HSP90 and checkpoint-dependent lengthening of the G2 phase observed in plant cells under hypoxia and cold

Summary. Proliferating cells of Allium cepa L. roots became adapted to hypoxia (5% oxygen) and cold (10°C) by acquiring new steady-state kinetics of growth. The cell cycle time increased from the 17.6 h in control meristems up to 29.7 and 69.0 h under hypoxia and cold conditions, respectively. Acclimation of the proliferating cells was stress specific. No acclimation took place after 24 h of heat treatment (40°C). Under cold treatment, all cycle phases enlarged uniformly. However, under hypoxia, while the G₁ and S cycle phases roughly doubled in their timing, the expected checkpoint-dependent lengthening of G₂ did not take place. This failure in lengthening G₂ in response to hypoxia correlated with a failure in the overinduction of a single peptide with a molecular mass of about 134 kDa which is among those recognised by an HSP90 antibody. Moreover, the presence of this large peptide of the HSP90 family proved to be a marker for cell proliferation. It was always absent from the contiguous differentiated cells of the root. Lastly, the mitochondrial chaperonin recognized by an HSP60 antibody in these roots not involved in photosynthesis was always higher in the proliferating than in the nonproliferating cells.     

Heterogeneity in G2 duration during lateral root development

Developing lateral roots of V. faba were treated with 5-aminouracil for up to 6 hours using the 5-AU inhibition method discussed in this paper; the duration of G₂+mitosis/2 and the percentages of slow dividing cells were estimated from the fall in MI observed in just emerged meristems, very large primordia and large primordia. The results indicate that during the period of development studied here there are two subpopulations of dividing cells: 1) fast dividing population which makes up about 84 % of the dividing cells and which has a G₂+mitosis/2 duration of about 3.3 hours, and 2) a slow dividing population which constitutes about 16 % of the dividing cells and which has a G₂ duration in excess of 12 hours. This heterogeneity is discussed in relationship to the behaviour of different populations of proliferating cells during root morphogenesis.     

Effect of OA-inhibitor of protein phosphatases PP1 and PP2A — on initiation of DNA replication and mitosis in <Emphasis Type="Italic">Vicia faba</Emphasis> root meristems

According to the principal control point (PCP) hypothesis, experiments with excised, carbohydrate-starved stationary root meristems of Vicia faba var. minor have demonstrated that cells which previously divided asynchronously were preferentially blocked in G₁ (PCP1) and G₂ (PCP2) phases. When stationary phase meristems are supplied with exogenous carbohydrate (2 % sucrose), the G1- and G2-arrested cells start out DNA replication and mitotic divisions, respectively. The resumption of DNA synthesis and mitosis is not immediate and the delays of G₁- and G₂-arrested cells are found different. Using this model, we examined the effects of 4 pulse incubations with okadaic acid (OA), a specific inhibitor of PP1 and PP2A, on the duration of intervals elapsing between the provision of sucrose and the first appearance of S- and M-phase cells. We have found that depending on the period during which OA had been applied, the release from G1 and G2 phase arrest-points becomes prolonged, showing different time-course modifications. The obtained data provide evidence that activation of PP1 and PP2A is required to allow the cells for both PCP1→S and PCP2→M transitions in root meristems of V. faba.     

Replication and G2 checkpoints: their response to caffeine

Under long hydroxyurea treatments, evidence was obtained for the sequential activation of four checkpoints located between the onset of S phase and mitosis in Allium cepa L. root meristems. Bi-parametric flow cytometry (Br-DNA/total DNA) showed that cells initially accumulated at early S phase but, after a delay, they resumed replication and paused again at mid S phase. Cells not only overrode this second replication block but also any G₂ checkpoint they encountered. Thus, a late mitotic wave was produced in the presence of hydroxyurea. The wave was formed by cells that had apparently completed their replication (normal mitoses), while others displayed anaphases/telophases with less than the expected DNA content and with chromosomal breaks (aberrant mitoses). The presence of aberrant mitoses is direct evidence for the undue override of the two G₂ checkpoints responsible for surveillance of completion of DNA synthesis and repair, respectively. Caffeine selectively abrogated the G₂ block produced by the checkpoint that controls post-replication DNA repair, as it advanced the entry of cells into an aberrant mitosis. However, caffeine proved not to be the universal checkpoint-evading agent as postulated. Caffeine did not modify the spontaneous override of the replication checkpoints. Moreover, it seems to enforce the checkpoint that controls the completion of DNA synthesis, as the appearance of the late wave of normal mitoses produced in the presence of hydroxyurea was prevented by the use of caffeine. Received: 21 February 2000 / Accepted: 31 July 2000     

