The early effects of ouabain on potassium metabolism and rate of proliferation of mouse lymphoblasts

Murine lymphoblasts grown in suspension culture in the presence of ouabain showed a dose dependent and sequential decrease in ⁸⁶Rb⁺ (K⁺ analogue) influx, cellular potassium content, and growth rate. An increase in eosin staining and a decrease in cell number was observed after two hours in the presence of 1 mM ouabain; 1 μM ouabain was without effect on any of the parameters measured. Ouabain inhibition was rapidly and completely reversible at concentrations that were not cytotoxic.     

Regulation of cAMP on the first mitotic cell cycle of mouse embryos

Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo.     

On the transition from the meiotic to mitotic cell cycle during early mouse development

Here, we outline the mechanisms involved in the regulation of cell divisions during oocyte maturation and early cleavages of the mouse embryo. Our interest is focused on the regulation of meiotic M-phases and the first embryonic mitoses that are differently tuned and are characterized by specifically modified mechanisms, some of which have been recently identified. The transitions between the M-phases during this period of development, as well as associated changes in their regulation, are of key importance for both the meiotic maturation of oocytes and the further development of the mammalian embryo. The mouse is an excellent model for studies of the cell cycle during oogenesis and early development. Nevertheless, a number of molecular mechanisms described here were discovered or confirmed during the study of other species and apply also to other mammals including humans.     

Effect of an erythrocytic chalone on the postsynthetic period of the erythroblastic mitotic cell cycle in mouse bone marrow

The effect of the erythrocytic chalone on the beginning, middle and end of the G2-period of the mitotic cycle has been studied in the erythroblastic cells of the mouse bone marrow. The end of S(-) and the beginning of G2(-) periods have been demonstrated to be the most sensitive to the effect in question. The erythrocytic chalone inhibits mitotic activity by decreasing the amount of prophases and inhibits incorporation of radioactive glycine into the dividing erythroblastic cells. The mechanism inhibiting their mitotic activity is discussed.     

Adaptation of potassium metabolism and restoration of mitosis during prolonged treatment of mouse lymphoblasts with ouabain

The effects of ouabain on the growth of murine lymphoblasts in vitro have been studied. Exposure of cells to ouabain (0.1 mM) initially inhibited ⁸⁶Rb⁺ uptake rate, reduced the intracellular potassium concentration, and decreased population growth rates. Continued exposure to the same ouabain concentration resulted in an increase of ⁸⁶Rb⁺ uptake rate, intracellular potassium content and population growth rates to control values (adaptation). When treated cells were resuspended in medium free of ouabain after 12 to 15 hours of ouabain treatment, ⁸⁶Rb⁺ uptake rates and intracellular potassium levels exceeded those of untreated cells. Adaptation was inhibited by cycloheximide (3 μg/ml) and by actinomycin D (0.05 μg/ml). Kinetic analysis of transport suggested that while the total capacity of the Na⁺, K⁺ transport system increased, the affinity for both the cation (⁸⁶Rb⁺) and ouabain decreased.     

The Arabidopsis BET bromodomain factor GTE4 regulates the mitotic cell cycle

Proteins containing bromodomains are capable of binding to acetylated histone tails and have a role in recognizing and deciphering acetylated chromatin. Plant BET proteins contain one bromodomain. Twelve BET-encoding genes have been identified in the Arabidopsis genome. Two of these genes have been functionally characterized, one shows a role in seed germination, the other is involved in the establishment of leaf shape. Recently, we characterized a third AtBET gene, named GTE4. We demonstrated that GTE4 is involved in the activation and maintenance of cell division in the meristems and by this controls cell numbers in differentiated organs. Moreover, the quiescent center (QC) identity is partially lost in the apex of the primary root of gte4 mutant, and there is a premature switch from mitosis to endocycling. Genes involved in the retinoblastoma (RB)-E2F pathway, which is important for coupling cell division and cell differentiation in plants and animals, were either up or downregulated in the gte4 mutant. In this report we also show that the defect in germination observed in gte4 mutant seeds is not rescued by the action of GA₃. Further the root pole of the mutant embryo shows irregular cytokinesis in the procambial stem cells, and the QC of the lateral root shows a partial, but not transient, loss of QC identity. These additional results reinforce the importance of GTE4 in the control of cell proliferation.Key words: arabidopsis, BET bromodomain, cell cycle, E2F, germination     

Regulation of sister chromatid cohesion during the mitotic cell cycle

Orderly execution of two critical events during the cell cycle––DNA replication and chromosome segregation––ensures the stable transmission of genetic materials. The cohesin complex physically connects sister chromatids during DNA replication in a process termed sister chromatid cohesion. Timely establishment and dissolution of sister chromatid cohesion is a prerequisite for accurate chromosome segregation, and is tight regulated by the cell cycle machinery and cohesin-associated proteins. In this review, we discuss recent progress in the molecular understanding of sister chromatid cohesion during the mitotic cell cycle.     

