S-phase Nuclei Research Articles

Visualizing Active Replication Regions in S-Phase Chromosomes.

A basic question of cell biology is how DNA folds to chromosome. A number of recently accumulated evidences have suggested that folding of chromosome proceeds tightly coupled with DNA replication progresses. Drug-induced PCC is a useful tool for visualization of the interphase nuclei, in particular, S-phase, as S-phase prematurely condensed chromosomes (S-phase PCC). Active replicating DNA is labeled directly with Cy3-dUTP by bead loading method, and then S-phase nuclei is immediately condensed prematurely by calyculin A to obtain S-phase PCC. Active replicating regions on S-PCC are observed under a scanning confocal microscope. Cy3-dUTP-labeled S-phase PCCs clearly reveal the drastic transitional change of chromosome formation through S-phase, starting from a "cloudy nebula" to numerous numbers of "beads on a string" and finally to "striped arrays of banding structured chromosome" known as G- or R-banding pattern. The number, distribution, and shape of replication foci were also measured in individual subphase of S-phase; maximally ~1400 foci of 0.35 μm average radius size were scored at the beginning of S-phase, and the number is reduced to ~100 at the end of S-phase. Drug-induced PCC clearly provided the new insight that eukaryote DNA replication is tightly coupled with the chromosome condensation/compaction for construction of eukaryote higher-ordered chromosome structure.

Planarian stem cells specify fate yet retain potency during the cell cycle.

Planarian whole-body regeneration is enabled by stem cells called neoblasts. At least some neoblasts are individually pluripotent. Neoblasts are also heterogeneous, with subpopulations of specialized neoblasts having different specified fates. Fate specification in neoblasts is regulated by fate-specific transcription factor (FSTF) expression. Here, we find that FSTF expression is common in neoblast S/G2/M cell-cycle phases but less common in G1. We find that specialized neoblasts can divide to produce progeny with asymmetric cell fates, suggesting that they could retain pluripotency. Furthermore, no known neoblast class was present in all neoblast colonies, suggesting that pluripotency is not the exclusive property of any known class. We tested this possibility with single-cell transplantations, which indicate that at least some specialized neoblasts are likely clonogenic. On the basis of these findings, we propose a model for neoblast pluripotency in which neoblasts can undergo specialization during the cell cycle without loss of potency.

Whole-Mount Immunostaining for the Identification of Histone Modifications in the S-Phase Nuclei of Arabidopsis Roots.

This chapter describes a method used to analyze the behavior of histone modifications in S phase in Arabidopsis using a whole-mount immunostaining technique. Previous studies have demonstrated that dramatic changes in local chromatin structure are required for the initiation and progression of DNA replication, and that histone modifications play an essential role in the determination of chromatin structure in S phase. Since euchromatic and heterochromatic regions are replicated in distinct S-phase stages, it is important to identify histone modifications at each stage. Here, we introduce a protocol for whole-mount immunostaining combined with 5-ethynyl-2'-deoxyuridine (EdU) staining, which enables the visualization of spatial patterns in histone modifications in the early and late S-phase nuclei of Arabidopsis roots.

The Hedgehog receptor Patched1 regulates proliferation, neurogenesis, and axon guidance in the embryonic spinal cord

The formation of the vertebrate nervous system depends on the complex interplay of morphogen signaling pathways and cell cycle progression to establish distinct cell fates. The Sonic hedgehog (Shh) signaling pathway is well understood to promote ventral cell fates in the developing spinal cord. A key regulator of Shh signaling is its receptor Patched1 (Ptch1). However, because the Ptch1 null mutation is lethal early in mouse embryogenesis, its role in controlling cell cycle progression, neurogenesis, and axon guidance in the developing spinal cord is not fully understood. An allele of Ptch1 called Wiggable (Ptch1Wig), which was previously shown to enhance Shh signaling, was used to test its ability to regulate neurogenesis and proliferation in the developing spinal cord. Ptch1Wig/Wig mutants displayed enhanced ventral proneural gene activation, and aberrant proliferation of the neural tube and floor plate cells, the latter normally being a quiescent population. The expression of the cell cycle regulators p27Kip1 and p57Kip2 were expanded in Ptch1Wig/Wig mutant spinal cords, as was the number of mitotic and S-phase nuclei, suggesting enhanced cell cycle progression. However, Ptch1Wig/Wig mutants also showed enhanced apoptosis in the ventral embryonic spinal cord, which resulted in thinner spinal cords at later embryonic stages. Commissural axons largely failed to cross the floor plate of Ptch1Wig/Wig mutant embryos, suggesting enhanced Shh signaling in these mutants led to a dorsal expansion of the chemoattraction front. These findings are consistent with a role of Ptch1 in regulating neurogenesis and proliferation of neural progenitors, and in restricting the influence of Shh signaling in commissural axon guidance to the floor plate.

