Extended Cell Cycle Research Articles

Initial characterization of gap phase introduction in every cell cycle of C. elegans embryogenesis.

Early embryonic cell cycles usually alternate between S and M phases without any gap phase. When the gap phases are developmentally introduced in various cell types remains poorly defined especially during embryogenesis. To establish the cell-specific introduction of gap phases in embryo, we generate multiple fluorescence ubiquitin cell cycle indicators (FUCCI) in C. elegans. Time-lapse 3D imaging followed by lineal expression profiling reveals sharp and differential accumulation of the FUCCI reporters, allowing the systematic demarcation of cell cycle phases throughout embryogenesis. Accumulation of the reporters reliably identifies both G1 and G2 phases only in two embryonic cells with an extended cell cycle length, suggesting that the remaining cells divide either without a G1 phase, or with a brief G1 phase that is too short to be picked up by our reporters. In summary, we provide an initial picture of gap phase introduction in a metazoan embryo. The newly developed FUCCI reporters pave the way for further characterization of developmental control of cell cycle progression.

Growth disadvantage associated with centrosome amplification drives population-level centriole number homeostasis.

The centriole duplication cycle normally ensures that centriole number is maintained at two centrioles per G1 cell. However, some circumstances can result in an aberrant increase in centriole number—a phenotype that is particularly prevalent in several types of cancer. Following an artificial increase in centriole number without tetraploidization due to transient overexpression of the kinase PLK4, human cells return to a normal centriole number during the proliferation of the population. We examine the mechanisms responsible for this return to normal centriole number at the population level in human retinal pigment epithelial cells. We find that the return to normal centriole number in the population of induced cells cannot be explained by limited duplication of centrioles, instability of extra centrioles, or by grossly asymmetric segregation of extra centrioles in mitosis. However, cells with extra centrioles display heterogenous phenotypes including extended cell cycle arrest, longer interphase durations, and death, which overall results in a proliferative disadvantage relative to normal cells in the population. Although about half of cells with extra centrioles in a population were able to divide, the extent of the disadvantages conferred by other fates is sufficient to account for the observed rate of return to normal centriole number. These results suggest that only under conditions of positive selection for cells with extra centrioles, continuous generation of such centrioles, or alleviation of the disadvantageous growth phenotypes would they be maintained in a population.

Adult Neural Stem Cells: Born to Last.

The generation of new neurons is a lifelong process in many vertebrate species that provides an extra level of plasticity to several brain circuits. Frequently, neurogenesis in the adult brain is considered a continuation of earlier developmental processes as it relies in the persistence of neural stem cells, similar to radial glia, known as radial glia-like cells (RGLs). However, adult RGLs are not just leftovers of progenitors that remain in hidden niches in the brain after development has finished. Rather, they seem to be specified and set aside at specific times and places during embryonic and postnatal development. The adult RGLs present several cellular and molecular properties that differ from those observed in developmental radial glial cells such as an extended cell cycle length, acquisition of a quiescence state, a more restricted multipotency and distinct transcriptomic programs underlying those cellular processes. In this minireview, we will discuss the recent attempts to determine how, when and where are the adult RGLs specified.

Divergent Aging of Isogenic Yeast Cells Revealed through Single-Cell Phenotypic Dynamics

Although genetic mutations that alter organisms' average lifespans have been identified in aging research, our understanding of the dynamic changes during aging remains limited. Here, we integrate single-cell imaging, microfluidics, and computational modeling to investigate phenotypic divergence and cellular heterogeneity during replicative aging of single S.cerevisiae cells. Specifically, we find that isogenic cells diverge early in life toward one of two aging paths, which are characterized by distinct age-associated phenotypes. We captured the dynamics of single cells along the paths with a stochastic discrete-state model, which accurately predicts both the measured heterogeneity and the lifespan of cells on each path within a cell population. Our analysis suggests that genetic and environmental factors influence both a cell's choice of paths and the kinetics of paths themselves. Given that these factors are highly conserved throughout eukaryotes, divergent aging might represent a general scheme in cellular aging of other organisms.

In vitro epidermis model mimicking IGF-1-specific age-related decline.

