Cell Cycle Elongation Research Articles

Neopetrosidines A–D, pyridine alkaloids isolated from the marine sponge Neopetrosia chaliniformis and their cell cycle elongation activity

Natural products that inhibit cell cycle progression show promise as anticancer agents and chemical probes. In our research on biologically active natural products that affect cell cycle progression of HeLa/fluorescent ubiquitination-based cell cycle indicator (Fucci)2 cells, the extract of the marine sponge Neopetrosia chaliniformis was revealed to inhibit cell proliferation. Purification of the extract afforded four new pyridine alkaloids, neopetrosidines A–D (1–4). Their structures were elucidated by the interpretation of spectroscopic data and chemical degradation. Compounds 1–4 were found to inhibit cell proliferation of HeLa/Fucci2 cells, and time-lapse imaging showed that 1 exerts its effect by increasing the duration of the cell cycle. Furthermore, we show that 1 perturbs bioenergetics to exhibit a cytostatic effect by reducing the mitochondrial membrane potential.

The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila through multiple modalities

Early embryos must rapidly generate large numbers of cells to form an organism. Many species accomplish this through a series of rapid, reductive, and transcriptionally silent cleavage divisions. Previous work has demonstrated that the number of divisions before both cell cycle elongation and zygotic genome activation (ZGA) is regulated by the ratio of nuclear content to cytoplasm (N/C). To understand how the N/C ratio affects the timing of ZGA, we directly assayed the behavior of several previously identified N/C ratio-dependent genes using the MS2-MCP reporter system in living Drosophila embryos with altered ploidy and cell cycle durations. For every gene that we examined, we found that nascent RNA output per cycle is delayed in haploid embryos. Moreover, we found that the N/C ratio influences transcription through three overlapping modes of action. For some genes (knirps, fushi tarazu, and snail), the effect of ploidy can be primarily attributed to changes in cell cycle duration. However, additional N/C ratio-mediated mechanisms contribute significantly to transcription delays for other genes. For giant and bottleneck, the kinetics of transcription activation are significantly disrupted in haploids, while for frühstart and Krüppel, the N/C ratio controls the probability of transcription initiation. Our data demonstrate that the regulatory elements of N/C ratio-dependent genes respond directly to the N/C ratio through multiple modes of regulation.

Heterotrimeric G-protein, Gi1, is involved in the regulation of proliferation, neuronal migration, and dendrite morphology during cortical development in vivo.

Heterotrimeric G-proteins are composed of α, β, and γ subunits, and function as signal transducers. Critical roles of the α-subunits of Gi/o family heterotrimeric G-proteins, Gαi2, and Gαo1, have so far been reported in brain development and neurodevelopmental disorders. In this study, we tried to clarify the role of Gαi1, α-subunit of another Gi/o family member Gi1, during corticogenesis, based on the recent identification of its gene abnormalities in neurodevelopmental disorders. In western blot analyses, Gαi1 was found to be expressed in mouse brain in a developmental stage-dependent manner. Morphological analyses revealed that Gαi1 was broadly distributed in cerebral cortex with relatively high expression in the ventricular zone (VZ) at embryonic day (E) 14. Meanwhile, Gαi1 was enriched in membrane area of yet unidentified early mitotic cells in the VZ and the marginal zone at E14. Acute knockdown of Gαi1 with in utero electroporation in cerebral cortex caused cell cycle elongation of the neural progenitor cells and promoted their cell cycle exit. Gαi1-deficient cortical neurons also exhibited delayed radial migration during corticogenesis, with abnormally elongated leading processes and hampered nucleokinesis. In addition, silencing of Gαi1 prevented basal dendrite development. The migration and dendritic phenotypes were at least partially rescued by an RNAi-resistant version of Gαi1. Collectively, these results strongly suggest a crucial role of Gi1 in cortical development, and disturbance of its function may cause deficits in synaptic network formation, leading to neurodevelopmental disorders.