Visualizing the dynamics of histone variants in the S-phase nucleus

Histone variants constitute a fundamental feature of the epigenome. However, their dynamics during normal and challenged DNA replication and their distribution in the three-dimensional space of the nucleus remain poorly characterized. A recent study employed stochastic optical reconstruction microscopy (STORM) to obtain a high-resolution view of the spatial distribution of H3 histone variants in the nucleus and related this to the timing of DNA replication.

Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans

BackgroundThe proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E.ResultsTo study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of-function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development.ConclusionsIn the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor cells is distinct from somatic cells, as it does not heavily rely on cyclic accumulation of classic cell cycle proteins.

EGFR Mutations Compromise Hypoxia-Associated Radiation Resistance through Impaired Replication Fork-Associated DNA Damage Repair.

EGFR signaling has been implicated in hypoxia-associated resistance to radiation or chemotherapy. Non-small cell lung carcinomas (NSCLC) with activating L858R or ΔE746-E750 EGFR mutations exhibit elevated EGFR activity and downstream signaling. Here, relative to wild-type (WT) EGFR, mutant (MT) EGFR expression significantly increases radiosensitivity in hypoxic cells. Gene expression profiling in human bronchial epithelial cells (HBEC) revealed that MT-EGFR expression elevated transcripts related to cell cycle and replication in aerobic and hypoxic conditions and downregulated RAD50, a critical component of nonhomologous end joining and homologous recombination DNA repair pathways. NSCLCs and HBEC with MT-EGFR revealed elevated basal and hypoxia-induced γ-H2AX-associated DNA lesions that were coincident with replication protein A in the S-phase nuclei. DNA fiber analysis showed that, relative to WT-EGFR, MT-EGFR NSCLCs harbored significantly higher levels of stalled replication forks and decreased fork velocities in aerobic and hypoxic conditions. EGFR blockade by cetuximab significantly increased radiosensitivity in hypoxic cells, recapitulating MT-EGFR expression and closely resembling synthetic lethality of PARP inhibition.Implications: This study demonstrates that within an altered DNA damage response of hypoxic NSCLC cells, mutant EGFR expression, or EGFR blockade by cetuximab exerts a synthetic lethality effect and significantly compromises radiation resistance in hypoxic tumor cells. Mol Cancer Res; 15(11); 1503-16. ©2017 AACR.

A flow cytometric method for estimating S-phase duration in plants

The duration of the DNA synthesis stage (S phase) of the cell cycle is fundamental in our understanding of cell cycle kinetics, cell proliferation, and DNA replication timing programs. Most S-phase duration estimates that exist for plants are based on indirect measurements. We present a method for directly estimating S-phase duration by pulse-labeling root tips or actively dividing suspension cells with the halogenated thymidine analog 5-ethynl-2'-deoxyuridine (EdU) and analyzing the time course of replication with bivariate flow cytometry. The transition between G1 and G2 DNA contents can be followed by measuring the mean DNA content of EdU-labeled S-phase nuclei as a function of time after the labeling pulse. We applied this technique to intact root tips of maize (Zea mays L.), rice (Oryza sativa L.), barley (Hordeum vulgare L.), and wheat (Triticum aestivum L.), and to actively dividing cell cultures of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) and rice. Estimates of S-phase duration in root tips were remarkably consistent, varying only by ~3-fold, although the genome sizes of the species analyzed varied >40-fold.

Sendai Virus Induces Persistent Olfactory Dysfunction in a Murine Model of PVOD via Effects on Apoptosis, Cell Proliferation, and Response to Odorants.

