IMAGE ANALYSIS OF CHROMATIN IN CELLS OF PREIMPLANTATION MOUSE EMBRYOS

Mouse two-celled embryos and blastulae were Feulgen stained and the DNA content of their nuclei was measured with an integrating microdensitometer. The cells considered on the basis of their nuclear DNA content to be in G₁, S, and G₂ phases of the cell cycle were selected and their total chromatin area and chromatin areas at different gray levels were measured by the image analyzing computer, Quantimet. The measurements were aimed at quantitation of several features of the chromatin morphology of cells in different functional states. The total area of chromatin was found to increase, and the mean density of chromatin to decrease, from the G₁ to the G₂ phase of the cell cycle in both two-celled embryos and blastulae. The area of chromatin decreased, and the mean density of chromatin increased, as embryos developed from two-celled to blastula stage. It was concluded that nuclear morphology in preimplantation mouse embryos depends on both the phase of the cell cycle and the stage of development. The method of image analysis described was found to be useful for quantitation of changes in chromatin morphology.     

Change of chromatin morphology during the cell cycle detected by means of automated image analysis

Mouse embryo fibroblasts growing asynchronously in vitro stained with Feulgen method and their nuclear chromatin was analysed by means of the image analysing computer Quantimet 720D. Cells with 2C, 3C and 4C content of DNA were considered as being in G₁, middle S and G₂ phase of cell cycle, respectively. It was found that the projected area of nuclei increases during the cell cycle and that the mean optical density of chromatin increases from G₁ through S to G₂ phase. The curves showing the areas of chromatin at different optical density thresholds are different for cells in G₁, S and G₂ phase. The results demonstrate cyclic changes in chromatin morphology in the interphase nuclei during the cell cycle.     

Dynamic Organization of Plant Microtubules at the Three Distinct Transition Points During the Cell Cycle Progression of Synchronized Tobacco BY-2 Cells

Dynamic changes of microtubule (MT) configuration have been examined during the cell cycle progression in tobacco BY-2 cells, which have been highly synchronized by aphidicolin treatment. Although it has been shown previously that four cell cycle stages display characteristic features of MTs (Hasezawa et al., 1991), distinct changes of MT configuration were observed at the interfaces of G₂/M, M/G₁ and G₁/S, and the frequency of appearance of such distinct structures were quantitatively examined. Among others, it is the first observation that at M/G₁ disintegrating phragmoplasts coexisted with short MTs in the perinuclear envelopes, but the MTs disappeared in the later stage, when cortical MTs were organizing. Thus it is supposed that cortical MTs originate from the transiently observed short MTs in the perinuclear region. This observation offered also an experimental system to analyze the molecular changes of MTs at the three interfaces during cell cycle progression in plant cells, as the mass culture of tobacco BY-2 cells is readily available.     

Characterization of the cell cycle of cultured human diploid cells: Effects of aging and hydrocortisone

Age-related changes in the cytokinetics of human diploid cells in vitro have been compared in normal cultures and in cultures in which lifespan has been prolonged by the addition of hydrocortisone to the medium. For both cultures, with advancing age the fraction of cells in the actively proliferating pool decreased and the intercellular variation in cell cycle times increased. The average cell cycle time was prolonged during aging due almost entirely to changes in the duration of G₁. The duration of S remained constant, while a small delay in G₂ was observed in late passage cells near the end of their lifespan. Although the same pattern of change in proliferative parameters occurred in both control and hydrocortisone-treated cultures, the changes were somewhat delayed in the presence of the steroid. The results are interpreted in terms of several cell cycle models and suggest that the events controlling cell proliferation are sensitive to hydrocortisone modulation during the G₁ and possibly the G₂ periods.     

Constitutive Expression of Human Ribosomal Protein L7 Arrests the Cell Cycle in G1and Induces Apoptosis in Jurkat T-Lymphoma Cells

Protein L7 is involved in translational control in eucaryotic cells as indicated by its association with ribosomes, its capability to inhibit specifically the cell-free translation of distinct mRNAs, and its interference with the synthesis of two major nucleus-associated proteins in L7 cDNA-transfected Jurkat T-lymphoma cells [F. Neumannet al.(1995)Nucleic Acids Res.23, 195]. In this report we show that the constitutive expression of protein L7 in Jurkat cells leads to an arrest in G₁of the cell cycle and induces apoptosis as a consequence of cell-to-cell contact. Treatment of the L7 transfectants with the inhibitor of translation cycloheximide, at doses which do not affect untransfected cells, enhances their sensitivity to the induction of apoptosis. These results suggest that L7 can interfere with the translation of proteins which control cell cycle progression and/or the initiation of the apoptotic pathway.     

Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion

Coordinated cell proliferation and ability to form intercellular seals are essential features of epithelial tissue function. Tight junctions (TJs) classically act as paracellular diffusion barriers. More recently, their role in regulating epithelial cell proliferation in conjunction with scaffolding zonula occludens (ZO) proteins has come to light. The kidney collecting duct (CD) is a model of tight epithelium that displays intense proliferation during embryogenesis followed by very low cell turnover in the adult kidney. Here, we examined the influence of each ZO protein (ZO-1, -2 and -3) on CD cell proliferation. We show that all 3 ZO proteins are strongly expressed in native CD and are present at both intercellular junctions and nuclei of cultured CD principal cells (mCCD_cl1). Suppression of either ZO-1 or ZO-2 resulted in increased G₀/G₁ retention in mCCD_cl1 cells. ZO-2 suppression decreased cyclin D1 abundance while ZO-1 suppression was accompanied by increased nuclear p21 localization, the depletion of which restored cell cycle progression. Contrary to ZO-1 and ZO-2, ZO-3 expression at intercellular junctions dramatically increased with cell density and relied on the presence of ZO-1. ZO-3 depletion did not affect cell cycle progression but increased cell detachment. This latter event partly relied on increased nuclear cyclin D1 abundance and was associated with altered β1-integrin subcellular distribution and decreased occludin expression at intercellular junctions. These data reveal diverging, but interconnected, roles for each ZO protein in mCCD_cl1 proliferation. While ZO-1 and ZO-2 participate in cell cycle progression, ZO-3 is an important component of cell adhesion.     

Regulation of mitotic activity and the cell cycle in primary chick muscle cells by neurotransferrin

We previously demonstrated that neurotransferrin (NTF), a transferrin extracted from adult chicken peripheral nerves, promotes growth of primary chick muscle cells in the absence of embryo extract. NTF was shown to stimulate DNA synthesis and cell proliferation. In the present study, we demonstrate that NTF is a mitogen using two independent methods; counts of orcein-stained mitotic figures and analysis of cell cycle kinetics with a fluorescence-activated cell sorter. In low-density cultures mitotic activity increases with increasing doses of NTF followed by a plateau at concentrations greater than 6 μg/ml. Residual, embryonic mitotic activity progressively declines with time after plating muscle cells in the absence of NTF. Absence of NTF for 2 days causes cells to lose irreversibly their myogenic potential. In the presence of NTF, mitotic activity increases for 2 days followed by a decline concurrent with myoblast fusion and formation of myotubes. Cell cycle analysis showed that NTF addition causes cell populations to shift from G_t to S and G₂ + M within 18.5 hr. Muscle cells, plated at high densities in the absence of NTF, show mitotic activities similar to those plated at low densities in the presence of NTF. Addition of NTF to high-density cultures is ineffective in stimulating mitosis. These studies show that at typical cell plating densities, NTF is a required mitogen for primary chick muscle cell cultures.     

Action of FR901228, a Novel Antitumor Bicyclic Depsipeptide Produced by Chromo-bacterium violaceum No. 968, on Ha-ras Transformed NIH3T3 Cells

FR901228, a novel antitumor antibiotic, reversed the transformed morphology of the Ha-ras transformants, Ras-1 cells, and inhibited their growth. The reduction of c-myc expression was observed in FR901228-treated Ras-1 cells by RNA dot-blot hybridization. This reduction of c-myc expression and morphological reversion of the transformed cells to normal were correlated with growth inhibition (G₀/G₁ arrest in cell cycle).     

Interaction of the Circadian Cycle with the Cell Cycle in Pyrocystis fusiformis

Dividing pairs or single cells of the large dinoflagellate, Pyrocystis fusiformis Murray, were isolated in capillary tubes and their morphology was observed over a number of days, either in a light-dark cycle or in constant darkness. Morphological stages were correlated with the first growth stage, G₁, DNA synthesis, S, the second growth stage, G₂, mitosis, M, and cytokinesis, C, segments of the cell division cycle. The S phase was identified by measuring the nuclear DNA content of cells of different morphologies by the fluorescence of 4′, 6-diamidino-2-phenylindole dichloride.

