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Chinese hamster ovary cell mitosis and its response to ionizing radiation: a morphological analysis of the living cell.

Repeated microscopic observations of exponentially growing Chinese hamster ovary cells were made and the times and mitotic stages were recorded in control and irradiated cultures at 37 degrees C. As determined by autoradiography, the time from the end of S phase to early prophase (the G2 phase) was 46 min, to breakdown of the nuclear envelope was 91 min, and to restoration of the nuclear envelope was 116 min. The time spent in morphologically distinguishable phases of mitosis and the effects of 0.5, 1.0, 1.5, 2.0, and 4.0 Gy of gamma or X radiation on cells at each phase were determined. Affected cells were found to be delayed without or with reversion to an earlier mitotic stage before recovering and advancing through mitosis. Cells were timed in the five steps comprising delay with reversion: inertia, cessation I, regression, cessation II, and reprogression. No cells treated in late prophase, i.e., within 8-10 min of nuclear envelope breakdown, were delayed by the doses used; therefore the critical or transition point must be situated in middle prophase. Cells irradiated in this stage were not delayed by 0.5 or 1.0 Gy, but suffered a dose-dependent delay with or without reversion after 1.5, 2.0, and 4.0 Gy. Cells irradiated in early prophase and very late interphase responded similarly, but a greater percentage of the latter reverted.

Animals↗

[The effect of picolinic acid and iron ions on the proliferation of SPEV cells].

The effect of picolinic acid (PA) on SPEV cell proliferation is found to be different from that on normal and virus transformed NRC cells, and on spontaneously transformed CHO cells. It is shown that SPEV cells are arrested by PA at the end of G1-phase and at the beginning of S-phase and probably in G2-phase of the cell cycle. Ferrous ions remove the G1/S block induced by PA to permit the cell transfer through S-phase. On the one hand, PA chelates ferrous ions from the cells, and on the other one it inhibits the replicative DNA synthesis. It can be suggested that PA may arrest the SPEV cell growth affecting the iron-depend stable radical formation which is introduced into the active centre of ribonucleotiDE reduCTase. This results in the lower enzyme activity.

Animals↗

[Energy characteristics of the transport of monovalent cations in ascites tumor cells].

The intensity of O2 adsorption by ascite tumour cells does not practically depend on the monovalent cation concentration gradient between the cells and the incubation medium, whereas the rate of glycolysis decreases simultaneously with the diminution of the concentration gradient. In synchronized cultures at the beginning of the mitotic cycle, the bulk of ATP resynthesized via glycolysis is utilized for the synthesis of biopolymers, whereas that at the end of the S-phase and in the G2-phase--for cation transport across plasma membranes. From 35 to 100% of the whole amount of ATP resynthesized via glycolysis is utilized for transport purposes. The experimental results and theoretical calculations suggest that in glucose-containing media Na+ transport increases from 0.75 to 1.78 pmol/hour on a per cell basis. The activation of Na+ transport is due to the exchange of protons formed via glucose conversion into lactate for Na+, i.e., to the stimulation of Na+/H+ antiport. The permeability of plasma membranes for K+ increases 2.75-fold, while the passive flux of Na+ diminishes. It is concluded that the observed increase in the Na+/K+ ratio in ascite tumour cells is connected with their enhanced ability to synthesize lactic acid. Presumably, glycolysis is one of regulatory mechanisms of intracellular ratios of monovalent cations.

Animals↗

[Various biochemical and biophysical properties of a subpopulation of rat thymocytes isolated in a Ficoll gradient].

Subpopulations of rat thymocytes were obtained by cell precipitation in a single-stage transient phycoll gradient (isorapid precipitation). The fraction obtained were characterized in terms of the size, intensity of UV fluorescence, protein content and nucleoid mobility. The radius of thymocytes and UV fluorescence intensity monotonously increased during the transfer from the upper to lower fractions. The cells of medium-size fractions were characterized by the minimum content of cytoproteins and maximum mobility of nucleoid. In order to evaluate the density of thymocytes in the fractions, use was made of an additional cell precipitation in the phycoll-paque stage rho = 1.077. Incubation of thymocytes in medium 199 with a 10% rat serum led to the increased cell density in all the fractions, particularly in medium-size ones. Gamma-irradiation of thymocytes in a dose of 5 Gy appreciably accelerated the process under consideration. It is assumed that the medium-size fractions are enriched with thymocytes in G1 S phase, while lower fractions in G2 phase of the mitotic cycle. At the same time the fractions obtained appear to contain the subpopulations of thymocytes undergoing different stages of differentiation.

Animals↗

Flow cytometric analysis of bone marrow in a patient with resistant neuroblastoma.

Using flow cytometric analysis, we followed changes in a patient's bone marrow which was infiltrated with cells resistant to chemotherapy. Samples taken prior to, during, and after treatment with dianhydrogalactitol (2 days), cytosine arabinoside and hydroxyurea (2 days), and Adriamycin (2 days) contained an aneuploid population of cells which exhibited changes in ploidy, viability, and distribution in cell cycle compartment. The restricted changes in cell ploidy and viability of tumor cells encountered as well as limited increases in S phase on day 4 and G2 phase on day 6 with a rebound on day 9 of G1 phase cells suggest the tumor to be sensitive to dianhydrogalactitol but not to the other agents used.

Antineoplastic Agents↗

Cell cycle control of apoptosis in human leukemic T cells.

Eukaryotic cell cycle is regulated by feedback controls that can arrest the progress of the cell cycle at specific checkpoints. Failure of feedback controls may predispose cells to undergo apoptotic death in response to a variety of stimuli. Transformed cell lines often carry defects in one or more proteins that regulate cell cycle, and therefore can be used as a model to study how cell cycle controls cell death. The present study investigated whether TCR/CD3-induced apoptosis of human leukemic T cells is cell cycle dependent. Studies of Jurkat and Sup-T13, two human leukemic T cell lines, demonstrated that anti-CD3 Ab-driven activation results in a transient, reversible inhibition of DNA synthesis in Jurkat cells, but results in a persistent, irreversible inhibition of DNA synthesis in Sup-T13 cells. Anti-CD3 mAb blocked Jurkat cells at the G1/S interface without inducing cell death. In contrast, anti-CD3 mAb allowed Sup-T13 cells to enter S phase but inhibited progression into G2 phase and triggered cell death. Electron microscopy showed that anti-CD3 mAb-stimulated Sup-T13 cells acquired the characteristic morphology of apoptosis. The nuclear DNA appeared as oligonucleosome-sized fragments by gel electrophoresis, and as an apoptotic peak displaying lower fluorescence than the DNA content of cells in G1 phase by DNA flow microfluorometry. DNA fragmentation correlated with entry into S phase. The DNA synthesis inhibitor aphidicolin arrested Sup-T13 cells at the G1/S interface and prevented the apoptosis. Therefore, DNA synthesis is required for executing the apoptosis program initiated by TCR/CD3 stimulation of Sup-T13 cells. These data are consistent with the hypothesis that the suicide of activated cycling T cells upon encountering autoantigens may be one of the mechanisms for the immune system to prevent autoimmunity.

Apoptosis↗

Cholesterol starvation decreases p34(cdc2) kinase activity and arrests the cell cycle at G2.