Multiple G proteins compete for binding with the human gonadotropin releasing hormone receptor

The GnRH receptor is coupled to G proteins of the families G_q and G₁₁. G_q and G₁₁ coupling leads to intracellular signaling through the phospholipase C pathway. GnRHR coupling to other G proteins is controversial. This study provides evidence that G protein families G_s, G_i, G_q and G₁₁ complete for binding with the GnRHR. We quantified interactions of over-expressed G proteins with GnRHR by a competitive binding approach, using measurements of second messengers, IP and cAMP. Transient co-transfection of HEK293 cells with human WT GnRHR and with stimulatory and inhibitory G proteins (G_q, G₁₁ and G_s, G_i) led to either production or inhibition of total inositol phosphate (IP) production, depending on the G protein that was over-expressed. Studies were conducted in different human (COS7, HeLa) and rodent-derived (CHO-K1, GH₃) cell lines in order to confirm that G protein promiscuity observed with the GnRHR was not limited to a particular cell type.     

Retinoic Acid-Induced Differentiation of Human Neuroblastoma SH-SY5Y Cells Is Associated with Changes in the Abundance of G Proteins

Abstract: Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH- SY5Y cells: Gaα, G_iα1, G_jα2, G_cα, G_zα, and Gβ. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 μmol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of μ-opioid binding sites, the levels of the inhibitory G proteins G_iα1 and G_jα1 were found to be significantly increased. This coordinate up-reg- ulation is accompanied by functional changes in μ-opioid receptor-stimulated Iow-K_m GTPase, μ-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5′-(βγ-imido)triphosphate [Gpp(NH)p; 10 nmol/ L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prosta- glandin E₁ (PGE₁) receptors and G_sα, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE₁ binding sites and PGE₁ stimulated adenylate cyclase activity, but significantly reduced amounts of G_zα were found. This down- regulation is paralleled by a decrease in the stimulatory activity of G_zα as assessed in S49 cyc- reconstitution assays. However, the reduction in G_aα levels had no effect on both intrinsic and receptor-independent-activated [Gpp(NH)p or forskolin; 100 μtmol/L each] adenylate cyclase, suggesting that the amount of G_zα is in excess over the functional capacity of adenylate cyclase in SH-SY5Y cell membranes. Additional quantitative changes were found for G_zα, G_cα, and Gβ subunits. In contrast, neuronal differentiation in the presence of 12-O-tetradecanoylphor- bol 13-acetate (16 nmol/L; 6 days) failed to affect G protein abundance. Our results provide evidence for a specific RA effect on the abundance of distinct G protein sub- units in human SH-SY5Y neuroblastoma cells. These alterations might contribute to functional changes in transmembrane signaling pathways associated with RA-in- duced neuronal differentiation of the cells.     

Growth and cell cycle of Ulva compressa (Ulvophyceae) under LED illumination

The cell‐cycle progression of Ulva compressa is diurnally gated at the G₁ phase in accordance with light–dark cycles. The present study was designed to examine the spectral sensitivity of the G₁ gating system. When blue, red, and green light‐emitting diodes (LEDs) were used for illumination either alone or in combination, the cells divided under all illumination conditions, suggesting that all colors of light were able to open the G₁ gate. Although blue light was most effective to open the G₁ gate, red light alone or green light alone was also able to open the G₁ gate even at irradiance levels lower than the light compensation point of each color. Occurrence of a period of no cell division in the course of a day suggested that the G₁ gating system normally functioned as under ordinary illumination by cool‐white fluorescent lamps. The rise of the proportion of blue light to green light resulted in increased growth rate. On the other hand, the growth rates did not vary regardless of the proportion of blue light to red light. These results indicate that the difference in growth rate due to light color resulted from the difference in photosynthetic efficiency of the colors of light. However, the growth rates significantly decreased under conditions without blue light. This result suggests that blue light mediates cell elongation and because the spectral sensitivity of the cell elongation regulating system was different from that of the G₁ gating system, distinct photoreceptors are likely to mediate the two systems.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE RADIATIONS ON CELL CYCLE KINETICS IN EXCISED ROOT MERISTEMS OF PISUM SATIVUM

Near-ultraviolet and visible radiations increased the duration of the mitotic cycle in excised pea root meristems primarily by lengthening the duration of the pre-DNA synthetic period (G₁). All radiations tested shortened the duration of the post-DNA synthetic period (G₂). The most pronounced effects were exhibited by green radiation, which lengthened the duration of the cell cycle, G₁, DNA synthesis (S), and mitosis (M), and shortened the duration of G₂. Progression of cells arrested by starvation in G₁ and G₂ into DNA synthesis and mitosis was also affected by light treatments. Green radiation appeared to arrest a group of cells in DNA synthesis as well as in G₁ and G₂. Meristems receiving green and near-ultraviolet radiations exhibited the most rapid progression of G₁ cells through S and G₂.     

Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells

Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα_s, Gα_q or Gα₁₂ to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24 h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G_s, 6 genes were solely mediated through G_q, and 3 genes were only controlled through G₁₂. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G_s and G_q, 3 genes were regulated by both G_s and G₁₂, and 3 genes were controlled by G_s, G_q and G₁₂. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.     

Conjugation Between G1 and G2 Phase Cells in Paramecium tetraurelia1

Cells of Paramecium tetraurelia, stock hr^d, cultured in a micro-capillary containing 1 μl fresh culture medium, expressed mating activity through the whole cell cycle. Mating-reactive G₂ phase cells can conjugate with cells of other phases. The G₂ phase cells, which have double (4C) the normal micronuclear DNA content, undergo pre-meiotic DNA synthesis when conjugated with G₁ phase cells. The micronucleus of the progeny from the cross between a G₁ and a G₂ cell becomes triploid.     

Cell cycles in embryos of the silkworm,Bombyx mori: G2-arrest at diapause stage

Summary In the silkworm, Bombyx mori, diapause occurs at a specific embryonic stage, i.e. after formation of the germ band with cephalic lobes and telson and sequential mesoderm segmentation. As long as the eggs are incubated at 25° C, cell divisions and morphological development of the embryos cease. To examine changes in percentage of embryonic cells in the G₁, S and G₂ phases during embryogenesis, nuclear fractions were isolated from embryos, stained with propidium iodide and then subjected to flow cytometric analysis. The percentages of embryonic cells in G₁, S and G₂ were 10, 35 and 55%, respectively, at the stage of formation of cephalic lobes, whilst 98% of cells were in G₂ at diapause stage. After termination of diapause by acclimation at 5° C or by a combination of chilling and HCl, cell division resumed in the embryos. During this period, the cells rapidly entered S phase through G₁ from G₂, suggesting that their G₁ phase was short. In eggs in which diapause was averted by HCl-treatment after incubation at 25° C for 20 h after oviposition, embryonic development proceeded continuously for 9.5 days at 25° C until hatching. Along with this development, the G₁ fraction increased to levels of about 90%. These results indicate that embryonic cells are arrested in G₂ at diapause and suggest that, concomitant with further embryonic development, cell cycles become slower in proportion to an increasing length of G₁. Finally, most of the cells may be arrested in G₁, while there is only a small fraction of cells continuously cycling. Offprint requests to: T. Yaginuma     

Cell Cycle Dynamics of Cultured Coral Endosymbiotic Microalgae (Symbiodinium) Across Different Types (Species) Under Alternate Light and Temperature Conditions

Dinoflagellates of the genus Symbiodinium live in symbiosis with many invertebrates, including reef‐building corals. Hosts maintain this symbiosis through continuous regulation of Symbiodinium cell density via expulsion and degradation (postmitotic) and/or constraining cell growth and division through manipulation of the symbiont cell cycle (premitotic). Importance of premitotic regulation is unknown since little data exists on cell cycles for the immense genetic diversity of Symbiodinium. We therefore examined cell cycle progression for several distinct SymbiodiniumITS2‐types (B1, C1, D1a). All types exhibited typical microalgal cell cycle progression, G₁ phase through to S phase during the light period, and S phase to G₂/M phase during the dark period. However, the proportion of cells in these phases differed between strains and reflected differences in growth rates. Undivided larger cells with 3n DNA content were observed especially in type D1a, which exhibited a distinct cell cycle pattern. We further compared cell cycle patterns under different growth light intensities and thermal regimes. Whilst light intensity did not affect cell cycle patterns, heat stress inhibited cell cycle progression and arrested all strains in G₁ phase. We discuss the importance of understanding Symbiodinium functional diversity and how our findings apply to clarify stability of host‐Symbiodinium symbioses.     