Cells changed from one morphological stage to the next only during the night phase of the circadian cycle, both under light-dark conditions and in continuous darkness. Cells in all segments of the cell division cycle displayed a circadian rhythm in bioluminescence. These findings are incompatible with a mechanism for circadian oscillations that invokes cycling in G_q, an hypothesized side loop from G₁. All morphological stages, not only division, appear to be phased by the circadian clock.

     

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G₁ phase due to a G₁ specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G₁ phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.     

Factors that influence the development of cultured neurons from the brain of the mothManduca sexta

Summary During metamorphic adult development, neurons and glial cells in the developing olfactory (antennal) lobes of the moth undergo characteristic and extensive changes in shape. These changes depend on an interplay among these two cell types and ingrowing sensory axons. All of the direct cellular interactions occur against a background of changing steroid hormone titers. Antennal-lobe (AL) neurons dissociated from stage-5 (of 18 stages) metamorphosing animals survive at least 3 wk in primary cell culture. We describe here the morphological influences on AL neurons of (1) exposure to the steroid hormone 20-hydroxyecdysone, (2) exposure to sensory axons, and (3) interactions among the AL neurons. Cultured AL neurons respond only weakly, if at all, to 20-hydroxyecdysone. They do, however, show greater total outgrowth and branching when they had been exposed in vivo to sensory axons. Because there is no direct contact between some of the neuronal types and the sensory axons at the time of dissociation, the increase in outgrowth must have been mediated via a diffusible factor(s). When AL cells (neurons and glia) are plated at high density in low volumes of medium, or when the cells are plated at low density but in the presence of medium conditioned by high-density cultures, neurite outgrowth and cell survival are increased. Nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor-basic (bFGF), transforming growth factor-β (TGF _β ) and insulin-like growth factor (ILGF) had no obvious effect on neuronal morphology and thus are unlikely to underlie these effects. Our results suggest that the mature shape of AL neurons depends on developmental interactions among a number of diffusible factors.     

The effect of the cell microenvironment on cell functions associated with tumor promotion and progression

To study the role of the cellular microenvironment in tumor promotion and progression, transformed cells of rat embryonic fibroblasts (CL-1 clone) were transplanted into transgenic immunodeficient mice and cells of the formed tumor were converted into culture (CL-1-1 cells). Cells before and after transplantation were compared in morphology, growth rate, and permeability of intercellular gap junction. CL-1-1 cells were shown to have a changed morphology, to grow faster than the CL-1 cell, and to have no contact inhibition. In the G₁ phase of the cell cycle, there were many more CL-1 than CL-1-1 cells, whereas, in the G₂ and M phases, CL-1-1 cells were predominant. The permeabilities of the gap junction in the CL-1 and CL-1-1 cells were approximately the same. It was concluded that the cell microenvironment can stimulate tumor promotion and tumor progression upon transplantation of transformed cells into immunodeficient animals.     

Regulation of NIH-3T3 cell G1 phase transit by serum during exponential growth

The proliferation rate of mammalian cells is regulated normally in the G₁ phase of the cell cycle. During this phase, it is convenient to assign positive and negative roles to the molecular programs that regulate the duration of G₁ and the phase transition from G₁ to S phase. Density-dependent inhibition of cellular proliferation results in an increase in the duration of G₁. This form of regulation is due to both secreted factors and cell—cell contact. Serum is mitogenic to a variety of mammalian cell types. Because quiescent cells enter S phase as a result of serum addition to culture media, serum is usually regarded as a source of positive regulatory growth factors. We have measured the length of the G₁, S and G₂+ M phases of NIH 3T3 cells during exponential growth as a function of cell density and serum concentration. The G₁ length increases during exponential growth as a function of density while S and G₂+ M are relatively constant. Further, this increase in G₁ phase time, or density mediated negative regulation, is inhibited by increasing serum concentration. This phenotype is saturable between 10% to 20% serum. Serum concentrations above 2.5% are able to increase the rate of cell cycling (decrease the G₁ phase time) by inhibiting density dependent negative regulation of NIH 3T3.     