As a major component of mammalian cell plasma membranes, cholesterol is essential for cell growth. Accordingly, the restriction of cholesterol provision has been shown to result in cell proliferation inhibition. We explored the potential regulatory role of cholesterol on cell cycle progression. MOLT-4 and HL-60 cell lines were cultured in a cholesterol-deficient medium and simultaneously exposed to SKF 104976, which is a specific inhibitor of lanosterol 14-alpha demethylase. Through HPLC analyses with on-line radioactivity detection, we found that SKF 104976 efficiently blocked the [(14)C]-acetate incorporation into cholesterol, resulting in an accumulation of lanosterol and dihydrolanosterol, without affecting the synthesis of mevalonic acid. The inhibitor also produced a rapid and intense inhibition of cell proliferation (IC(50) = 0.1 microM), as assessed by both [(3)H]-thymidine incorporation into DNA and cell counting. Flow cytometry and morphological examination showed that treatment with SKF 104976 for 48 h or longer resulted in the accumulation of cells specifically at G2 phase, whereas both the G1 traversal and the transition through S were unaffected. The G2 arrest was accompanied by an increase in the hyperphosphorylated form of p34(cdc2) and a reduction of its activity, as determined by assaying the H1 histone phosphorylating activity of p34(cdc2) immunoprecipitates. The persistent deficiency of cholesterol induced apoptosis. However, supplementing the medium with cholesterol, either in the form of LDL or free cholesterol dissolved in ethanol, completely abolished these effects, whereas mevalonate was ineffective. Caffeine, which abrogates the G2 checkpoint by preventing p34(cdc2) phosphorylation, reduced the accumulation in G2 when added to cultures containing cells on transit to G2, but was ineffective in cells arrested at G2 by sustained cholesterol starvation. Cells arrested in G2, however, were still viable and responded to cholesterol provision by activating p34(cdc2) and resuming the cell cycle. We conclude that in both lymphoblastoid and promyelocytic cells, cholesterol availability governs the G2 traversal, probably by affecting p34(cdc2) activity.

CDC2 Protein Kinase↗

Mos positively regulates Xe-Wee1 to lengthen the first mitotic cell cycle of Xenopus.

Several key developmental events occur in the first mitotic cell cycle of Xenopus; consequently this cycle has two gap phases and is approximately 60-75 min in length. In contrast, embryonic cycles 2-12 consist only of S and M phases and are 30 min in length. Xe-Wee1 and Mos are translated and degraded in a developmentally regulated manner. Significantly, both proteins are present in the first cell cycle. We showed previously that the expression of nondegradable Mos, during early interphase, delays the onset of M phase in the early embryonic cell cycles. Here we report that Xe-Wee1 is required for the Mos-mediated M-phase delay. We find that Xe-Wee1 tyrosine autophosphorylation positively regulates Xe-Wee1 and is only detected in the first 30 min of the first cell cycle. The level and duration of Xe-Wee1 tyrosine phosphorylation is elevated significantly when the first cell cycle is elongated with nondegradable Mos. Importantly, we show that the tyrosine phosphorylation of Xe-Wee1 is required for the Mos-mediated M-phase delay. These findings indicate that Mos positively regulates Xe-Wee1 to generate the G2 phase in the first cell cycle and establish a direct link between the MAPK signal transduction pathway and Wee1 in vertebrates.

Animals↗

Checkpoint control of the G2/M phase transition during the first mitotic cycle in mammalian eggs.

The high incidence of chromosomally abnormal human embryos is frequently assumed to be due to a lack of checkpoint controls operating during early embryogenesis. In our study we have analysed when these mechanisms first become functional. Mouse oocytes treated in late metaphase I with either of two different cyclin-dependent kinase inhibitors [butyrolactone 1 (BL1) or 6-dimethylaminopurine (6-DMAP)] form nuclei in the cytoplasm. BL1-treated eggs enter S-phase at 16-18 h post-treatment and, after completion of DNA synthesis, cleave to 2-cell stage embryos. 6-DMAP treatment results in the rapid initiation of DNA synthesis, its completion by 12 h and then arrest in the G2 phase. Thus, two different cell cycle stages can be obtained at the same time point after the initiation of treatment: G1- after BL1 and G2-staged nuclei after 6-DMAP treatment. That this approach greatly facilitates cell cycle studies has been shown by analysing checkpoint function during the first division. Whilst G2-staged eggs enter M phase within 2-3 h when 6-DMAP is washed out, the onset of M phase is delayed after their fusion to G1 (BL1) cells. Here M phase occurs only after the less advanced nucleus completes DNA replication. Our results indicate that checkpoints in mammalian eggs are functional during the first mitotic cycle.

4-Butyrolactone↗

Lack of inverse dose-rate effect and binding of the retinoblastoma gene product in the nucleus of human cancer T-47D cells arrested in G2 by ionizing radiation.

PURPOSE: To investigate the radiosensitivity of human breast cancer cells, T-47D, irradiated with low dose-rates and to study activation of the retinoblastoma gene product in the G1 and G2 phases during irradiation. MATERIALS AND METHODS: Cells were irradiated with (60)Co gamma-rays with dose-rates of 0.37 and 0.94 Gy h(-1). Cell survival was measured as the ability of cells to form colonies. Cells were extracted, fixed and stained for simultaneous measurements of nuclear-bound pRB content and DNA content. Cell nuclei were stained with monoclonal antibody PMG3-245 and Hoechst 33258 was used for additional staining of DNA. Two-parametric flow cytometry measurements of pRB and DNA content were performed using a FACSTAR(PLUS) flow cytometer. RESULTS: It was observed that irradiated cells were arrested in G2. No increase in radiation sensitivity was observed when the cells accumulated in G2. Irradiation of cells at both 0.37 and 0.94 Gy h(-1) resulted in exponential dose-survival curves with nearly equal alpha values, i.e. the same radiosensitivity. However, the retinoblastoma gene product was bound in the nucleus, i.e. hypophosphorylated, in about 15% of the cells arrested in G2. CONCLUSIONS: T47-D cells accumulate in G2 during low dose irradiation, but no inverse dose-rate effect, i.e. a more efficient inactivation of cells at lower than at higher dose-rates, was observed. A population of arrested G2 cells has pRB protein bound in the nucleus, and pRB therefore could play a role in protecting cells against radiation-induced cell death in G2.

Bisbenzimidazole↗

Differential phosphorylation of nuclear nonhistone high mobility group proteins HMG 14 and HMG 17 during the cell cycle.

The phosphorylation of the high-mobility group (HMG) proteins at different stages of the cell cycle was studied in synchronized HeLa cells. HMG proteins were extracted and analyzed by NaDodSO4/polyacrylamide gel electrophoresis. Although the molecular weight distribution of HMGs remains unchanged, their total amounts increase by as much as 20-25% in the G1 and S phases when compared with amounts in G2. However, the most significant finding is that there is a 7-fold increase of 32P incorporation into HMG 14 in the G2 phase compared with that in G1, and a 2-fold increase of 32P incorporation into HMG 17 in early S phase relative to the incorporation in the G1 and G2 stages. In contrast, HMG 1 and HMG 2 are not phosphorylated. The clear demonstration of differential phosphorylation of HMG 14 and 17 at specific stages of the cell cycle warrants a serious consideration of their role in tissue-specific maintenance of the altered chromatin structure characteristic of potentially active or actively transcribed chromatin domains.

Cell Cycle↗

Inhibition of human neuroblastoma cell growth by CAY10404, a highly selective Cox-2 inhibitor.

