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At least 883 records · Page 49Linked to original sources
PubMed2005

Appearance of actin microfilament 'twin peaks' in mitosis and their function in cell plate formation, as visualized in tobacco BY-2 cells expressing GFP-fimbrin.

The actin cytoskeleton of higher plants plays an essential role in plant morphogenesis and in maintaining various cellular activities. In this study we established a tobacco BY-2 cell line, stably transformed with a GFP-fimbrin actin-binding domain (ABD) 2 construct, that allows visualization of actin microfilaments (MFs) in living cells. Using this cell line, designated BY-GF11, we performed time-sequential observations of MF dynamics during cell-cycle progression. Detailed analyses revealed the appearance of a broad MF band in the late G2 phase that separated to form a structure corresponding to the so-called actin-depleted zone (ADZ) in mitosis. In BY-GF11, the MF structure at the cell cortex in mitosis appeared to form two bands rather than the ADZ. Measurements of fluorescent intensities of the cell cortex indicated an MF distribution that resembled two peaks, and we therefore named the structure MF 'twin peaks' (MFTP). The cell plate formed exactly within the valley between the MFTP at cytokinesis, and this cell-plate guidance was distorted by disruption of the MFTP by an inhibitor of actin polymerization. These results suggest that the MFTP originates from the broad MF band in the G2 phase and functions as a marker of cytokinesis.

Actin Cytoskeleton↗
PubMed1990

Carrier detection in xeroderma pigmentosum.

We were able to detect clinically normal carriers of xeroderma pigmentosum (XP) genes with coded samples of either peripheral blood lymphocytes or skin fibroblasts, using a cytogenetic assay shown previously to detect individuals with cancer-prone genetic disorders. Metaphase cells of phytohemagglutinin-stimulated T-lymphocytes from eight individuals who are obligate heterozygotes for XP were compared with those from nine normal controls at 1.3, 2.3, and 3.3 h after x-irradiation (58 R) during the G2 phase of the cell cycle. Lymphocytes from the XP heterozygotes had twofold higher frequencies of chromatid breaks or chromatid gaps than normal (P less than 10(-5)) when fixed at 2.3 or 3.3 h after irradiation. Lymphocytes from six XP homozygotes had frequencies of breaks and gaps threefold higher than normal. Skin fibroblasts from an additional obligate XP heterozygote, when fixed approximately 2 h after x-irradiation (68 R), had a twofold higher frequency of chromatid breaks and a fourfold higher frequency of gaps than fibroblasts from a normal control. This frequency of aberrations in cells from the XP heterozygote was approximately half that observed in the XP homozygote. The elevated frequencies of chromatid breaks and gaps after G2 phase x-irradiation may provide the basis of a test for identifying carriers of the XP gene(s) within known XP families.

Adolescent↗
PubMed1982

[Possible mechanisms of the occurrence of chromosome aberrations. I. Patterns in the occurrence of aberrations induced by ultraviolet irradiation].

The frequency of chromosome aberrations induced by UV light at wavelengths 254, 265, 280 and 302 using doses 2-10 J/m2 in the primary culture of mouse embryonic fibroblasts during the G1, S and G2 phases was studied at metaphase of the first mitosis. Two classes of chromosome aberrations were distinguished. These classes differ in the time intervals of the final establishment of the cell cycle. The aberrations of the class 1 emerge before the beginning of prometaphase (possibly, at interphase). Formation of the second class aberrations is completed during the metaphase. It is shown that the class 1 aberrations occur with almost the same rate in approx. 7% of cells, irrespective of the cell cycle, irradiation dose and wavelength. It is suggested that these aberrations arise as a result of indirect UV action on the chromosome structures; the mechanism of their emergence does not depend on DNA replication. The class 2 aberrations do not appear after UV irradiation during the post-DNA-synthetic G2 phase of the cell cycle. However, after UV treatment at the G1 or S periods, they represent the majority of aberrations and their rate increases almost monotonously with the radiation dose. The UV action spectrum for these aberrations coincides with the adsorption spectrum of thymidine and the action spectrum for DNA cross-links. Thus, it may be inferred that formation of DNA cross-links following thymine dimerization is the first step in formation of UV-induced aberrations of the class 2. The passage of cells through DNA replication is a very important step in the process of their emergence.