Efficient differentiation of proadipocyte stem cells on nonadherent surfaces: Evidence for differentiation without DNA synthesis

The differentiation of low density BALB/3T3 T proadipocytes that are cultured in standard tissue culture flasks can be induced by heparinized medium containing human plasma. It has been shown that under these conditions, cells first growth-arrest at a distinct state in the G₁ phase of the cell cycle, designated G_D, and thereafter differentiate within 8 to 12 days. In this paper, we report that the kinetics of proadipocyte differentiation can be significantly accelerated by culture of cells in differentiation-promoting medium on non-adherent surfaces, such as agarose-coated plates or bacteriological Petri dishes. Data also show that in a nonadherent microenvironment extensive differentiation can occur in the absence of DNA synthesis. This was established most convincingly by the demonstration that placement of mitotic cells in heparinized medium containing human plasma and hydroxyurea on agarose-coated Petri dishes induced 70–80% of the cells to G_D arrest and differentiate without traversing the S phase of the cell cycle. It is concluded that under appropriate microenvironmental conditions metabolic events that occur solely in the late M or early G₁ phase of the cell cycle can mediate the integrated control of proadipocyte proliferation and differentiation.     

Conjugation in Tetrahymena: the relationship between the division cycle and cell pairing

Conjugation, a sexual stage in the life cycle of Tetrahymena, is marked by the pairing of two cells of opposite mating types. Pairing establishes cytoplasmic continuity between the two cells and initiates the complex of nuclear events involved in sexual exchange. After mixing cells of opposite mating types in nonnutrient medium, a 3-hr refractory period ensues before pairing begins.A wave of cell division occurs concurrently with the onset of pairing. However, although all cells pair, the population does not double. This indicates that some cells do not divide and yet are capable of pairing. Apparently division per se is not required for pairing but does occur in most of the cells.Autoradiographic analysis demonstrates that the cells that divide before pairing were at a stage in the cell cycle beyond the initiation of macronuclear replication at the time they were transferred to nonnutrient medium. Cells that did not divide were in G₁ at the time of shift-down. Thus, neither replication nor division is required to be able to fuse. However, since fusion occurs only in G₁ and most cells are not in G₁ at the time of shift-down, a traverse of the cell cycle is required.Shift-down induces G₁ arrest and preparations for the mating reaction. Mixing the cells induces a synchronous wave of division for cells beyond the $\text{G}{}_1\text{S}$ interface. Preparations for the mating reaction occur independently of but simultaneous with the preparations for cell division.     

Measles Virus Infection Induces Terminal Differentiation of Human Thymic Epithelial Cells

Measles virus infection induces a profound immunosuppression that may lead to serious secondary infections and mortality. In this report, we show that the human cortical thymic epithelial cell line is highly susceptible to measles virus infection in vitro, resulting in infectious viral particle production and syncytium formation. Measles virus inhibits thymic epithelial cell growth and induces an arrest in the G₀/G₁ phases of the cell cycle. Moreover, we show that measles virus induces a progressive thymic epithelial cell differentiation process: attached measles virus-infected epithelial cells correspond to an intermediate state of differentiation while floating cells, recovered from cell culture supernatants, are fully differentiated. Measles virus-induced thymic epithelial cell differentiation is characterized by morphological and phenotypic changes. Measles virus-infected attached cells present fusiform and stellate shapes followed by a loss of cell-cell contacts and a shift from low- to high-molecular-weight keratin expression. Measles virus infection induces thymic epithelial cell apoptosis in terminally differentiated cells, revealed by the condensation and degradation of DNA in measles virus-infected floating thymic epithelial cells. Because thymic epithelial cells are required for the generation of immunocompetent T lymphocytes, our results suggest that measles virus-induced terminal differentiation of thymic epithelial cells may contribute to immunosuppression, particularly in children, in whom the thymic microenvironment is of critical importance for the development and maturation of a functional immune system.     

Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

Invasive cancer cells are a critical target in order to prevent metastasis. In the present report, we demonstrate real-time visualization of cell cycle kinetics of invading cancer cells in 3-dimensional (3D) Gelfoam® histoculture, which is in vivo-like. A fluorescence ubiquitination cell cycle indicator (FUCCI) whereby G₀/G₁ cells express a red fluorescent protein and S/G₂/M cells express a green fluorescent protein was used to determine the cell cycle position of invading and non-invading cells. With FUCCI 3D confocal imaging, we observed that cancer cells in G₀/G₁ phase in Gelfoam® histoculture migrated more rapidly and further than cancer cells in S/G₂/M phases. Cancer cells ceased migrating when they entered S/G₂/M phases and restarted migrating after cell division when the cells re-entered G₀/G₁. Migrating cancer cells also were resistant to cytotoxic chemotherapy, since they were preponderantly in G₀/G₁, where cytotoxic chemotherapy is not effective. The results of the present report suggest that novel therapy targeting G₀/G₁ cancer cells should be developed to prevent metastasis.