Cell cycle arrest in replicative senescence is not an immediate consequence of telomere dysfunction

In replicative senescence, cells with critically-short telomeres activate a DNA-damage response leading to cell-cycle arrest, while those without telomere dysfunction would be expected to cycle normally. However, population growth declines more gradually than such a simple binary switch between cycling and non-cycling states would predict. We show here that late-passage cultures of human fibroblasts are not a simple mixture of cycling and non-cycling cells. Rather, although some cells had short cycle times comparable to those of younger cells, others continued to divide but with greatly extended cycle times, indicating a more-gradual approach to permanent arrest. Remarkably, in late passage cells, the majority showed prominent DNA-damage foci positive for 53BP1, yet many continued to divide. Evidently, the DNA-damage-response elicited by critically-short telomeres is not initially strong enough for complete cell-cycle arrest. A similar continuation of the cell cycle in the face of an active DNA-damage response was also seen in cells treated with a low dose of doxorubicin sufficient to produce multiple 53BP1 foci in all nuclei. Cell cycle checkpoint engagement in response to DNA damage is thus weaker than generally supposed, explaining why an accumulation of dysfunctional telomeres is needed before marked cell cycle elongation or permanent arrest is achieved.

Histone concentration regulates the cell cycle and transcription in early development.

The early embryos of many animals, including flies, fish and frogs, have unusually rapid cell cycles and delayed onset of transcription. These divisions are dependent on maternally supplied RNAs and proteins including histones. Previous work suggests that the pool size of maternally provided histones can alter the timing of zygotic genome activation (ZGA) in frogs and fish. Here, we examine the effects of under- and overexpression of maternal histones in Drosophila embryogenesis. Decreasing histone concentration advances zygotic transcription, cell cycle elongation, Chk1 activation and gastrulation. Conversely, increasing histone concentration delays transcription and results in an additional nuclear cycle before gastrulation. Numerous zygotic transcripts are sensitive to histone concentration, and the promoters of histone-sensitive genes are associated with specific chromatin features linked to increased histone turnover. These include enrichment of the pioneer transcription factor Zelda, and lack of SIN3A and associated histone deacetylases. Our findings uncover a crucial regulatory role for histone concentrations in ZGA of Drosophila.

Chk1 Inhibition of the Replication Factor Drf1 Guarantees Cell-Cycle Elongation at the Xenopus laevis Mid-blastula Transition

SummaryThe early cell divisions of many metazoan embryos are rapid and occur in the near absence of transcription. At the mid-blastula transition (MBT), the cell cycle elongates and several processes become established including the onset of bulk transcription and cell-cycle checkpoints. How these events are timed and coordinated is poorly understood. Here we show in Xenopus laevis that developmental activation of the checkpoint kinase Chk1 at the MBT results in the SCFβ-TRCP-dependent degradation of a limiting replication initiation factor Drf1. Inhibition of Drf1 is the primary mechanism by which Chk1 blocks cell-cycle progression in the early embryo and is an essential function of Chk1 at the blastula-to-gastrula stage of development. This study defines the downregulation of Drf1 as an important mechanism to coordinate the lengthening of the cell cycle and subsequent developmental processes.

The Relationship between Cell Number, Division Behavior and Developmental Potential of Cleavage Stage Human Embryos: A Time-Lapse Study.