Acriflavine↗
PubMedDate not supplied

[Spatial-temporal patterns of the cell cycles in embryos of the clawed toad during gastrulation and neurulation].

The dynamics of DNA synthesis and duration of G2-phases in different dorso-ventral zones and cell layers were studied using 3H-thymidine in the X. laevis embryos during gastrulation and neurulation. Gradients of the index of labelled nuclei and of the rate of 3H-thymidine incorporation decreasing in the ventro-dorsal direction were observed during the whole period of gastrulation. In the end of gastrulation a wave of increase of the index of labelled nuclei and of the rate of 3H-thymidine incorporation proceeds in the same direction and, as a result, both the gradients are inverted. A high positive correlation was found between the simultaneously registered indices of labelled nuclei in different cell layers of the same dorso-ventral zones. The data were obtained suggesting the ventro-dorsal migration of cells in G2-phase. A conclusion is drawn that the regulation of cell cycles during gastrulation--neurulation obeys integral laws among which the waves of cell entrance in S-phase play an important role.

Animals↗
PubMed1999

Identification of the Xenopus 20S proteasome alpha4 subunit which is modified in the meiotic cell cycle.

The proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. To investigate the regulatory mechanism for the 26S proteasome in cell-cycle events, we purified this proteasome from immature and mature oocytes, and compared its subunits. Immunoblot analysis of 26S proteasomes showed a difference in the subunit of the 20S proteasome. A monoclonal antibody, GC3beta, cross-reacted with two bands in the 26S proteasome from immature oocytes (in G2-phase); however, the upper band was absent in the 26S proteasome from mature oocytes (in M-phase). These results suggest that changes in the subunits of 26S proteasomes are involved in the regulation of the meiotic cell cycle. Here we describe the molecular cloning of one of the alpha subunits of the 20S proteasome from a Xenopus ovarian cDNA library using an anti-GC3beta monoclonal antibody. From the screening, two types of cDNA are obtained, one 856bp, the other 984bp long. The deduced amino-acid sequences comprise 247 and 248 residues, respectively. These deduced amino-acid sequences are highly homologous to those of alpha4 subunits of other vertebrates. Phosphatase treatment of 26S proteasome revealed the upper band to be a phosphorylated form of the lower band. These results suggest that a part of the alpha4 subunit of the Xenopus 20S proteasome, alpha4_xl, is phosphorylated in G2-phase and dephosphorylated in M-phase.

Amino Acid Sequence↗
PubMed1982

Production of plasminogen activator by synchronized normal and Rous sarcoma virus-transformed chicken fibroblasts in culture.

We studied intracellular activity of the plasminogen activator within the cell cycle of chemically synchronized normal and RSV-transformed chick fibroblasts in culture. Consideration has also been given to the relationship between the plasminogen activator activity and cycles of DNA synthesis or mitosis in cycling fibroblasts after viral infection. The plasminogen activator activity of the cell lysates was assayed on [125I]fibrin-coated Petri dishes and was expressed as the radioactivity released from the plates. Normal fibroblasts produced detectable levels of plasminogen activator in the S-phase and late G2-phase or mitosis of the cell cycle. In contrast, RSV-transformed cells produced high levels of this activator throughout the entire cell cycle although this activity fluctuated and reached a maximum in the G2-M periods. We also found that the level of plasminogen activator activity in the transformed fibroblasts is influenced by the cycles of DNA synthesis and that cell division is required for the appearance of plasminogen activator activity in the 'de novo' virus-infected cultures.

Animals↗
PubMed2005

A temperature-sensitive mutation in the WD repeat-containing protein Smu1 is related to maintenance of chromosome integrity.

Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibits chromosomal instability and cell cycle arrest at S and G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39 degrees C. To identify the causative mutation, we fused tsTM18 cells with normal human cells to generate hybrids carrying fragments of human chromosomes. Analysis of chromosome content of temperature-resistant transformants and introduction of a bacterial artificial chromosome containing part of human chromosome 9 led to isolation of the human SMU1 gene. Comparison of sequences of the Smu1 gene from wild-type and mutant cells revealed that the mutant phenotype is caused by a G-to-A transition that yields a gly-to-arg substitution at position 489 in hamster Smu1. The substituted glycine is located in the WD-repeat domain of Smu1. Single-stranded DNA accumulated in the nuclei of mutant cells at 39 degrees C. Furthermore, cdc2 kinase was not activated during G2 phase, and there was no chromosome segregation due to incomplete assembly of the spindle during M phase. Thus, Smu1 appears to be involved directly or indirectly in DNA replication, activation of cdc2 kinase, spindle assembly, and maintenance of chromosome integrity, reflecting the important roles of Smu1 in cellular function.

Amino Acid Sequence↗
PubMed2005

The Plk3-Cdc25 circuit.

Polo-like kinases (Plks) are key regulators of the cell cycle, especially in the G2 phase and mitosis. They are incorporated into signaling networks that regulate many aspects of the cell cycle, including but not limited to centrosome maturation and separation, mitotic entry, chromosome segregation, mitotic exit, and cytokinesis. The Plks have well conserved 30-amino-acid elements, designated polo boxes (PBs), located in their carboxyl-termini, which with their flanking regions constitute a functional Polo-box domain (PBD). Members of the Plk family exist in a variety of organisms including Polo in Drosophila melanogaster; Cdc5 in Saccharomyces cerevisiae; Plo1 in Schizosaccharomyces pombe; Plx1 in Xenopus laevis; and Plk1, Snk/Plk2, Fnk/Prk/Plk3, and Sak in mammals. Polo, Cdc5, and Plo1 are essential for viability. The Plks can be separated into two groups according to their functions. The first group (Polo, Cdc5, plo1, Plx1, and Plk1) primarily performs mitotic functions, whereas the second group (Plk2 and Plk3) appears to have additional functions during the G1, S, and G2 phases of the cell cycle. Several contributions to this issue will discuss different aspects of Plk involvement in cell-cycle regulation. This review, therefore, will focus on the role of Plk3 in regulating Cdc25 phosphatase function and its effect on the cell cycle.

Animals↗
PubMed1983

Effects of Hoechst 33258 on different cell cycle events. I. Inhibition of synthetic activities in bone-marrow cells of the mole rat Bandicota bengalensis.

A study of the effects of the A-T base-specific ligand Hoechst 33258 on the cell cycle events of the bone-marrow cells of the mole rat Bandicota bengalensis reveals that (1) the G2 phase is prolonged by 2 h; (2) the chromosomes are undercondensed to varying degrees in about 30% metaphases; (3) DNA as well as RNA synthesis is partially inhibited. Study on DNA fibre autoradiograms shows that despite the inhibition of DNA synthesis the average replicon size and rate of fork migration remain unchanged, but there is a marked asynchrony of initiation of replicons in replicon clusters in H-treated cells in comparison with control. It is suggested that these inhibitory effects result from the stabilization of the double-stranded organization of DNA (dsDNA) by the drug so that the unwinding of DNA at the replication or transcription origin sites is greatly impaired. It is likely that this introduces asynchrony in the initiation of replicons within replicon clusters which may retard the overall rate of replication. In addition, it appears that the reduction in transcription is at least one cause for the prolongation of G2 phase.

Animals↗
PubMed1996

Differential effects of dose rate and superfractionation on survival and cell cycle of V79 cells from spheroid and monolayer culture.