Neuroblastomas constitute about 10% of childhood cancers and are responsible for 15% of pediatric cancer mortality. We evaluated the efficacy and the mechanism of cell death induced by CAY10404, a selective cyclooxygenase-2 (Cox-2) inhibitor in four human neuroblastoma cell lines (SH-EP, SH-SY5Y, SK-N-MC and MSN). Treatment with CAY10404 in the range of 15-115 microM revealed a dose-dependent decrease in cell number and an average IC50 (inhibitory concentration 50%) of 60 microM. About 20-30% of the cells were terminal deoxynucleotidyltransferase-mediated UTP nick-end-labeling (TUNEL) positive 48 h after treatment. Western blot analysis of CAY10404-treated cells showed poly(ADP-ribose) polymerase (PARP) cleavage and cleaved caspase-3 signifying caspase activity and apoptotic cell death. Inhibitor-of-apoptosis proteins including X-linked inhibitor-of-apoptosis protein (XIAP) and survivin did not change significantly after CAY10404 treatment. Fluorescence activated cell sorter (FACS) analysis performed in two different cell lines 48 h following CAY10404 treatment showed a reduction in the number of cells in the G1 phase of the cell cycle and an increase in the number of cells in the G2 phase. When radioresistant SH-EP cells were treated with CAY10404, a 49% decrease in cell viability was observed relative to DMSO-treated cells; pretreatment with CAY10404 followed by ortho-voltage irradiation further enhanced cell death (58%) suggesting radiosensitization by CAY10404.

Apoptosis↗

Influence of p53 and p21Waf1 expression on G2/M phase arrest of colorectal carcinoma HCT116 cells to proteasome inhibitors.

Ubiquitin-mediated protein degradation in vertebrates has been implicated in cell cycle control. In this report we explored the effects of proteasome inhibitors (MG132, lactacystin and ALLN) on cell cycle distribution. Colorectal carcinoma HCT116 cells were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in a dose-dependent manner. MG132 arrested HCT116 cells at G2/M phase, which was associated with drug-induced blockade of p53 degradation and/or induction of p53-related gene expression along with the accumulation of cyclin B, cyclin A and p21. MG132 treated HCT116 (wild-type) had a similar cell cycle distribution as the MG132 treated HCT116 (p53-/-) and HCT116 (p21-/-) cells, suggesting that p53 and p21 may not be essential for MG132-induced G2/M phase arrest. The release experiments from nocodazole-induced mitotic phase cells indicated that MG132 inhibits the proliferation of HCT116 cells via arrest in the G2 phase. In addition, when HCT116 cells were exposed to combination of sodium butyrate and MG132 enhanced cell growth inhibition and induction of apoptosis were observed.

Acetylcysteine↗

Increased G2 chromosomal radiosensitivity in cancer patients: the role of cdk1/cyclin-B activity level in the mechanisms involved.

PURPOSE: To test the hypothesis that deficient DNA repair as measured by increased G2 chromosomal radiosensitivity results from up-regulation of cdk1/cyclinB and cell cycle control mechanisms during the G2 to M transition. MATERIALS AND METHODS: A total of 185 cancer patients and 25 normal individuals were tested for G2 chromosomal radiosensitivity. The chromatid breaks were analysed in metaphase using the G2 assay or directly in G0 and G2 phase using premature chromosome condensation (PCC). The activity of cdk1/cyclinB, a key regulator of the G2 to M-phase transition, was measured by histone H1 kinase activity and correlated with the development of chromatid breaks after irradiation of cell lines in vitro. RESULTS: Based on the G2 assay, cancer patients on average showed increased chromosomal radiosensitivity above controls. When the analysis was carried out directly in G0 or G2 lymphocytes using PCC, no differences in the induction of chromosomal damage and its repair were observed between G2 assay-sensitive and G2-normal donors. Using the G2 assay to test G2 radiosensitivity in various cell lines, it was found that the higher the cdk1/cyclinB activity level of the cell line tested, the higher the yield of chromatid breaks scored. Furthermore, when mitotic cells from these cell lines were used for PCC induction in irradiated G2 lymphocytes it was observed that the higher the cdk1/cyclinB activity level of mitotic cells used, the higher was the induced yield of chromatid breaks. CONCLUSION: The cdk1/cyclin-B activity levels during the G2 to M transition impair DNA repair processes and play a major role in the yield of chromatid breaks induced after G2-irradiation. Regulation of cdk1/cyclinB complex activity rather than deficient repair enzymes of DNA damage may underlie the mechanisms of G2 radiosensitivity.

CDC2 Protein Kinase↗

Chromosomal aberration types in cells at the second division after irradiation in G1 or G2.