BackgroundViral infection is a common cause of olfactory dysfunction. The complexities of studying post-viral olfactory loss in humans have impaired further progress in understanding the underlying mechanism. Recently, evidence from clinical studies has implicated Parainfluenza virus 3 as a causal agent. An animal model of post viral olfactory disorders (PVOD) would allow better understanding of disease pathogenesis and represent a major advance in the field.ObjectiveTo develop a mouse model of PVOD by evaluating the effects of Sendai virus (SeV), the murine counterpart of Parainfluenza virus, on olfactory function and regenerative ability of the olfactory epithelium.MethodsC57BL/6 mice (6–8 months old) were inoculated intranasally with SeV or ultraviolet (UV)-inactivated virus (UV-SeV). On days 3, 10, 15, 30 and 60 post-infection, olfactory epithelium was harvested and analyzed by histopathology and immunohistochemical detection of S-phase nuclei. We also measured apoptosis by TUNEL assay and viral load by real-time PCR. The buried food test (BFT) was used to measure olfactory function of mice at day 60. In parallel, cultured murine olfactory sensory neurons (OSNs) infected with SeV or UV-SeV were tested for odorant-mixture response by measuring changes in intracellular calcium concentrations indicated by fura-4 AM assay.ResultsMice infected with SeV suffered from olfactory dysfunction, peaking on day 15, with no loss observed with UV-SeV. At 60 days, four out of 12 mice infected with SeV still had not recovered, with continued normal function in controls. Viral copies of SeV persisted in both the olfactory epithelium (OE) and the olfactory bulb (OB) for at least 60 days. At day 10 and after, both unit length labeling index (ULLI) of apoptosis and ULLI of proliferation in the SeV group was markedly less than the UV-SeV group. In primary cultured OSNs infected by SeV, the percentage of cells responding to mixed odors was markedly lower in the SeV group compared to UV-SeV (P = 0.007).ConclusionWe demonstrate that SeV impairs olfaction, persists in OE and OB tissue, reduces their regenerative ability, and impairs the normal physiological function of OSNs without gross cytopathology. This mouse model shares key features of human post-viral olfactory loss, supporting its future use in studies of PVOD. Further testing and development of this model should allow us to clarify the pathophysiology of PVOD.

The Enigma of Progressively Partial Endoreplication: New Insights Provided by Flow Cytometry and Next-Generation Sequencing.

In many plant species, somatic cell differentiation is accompanied by endoreduplication, a process during which cells undergo one or more rounds of DNA replication cycles in the absence of mitosis, resulting in nuclei with multiples of 2C DNA amounts (4C, 8C, 16C, etc.). In some orchids, a disproportionate increase in nuclear DNA contents has been observed, where successive endoreduplication cycles result in DNA amounts 2C + P, 2C + 3P, 2C + 7P, etc., where P is the DNA content of the replicated part of the 2C nuclear genome. This unique phenomenon was termed “progressively partial endoreplication” (PPE). We investigated processes behind the PPE in Ludisia discolor using flow cytometry (FCM) and Illumina sequencing. In particular, we wanted to determine whether chromatin elimination or incomplete genome duplication was involved, and to identify types of DNA sequences that were affected. Cell cycle analysis of root tip cell nuclei pulse-labeled with EdU revealed two cell cycles, one ending above the population of nuclei with 2C + P content, and the other with a typical “horseshoe” pattern of S-phase nuclei ranging from 2C to 4C DNA contents. The process leading to nuclei with 2C + P amounts therefore involves incomplete genome replication. Subsequent Illumina sequencing of flow-sorted 2C and 2C + P nuclei showed that all types of repetitive DNA sequences were affected during PPE; a complete elimination of any specific type of repetitive DNA was not observed. We hypothesize that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development.

Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

Cytophysiological characteristics of Arabidopsis thaliana cultivated cells with disable perception of ethylene signal by the ETR1 receptor

Contradictory data about ethylene influence on cell growth and division prompted us to investigate cytophysiological characteristics of suspension cultures of Arabidopsis thaliana of wild type Col-0 and ert1-1 mutant carrying a point mutation in the site of ethylene binding by the ETR1 receptor. Some cytophysiological characteristics of the etr1-1 cultivated cells differed from those of Col-0: the growth rate of mutant cells was less and cell sizes were smaller, the culture was committed to the formation of tracheary elements (TE), had a pronounced modal class of nuclei (54%) with the amount of DNA 8C and a tendency to expand the ploidy toward 32C. Despite the absence of ethylene perception by the ETR1 receptor, the cell culture of mutant responded to treatment with ethylene by growth acceleration, an increase in cell viability and in the number of cells in the S-phase of the cell cycle. The inhibitor of ethylene binding to receptors, 1-methylcyclopropene, suppressed growth and viability of the cells of both genotypes. In the etr1-1 cell culture, the inhibitor reduced the number of S-phase nuclei and activated TE formation. All data obtained indicate that ethylene perception and transduction of ethylene signal are required for the maintenance of cell viability and active in vitro growth. It is supposed that the functional activity of the ETR1 receptor is necessary for optimal cell expansion, whereas other receptors are responsible for cell proliferation.

