foci in G1 reflecting the assembly of histone gene regulatory complexes in situ. In contrast to somatic cells where duplication of p220(NPAT) foci is evident in S phase, the increase in the number of p220(NPAT) foci in ES cells appears to precede the onset of DNA synthesis as measured by BrdU incorporation. Phosphorylation of p220(NPAT) at CDK dependent epitopes is most pronounced in S phase when cells exhibit elevated levels of cyclins E and A. Our data indicate that subnuclear organization of the HiNF-P/p220(NPAT) pathway is rapidly established as ES cells emerge from mitosis and that p220(NPAT) is subsequently phosphorylated in situ. Our findings establish that the HiNF-P/p220(NPAT) gene regulatory pathway operates in a cell cycle dependent microenvironment that supports expression of DNA replication-linked histone genes and chromatin assembly to accommodate human stem cell self-renewal.  相似文献   

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