Cultures of JU-56 cells were irradiated in either G1 or G2 and examined either in their first post-irradiation metaphase (in diploids) or in their second post-irradiation metaphase (in colcemid-induced tetraploids). The timing of fixation, together with tritiated thymidine pulse labelling, allowed selection for scoring only metaphases of cells that were in the G1 or G2 phase of the cell cycle during irradiation. With G1 irradiation it was found that many of the aberrations observed at the first division were not present (as derivatives) at the second division, and also that new aberrations were found at the second division, which were not derived from aberrations at the first division. Clonogenic survival was also measured in populations of cells irradiated in G1. It was found that cells containing chromosomal aberrations at the first division were not numerous enough to explain lack of survival. When frequencies of aberrations following G2 irradiation scored at the second division were compared with those scored at the first, there was a significant increase in dicentrics as compared with their progenitor asymmetrical chromatid interchanges, and of mirror-image dicentrics as compared with their progenitor sister unions. A substantial number of sister unions were also observed at the second division. We conclude that some aberrations are lost during the interphase between the first and the second post-irradiation metaphase and that new chromosomal aberrations arise during the second post-irradiation interphase.

Animals↗

Roscovitine, a novel cyclin-dependent kinase inhibitor, characterizes restriction point and G2/M transition in tobacco BY-2 cell suspension.

Although the developmental programs of plants and animals differ, key regulatory components of their cell cycle have been conserved. Particular attention has been paid to the role of the complexes between highly conserved cyclin and cyclin-dependent kinases in regulating progression through the cell cycle. The recent demonstration that roscovitine is a potent and selective inhibitor of the animal cyclin-dependent kinases cdc2 (CDK1), CDK2 and CDK5 prompted an investigation into its effects on progression through the plant cell cycle. Roscovitine induced arrests both in late G1 and late G2 phase in BY-2 tobacco cell suspensions. Both block were fully reversible when roscovitine was used at concentrations similar to those used in the animal system. Stationary-phase cells subcultured in the presence of roscovitine were arrested at a 2C DNA content. This arrest was more efficient without exogenous addition of plant growth regulator. Roscovitine induced a block in G1 earlier than that induced by aphidicolin. S-phase synchronized cells treated with roscovitine were arrested at a 4C DNA content at the G2/ M transition. The expression analysis of a mitotic cyclin (NTCYC1) indicated that the roscovitine-induced G2 block probably occurs in late G2. Finally, cells in metaphase were insensitive to roscovitine. The purified CDK/cyclin kinase activities of late G1 and early M arrested cells were inhibited in vitro by roscovitine. The implications of these experimental observations for the requirement for CDK activity during progression through the plant cell cycle are discussed.

2,4-Dichlorophenoxyacetic Acid↗

Human immunodeficiency virus type 1 Vpr is a positive regulator of viral transcription and infectivity in primary human macrophages.

It is currently well established that HIV-1 Vpr augments viral replication in primary human macrophages. In its virion-associated form, Vpr has been suggested to aid efficient translocation of the proviral DNA into the cell nucleus. Although Vpr growth-arrests dividing T cells, the relevance of this biological activity in nondividing macrophages is unclear. Here we use Vpr-mutants to demonstrate that the molecular determinants involved in G2-arresting T cells are also involved in increasing viral transcription in macrophages, even though these cells are refractive to the diploid DNA status typical of G2 phase. Our results suggest that the two phenotypes, namely the nuclear localization and the G2-arrest activity of the protein, segregate functionally among the late and early functions of Vpr. The nuclear localization property of Vpr correlates with its ability to effectively target the proviral DNA to the cell nucleus early in the infection, whereas the G2-arrest phenotype correlates with its ability to activate viral transcription after establishment of an infection. These two functions may render Vpr's role essential and not accessory under infection conditions that closely mimic the in vivo situation, that is, primary cells being infected at low viral inputs.

Cell Nucleus↗