Day 3 cleavage embryo transfer is routine in many assisted reproductive technology centers today. Embryos are usually selected according to cell number, cell symmetry and fragmentation for transfer. Many studies have showed the relationship between cell number and embryo developmental potential. However, there is limited understanding of embryo division behavior and their association with embryo cell number and developmental potential. A retrospective and observational study was conducted to investigate how different division behaviors affect cell number and developmental potential of day 3 embryos by time-lapse imaging. Based on cell number at day 3, the embryos (from 104 IVF/intracytoplasmic sperm injection (ICSI) treatment cycles, n = 799) were classified as follows: less than 5 cells (< 5C; n = 111); 5–6 cells (5–6C; n = 97); 7–8 cells (7–8C; n = 442), 9–10 cells (9–10C; n = 107) and more than 10 cells (>10C; n = 42). Division behavior, morphokinetic parameters and blastocyst formation rate were analyzed in 5 groups of day 3 embryos with different cell numbers. In <5C and 5–6C embryos, fragmentation (FR; 62.2% and 30.9%, respectively) was the main cause for low cell number. The majority of 7–8C embryos exhibited obvious normal behaviors (NB; 85.7%) during development. However, the incidence of DC in 9–10C and >10C embryos increased compared to 7–8C embryos (45.8%, 33.3% vs. 11.1%, respectively). In ≥5C embryos, FR and DC significantly reduced developmental potential, whereas <5C embryos showed little potential irrespective of division behaviors. In NB embryos, the blastocyst formation rate increased with cell number from 7.4% (<5C) to 89.3% (>10C). In NB embryos, the cell cycle elongation or shortening was the main cause for abnormally low or high cell number, respectively. After excluding embryos with abnormal division behaviors, the developmental potential, implantation rate and live birth rate of day 3 embryos increased with cell number.

Abstract 5096: Quiescence induced checkpoint activation in the absence of CIZ1

Abstract The CIZ1 protein is required for timely entry to S phase following exit from quiescence (1). We show here that CIZ1 is also required for accurate entry into quiescence. Analysis of quiescence entry following contact inhibition in a CIZ1-deletion model has revealed a role in dampening a previously unreported quiescence checkpoint, evidenced by ubiquitous post-replicative phosphorylation of histone H2AX and activation of checkpoint kinases ATM and CHK1. Subsequent emergence of proliferating colonies indicates escape from checkpoint-mediated arrest, and begins to implicate inappropriate response to contact induced signaling as a novel endogenous driver of oncogenesis. Unscheduled checkpoint activation was also observed in bone marrow cells from juvenile CIZ1 null mice, and high frequency B-cell lymphoma in young adults in the absence of any additional oncogenic stimulation, supporting the idea that CIZ1 plays a very early role in tumorigenesis. Analysis of cell cycle progression using immunofluorescence and flow cytometry of primary fibroblasts from post-natal CIZ1 null mice revealed cell cycle elongation, with extended expression of cyclins D2 and E1, increased cyclin A2, and altered p21, p27 and CDK2, but no checkpoint activation. Thus, CIZ1 null cells can tolerate altered expression, targeting and recruitment of multiple cell cycle regulators. Checkpoint activation occurs only after exit from the cell cycle, reflecting aberrant response to contact induced signaling and failure to accurately transition into a stable quiescent state. Inappropriate expression of CIZ1 splice variants has been previously linked with both adult and pediatric cancers (2-4), and shown to play a proliferation-promoting role in lung (4) and breast (5) tumor models. Furthermore, CIZ1 b-variant has biomarker capability for the early detection of lung cancer (4). Despite these advances, the precise role of CIZ1 in the cell is not yet understood. The CIZ1 knock out mouse model described here, and a related model (6), indicate a tumor suppressor function, which is apparently at odds with the DNA replication- and proliferation-promoting roles reported previously. The current work helps to reconcile these observations by showing that CIZ1s established activity in the promotion of cyclin A-mediated initiation of DNA replication (1) is as a coordinator. This in fact, influences multiple cyclins and CDKis, however, impairment of this capability during cell cycle progression may not underlie CIZ1s role in tumorigenesis. Our central finding is that altered cell cycle homeostasis in CIZ1 null cells does not lead to checkpoint activation, until they are driven to contact induced quiescence. 1 Copeland et al (2010) J Cell Sci 123, 1108-15 2 Warder &amp; Keherly (2003) J Biomed Sci.10, 406-17 3 Rahman et al (2007) Human Mutation 28, 993-1004 4 Higgins et al (2012) Proc Natl Acad Sci U S A 109, E3128-35 5 den Hollander et al (2006) Cancer Research 66, 11021-30 6 Nishibe et al (2013) FEBS Lett 587, 1529-35 Citation Format: Rosemary H.C Wilson, Jo-An Roulson, Sumia Bageghni, Ghadeer Albadrani, Justin F-X Ainscough, Dawn Coverley. Quiescence induced checkpoint activation in the absence of CIZ1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5096. doi:10.1158/1538-7445.AM2014-5096

