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1.
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 (G 1and G 2) phases of the cell cycle. At the level used, BUdR by itself induces no aberrations. Illumination during the G 1 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 G 2 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 G 2-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. 相似文献
2.
The shape, distribution, and content of mitochondria in individual cells were examined during the cell cycle phases (G 0/G 1, S, G 2 mitosis) in living human fibroblasts by static cytofluorometry and fluorescence microscopy. The morphocytochemical evaluations were performed in cell cultures submitted to double supravital fluorochrome staining with Hoechst 33342 and DiOC 6 to label DNA and mitochondria, respectively. The staining modalities were based on the stability of mitochondrial labeling. The G 1 to early S phases were characterized by the presence of filamentous mitochondria, except during the early postmitotic period. During late S, G 2, and mitotic phases, mitochondrial mass reached its highest value and mitochondria became short and numerous. During the last stage of mitosis, mitochondria were distributed among daughter cells through a cytoplasmic bridge. 相似文献
3.
The cytotoxic and mutagenic effect of X-irradiation was determined with Chinese hamster ovary cells arrested in the G 0/G 1 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 G 1, show a similar does response for mutant frequency, while those irradiated at 10 h or later, i.e. in late G 1, S or G 2, 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 G 0/G 1 phase allow more time for such interactions in the absence of semiconservative DNA replication. 相似文献
4.
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 G 2+ 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 G 2+M phase was reduced by treatment with NG-mono-methyl-
-arginine. However, after treatment with exogenous nitric oxide or sodium nitroprusside, the G 0/G 1 phase increased, but the early-G 2+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 G 2+M phase. 相似文献
5.
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 G 1 phase and decreased to a minimum during S phase. Similarly, cAMP-dependent protein kinase activities peaked at both G 1 and G 2+M phases of the cell cycle, decreasing to a minimum at S phase. Addition of N6, O 2'-dibutyryl (Bt 2)-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 Bt 2-cAMP treated cells at G 1 was also larger than the control cells. The present study demonstrated a regulatory role of cAMP in the cell cycle progression in dinoflagellates. 相似文献
6.
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, G 2 and (G 1 + G 0) 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 G 2 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. 相似文献
7.
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 mm 2). Ten days later, a tumor sample (area of ∼5 mm 2) 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 G 1, S or G 2-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 G 2-M phase cells. The approximate average distribution patterns of G 1 and G 2-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 G 1, 21 HALO for S and 15 HALO for G 2-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. 相似文献
8.
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 G 1 and retained G 1-phase-specific hypophosphorylated retinoblastoma protein (pRb). We showed that suppression of traverse through G 1 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 G 2/M phase progression. FGF-2 acted solely to sustain hESC pluripotency rather than to promote cell cycle progression or inhibit apoptosis. Differentiation increased G 1-phase content, reinstated cyclin D activity, and restored the proliferative response to FGF-2. Treatment with CDK-2 inhibitor delayed hESC in G 1 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 G 1-specific, pRb-dependent restriction checkpoint in hESC closely associated with the regulation of pluripotency. 相似文献
9.
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 G 1 phase, cyclins C, D and E, or in S, G 2 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 G 1 phase, cyclin A in late G 1/early S phase and cyclin B1 in G 2. 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 G 1 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 G 1 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. 相似文献
10.
Abstract: Rat glioma mouse neuroblastoma hybrid neurotumor cells (NG108-15), synchronized by amino acid deprivation, showed a cell-cycle-dependent peak of activity of a ganglioside N-acetylgalactosaminyl transferase 14-24 h following release from the cell cycle block (S/G 2 phase). Maximal expression of two typical lysosomal hydrolases, N-acetyl-β-hexosaminidase and β-galactosidase, occurred between 18 and 21 h following release (S phase), declining to G 1 phase levels during the peak of N-acetylgalactosamine (GalNAc) transferase activity. In addition, glycosyltransferase activity in G 2 phase cells showed an increase in apparent V max (suggesting the presence of more enzyme/mg of cell protein) and apparent binding affinity for uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) (32 versus 14 M) when compared to transferase activity in the G 1 phase. However, the opioid peptide enkephalin [D-Ala 2, o-Leu 5], which inhibits ganglioside GalNAc transferase activity in unsynchronized NG108-15 cultures, was much more inhibitory in whole cells 8 h after release from the cell cycle block (G 1 phase) than in cells 20 h after release (G, phase), with 50% inhibition occurring at 2 ± 10 -9M and 2 ± 10 -7M, respectively. These results suggest that the GalNAc transferase activity is regulated in more than one way during the cell cycle, since both V max and K m changes are observed, and that the cyclic AMP-dependent mechanism by which opiates reduce transferase activity is receptor mediated and cell cycle dependent. 相似文献
12.
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 G 2 and S phase of the cell cycle. 相似文献
13.
The cytotoxic effect of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of cultured human diploid fibroblasts was examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-hour periods during the S phase with 5-bromodeoxyuridine (0.1–10 μM), followed by irradiation with near-UV (5–10 min). The 5-bromodeoxyuridine-plus-irradiation treatment was cytotoxic, while treatment with 5-bromodeoxyuridine alone or irradiation alone was not cytotoxic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 hours of S phase, whereas scarce lethality was observed in late S phase. The extent of substitution of 5-bromodeoxyuridine for thymidine in newly synthesized DNA was similar in every period of the S phase. Furthermore, no specific period during S phase was significantly more sensitive to treatment with respect to DNA damage, as determined by an induction of unscheduled DNA synthesis. These results suggest that a certain region or regions in the DNA of human diploid fibroblasts, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by 5-bromodeoxyuridine treatment plus near-UV irradiation for cell survival. 相似文献
14.
Using the C3H/10T 1/2 CL8 line of mouse embryo fibroblasts and three different methods of obtaining cell cycle synchrony, namely arginine or isoleucine deficiency and release from postconfluence inhibition of growth, a sensitive phase for oncogenic transformation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been found. This sensitive phase is located somewhere between the G1/S boundary and a point 4 hr prior to this marker. Methylation of cellular macromolecules by tritiated MNNG is not cycle-dependent in cells synchronized by arginine deficiency. The capacity of cells to repair DNA single strand breaks produced by MNNG was examined by alkaline sucrose sedimentation analysis in cells synchronized by arginine deficiency and treated with MNNG during phases of the cell cycle sensitive and insensitive to oncogenic transformation. Whereas DNA repair was found to be equally rapid in cells treated just before S phase (I), or just after commencement of DNA synthesis (III), transformation was maximal in I. By contrast, cells treated when blocked by arginine deficiency (II) repaired DNA slowly and were not sensitive to malignant transformation. Cells in I and II, which repaired DNA at very different rates, were equally sensitive to MNNG-induced lethality, while cells in III, which repaired DNA at the same rate as cells in I, suffered greater lethality. Thus, in this system it was concluded that there was no direct correlation between DNA repair, as measured by alkaline sucrose sedimentation analysis of prelabeled DNA, and malignant transformation or lethality produced by MNNG. In preliminary experiments malignant transformation induced by cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) has been found to occur mainly in S phase, indicating that diverse chemical oncogens may have different sites of action, or that activation of chemical oncogens is cell cycle-specific for some agents. 相似文献
15.
The cytotoxic and mutagenic effects of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of a chemically induced, transformed line (Hut-11A cells) derived from diploid human skin fibroblasts were examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-h periods during the S phase with 5-bromodeoxyuridine (BrdU) followed by irradiation with near-ultraviolet (UV). The BrdU-plus-irradiation treatment was cytotoxic and mutagenic, while treatment with BrdU alone or irradiation alone was neither cytotoxic nor mutagenic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 h of S phase, whereas scare lethality was observed in late S phase. The BrdU-plus-irradiation treatment induced ouabain- and 6-thioguanine-resistant mutants, while BrdU alone or irradiation alone was not mutagenic. Ouabain-resistant mutants were induced during early S phase by the BrdU-plus-irradiation treatment. 6-Thioguanine-resistant mutants, however, were induced during middle to late S phase. These results suggest that a certain region or regions in the DNA of Hut-11A cells, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by BrdU treatment plus near-UV irradiation for cell survival and that gene(s) responsible for Na+/K+ ATPase is replicated during early S phase and gene(s) for hypoxanthine phosphoribosyl transferase is replicated during middle to late S phase. 相似文献
16.