Recent developments concerning brachytherapy suggest conditions for an equivalence between the common continuous low dose rate (CLDR) exposure and pulsed irradiation regimens (PDR), provided that total dose is administered in the same overall time. The respective theoretical considerations have been based solely on the phenomenon of sublethal damage recovery. The present study, therefore, aimed to assess a possible influence of growth state/cell cycle progression when CLDR and different super fractionation protocols are compared. The respective experiments were performed with V79 cells that can be grown as a rapidly proliferating monolayer culture or as small spheroids (without hypoxia) where most of the cells are out of cycle. Differential changes in cell cycle distribution occurring during the compared exposure schemes and their impact on cell survival were expected to be expressed most clearly with this model system because of the short G1 phase. Cell irradiations were performed with brachytherapy sources either continuously (137Cs) or with high dose rate pulses (192Ir) at different (1 h and 4 h) pulse repetitions whereby the overall dose rate was kept constant to approximately 1 Gy/h. Cell survival curves were generated by sampling cells at different exposure times or number of pulses, respectively. For spheroid cells an unequivocal decrease of effectivity was demonstrated with decreasing dose per pulse, and the dose effect relation obtained with hourly pulses of 1 Gy was indistinguishable from the CLDR response. For monolayer cells, on the contrary, the scheme of hourly pulses was significantly more effective than the CLDR irradiation. As measured by flow cytometry, this different behaviour could be attributed to the accumulation of cycling cells in the radiosensitive G2/M phase (G2 block) during protracted exposure which was drastically more pronounced for the pulsed scheme compared to the CLDR condition. The observed principle phenomenon of a block to cell cycle progression from high dose rate pulses (at low overall dose rate) may be less expressed in (human) cells having a long G1 period, but if applicable to a clinical situation, an increase of acute effectiveness of a superfractionated brachytherapy protocol has to be considered.

Animals↗
PubMed1995

Epidermal growth factor rapidly impairs activation of p34cdc2 protein kinase in HeLa cells at the G2-M boundary.

Epidermal growth factor (EGF) has been shown rapidly to inhibit the transition from G2 phase to mitosis: beyond this transition point the cells are refractory to EGF (Kinzel et al., 1990, Cancer Res., 50:7932-7936). Using synchronized HeLa cells, EGF has now been shown to induce an overall decrease of the histone H1 kinase activity of p34cdc2 after 20 min of treatment, a time course which correlates with the number of cells in metaphase. The kinase level of actively mitotic cells is not altered by EGF. Neither the amount of p34cdc2 protein present nor that of Cyclin B in influenced by EGF, and the formation of the p34cdc2/Cyclin B complex is also unaffected. The use of antiphosphotyrosine antibodies, however, showed that p34cdc2 from cultures treated with EGF was more intensely stained than that of control cells, indicating that EGF treatment prevents the tyrosine dephosphorylation which is required for expression of the protein kinase activity of the complex. Taken together, the results show that EGF in HeLa cells very rapidly prevents the p34cdc2/Cyclin B complex from expressing kinase activity at the G2-M boundary, which appears to be the cause for the inhibition in G2 phase.

Blotting, Western↗
PubMed1997

p53, Cip1, and Gadd153 expression following treatment of A549 cells with natural and man-made vitreous fibers.

DNA damage induced by chemicals and ionizing radiation is associated with the expression of negative regulators of the cell cycle. The arrest of cells in G1 and G2 phases of the cell cycle provides time for DNA repair. Asbestos fibers are carcinogenic when inhaled by both humans and animals; however, the mechanism by which the fibers exert their effect is unknown. This work was undertaken to determine whether the expression of DNA damage-inducible genes differs between crocidolite, a fiber positive for lung tumors, and JM 100 glass microfiber, which is negative for lung tumors when inhaled by rats. Temporal and dose-related expressions of p53, Cip1, and Gadd153 proteins were determined in cultured A549 cells treated with either Union Internationale Contre le Cancer crocidolite or JM 100 for 20 hr and cultured in fresh media. Immunolabeled cells were analyzed by flow cytometry, and the increased number of protein-expressing cells was determined by subtracting the expression in unexposed cells from exposed cells. Crocidolite induced the expression of all three proteins with a maximum expression after approximately 18 hr in fresh media. At a similar time point, JM 100 did not markedly induce the three proteins. Crocidolite also induced a dose-dependent increase in the number of cells in the G2 phase of the cell cycle. These results show that asbestos behaves like ionizing radiation and genotoxic chemicals by inducing proteins associated with DNA damage and cell-cycle arrest. The clear difference in response between crocidolite and JM 100 may help elucidate the mechanism of action of toxic and nontoxic fibers.

Animals↗
PubMed1980

The activities of thymidine metabolising enzymes during the cell cycle of a human lymphocyte cell line LAZ-007 synchronised by centrifugal elutriation.