The effects of cyclosporins on the cell cycle of T-lymphoid cell lines

Cyclosporin A (CsA) is a cyclic endecapeptide of fungal origin displaying strong immunosuppressive properties. CsA and another active member of the cyclosporin (Cs) family, but not an inactive one, can interfere with the proliferation of some, but not all, T-lymphoid cell lines. Cells from Cs-sensitive lines accumulate in the G1 phase of the cell cycle. No effect is detected on the cycle of Cs-resistant lines. Both Cs-sensitive and Cs-resistant lines are arrested by another G1 blocker (actinomycin D) and DNA synthesis inhibitors (cytosine arabinoside, hydroxyurea), become multinucleated/polyploid when exposed to cytochalasin B (CB), are arrested in mitosis by colchicine and accumulate in G2 phase in the presence of Taxol. The effect of Cs is best evidenced when the drug is applied to cells which were already delayed in G1 by saturation density cultivation or serum deprivation. By the combined use of Cs and of other drugs working at a later phase of the cycle, results were obtained which suggest that the effect of Cs is either to delay very much the cells throughout the G1 phase or to arrest them at that G1 phase or at the following one. A correlation of the G1-blocking property of Cs with their immunosuppressive properties may be possible but is still speculative.     

Effect of hydrocortisone on the mitotic cycle of epithelial cells in the mouse pylorus]

It was shown with the aid of thymidine-H3 that the mitotic cycle of mucous-forming cells (superficial epithelial mucosal cells of the neck) of the stomach pyloric glands of mice lasted 13.5 hrs (G1+1/2M = 7.6 hrs, S = 5.3 hrs; G2+1/2M = 0.6 hrs). With the administration of a physiological dose of hydrocortisone (0.1 mg) the duration of the mitotic cycle of mucous-forming cells of the stomach pyloric glands increased by 6.7 hrs (G1+1/2M = 11.6 hrs, S = 7.8 hrs; G2+1/2M = 0.8 hrs). A high dose of the hormone had a similar effect and increased the presynthetic period to 12.9 hours and the postsynthetic one--to 2.3 hours.     

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNA and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [³H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K⁺ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K⁺, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K⁺ transport, controlling mitosis at distinct phases of the cell cycle.     

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNa and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [3H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K+ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K+, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K+ transport, controlling mitosis at distinct phases of the cell cycle.     

Localization of the nuclear matrix protein mitotin in mouse cells with a mitotic or endomitotic cell cycle.

Immunofluorescence staining was used to study the precise subcellular distribution of the nuclear matrix antigen, mitotin, in mouse cells characterized by either a mitotic or an endomitotic organization of the cell cycle. In mitotically dividing cells, mitotin showed a speckled distribution within interphase nuclei. In addition, some interphase cells exhibited a weak, focused signal adjacent to the nucleus, reflecting a possible staining of the centrosome region. Using digital contrast-enhanced immunofluorescence microscopy, a distinct association of mitotin to the centrosome, pole microtubules, and midbody could be revealed in cells at different stages of mitosis. In parallel, trophoblast giant cells characterized by an endomitotic cell cycle were derived from blastocyst outgrowths and analyzed likewise. In all giant cells examined so far, mitotin was restricted to the nuclear compartment alone, although different patterns of intranuclear staining could be detected. The present study provides further information about the precise localization of mitotin in mitotic cells, especially during mitosis. In view of the results, the staining pattern observed in endomitotic cells may allow for a better understanding of the origin and the organization of the endomitotic cell cycle.     

The centrosome cycle in the mitotic cycle of sea urchin eggs

When sea urchin eggs entering mitosis are exposed to an appropriate concentration of mercaptoethanol, the chromosome cycle is restrained while the centrosome cycle advances. The two poles of the mitotic apparatus separate into four poles, while the chromosomes remain in their metaphase arrangements until released by the removal of the mercaptoethanol. We follow the centrosomes through the stages of the generation of two poles by each original pole. In electron microscopic studies, the osmiophilic component of the centrosomes serves as an indicator of their changing forms as each pole generates two poles. In light microscopic studies, including observations of birefringence, the shapes of the polar ends of the spindles are taken as indicators of the shapes of the centrosomes. The successive stages of the centrosome cycle are (1) compact spherical centrosomes at the time of formation of the mitotic apparatus; (2) expansion and flattening of the centrosomes, leading to (3) formation of thin flat plates, perpendicular to the spindle axis. Corresponding to the extended flat shape of the centrosomes, the spindle poles are flat; microtubules 'point' to the centrosomal plate and not the centrioles. The centrioles are separated in the flattening of the centrosomes. (4) The flat plate divides into two and each of the two halves becomes more compact, defining two separate poles. Our findings resurrect and update Boveri's [5] observations and interpretations of the centrosome. Centrosomes have shapes. The shapes may be imparted to the microtubular structures that they generate. The formation of two separate centrosomes from one, in the formation of mitotic poles, is describable as a sequence of changes in shape.     

Use of flow cytometry in the measurement of cell mitotic cycle

Variations in many cellular characteristics during the cell cycle can be analyzed simply and directly by flow cytometry. Using multiparameter analysis of DNA content, RNA content, cell size and 5-bromodeoxyuridine (BrdUrd) incorporation, it is now possible to define cells' positions in the cell cycle with a precision previously unimaginable. It is also possible, by using the sorting function of the flow cytometer, to separate populations in different phases of the cell cycle for biological and biochemical studies. This review describes the technical aspects of flow cytometric instrumentation, DNA staining procedures, and the cytometric applications of both in cell cycle analysis including some of the more innovative, new approaches with antibody against BrdUrd.