Disrupted ERK Signaling during Cortical Development Leads to Abnormal Progenitor Proliferation, Neuronal and Network Excitability and Behavior, Modeling Human Neuro-Cardio-Facial-Cutaneous and Related Syndromes

Genetic disorders arising from copy number variations in the ERK (extracellular signal-regulated kinase) MAP (mitogen-activated protein) kinases or mutations in their upstream regulators that result in neuro-cardio-facial-cutaneous syndromes are associated with developmental abnormalities, cognitive deficits, and autism. We developed murine models of these disorders by deleting the ERKs at the beginning of neurogenesis and report disrupted cortical progenitor generation and proliferation, which leads to altered cytoarchitecture of the postnatal brain in a gene-dose-dependent manner. We show that these changes are due to ERK-dependent dysregulation of cyclin D1 and p27(Kip1), resulting in cell cycle elongation, favoring neurogenic over self-renewing divisions. The precocious neurogenesis causes premature progenitor pool depletion, altering the number and distribution of pyramidal neurons. Importantly, loss of ERK2 alters the intrinsic excitability of cortical neurons and contributes to perturbations in global network activity. These changes are associated with elevated anxiety and impaired working and hippocampal-dependent memory in these mice. This study provides a novel mechanistic insight into the basis of cortical malformation which may provide a potential link to cognitive deficits in individuals with altered ERK activity.

Role of the PI3K-TOR-S6K pathway in the onset of cell cycle elongation during Xenopus early embryogenesis

In the early embryogenesis of the frog, Xenopus laevis, cells proliferate by rapid and synchronous divisions, followed by cell cycle elongation and prolongation of the S phases, and then the appearance of the G2 and G1 phases after the midblastula transition (MBT). The beginning of cell cycle elongation was thought to depend on an increase in the nucleo-cytoplasmic (N/C) ratio in blastomeres and a decrease in cortical cytoplasmic factors necessary for cell cycle progression, although these factors are unknown. In the present study, we demonstrated that a regulatory subunit of PI3K (p85α) was localized in the cortical cytoplasm of the blastomere during the MBT. When the embryos were treated with a PI3K inhibitor, LY294002, or a TOR inhibitor, rapamycin, cell cycle elongation was initiated before the MBT. In addition, the inhibition of S6K expression by antisense morpholino oligo enhanced the initiation of cell cycle elongation. In contrast, the activation of PI3K-TOR by Rheb-S16H expression delayed the initiation of cell cycle elongation. These results indicate that a decrease in translational activity dependent on the PI3K-TOR-S6K pathway causes the initiation of cell cycle elongation at the onset of the MBT.

Cell Cycle Elongation Impairs Proliferation of Cerebellar Granule Cell Precursors in the Ts65Dn Mouse, an Animal Model for Down Syndrome

Mental retardation, the hallmark of Down syndrome (DS), has been attributed to the reduced number of neurons populating the DS brain. The Ts65Dn mouse model of DS displays several anomalies analogous to those in individuals with DS, including neurogenesis impairment. The goal of the current study was to determine whether cell cycle alterations underlie neurogenesis impairment in the cerebellum of the Ts65Dn mouse and to identify the molecular mechanisms responsible for this defect. In neonatal (2-day old) Ts65Dn mice, cerebellar granule cell precursors exhibited a reduced proliferation rate (-40%) and a notable elongation (+45%) of the cell cycle. Alteration of cell cycle rate was due to elongation of the G(2) and G(1) phases. Microarray screening of cell cycle regulatory genes showed that Ts65Dn mice had a decreased expression of Cyclin B1 and Skp2, two key regulators of G(2)/M and G(1)/S transition. Results point at cell cycle elongation as major determinant of neurogenesis reduction in the cerebellum of Ts65Dn mice and suggest that this defect is specifically linked to an altered expression of two cell-cycle regulatory genes, Cyclin B1 and Skp2. These findings may establish the basis for a therapeutic approach aimed at restoring neurogenesis in the DS brain.

Loss of protooncogene c-Myc function impedes G1 phase progression both before and after the restriction point.

c-myc is an important protooncogene whose misregulation is believed to causally affect the development of numerous human cancers. c-myc null rat fibroblasts are viable but display a severe (two- to threefold) retardation of proliferation. The rates of RNA and protein synthesis are reduced by approximately the same factor, whereas cell size remains unaffected. We have performed a detailed kinetic cell cycle analysis of c-myc(-/-) cells by using several labeling and synchronization methods. The majority of cells (>90%) in asynchronous, exponential phase c-myc(-/-) cultures cycle continuously with uniformly elongated cell cycles. Cell cycle elongation is due to a major lengthening of G(1) phase (four- to fivefold) and a more limited lengthening of G(2) phase (twofold), whereas S phase duration is largely unaffected. Progression from mitosis to the G1 restriction point and the subsequent progression from the restriction point into S phase are both drastically delayed. These results are best explained by a model in which c-Myc directly affects cell growth (accumulation of mass) and cell proliferation (the cell cycle machinery) by independent pathways.

Chk1 is activated transiently and targets Cdc25A for degradation at the Xenopus midblastula transition.

In Xenopus embryos, cell cycle elongation and degradation of Cdc25A (a Cdk2 Tyr15 phosphatase) occur naturally at the midblastula transition (MBT), at which time a physiological DNA replication checkpoint is thought to be activated by the exponentially increased nucleo-cytoplasmic ratio. Here we show that the checkpoint kinase Chk1, but not Cds1 (Chk2), is activated transiently at the MBT in a maternal/zygotic gene product-regulated manner and is essential for cell cycle elongation and Cdc25A degradation at this transition. A constitutively active form of Chk1 can phosphorylate Cdc25A in vitro and can target it rapidly for degradation in pre-MBT embryos. Intriguingly, for this degradation, however, Cdc25A also requires a prior Chk1-independent phosphorylation at Ser73. Ectopically expressed human Cdc25A can be degraded in the same way as Xenopus Cdc25A. Finally, Cdc25A degradation at the MBT is a prerequisite for cell viability at later stages. Thus, the physiological replication checkpoint is activated transiently at the MBT by developmental cues, and activated Chk1, only together with an unknown kinase, targets Cdc25A for degradation to ensure later development.

Onset of transcription in Patella vulgata coincides with cell cycle elongation and expression of tubulin genes.

In this study we show that the onset of embryonic transcription in the marine snail Patella vulgata coincides with the start of the sixth cleavage, when the cell-cycle elongates and divisions become asynchronous. Changes in mRNA content before and after onset of transcription were initially demonstrated by in vitro translation of isolated mRNA from different stages. Before the sixth cleavage, three major mRNAs encoding proteins of 36, 50 and 52 kDa were present. These proteins probably correspond to cyclin A and B and ribonucleotide reductase. After this stage, three major proteins with molecular weights of 36.5, 52.5 and 53 kDa were found after in vitro translation. Via hybrid selected translation and differential screening cDNAs corresponding to the 52.5 and 53 kDa proteins were cloned. The encoded proteins resemble tubulins from other animals to a high extent (between 96.5 and 93.1% identity for α-tubulin and 97.9 and 75.9% for β-tubulin). The 36.5 kDa protein is the previously described actin. Both tubulins were expressed at or shortly after the first asynchronous division after the fifth cleavage.