Abstract. The c-sis oncogene encoding the B-chain of platelet-derived growth factor (PDGF) may be involved in an autocrine growth stimulation of tumours expressing the PDGF receptor, such as glioblastomas and sarcomas. To investigate whether expression of c-sis RNA is regulated in a cell cycle dependent manner, human A172 glioblastoma cells were synchronized by either centrifugal elutriation or chemical blockage with the DNA synthesis inhibitors hydroxyurea or aphidicolin. In non-perturbed elutriated cells, c-sis RNA levels were lower in the S phase of the cell cycle than in the G 1 phase. In contrast, the chemically synchronized cells revealed a transient rise in c-sis RNA shortly after drug release, in early S phase. The RNA changes occurring after release from drug inhibition represent cell recovery from drug induced metabolic disturbances rather than true cell cycle dependent effects. 相似文献
17.
We investigate the effect of EGF on IP 3 production, PLCγ phosphorylation, calcium transients in rat hepatocytes isolated in quiescent liver (G 0 phase of cell cycle) and at 4 h (G 1 phase of cell cycle) and 24 h (M phase of cell cycle) after partial hepatectomy. Our results show that EGF does not utilize IP 3 and calcium as its signal transduction molecules when the hepatocytes are in vivo stimulated to entry in the cell cycle. In particular the growth factor does not phosphorylate PLCγ and induces a decrease in IP 3 content. These data suggest that EGF utilizes different signal transduction to send information from receptor to nucleus during PH with respect to the quiescent liver. 相似文献
18.
The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G 1 phase by anisoniycio or cycloheximide caused the arrest of cells in the G 1 phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G 1 phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G 1 phase caused the arrest of cells in the G 1 phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G 1 phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G 1 phase, and that the mRNA(s) that encode these proteins are also synthesized at the G 1 phase. 相似文献
19.
Rotenone is a heterocyclic compound widely used as an insecticide, acaricide and piscicide. Its toxicity is mainly caused by the inhibition of mitochondrial respiratory processes and ATP production, resulting in the generation of reactive oxygen species. Reactive oxygen species can interact with DNA, RNA and proteins, leading to cell damage, followed by death. We used the Comet assay, and we analyzed chromosome aberrations, in order to evaluate the genotoxic and clastogenic effects of rotenone on the different phases of the cell cycle. Cultured human lymphocytes were treated with 1.0, 1.5 and 2.0 microg/mL rotenone during the G1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle. Rotenone induced DNA damage and was clastogenic, but the clastogenicity was detected only with treatments conducted during the G1/S and S phases of the cell cycle. Rotenone also induced endoreduplication and polyploidy in treatments made during G1, while it significantly reduced the mitotic index in all phases of the cell cycle. 相似文献
20.
Abstract. A computer model of rat spermatogenesis was created, based on autoradiographic studies of durations of the phases of the cell cycle (G 1, S, G 2 and mitotic phases) of each germ-cell type. With this model it is possible to predict and to gain insight into the changes of the DNA content occurring during the normal process of spermatogenesis. the relative proportions of haploid, diploid, S phase and tetraploid germ cells with increasing age of the rats were calculated. Calculated and actual experimental flow cytometry data were compared to test the accuracy of the model, and these show good agreement. the present work demonstrates that single-parameter DNA analysis of testicular cells is primarily a reflection of germ cells in the spermatocyte and spermatid stages of development, and of non-germ cells. the FCM single-parameter DNA analysis of testicular cells is relatively insensitive to changes in the stem cell and spermatogonial stages of germ-cell development. 相似文献
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