The activities throughout the cell cycle of thymidine kinase (EC 2.7.1.21), dihydrothymine dehydrogenase (EC 1.3.1.2), thymidine phosphorylase (EC 2.4.2.4) and dTMP phosphatase (EC 3.3.3.35) were measured in the Epstein-Barr virally transformed human B lymphocyte line LAZ-007. Cells were synchronised at different stages of the cell cycle using the technique of centrifugal elutriation. The degree of synchrony in each cycle-stage cell population was determined by flow microfluorimetric analysis of DNA content and by measurement of thymidine incorporation into DNA. The activity of the anabolic enzyme thymidine kinase was low in the G1 phase cells, but increased manyfold during the S and G2 phases, reaching a maximum after the peak of DNA synthesis, then decreasing in late G2 + M phase. By contrast, the specific activities of the enzymes involved in thymidine and thymidylate catabolism, dihydrothymine dehydrogenase, thymidine phosphorylase and dTMP phosphatase remained essentially constant throughout the cell cycle, indicating that the fate of thymidine at different stages of the cell cycle is governed primarily by regulation of the level of the anabolic enzyme thymidine kinase and not by regulation of the levels of thymidine catabolising enzymes.

B-Lymphocytes↗
PubMed1997

Preferential cytoplasmic localization of p34cdc2 in recurrent human squamous cell carcinoma after radiotherapy.

Duration of the G2-phase delay and arrest after exposure to ionizing radiation is thought to influence radiosensitivity. The kinase activity of the p34cdc2-cyclin B complex and the p34cdc2-cyclin A complex is implicated in G2- to M-phase transition and in G2-phase arrest after exposure to ionizing radiation. We analyzed the expression level and the subcellular location of p34cdc2, cyclin A and cyclin B in head and neck squamous cell carcinoma (SCC) tumors; samples were obtained from patients with locally nonrecurrent and recurrent tumors that had been treated by surgery and radiotherapy. No significant difference was noticed in cyclin A, cyclin B and p34cdc2 expression. However, we noted a significant preferential cytoplasmic location of p34cdc2 in recurring tumors compared to the nonrecurring ones (P < 0.001). This abnormal location of p34cdc2 occurs even in primary tumors in patients with recurring tumors, suggesting that a default in the activation of p34cdc2 kinase could be implicated in clinical radioresistance.

CDC2 Protein Kinase↗
PubMed2003

Induction of apoptosis and expression of cell cycle regulatory proteins in response to a phytosphingosine derivative in HaCaT human keratinocyte cells.

Ceramide, a compound derived from sphingomyelin, a sphingolipid precursor, affects cell functions such as growth, differentiation, cell division and apoptosis. We have shown that the phytosphingosine derivative, tetra-acetyl phytosphingosine (TAPS), inhibits the growth of HaCaT cells mainly by inducing apoptosis. In this study, we investigated its effect on the cell cycle and on cell cycle regulatory proteins. We showed by flow cytometry and staining for BrdU and phosphorylated histone H3 that the cells accumulated in S phase and arrested in G2 phase and did not divide before undergoing apoptosis. The level of the pro-apoptotic regulator Bax peaked after 6 h and then returned to normal, whereas the level of the anti-apoptotic regulator Bcl-xL, which is presumably induced in order to inhibit apoptosis, started to increase at 6 h, and remained high for 24 h. Phosphorylation of Cdc2 on Tyr-15 greatly increased while p21 rose to a plateau at 8 h. Levels of p53 and Mad2 proteins were unchanged. Our observations suggest that TAPS induces apoptosis of the HaCaT cells at least in part via transient G2 arrest.

Apoptosis↗
PubMed1997

G1-phase arrest is not a prerequisite for encystment in Physarum.

Amoebae of the Myxomycete Physarum polycephalum form resistant, walled cysts when the food bacteria in a culture have been consumed. No G1 phase has been detected in the vegetative amoebal cell cycle, most of which comprises the G2 phase. Mature cysts are also in G2, but it has been reported that a G1 phase of roughly 24 h, followed by an S phase, is obligatory prior to encystment. We used flow cytometry to determine the distribution of DNA contents in amoebal cultures at intervals during vegetative growth and encystment. In all cultures, the cells were predominantly in G2 phase, and the percentage of cells with G1 DNA content remained very low. We conclude that an extended G1 phase of 24 h did not occur in our cultures and cannot be a prerequisite for encystment.

Animals↗
PubMed1998

Changes in the rate of RNA synthesis during the cell cycle.

BACKGROUND: Although the rate of RNA synthesis is known to drop at mitosis, the recent identification of 11 stages in the cell cycle (El-Alfy et al., 1994) makes it possible to measure the rate of this synthesis at each one of the stages and thus find out how it varies throughout the cell cycle. METHODS: Mice were injected intravenously with the RNA precursor, 3H-uridine; the duodenum was fixed 5-15 minutes later for embedment in Epon, and the duodenal crypts were cut in semithin serial sections for study of the rapidly dividing crypt columnar cells. Using Feulgen-stained sections, each cell nucleus was assigned to one of the 11 stages described in the cell cycle, and the same nucleus was identified in the next serial section that had been processed for radioautography, so that the overlying silver grains were enumerated. The count was taken as an index of the rate of RNA synthesis by this nucleus. RESULTS: Starting from stage I of the cell cycle (the period defined by the presence of a minimal amount of chromatin during which the S phase begins) and up to stage IV (when the S phase ends and the G2 phase begins), all or nearly all nuclei are synthesizing RNA with the rate peaking at stage III. During stages V to VIII (the period comprising the mitotic steps), the percentage of RNA-synthesizing nuclei decreases to over half at stage V (prophase), -10% at stages VIa (prometaphase) and VIb (metaphase) and none at stages VII (anaphase) and VIII (telophase). During stages IX-XI (which correspond to the G1 phase), the percentage rises sharply at stage XI to reach up to 100% at stages X and XI. Finally, on the average, 35% of nuclear silver grains are over the nucleolus (presumably representing ribosomal RNA precursors), whereas 65% are over the nucleoplasm (presumably representing mainly heterogeneous RNA precursors). CONCLUSIONS: Cells synthesize RNA during the interphase, but at a variable rate with a peak in S. The synthesis proceeds in a majority of the cells at prophase, but only in a few of them at prometaphase and metaphase, and in none at anaphase and telophase.

Animals↗
PubMed2003

A novel immunohistochemical method to estimate cell-cycle phase distribution in archival tissue: implications for the prediction of outcome in colorectal cancer.

An immunohistochemical method for assessing cell-cycle phase distribution in colorectal resection specimens would enable phase data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in colorectal cancer. In contrast to flow cytometry, an immunohistochemical method would also allow the phase distribution to be examined within morphologically heterogeneous regions of neoplasms. Paraffin sections of normal colon (n = 25), colonic adenoma (n = 15), and colonic adenocarcinoma (n = 30) were analysed by immunohistochemistry using antibodies against markers of cell-cycle entry, Mcm-2 and Ki67, and putative markers of the cell-cycle phase, cyclins D1 and E (putative markers of G1 phase), cyclin A (S phase), cytoplasmic cyclin B1 (G2 phase), and phosphohistone H3 (M phase). The phase specificity of each marker was assessed by examining the degree of co-expression of adjacent phase markers using double-antibody fluorescence confocal microscopy and by comparison with flow cytometric analysis performed on adjacent tissue sections. The S-phase specificity of detectable cyclin A was also assessed in combination with in situ DNA replication using fluorescence confocal microscopy. All cells expressing phase markers co-expressed Mcm-2. Adjacent phase markers were not significantly co-expressed, confirming the relative specificity of these markers in tissue sections of colon. Cell-cycle phase distribution, calculated by immunohistochemistry, compared well with phase analyses obtained by flow cytometry. No cells expressed cyclin A in the absence of active DNA replication. The S-phase labelling index, as defined by detectable cyclin A expression, showed a positive correlation with the Mcm-2 labelling index and increased in the progression from normal colon to adenocarcinoma. In conclusion, a combination of these cell-cycle phase markers can be used to calculate the distribution of cells throughout each phase of the cell cycle in colorectal tissue sections. Detectable cyclin A can be used as a surrogate marker of S phase and may be of value in predicting prognosis and response to adjuvant therapy.

Adenocarcinoma↗