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Replication of nucleolus-associated DNA during "G2 phase" in Physarum polycephalum.

In the myxomycete, Physarum polycephalum, the bulk of nuclear DNA replication occurs during a period of a few hours immediately following upon mitosis. During the remainder of the intermitotic period, incorporation of thymidine-(3)H continues at a low rate in the region of the nucleolus (radioautographs). A few nuclei incorporated thymidine-(3)H into the extranucleolar chromatin at a high rate at all times of the intermitotic period. These nuclei were exceptionally large and they frequently contained several small nucleoli of different sizes rather than the one, central nucleolus which is characteristic of a normal interphase nucleus.

Autoradiography↗

Reduction in the radiation-induced late S phase and G2 blocks in HL-60 cell populations by amiloride, an efficient inhibitor of the Na+/H+ transporter.

Recent investigations that showed that amiloride delayed or inhibited apoptosis indicated it might also attenuate cell cycle checkpoints activated by ionizing radiation. In this report, single- and dual-parameter flow cytometry were used to investigate the effects of amiloride on cell cycle progression, and the effectiveness of amiloride to attenuate the S and G2 phase checkpoint responses induced by 2.5, 5.0, and 7.5 Gy of gamma radiation. The late S-phase delay, noted at 8 h following irradiation, and a radiation-induced G2 block, which was maximum at 16 h after irradiation, were both significantly reduced in amiloride-treated samples. Attenuation of the radiation-induced late S phase and G2 blocks resulted in cell division without apparent apoptosis or necrosis over a 24-h period. Results presented indicate that amiloride reduces the radiation-induced G2 block in HL-60 cell populations almost equally well as caffeine and to a greater extent than staurosporine. Immunofluorescent detection and quantitation of cyclin B1 expression demonstrated that amiloride only significantly reduced cyclin B1 expression following 5.0 Gy, when there was a notable induction of a significant G2 delay, followed by a relatively rapid recovery in cycling potential. The results suggest that amiloride affects the radiation-triggered signaling cascades to alter the kinase activity of proteins associated with mitotic progression, particularly the cyclin B1-p34cdc2 complex. Alternatively, alterations in intracellular ion concentrations induced by amiloride may lead to changes in Ca2+-dependent signaling cascades and thereby decrease the radiation-mediated cell cycle perturbations.

Amiloride↗

The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2.

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

Animals↗

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation.

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

4-Butyrolactone↗

HOX11 interacts with protein phosphatases PP2A and PP1 and disrupts a G2/M cell-cycle checkpoint.

Hox11 is an orphan homeobox gene that controls the genesis of the spleen. HOX11 is also oncogenic, having been isolated from a chromosomal breakpoint in human T-cell leukaemia. Transgenic mice that redirected HOX11 to the thymus demonstrated cell-cycle aberration and progression to malignancy. We observed that the protein HOX11 interacted with protein serine-threonine phosphatase 2A catalytic subunit (PP2AC), as well as protein phosphatase 1 (PP1C) in mammalian cells. Inhibition of PP2A can regulate the cell cycle and control the activation of maturation-promoting factor in Xenopus oocytes. Microinjection of HOX11 into Xenopus oocytes arrested at the G2 phase of the cell cycle promoted progression to the M phase. G2 arrest can be induced by gamma-irradiation, but is eliminated by expression of HOX11 within a T-cell line. Thus HOX11 is a cellular oncogene that targets PP2A and PP1, both of which are targets for oncogenic viruses and chemical tumour promoters. This interaction suggests a mechanism by which a homeobox can alter the cell cycle.

Animals↗

The effect of adriamycin on the cell cycle traverse of a human lymphoid cell line.

The kinetic response of a human lymphoma cell line to adriamycin was analyzed by means of pulse cytophotometry. Depending on concentration and exposure time, cell cycle progression was delayed in G1, S, and G2 phases. There was a differential sensitivity for the interaction of adriamycin with the transit through these phases. G2 arrest could be induced by low concentrations of adriamycin, whereas the block in G1 was exerted only after long-term treatment with high concentrations and was completely reversible after drug removal. Delay in S-phase transit was transient in spite of continuous exposure to high concentrations of adriamycin. Thus, concentration and duration of treatment determined the magnitude of G2 arrest as well as onset and rate of G2 accumulation due to progression delay in G1 and S phases. Cell age had only little influence on the degree of subsequent G2 arrest. Irreversibility of the G2 block strongly suggests eventual cell death in G2 phase, which may be utilized as a predictive test for response to adriamycin in vivo.

Cell Division↗

Anomalous phase behavior of long chain saturated lecithin bilayers.

X-ray scattering has been performed on fully hydrated unoriented multilamellar vesicles of lecithins with even chain lengths n from 16 to 24 as a function of temperature in chain ordered phases. The longer chain lengths, n > or = 20, show anomalous behavior compared to the shorter chain lengths, n < 20. This report concentrates on n = 24. Although the history and time dependence shows that equilibrium was not always achieved, it appears that there is a second gel-like phase G2 below 40 degrees C. The G2 phase has a small tilt angle and opposite hexagonal symmetry breaking from the usual G1 gel phase. Also, as T is raised above 45 degrees C, the wide-angle data suggest the appearance of a phase with hexagonal chain packing and small chain tilt angle.

Lipid Bilayers↗

Synthesis and secretion of light-chain immunoglobulin in two successive cycles of synchronized plasmacytoma cells.

Suspension-cultured mouse plasmacytoma cells (MPC-11) were accumulated in the late G1 phase by exposure to isoleucine-deficient medium for 20-24 h. The arrested culture was fed with complete medium enabling the cells to continue the cell cycle synchronously, undergo mitosis, and enter a second cycle of growth. This method of synchronization left the protein-synthesizing ability intact as judged by the polysome profile and the capacity of the cells to incorporate labeled amino acids into protein after the restoration of isoleucine. After incubation in isoleucine-deficient medium and the addition of isoleucine to the culture, cells entered the S phase after a short lag, as judged by [3H]thymidine incorporation into nucleic acid and by spectrophotometric measurement of nuclear DNA. The cells were in mitosis between 12 and 18 h as judged by the increase in cell count and analysis of cell populations on albumin gradients. Synthesis and secretion of light-chain immunoglobulin were maximal in the late G1/early S phase of the first cycle. During late S phase, G2 phase, and mitosis, both synthesis and secretion were observed to be at a low level; however, immediately after motosis the cells which then entered the G1 phase apparently commenced synthesis of light chain immunoglobulin straight away, although secretion of labeled material remained at a low level.

Cell Count↗