Ageing is a complex multifaceted process affecting skin functionality and structure. Several 3D organotypic skin culture models have reproduced ageing by inducing replicative senescence, glycation or oxidative stress. Yet, very few models have focused on hormonal ageing and especially the insulin-like growth factor 1 (IGF-1) signalling pathway, which has been associated with longevity in animal studies and is necessary for the early stages of skin development. In this study, we built an organotypic epidermis model with targeted IGF-1 receptor knockdown to reproduce some aspects of hormonal ageing on skin. Our model displayed morphological and functional features of aged epidermis, which were mostly attributed to a loss of function of the Stratum basale. IGF-1 receptor knockdown keratinocytes depicted an extended cell cycle, reduced proliferation potential and reduced adhesion capacities and greater sensitivity to oxidative stress than control cells. Altogether, this model represents an essential tool for further investigations into the mechanisms linked to some aspects of hormonal decline or when screening for potent anti-ageing compounds.

Centromere inheritance through the germline.

The centromere directs chromosome segregation and genetic inheritance but is not itself heritable in a canonical, DNA-based manner. In most species, centromeres are epigenetically defined by the presence of a histone H3 variant centromere protein A (CENP-A), independent of underlying DNA sequence. Therefore, centromere inheritance depends on maintaining the CENP-A nucleosome mark across generations. Experiments in cycling somatic cells have led to a model in which centromere identity is maintained by a cell cycle-coupled CENP-A chromatin assembly pathway. However, the processes of animal gametogenesis pose unique challenges to centromere inheritance because of the extended cell cycle arrest and the massive genome reorganization in the female and male germline, respectively. Here, we review our current understanding of germline centromere inheritance and highlight outstanding questions.

A new twist to Sin3 complexes in pluripotent cells.

Sin3a is a central component of a class of histone deacetylase‐containing transcriptional co‐regulatory complexes. In this issue of The EMBO Journal, Streubel et al () purify Sin3a and identify a variant Sin3a complex containing Fam60a in undifferentiated embryonic stem cells (ESCs). Fam60a is a critical component of the ESC Sin3a complex since Fam60a knockdown leads to an extended G1 cell cycle phase and reduced ESC self‐renewal. These exciting results open up new questions about how biochemical differences between variant Sin3a complexes may facilitate alterations in cell‐specific function.

Effects of chronic exposure to acidic environment on the response of tumor cells to radiation

Purpose: The influence of short-term exposure to an acidic environment on the radiosensitivity of tumor cells has been extensively explored, but the implication of chronic exposure to an acidic environment for the response of tumor cells to radiation has not been fully elucidated. This study aimed to investigate the effects of chronic pre- and post-irradiation exposure of tumor cells to an acidic environment on the radiation-induced clonogenic death.Materials and methods: Rat gliosarcoma cells were used throughout the in vitro study. Cells were exposed to pH 6.6 medium for varying durations of up to 4 days before and after X-irradiation. Cell viability, apoptosis, clonogenic cell death and cell cycle distribution were observed.Results: Incubation of tumor cells in pH 6.6 medium for 2 or 4 days extended cell cycle, decreased cell viability, and induced apoptotic and clonogenic cell death. The radiation-induced clonogenic death was increased by 2- or 4-day pre-irradiation exposure of tumor cells to pH 6.6 medium, whereas it was reduced by 4-day post-irradiation exposure to an acidic medium.Conclusion: Prolonged exposure to an acidic environment enhanced the sensitivity of tumor cells to subsequent X-irradiation. However, the radiosensitization by pre-irradiation exposure was almost completely nullified by prolonged post-irradiation exposure to an acidic environment.

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma.

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulate retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.

Environmental stress speeds up DNA replication in Pseudomonas putida in chemostat cultivations.