Dopaminergic and adrenergic toxicities on SK-N-MC human neuroblastoma cells are mediated through G protein signaling and oxidative stress

Dopamine and norepinephrine are neurotransmitters which participate in various regulatory functions of the human brain. These functions are lost in neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease. In this study, we used SK-N-MC neuroblastoma cells to investigate the cytotoxicities of high concentrations of dopamine and norepinephrine on neuronal cells. Dopamine, norepinephrine, as well as their corresponding synthetic agonists (SKF38393 and isoproterenol, respectively) triggered SK-N-MC cell death when applied at 50–100 μM persistently for 2 days. This catecholamine-induced cell death appears to be neuronal specific, as demonstrated by their inabilities of triggering apoptosis of A549 lung carcinoma cells and Cos-7 kidney fibroblasts. By pretreating SK-N-MC cells with target-specific inhibitors before administration of catecholamine, components of G protein signaling (i.e. G _s /cAMP/PKA), monoamine oxidases, nitric oxide synthase, c-Jun N-terminal kinase and oxidative stress were found to be involved in this dopamine/norepinephrine-induced cytotoxicity, which subsequently led to caspase-dependent and -independent apoptotic responses as well as DNA degradation. In contrast, agonists of G _i -coupled dopamine receptors and adrenergic receptors (quinpirole and UK14,304, respectively) were incapable of triggering apoptosis of SK-N-MC cells. Our results suggest that both G protein (G _s )-mediated signaling cascade and oxidative stress participate in the dopamine/norepinephrine-induced neuronal apoptosis. Anthony Chan and Ng Contributed equally to this work.     

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.     

Receptor-Mediated Activation of G Proteins Is Increased in Postmortem Brains of Bipolar Affective Disorder Subjects

Abstract: Guanine nucleotide binding proteins (G proteins) have been implicated in the pathophysiology of bipolar affective disorder. In the present investigation receptor-mediated G protein activation and changes in G protein trimeric state were examined in frontal cortical membranes obtained from postmortem brains of bipolar affective disorder subjects and from age-, sex-, and postmortem interval-matched controls. Stimulation of cortical membranes with serotonin, isoproterenol, or carbachol increased guanosine 5′-O-(3-[³⁵S]thiophosphate) ([³⁵S]GTPγS) binding to specific G_α proteins in a receptor-selective manner. The abilities of these receptor agonists to stimulate the binding of [³⁵S]GTPγS to the G_α proteins was enhanced in membranes from bipolar brains. Immunoblot analyses showed increases in the levels of membrane 45- and 52-kDa G_αs proteins but no changes in the amounts of G_αi, G_αo, G_αz, G_αq/11, or G_β proteins in membrane or cytosol fractions of bipolar brain homogenates. Pertussis toxin (PTX)-activated ADP-ribosylations of G_αi and G_αo were enhanced by ～80% in membranes from bipolar compared with control brains, suggesting an increase in the levels of the trimeric state of these G proteins in bipolar disorder. Serotonin-induced, magnesium-dependent reduction in PTX-mediated ADP-ribosylation of G_αi/G_αo in cortical membranes from bipolar brains was greater than that observed in controls, providing further evidence for enhanced receptor-G protein coupling in bipolar brain membranes. In addition, the amounts of G_β proteins that coimmunoprecipitated with the G_α proteins were also elevated in bipolar brains. The data show that in bipolar brain membrane there is enhanced receptor-G protein coupling and an increase in the trimeric state of the G proteins. These changes may contribute to produce exaggerated transmembrane signaling and to the alterations in affect that characterize bipolar affective disorder.     

Activation of Type II Adenylyl Cyclase by the Cloned μ-Opioid Receptor: Coupling to Multiple G Proteins

Abstract: Opioid receptors are multifunctional receptors that utilize G proteins for signal transduction. The cloned δ-opioid receptor has been shown recently to stimulate phospholipase C, as well as to inhibit or stimulate different isoforms of adenylyl cyclase. By using transient transfection studies, the ability of the cloned μ-opioid receptor to stimulate type II adenylyl cyclase was examined. Coexpression of the μ-opioid receptor with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the μ-selective agonist, [d -Ala², N-Me-Phe⁴,Gly⁵-ol]enkephalin, to stimulate cyclic AMP accumulation in a dose-dependent manner. The opioid-induced stimulation of type II adenylyl cyclase was mediated via pertussis toxin-sensitive G_i proteins, because it was abolished completely by the toxin. Possible coupling between the μ-opioid receptor and various G protein α subunits was examined in the type II adenylyl cyclase system. The opioid-induced response became pertussis toxin-insensitive and was enhanced significantly upon co-expression with the α subunit of G_z, whereas those of G_q, G₁₂, or G₁₃ inhibited the opioid response. When pertussis toxin-sensitive G protein α subunits were tested under similar conditions, all three forms of α_i and both forms of α_o were able to enhance the opioid response to various extents. Enhancement of type II adenylyl cyclase responses by the co-expression of α subunits reflects a functional coupling between α subunits and the μ-opioid receptor, because such potentiations were not observed with the constitutively activated α subunit mutants. These results indicate that the μ-opioid receptor can couple to G_i1–3, G_o1–2, and G_z, but not to G_s, G_q, G₁₂, G₁₃, or G_t.