Nuclear MMP‐9 role in the regulation of rat skeletal myoblasts proliferation

Matrix metalloproteinases (MMPs) are the key enzymes responsible for the remodelling of extracellular matrix. Two of them, namely MMP-2 and MMP-9 (gelatinases A and B, respectively), are expressed in skeletal muscles and are involved in their regeneration after the injury. Although MMPs are primarily known to act extracellularly, recent studies have shown that some of them are also found within the cell. In this study, we examine intracellular localisation of gelatinases during myoblasts differentiation in vitro, focussing the impact of MMPs inhibition on the myoblasts proliferation and function. We show that MMP-9 localises within the S-phase nuclei of in vitro differentiating myoblasts. The inhibition of MMPs activity achieved by either doxycycline (a non-competitive inhibitor of collagenases), TIMP-1 (tissue inhibitor of metalloproteinases 1) or neutralising anti-MMP-9 antibody affects nuclear localisation of this gelatinase, and impacts at myoblasts proliferation. During myoblasts differentiation, MMP-9 that is localised in nuclei might be involved in the processes regulating cell cycle progression.

Sorafenib prolongs liver regeneration after hepatic resection in rats

BackgroundThe multikinase inhibitor sorafenib inhibits angiogenesis and tumor cell proliferation. Sorafenib targets signaling pathways involved in liver regeneration. Previous works on regenerating mouse liver show differing results. We asked to which degree different lengths of sorafenib treatment would influence liver regeneration after hepatic resection in rats. MethodsFischer-344 rats received intragastric injections of sorafenib (5–15 mg/kg/d), underwent a two-thirds partial hepatectomy (PH), and were sacrificed at different time points thereafter. Sorafenib treatment was stopped 0, 3, or 14 d after PH. Serum levels of aminotransferases and labeling indices of S-phase nuclei (bromodeoxyuridine and MIB-5) were analyzed, body and liver weights measured, and levels of phospho-ERK determined by Western blot. ResultsSorafenib increased aminotransferases and the number of S-phase nuclei at baseline, but decreased liver weights and levels of phospho-ERK 24 h after PH. The number of S-phase nuclei and mitotic indices decreased 48 h after PH and increased 7 d after PH in animals on sorafenib treatment. Relative liver weights were restored 5 d after PH in control rats, at 7 d in animals receiving sorafenib prior to surgery, at 10 d in rats where sorafenib was stopped 3 d after surgery, and after 14 d in rats on continuous treatment. ConclusionsIn this rat model, the regenerating liver adapted to the proliferation-inhibitory effect of sorafenib during continuous treatment. Sorafenib given after hepatic resection did not completely inhibit liver regeneration, but it prolonged the regenerative phase in proportion to the length of treatment.

Bone marrow mesenchymal stem cell from chronic hepatitis B patients differentiation into hepatocyte-like cells

The aim of this study was to observe the biological characteristics and hepatic differentiation potential of bone marrow mesenchymal stem cells (BM-MSCs) from chronic hepatitis B patients in vitro. MSCs were isolated from bone marrow of 15 chronic hepatitis B patients and 11 normal donors by density gradient centrifugation. The adipogenic and osteogenic differentiation potential, immunophenotyping and DNA contents were detected. Chronic hepatitis B patients BM-MSCs were induced to hepatocytes. Hepatic markers were detected by immunohistochemical analysis. Periodic acideschiff staining was used to detect for glycogen production. The results showed that both MSCs had adipogenic and osteogenic differentiation potential, nearly all the BM-MSCs expressed CD105, CD166, CD44 and CD29. Compared with normal donors, the primary passage time of BM-MSCs from chronic hepatitis B patients were much longer, and the S-phase nuclei were lower. Chronic hepatitis B patients BM-MSCs were induced to hepatocyte-like cell, which express the hepatic-specific markers and have the glycogen production function. Though it was difficult to cultivate BM-MSCs from chronic hepatitis patients in vitro and they had deficient proliferation, they could differentiate into functional hepatocyte-like cells. Key words: Hepatitis B, mesenchymal stem cell, bone marrow, hepatocyte-like cell, glycogen.