Cellular response to different types of stress is the hallmark of the cell's strategy for survival. How organisms adjust their cell cycle dynamics to compensate for changes in environmental conditions is an important unanswered question in bacterial physiology. A cell using binary fission for reproduction passes through three stages during its cell cycle: a stage from cell birth to initiation of replication, a DNA replication phase and a period of cell division. We present a detailed analysis of durations of cell cycle phases, investigating their dynamics under environmental stress conditions. Applying continuous steady state cultivations (chemostats), the DNA content of a Pseudomonas putida KT2440 population was quantified with flow cytometry at distinct growth rates. Data-driven modeling revealed that under stress conditions, such as oxygen deprivation, solvent exposure and decreased iron availability, DNA replication was accelerated correlated to the severity of the imposed stress (up to 1.9-fold). Cells maintained constant growth rates by balancing the shortened replication phase with extended cell cycle phases before and after replication. Transcriptome data underpin the transcriptional upregulation of crucial genes of the replication machinery. Hence adaption of DNA replication speed appears to be an important strategy to withstand environmental stress.

The SWI/SNF ATPases Are Required for Triple Negative Breast Cancer Cell Proliferation.

The Brahma (BRM) and Brahma-related Gene 1 (BRG1) ATPases are highly conserved homologs that catalyze the chromatin remodeling functions of the multi-subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in many cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor's receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence, or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in the loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells.

Uncoupling different characteristics of the C. elegans E lineage from differentiation of intestinal markers.

In the 4-cell C. elegans embryo, a signal from P2 to its anterior sister, EMS, specifies the posterior daughter of EMS, E, as the sole founder cell for intestine. The P2-to-EMS signal restricts high level zygotic expression of the redundant GATA transcription factors, END-1 and END-3, to only the E lineage. Expression of END-1 or END-3 in early blastomeres is sufficient to drive intestinal differentiation. We show here that a number of E lineage characteristics, which are also regulated through P2-EMS signaling, can be uncoupled from intestine development, and each with a different sensitivity to specific perturbations of the P2-EMS signal. For example, we show that the extended cell cycle in Ea and Ep depends on the P2-induced high level expression of the cell cycle regulator, WEE-1.1, in E. A mutation in wee-1.1 results in shortened Ea and Ep cell cycles, but has no effect upon intestinal differentiation or embryogenesis. Furthermore, it has been shown previously that the total number of E lineage cell divisions is regulated by a mechanism dependent upon E being specified as the intestinal founder cell. We now show, however, that cell division counting can be uncoupled from intestine differentiation in the E lineage. Many mutations in P2-EMS signal genes exhibit nonfully-penetrant defects in intestinal differentiation. When embryos with those mutations generate intestinal cells, they often make too many intestinal cells. In addition, at the level of individual embryos, expression of end-1 and end-3, and another very early E-specific zygotic gene, sdz-23, exhibit stochastic and discordant defects in P2-EMS signaling mutants. We show here that sdz-23 is expressed close to wildtype levels in embryos deleted of both end-1 and end-3. sdz-23 does not appear to function in intestine development, raising the intriguing possibility that the P2-EMS interaction has downstream molecular consequences within the E lineage independent of end-1/3 and intestinal development.

Impact of cell cycle delay on micronucleus frequency in TK6 cells

Previous studies with TK6 cells have shown that extending the recovery period after pulse treatment allows for greater micronucleus expression for some compounds. This study explores the role of cell cycle delay in micronucleus expression after pulse treatment with three model genotoxins [mitomycin C, etoposide (ETOP), vinblastine]. Cells were treated for 4 hr and allowed to recover for 36 hr with samples removed at various time points during the recovery period and analyzed for cell cycle distribution, apoptosis and micronucleus frequency. Our results show that mitomycin C causes cell cycle delay for 20 hr after pulse treatment and cell cycle perturbation is no longer evident after 36 hr of recovery. The micronucleus frequency of cells sampled at 36 hr is doubled when compared with cells sampled at 20 hr after mitomycin C removal. When cells were treated with indirect acting genotoxins (ETOP, vinblastine), cell cycle perturbation was not observed at the 20 hr time point. Micronucleus frequency after treatment with either ETOP or vinblastine did not differ between the 20 hr and the 36 hr time point. All three compounds induced similar levels of apoptosis ranging from 4.5 to 5.6% with maximum induction occurring at the 36-hr time point. We conclude that TK6 cells exhibit extended cell cycle arrest after exposure to MMC and can go on to express micronuclei, after overcoming cell cycle arrest.

TheArabidopsis thalianaCheckpoint Kinase WEE1 Protects against Premature Vascular Differentiation during Replication Stress

A sessile lifestyle forces plants to respond promptly to factors that affect their genomic integrity. Therefore, plants have developed checkpoint mechanisms to arrest cell cycle progression upon the occurrence of DNA stress, allowing the DNA to be repaired before onset of division. Previously, the WEE1 kinase had been demonstrated to be essential for delaying progression through the cell cycle in the presence of replication-inhibitory drugs, such as hydroxyurea. To understand the severe growth arrest of WEE1-deficient plants treated with hydroxyurea, a transcriptomics analysis was performed, indicating prolonged S-phase duration. A role for WEE1 during S phase was substantiated by its specific accumulation in replicating nuclei that suffered from DNA stress. Besides an extended replication phase, WEE1 knockout plants accumulated dead cells that were associated with premature vascular differentiation. Correspondingly, plants without functional WEE1 ectopically expressed the vascular differentiation marker VND7, and their vascular development was aberrant. We conclude that the growth arrest of WEE1-deficient plants is due to an extended cell cycle duration in combination with a premature onset of vascular cell differentiation. The latter implies that the plant WEE1 kinase acquired an indirect developmental function that is important for meristem maintenance upon replication stress.

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate-dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21(Cip1) accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.

Cdk1 is sufficient to drive the mammalian cell cycle

Unicellular organisms such as yeasts require a single cyclin-dependent kinase, Cdk1, to drive cell division. In contrast, mammalian cells are thought to require the sequential activation of at least four different cyclin-dependent kinases, Cdk2, Cdk3, Cdk4 and Cdk6, to drive cells through interphase, as well as Cdk1 to proceed through mitosis. This model has been challenged by recent genetic evidence that mice survive in the absence of individual interphase Cdks. Moreover, most mouse cell types proliferate in the absence of two or even three interphase Cdks. Similar results have been obtained on ablation of some of the activating subunits of Cdks, such as the D-type and E-type cyclins. Here we show that mouse embryos lacking all interphase Cdks (Cdk2, Cdk3, Cdk4 and Cdk6) undergo organogenesis and develop to midgestation. In these embryos, Cdk1 binds to all cyclins, resulting in the phosphorylation of the retinoblastoma protein pRb and the expression of genes that are regulated by E2F transcription factors. Mouse embryonic fibroblasts derived from these embryos proliferate in vitro, albeit with an extended cell cycle due to inefficient inactivation of Rb proteins. However, they become immortal on continuous passage. We also report that embryos fail to develop to the morula and blastocyst stages in the absence of Cdk1. These results indicate that Cdk1 is the only essential cell cycle Cdk. Moreover, they show that in the absence of interphase Cdks, Cdk1 can execute all the events that are required to drive cell division.

The disarrayed mutation results in cell cycle and neurogenesis defects during retinal development in zebrafish

BackgroundThe vertebrate retina is derived from proliferative neuroepithelial cells of the optic cup. During retinal development, cell proliferation and the processes of cell cycle exit and neurogenesis are coordinated in neuroepithelial progenitor cells. Previous studies have demonstrated reciprocal influences between the cell cycle and neurogenesis. However the specific mechanisms and exact relationships of cell cycle regulation and neurogenesis in the vertebrate retina remain largely unknown.ResultsWe have isolated and characterized a zebrafish mutant, disarrayed (drya64), which exhibits retinal defects in cell cycle regulation and neurogenesis. By 42 hours post fertilization, disarrayed mutants show small eyes and a reduced forebrain. Other aspects of development appear normal. Although retinogenesis is delayed, mutant retinal cells eventually differentiate to all major cell types. Examination of the disarrayed mitotic cycle using BrdU and direct imaging techniques revealed that retinal neuroepithelial cells have an extended cell cycle period and reduced rate of cell cycle exit and neurogenesis, despite the fact that neurogenesis initiates at the appropriate time of development. Genetic mosaic analyses indicate that the cell cycle phenotype of disarrayed is cell-non-autonomous.ConclusionThe disarrayed mutant shows defects in both cell cycle regulation and neurogenesis and provides insights into the coordinated regulation of these processes during retinal development.

The archaeal cell cycle: current issues.

The recently discovered structural similarities between the archaeal Orc1/Cdc6 and bacterial DnaA initiator proteins for chromosome replication have exciting implications for cell cycle regulation. Together with current attempts to identify archaeal chromosome replication origins, the information is likely to yield fundamental insights into replication control in both archaea and eukaryotes within the near future. Several proteins that affect, or are likely to affect, chromatin structure and genome segregation in archaea have been described recently, including Sph1 and 2, ScpA and B, Sir2, Alba and Rio1p. Important insights into the properties of the MinD and FtsZ cell division proteins, and of putative cytoskeletal elements, have recently been gained in bacteria. As these proteins also are present among archaea, it is likely that the new information will also be essential for understanding archaeal genome segregation and cell division. A series of interesting cell cycle issues has been brought to light through the discovery of the novel Nanoarchaeota phylum, and these are outlined briefly. Exciting areas for extended cell cycle investigations of archaea are identified, including termination of chromosome replication, application of in situ cytological techniques for localization of cell cycle proteins and the regulatory roles of GTP-binding proteins and small RNAs.

Mutations in DNA replication genes reduce yeast life span.

Surprisingly, the contribution of defects in DNA replication to the determination of yeast life span has never been directly investigated. We show that a replicative yeast helicase/nuclease, encoded by DNA2 and a member of the same helicase subfamily as the RecQ helicases, is required for normal life span. All of the phenotypes of old wild-type cells, for example, extended cell cycle time, age-related transcriptional silencing defects, and nucleolar reorganization, occur after fewer generations in dna2 mutants than in the wild type. In addition, the life span of dna2 mutants is extended by expression of an additional copy of SIR2 or by deletion of FOB1, which also increase wild-type life span. The ribosomal DNA locus and the nucleolus seem to be particularly sensitive to defects in dna2 mutants, although in dna2 mutants extrachromosomal ribosomal circles do not accumulate during the aging of a mother cell. Several other replication mutations, such as rad27 Delta, encoding the FEN-1 nuclease involved in several aspects of genomic stability, also show premature aging. We propose that replication fork failure due to spontaneous, endogenous DNA damage and attendant genomic instability may contribute to replicative senescence. This may imply that the genomic instability, segmental premature aging symptoms, and cancer predisposition associated with the human RecQ helicase diseases, such as Werner, Bloom, and Rothmund-Thomson syndromes, are also related to replicative stress.

TGFbeta1 induces a cell-cycle-dependent increase in motility of epithelial cells.

We have previously shown that addition of type 1 transforming growth factor-beta (TGFbeta1) to an exponentially growing population of mink lung CCl64 cells increases their average intermitotic time from 14.4 to 20.3 hours, predominantly by extending G1 from 7.5 to 13.5 hours. Here we have used the DRIMAPS system (digitally recorded interference microscopy with automatic phase-shifting) for obtaining data on cellular mass distribution, cell motility and morphology. We found no significant change in the cells' rate of mass increase following TGFbeta1 treatment, which implies that the treated cells attained a higher mass during their extended cell cycle and this was confirmed by direct measurement of cell size. However, the cells showed a dramatic motile response to treatment: TGFbeta1-treated cells had a significantly higher time-averaged speed of 36.2 microm hour-1 compared to 14.5 microm hour-1 for the control cells. The time course of the response was gradual, reaching a maximum mean speed of 52.6 microm hour-1 after 15 hours exposure. We found that the gradual onset of the response was probably not due to a slow accumulation of a secondary factor but because cells were dividing throughout the experiment and most of the response to TGFbeta1 occurred only after the first cell division in its presence. Thus, taking only those cells that had not yet divided, the time-averaged speed of treated cells (26.1 micrometer hour-1) was only moderately higher than that of untreated cells (14.9 micrometer hour-1) whereas, for those cells that had divided, the difference in speed between treated cells (45.1 micrometer hour-1) and untreated cells (14.1 microm hour-1) was much greater. Increased speed was a consequence of enhanced protrusion and retraction of the cell margin coupled with an increase in cell polarity. TGFbeta1 also increased the mean spreading of the cells, measured as area-to-mass ratio, from 3.2 to 4.4 micrometer2 pg-1, and the intracellular mass distribution became more asymmetric. The observations indicate that a G2 signal may be necessary to reach maximal motility in the presence of TGFbeta1.