Iron-regulatory gene expression during liver regeneration

Background. In rat, the first 18–24 h after partial hepatectomy (PH) are characterized by an acute-phase reaction, after which liver regeneration predominates. Interleukin-6 (IL-6) induces the iron hormone hepcidin, which blocks iron uptake and may compromise iron uptake in the growing liver. The expressions of hepcidin and the iron-regulatory pathway of hepcidin gene expression during the late phase of liver regeneration are unknown. Aim. To characterize the expression pattern of hepcidin and the iron-sensing pathway of hepcidin regulation during liver regeneration. Methods. Rats were subjected to PH or sham operation. Liver weights, number of S-phase nuclei, and serum levels of iron and IL-6 were determined. Messenger-RNA levels of hepcidin, ferritin, hemojuvelin, transferrin receptor 1 and 2, HFE, divalent metal transporter 1, ferroportin, and ceruloplasmin were determined with qPCR at different time points. Protein levels of STAT3 and SMAD4 were determined with western blot. Results. During the acute-phase response, IL-6 release induced STAT3 protein and hepcidin mRNA, whereas mRNA levels of proteins in the iron-sensing pathway (HFE, hemojuvelin, and transferrin receptor 2) decreased. The mRNA levels of proteins involved in cellular iron uptake were increased and cellular iron export unchanged. During liver regeneration >24 h after PH, gene expressions in the iron-sensing pathway were continuously suppressed and hepcidin mRNA levels declined 3–7 days after surgery. Conclusions. Hepcidin gene expression peaks during the acute-phase response, but a sustained down-regulation of the iron-sensing pathway of hepcidin regulation gradually reduces hepcidin gene expression until regeneration is complete, thereby promoting iron mobilization to the regenerating liver.

Trypanosomal histone γH2A and the DNA damage response

DNA damage and repair in trypanosomatids impacts virulence, drug resistance and antigenic variation but, currently, little is known about DNA damage responses or cell cycle checkpoints in these divergent protozoa. One of the earliest markers of DNA damage in eukaryotes is γH2A(X), a serine phosphorylated histone H2A (variant). Here, we report the identification and initial characterization of γH2A in Trypanosoma brucei. We identified Thr130 within the replication-dependent histone H2A as a candidate phosphorylation site and found that the abundance of this trypanosomal γH2A increased in vivo in response to DNA damage. Nuclear γH2A foci mark the sites of putative natural replication fork stalling, sites of meganuclease-induced DNA double strand breaks and sites of methyl methanesulphonate-induced DNA damage. Naturally occurring and meganuclease-induced γH2A and RAD51 double-positive repair foci are typically found in S-phase or G2 nuclei. The results link trypanosomal γH2A, with an unusual histone modification motif, to DNA damage sensing and mitotic checkpoint signaling.

Genotoxicity screening via the γH2AX by flow assay

The measurement of serine139-phosphorylated histone H2AX (γH2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for γH2AX detection (hereafter termed the γH2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in γH2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold γH2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the γH2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). γH2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that γH2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for “biologically-irrelevant” positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on γH2AX were atypical and limited to a small sub-population of S-phase nuclei. Nevertheless, the γH2AX by flow assay represents a novel genotoxicity assay with the potential to flag both pro- and proximate genotoxins.

Purinergic Signals Regulate Daily S-Phase Cell Activity in the Ciliary Marginal Zone of the Zebrafish Retina

Regeneration and growth that occur in the adult teleost retina have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. Here, it is reported that S-phase cell number, in the ciliary marginal zone (CMZ) of the adult zebrafish retina, exhibits day-night variations with a mid-light phase peak. Oscillations persist for 24 h in constant darkness (DD), suggesting control by a circadian component. However, variations in the S-phase nuclei number were rapidly dampened and not present during and after a second day in DD. An ADPβS treatment significantly enhanced S-phase activity at night to mid-light levels, as assessed by in vivo BrdU incorporation in a 2-h interval. Moreover, daylight increase in S-phase cell number was completely abolished when extracellular nucleotide levels or their extracellular hydrolysis by ectonucleoside triphosphate diphosphohydrolases (NTPDases) were significantly disrupted or when a selective antagonist of purinergic P2Y1 receptors was intraocularly injected before BrdU exposure. Extracellular nucleotides and NTPDase action were also important for maintaining nocturnal low levels of S-phase activity in the CMZ. Finally, we showed that mRNAs of NTPDases 1, 2 (3 isoforms), and 3 as well as of P2Y1 receptor are present in the neural retina of zebrafish. NTPDase mRNA expression exhibited a 2-fold increment in light versus dark conditions as assessed by quantitative RT-PCR, whereas P2Y1 receptor mRNA levels did not show significant day-night variations. This study demonstrates a key role for nucleotides, principally ADP as a paracrine signal, as well as for NTPDases, the plasma membrane-bound enzymes that control extracellular nucleotide concentration, for inducing S-phase cell entry in the CMZ-normally associated with retinal growth-throughout the light-dark cycle.

Cdc45 Limits Replicon Usage from a Low Density of preRCs in Mammalian Cells

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase.