Cell Cycle Control Mechanisms Research Articles

SUMO1 modification stabilizes CDK6 protein and drives the cell cycle and glioblastoma progression.

Ubiquitination governs oscillation of cyclin-dependent kinase (CDK) activity through a periodic degradation of cyclins for orderly cell cycle progression; however, the mechanism that maintains the constant CDK protein levels throughout the cell cycle remains unclear. Here we show that CDK6 is modified by small ubiquitin-like modifier-1 (SUMO1) in glioblastoma, and that CDK6 sumoylation stabilizes the protein and drives the cell cycle for the cancer development and progression. CDK6 is also a substrate of ubiquitin; however, CDK6 sumoylation at Lys 216 blocks its ubiquitination at Lys 147 and inhibits the ubiquitin-mediated CDK6 degradation. Throughout the cell cycle, CDK1 phosphorylates the SUMO-specific enzyme, ubiquitin-conjugating enzyme9 (UBC9) that in turn mediates CDK6 sumoylation during mitosis; CDK6 remain sumoylated in G1 phase and drives the cell cycle through G1/S transition. Thus, SUMO1-CDK6 conjugation constitutes a mechanism of cell cycle control and inhibition of this sumoylation pathway may provide a strategy for treatment of glioblastoma.

The plant cell cycle in context.

Plants have a distinctive mode of continuous development, involving the repeated initiation and growth of new organs throughout the lifespan of the organism. Their sessile nature means that this growth must be responsive not only to developmental cues but also to changes in the prevailing conditions. Since almost all growth ultimately derives from the process of cell division, the control of the cell cycle is at the heart of understanding patterning, growth, and development in plants. The unique features of plant development are reflected in distinct modes and molecular mechanisms of cell cycle control, using underpinning conserved control modules in distinct ways. The development of our understanding of these distinct aspects of plant cell cycle control is reviewed here.

Cell cycle regulation during viral infection.

To replicate their genomes in cells and generate new progeny, viruses typically require factors provided by the cells that they have infected. Subversion of the cellular machinery that controls replication of the infected host cell is a common activity of many viruses. Viruses employ different strategies to deregulate cell cycle checkpoint controls and modulate cell proliferation pathways. A number of DNA and RNA viruses encode proteins that target critical cell cycle regulators to achieve cellular conditions that are beneficial for viral replication. Many DNA viruses induce quiescent cells to enter the cell cycle; this is thought to increase pools of deoxynucleotides and thus, facilitate viral replication. In contrast, some viruses can arrest cells in a particular phase of the cell cycle that is favorable for replication of the specific virus. Cell cycle arrest may inhibit early cell death of infected cells, allow the cells to evade immune defenses, or help promote virus assembly. Although beneficial for the viral life cycle, virus-mediated alterations in normal cell cycle control mechanisms could have detrimental effects on cellular physiology and may ultimately contribute to pathologies associated with the viral infection, including cell transformation and cancer progression and maintenance. In this chapter, we summarize various strategies employed by DNA and RNA viruses to modulate the replication cycle of the virus-infected cell. When known, we describe how these virus-associated effects influence replication of the virus and contribute to diseases associated with infection by that specific virus.

DNA topoisomerase II‐dependent control of the cell cycle progression in root meristems of Allium cepa

The catalytic ability of DNA topoisomerases (Topo) to generate short-term DNA breaks allow these enzymes to play crucial functions in managing DNA topology during S-phase replication, transcription, and chromatin-remodelling processes required to achieve commitment for the onset and transition through mitosis. Our experiments on root meristem cells of onion (Allium cepa) were designed to gain insight into the contribution of Topo II to plant-specific progression throughout interphase and mitosis. Irrespective of the position of the cell in interphase, the immunofluorescence of Topo II revealed similar nuclear labelling pattern with well defined signals dispersed in the nucleoplasm and the cortical zone of the nucleolus. Only weak labelling was detected in metaphase and anaphase chromosomes. Experiments with two potent anti-Topo II agents, doxorubicin (DOX, an anthracycline) and a bisdioxopiperazine derivative, ICRF-193, suggest that the inhibition-mediated increase in Topo II immunofluorescence may represent a compensatory mechanism, by which an up-regulated expression of the enzyme tends to counteract the drug-induced loss of indispensable catalytic and relaxation functions. γ-H2AX immunolabelling seems to indicate that both DOX- and ICRF-193-induced alterations in cell cycle progression reflect primarily the activity of the G2/M DNA damage checkpoint. Our findings provide evidence for the plant-specific cell cycle control mechanism induced by Topo II inhibitors under DNA stress conditions.

Loss Of NIPA Leads To Defects In The Repair Of DNA Double Strand Breaks In Mice

Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry.Using a conditional knockout strategy we inactivated the gene encoding Nipa. Nipa-deficient animals are viable, but show a lower birth rate and a reduced body weight. Furthermore, Nipa-deficient males were sterile due to a block of spermatogenesis during meiotic prophase. Virtually no spermatocytes progress beyond a late-zygotene to early-pachytene stage and reach an aberrant stage, with synaptonemal complex disassembly and incomplete synapsis. Nipa-/- females are sub-fertile with an early and severe meiotic defect during embryogenesis with extensive apoptosis in early prophase (E13.5-E14.5).Here we report, that Nipa-/- meiocytes exhibit persistent cytological markers for DNA double strand break repair proteins (like DMC1, RAD51) in meiotic prophase with more than twice as many DMC1 foci as control animals. Kinetic analysis of the first wave of spermatogenesis showed increased DMC1/RAD51 foci in Nipa-/- cells as soon as early-pachynema cells appear (13-14 days post partum). Moreover, we show that Nipa deficiency does not lead to a defect in meiotic sex chromosome inactivation despite epithelial stage IV apoptosis. Nipa-deficient spermatocytes exhibit numerous abnormalities in staining of chromosome axis associated proteins (like SYCP3 and STAG3) indicating that chromosome axis defects were associated with compromised chromosome axis integrity leading to overt chromosome fragmentation.Further in vitro analyses with bleomycin treated MEFs displayed high pH2AX levels in cells lacking NIPA. Repair of DNA DSB seemed to be abolished in these cells as the pH2AX-level were sustained and still visible after 90 min of timecourse, where wildtype cells already repaired sides of DNA Damage. Consistent with these findings NIPA-deficient spleen cells showed compromised DNA Damage repair measured in a comet assay with a significantly longer olive tail moment in NIPA knockout cells under repair conditions.Taken together, the phenotype of Nipa-knockout mice is a definitive proof of the meiotic significance of NIPA and our results show a new, unsuspected role of NIPA in chromosome stability and the repair of DNA double strand breaks. Disclosures:No relevant conflicts of interest to declare.

Dissimilar effects of β-lapachone- and hydroxyurea-induced DNA replication stress in root meristem cells of Allium cepa

Two anticancer drugs, β-lapachone (β-lap, a naphthoquinone) and hydroxyurea (HU, an inhibitor of ribonucleotide reductase), differently affect nuclear morphology and cell cycle control mechanisms in root meristem cells of Allium cepa. The 18 h treatment with 100 μM β-lap results in a lowered number of M-phase cells, increased occurrence of mitotic abnormalities, including over-condensation of chromosomes, their enhanced stickiness, formation of anaphase bridges, micronucleation and reduced mitotic spindles. Following prolonged incubations using high doses of β-lap, cell nuclei reveal dark-red fluorescence evenly distributed in chromatin surrounding the unstained regions of nucleoli. Both drugs generate H2O2 and induce DNA double strand breaks, which is correlated with γ-phoshorylation of H2AX histones. However, the extent of H2AX phosphorylation (including the frequency of γ-H2AX foci and the relative number cells creating phospho-H2AX domains) is considerably reduced in root meristem cells treated jointly with the β-lap/HU mixture. Furthermore, various effects of caffeine (an inhibitor of ATM/ATR cell cycle checkpoint kinases) on β-lap- and HU-induced γ-phoshorylation of H2AX histones and the protective activity of HU against β-lap suggest that their genotoxic activities are largely dissimilar. β-Lap treatment results in the induction of apoptosis-like programmed cell death, while HU treatment leads to cell adaptation to replication stress and promotion of abnormal nuclear divisions with biphasic interphase/mitotic states of chromatin condensation.

Human Papilloma Virus: An Insight in Role in Oral Carcinogenesis

The incidence of head and neck squamous cell carcinoma has been gradually increasing over the last three decades. Recent data have now attributed a viral aetiology to a subset of head and neck cancers. Human papilloma virus-16 genotype is present in many oral and oropharyngeal squamous cell carcinoma. Several studies indicate that oral HPV infection is likely to be sexually acquired. The HPV-associated cytopositive oral and oropharyngeal carcinoma is thought to be initiated and maintained by high-risk HPV E6/E7 oncoprotein-induced dysregulation of cell cycle control mechanisms, leading to genomic instability. Loss of cell cycle and apoptosis control, therefore, constitutes an early and central event in HPV-mediated carcinogenesis. It has an essential aetiologic role in the development of oropharyngeal squamous cell carcinoma in a subset of subjects who are typically younger, are more engaged with high-risk sexual behaviour and were found to have higher human papilloma virus -16 serum antibody titer.

Novel HSP90 Inhibitor NVP-HSP990 Targets Cell-Cycle Regulators to Ablate Olig2-Positive Glioma Tumor–Initiating Cells

Genetic heterogeneity and signaling alterations diminish the effectiveness of single-agent therapies in glioblastoma multiforme (GBM). HSP90 is a molecular chaperone for several signaling proteins that are deregulated in glioma cells. Thus, HSP90 inhibition may offer an approach to coordinately correct multiple signaling pathways as a strategy for GBM therapy. In this study, we evaluated the effects of a novel HSP90 inhibitor, NVP-HSP990, in glioma tumor-initiating cell (GIC) populations, which are strongly implicated in the root pathobiology of GBM. In GIC cultures, NVP-HSP990 elicited a dose-dependent growth inhibition with IC50 values in the low nanomolar range. Two GIC subgroups with different responses were observed with an Olig2-expressing subset relatively more sensitive to treatment. We also showed that Olig2 is a functional marker associated with cell proliferation and response to NVP-HSP990, as NVP-HSP990 attenuated cell proliferation in Olig2-high GIC lines. In addition, NVP-HSP990 disrupted cell-cycle control mechanism by decreasing CDK2 and CDK4 and elevating apoptosis-related molecules. Mechanistic investigations revealed molecular interactions between CDK2/CDK4 and Olig2. Inhibition of CDK2/CDK4 activity disrupted Olig2-CDK2/CDK4 interactions and attenuated Olig2 protein stability. In vivo evaluation showed a relative prolongation of median survival in an intracranial model of GIC growth. Our results suggest that GBM characterized by high-expressing Olig2 GIC may exhibit greater sensitivity to NVP-HSP990 treatment, establishing a foundation for further investigation of the role of HSP90 signaling in GBM.

Clonally Diverse T Cell Homeostasis Is Maintained by a Common Program of Cell-Cycle Control

Lymphopenia induces T cells to undergo cell divisions as part of a homeostatic response mechanism. The clonal response to lymphopenia is extremely diverse, and it is unknown whether this heterogeneity represents distinct mechanisms of cell-cycle control or whether a common mechanism can account for the diversity. We addressed this question by combining in vivo and mathematical modeling of lymphopenia-induced proliferation (LIP) of two distinct T cell clonotypes. OT-I T cells undergo rapid LIP accompanied by differentiation that superficially resembles Ag-induced proliferation, whereas F5 T cells divide slowly and remain naive. Both F5 and OT-I LIP responses were most accurately described by a single stochastic division model where the rate of cell division was exponentially decreased with increasing cell numbers. The model successfully identified key biological parameters of the response and accurately predicted the homeostatic set point of each clone. Significantly, the model was successful in predicting interclonal competition between OT-I and F5 T cells, consistent with competition for the same resource(s) required for homeostatic proliferation. Our results show that diverse and heterogeneous clonal T cell responses can be accounted for by a single common model of homeostasis.

Plasmid-based E6-specific siRNA and co-expression of wild-type p53 suppresses the growth of cervical cancer in vitro and in vivo

The E6 protein of the oncogenic HPV-16 functions by interfering with the normal cell cycle control mechanisms, particularly those controlled by p53. In this study, we developed a dual expression plasmid that coexpressed-E6-specific siRNA and wild type p53, and to evaluate its effects on cervical cancer growth. We found that simultaneous expression of pSi-E6-P53 caused a robust suppression of tumor growth when compared to the controls either E6-specific siRNA or p53 alone. In conclusion, our findings demonstrate that a combined strategy of co-expressed E6-specific siRNA and p53 synergistically and more effectively suppressed cervical tumor growth when compared with single treatment.

Robustness and adaptation reveal plausible cell cycle controlling subnetwork in Saccharomyces cerevisiae

Biological systems are often organized spatially and temporally by multi-scale functional subsystems (modules). A specific subcellular process often corresponds to a subsystem composed of some of these interconnected modules. Accurate identification of system-level modularity organization from the large scale networks can provide valuable information on subsystem models of subcellular processes or physiological phenomena. Computational identification of functional modules from the large scale network is the key approach to solve the complexity of modularity in the past decade, but the overlapping and multi-scale nature of modules often renders unsatisfactory results in these methods. Most current methods for modularity detection are optimization-based and suffered from the drawback of size resolution limit. It is difficult to trace the origin of the unsatisfactory results, which may be due to poor data, inappropriate objective function selection or simply resulted from natural evolution, and hence no system-level accurate modular models for subcellular processes can be offered. Motivated by the idea of evolution with robustness and adaption as guiding principles, we propose a novel approach that can identify significant multi-scale overlapping modules that are sufficiently accurate at the system and subsystem levels, giving biological insights for subcellular processes. The success of our evolution strategy method is demonstrated by applying to the yeast protein-protein interaction network. Functional subsystems of important physiological phenomena can be revealed. In particular, the cell cycle controlling network is selected for detailed discussion. The cell cycle subcellular processes in yeast can be successfully dissected into functional modules of cell cycle control, cell size check point, spindle assembly checkpoint, and DNA damage check point in G2/M and S phases. The interconnections between check points and cell cycle control modules provide clues on the signal stimulus entries of check points into the cell cycle, which are consistent with experimental findings. This evolution strategy method can be applied adequately to extract the plausible yeast cell cycle subnetworks from the whole network. Connections between modules in the obtained cell cycle subnetworks reveal significant cell cycle control mechanisms. This method can also be useful when applied to other biological systems at various temporal and spatial scales for example, the gene transcription networks, and biological systems from mesoscopic scale, e.g cortical network in brain, to subcellular molecular networks.

Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

BackgroundRetroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway.ResultsThe comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1.ConclusionsBoth Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway.

A Mouse Model of Hematopoietic Stem Cell Failure Associated with p53-Loss and Defective Fbw7-Dependent Cyclin E Regulation.

AbstractAbstract 2297Recent findings have challenged the notion that increased proliferation of hematopoietic stem cells (HSCs) necessarily restricts their self-renewal capacity. We have studied the physiologic consequences to HSCs of ablating a key cell cycle regulatory mechanism, Fbw7-dependent cyclin E ubiquitination, using germline knock-in of a cyclin ET74A T393A allele. Fbw7 is a tumor suppressor that regulates the abundance of several oncoprotein substrates by ubiquitin-mediated proteolysis, including cyclin E, Notch, and c-Myc. Cyclin E overexpression in vivo is associated with increased proliferation in some cellular contexts as well as a variety of deleterious consequences, including genomic instability, senescence, or apoptosis. In HSCs, Fbw7-loss has been shown to induce self-renewal and multi-lineage reconstitution defects, and the effect of Fbw7-loss in HSCs has been ascribed to dysregulated Myc and Notch expression. Using the cyclin ET74A T393A mouse model, we tested the hypothesis that impaired Fbw7-mediated regulation of cyclin E, specifically, promotes HSC exhaustion due to loss of self-renewal capacity. We first examined bone marrow HSC counts and their cell cycle kinetics in cyclin E knock-in and wild-type control mice at steady state and following hematologic injury induced by 5-fluorouracil treatment. We found that cyclin E dysregulation reduces numbers of quiescent HSCs and increases cells in S/G2/M-phases, while decreasing total numbers of HSCs, phenotypes made more severe after recovery from hematologic stress. Using bromodeoxyuridine labeling studies, we found that excess cyclin E activity causes DNA hyper-replication in cyclin ET74A T393A HSCs in a cell autonomous manner. By enumerating multi-potent progenitors (MPPs), we ruled out increased rate of transit from HSC-to-MPP as a cause of the apparent exhaustion of cyclin E knock-in HSCs. Thus, dysregulated cyclin E in HSCs promotes both increased proliferation and depletion of the HSC pool. Serial transplantation further revealed peripheral blood reconstitution defects associated with cyclin ET74A T393A HSCs. Recently, we have found that p53 is activated by dysregulated cyclin E in hematopoietic cells in vivo, in association with phosphorylation of both p53 and Chk1 proteins, resembling a DNA damage-type response. Interestingly, p53-loss has been found to be associated with a gain of HSC self-renewal activity. We therefore hypothesized that p53-loss would rescue the self-renewal defect of cyclin E knock-in HSCs. Surprisingly, we discovered that cyclin ET74A T393A; p53-null HSCs showed evidence of significantly worse self-renewal and peripheral reconstitution, compared to p53-null HSCs, defects that are more severe than those associated with impaired Fbw7-mediated cyclin E control in the setting of wild-type p53 (Chi-squared test, p<0.0001). Thus, our data are consistent with the concept that intact p53 function, in the setting of oncogenic insult, can preserve partial HSC self-renewal capacity, and its loss in vivo is detrimental to HSC viability when accompanied by defects in cell cycle control mechanisms. Disclosures:No relevant conflicts of interest to declare.

A Structural Systems Biology Approach for Quantifying the Systemic Consequences of Missense Mutations in Proteins

Gauging the systemic effects of non-synonymous single nucleotide polymorphisms (nsSNPs) is an important topic in the pursuit of personalized medicine. However, it is a non-trivial task to understand how a change at the protein structure level eventually affects a cell's behavior. This is because complex information at both the protein and pathway level has to be integrated. Given that the idea of integrating both protein and pathway dynamics to estimate the systemic impact of missense mutations in proteins remains predominantly unexplored, we investigate the practicality of such an approach by formulating mathematical models and comparing them with experimental data to study missense mutations. We present two case studies: (1) interpreting systemic perturbation for mutations within the cell cycle control mechanisms (G2 to mitosis transition) for yeast; (2) phenotypic classification of neuron-related human diseases associated with mutations within the mitogen-activated protein kinase (MAPK) pathway. We show that the application of simplified mathematical models is feasible for understanding the effects of small sequence changes on cellular behavior. Furthermore, we show that the systemic impact of missense mutations can be effectively quantified as a combination of protein stability change and pathway perturbation.

HPV TESTING IN CERVICAL CANCER PREVENTION

Despite the introduction of HPV vaccines and its clinical application expanded during the last decade with increasing vaccine coverage, cervical cancer prevention still requires continuing and widespread screening activity. Based on the knowledge about the impact of HPV on cell cycle control mechanisms, tests for detection of HPV DNA, host cellular proteins or HPV proteins have been developed and introduced to clinical application. Among them, tests able to detect DNA of high-risk HPVs have been widely used worldwide and start to be evidenced as a valuable screening test in cervical cancer prevention. Hybrid Capture II (HCII, Qiagen, USA) using DNA hybridization and signals amplification technique is the FDA and EC approved test, which can detect and differentiated any type from 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) and 5 low-risk HPV types (6, 11, 42, 43, 44). The introduction of a newly developed HPV test using simpler technology and affordable cost - careHPV - and its commercialization in the very near future, in combination with visual inspection with acetic acid and/or colposcopy will allow the implementation of new approaches with higher coverage and a more effective strategy of screening – diagnosis – management.

The PfNF‐YB transcription factor is a downstream target of melatonin and cAMP signalling in the human malaria parasite Plasmodium falciparum

Plasmodium falciparum causes the most severe form of malaria and is responsible for the majority of deaths worldwide. The mechanism of cell cycle control within intra-erythrocytic stages has been examined as a potential means of a promising way to identifying how to stop parasite development in red blood cells. Our group determined that melatonin increases parasitemia in P.falciparum and P.chabaudi through a complex signalling cascade. In vertebrates, melatonin controls the expression of transcription factors, leading us to postulate rather that the indoleamine would affect PfNF-YB expression in human malaria parasites. We show here that PfNF-YB transcription factor is highly expressed and colocalized in the nucleus in mature parasites during intra-erythrocytic stages, thus suggesting an important role in cell division. Moreover, we demonstrate for the first time that melatonin and cAMP modulate the PfNF-YB transcription factor expression in P.falciparum at erythrocytic stages. In addition, PfNF-YB is found to be more ubiquitinated in the presence of melatonin. Finally, the proteasome inhibitor bortezomib is able to modulate PfNF-YB expression as well. Taken together, our dada reinforce the role played by melatonin in the cell cycle control of P.falciparum and point this indolamine as a target to develop new antimalarial drugs.

Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells

Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs.

Elevated Cyclin G2 Expression Intersects with DNA Damage Checkpoint Signaling and Is Required for a Potent G2/M Checkpoint Arrest Response to Doxorubicin

To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.

Non-Hodgkin Lymphoma in Early Life

The non-Hodgkin lymphomas (NHLs) are a heterogeneous group of B-cell and T-cell neoplasms that vary by morphological appearance, etiology, biology, and clinical manifestations. Worldwide, the incidence of NHL began to increase circa 1950 (1,2), approximately doubling in the United States between 1975 and 2000 (3,4), and then stabilizing with an age-standardized rate near 20 per 100 000 person-years (3). The age-standardized incidence rate, however, is an age-specific weighted average that may mask important age-related trends (5,6), especially among younger persons. Additionally, whereas recent trends for NHL overall may be related to HIV/AIDS and changes in classification systems or diagnostic practice patterns (2), perinatal and family risk factors also may be important for NHL in early life. Therefore, in this issue of the Journal, Crump et al. (7) conducted a national cohort study in Sweden among young persons aged 0–37 years, who were newly diagnosed with NHL between 1973 and 2008 and followed through 2009. The authors linked Swedish birth and cancer registries data for NHL, family characteristics, and the perinatal period (variously defined as the days immediately before and after birth). Linkage to the national birth and cancer registries was nearly 100% complete. More than 99% of the original cohort remained after exclusions for missing data regarding gestational age at birth and/or birth weight. There were 936 NHL patients with 66.3 million person-years of follow-up. The overall incidence rate was 1.4 per 100 000 person-years (1.7 for men and boys and 1.1 for women and girls). Age-specific incidence rates rose with advancing age at diagnosis, like rates of epithelial carcinomas (8). For a similar period, incidence rate patterns were slightly higher among young persons in the United States than in Sweden (Figure 1), using the National Cancer Institute's Surveillance, Epidemiology, and End Results 9 Registries Database (SEER, 1973–2008) (9). There were 15 089 NHL patients with 527 million person-years of follow-up among persons of the youngest nine of 19 SEER age groups (ie, ages 00, 01–04, 05–09, … 35–39 years). The age-standardized incidence rate (adjusted to the World Standard Million population) for these nine age intervals combined was 2.37 per 100 000 person-years (3.15 for men and boys and 1.58 for women and girls) (Figure 1, A). The estimated annual percentage change for the age-standardized rate was 1.26% and 1.91% among men and boys and women and girls, respectively. Age-specific rates rose continuously with advancing aging (Figure 1, B), as in Sweden. Figure 1 Age-standardized incidence rates and age-specific incidence rates, by sex, for non-Hodgkin lymphoma among Americans aged 0–39 years. Incidence rates and 95% confidence intervals (shaded areas) are based on November 2010 data from the National ... In the Swedish study (7), relative risks of non-Hodgkin lymphoma were presented as hazard ratios (HRs) unadjusted and adjusted for sex, birth year, fetal growth, gestational age at birth, twinning, birth order, maternal age at delivery, maternal and paternal education level, and family history of NHL. Independent risk factors were family history of NHL, high fetal growth, older maternal age, low birth order, and male sex. Based on small numbers, the strongest risk factor was for family history of NHL in either a sibling (HR = 9.84) or parent (HR = 2.36) with a greater risk for a same-sex sibling or parent. High fetal growth weight that was two standard deviations or more from the reference birth weight, by gestational age and sex, was associated with increased risk of NHL (HR = 1.64). Maternal age was an important confounder for low birth order with an inverse relationship between NHL and birth order after adjusting for maternal age. Male sex was an age-specific effect modifier, with a twofold increase in NHL among boys who were aged younger than 15 years but not older. In the United States, there was also a relative excess of NHL among younger men compared with women (Figure 1, B) with a male to female incidence rate ratio of 2.7 (95% CI = 1.9 to 3.7) between ages 5–9 years and 2.3 (95% CI = 1.7 to 3.0) between ages 10–14 years. When incidence of NHL subtypes was examined (7), family history and older maternal age were associated with increased risk for diffuse B-cell subtypes. Family history was not estimable for the other subtypes. Older maternal age was associated with a borderline increased risk for T-cell tumors. A reciprocal birth cohort effect was observed with decreasing risk by year of birth for diffuse B-cell and increasing risk for other B-cell and T-cell tumors. Although it is important to determine whether the etiology of NHL differs by subtype, subtype trends in this study are somewhat uncertain because of small sample sizes and missing data (35% missing subtype, 332 of 936 subjects). Additionally, although the distinction between B-cell and T-cell tumors is generally straightforward, the categorization for B-cell and T-cell subtypes has evolved over time, raising concern for calendar period effects due to changing classification systems or improved diagnostic techniques. The authors attempted to rule out calendar period effects and to confirm birth cohort or exposure effects by accommodating different patterns of missingness. First, they distributed the subjects with missing subtype data in the observed proportions of known subjects with diffuse B-cell tumors (50%), other B-cell tumors (40%), and T-cell tumors (10%) and then recalculated the subtype trends. Second, missing subtype data were redistributed by the observed proportions of known subjects for each year of birth, age at diagnosis, and sex. Birth cohort effects persisted and remained statistically significant in these sensitivity analyses. Alternatively, population-based birth cohort effects could have been assessed with age–period–cohort models (10). Notwithstanding the well-documented identifiability issues for the age–period–cohort framework (11), cohort deviations or changes are estimable functions that have the added value of being fully adjusted for age and period effects. In the United States SEER population (Figure 2), we could show statistically significant decelerating (concave parabola) cohort deviations or changes for diffuse B-cell tumors (P < .001) and accelerating (convex parabola) deviations for T-cell tumors (P < .001) but not for other B-cell tumors (P = .28). Figure 2 Birth cohort deviations in US incidence of non-Hodgkin lymphoma (NHL) by histopathologic subtype. The National Cancer Institute's Surveillance, Epidemiology, and End Results 9 Registries Database (SEER 9, 1973–2008) was used to determine age–period–cohort ... In conclusion, the study by Crump et al. (7) is a national cohort study of perinatal and family characteristics associated with the diagnosis of NHL among children and young adults in Sweden. Limitations included the unavailability of many host and environmental risk factors for NHL such as infectious exposures, radiation, environmental contaminants, and primary or inherited immune deficiencies. Nonetheless, their large-scale and population-based design minimized selection bias and maximized generalizable conclusions for associations between NHL in early life and family history, high fetal growth weight, older maternal age, low birth order, and male sex. However, risk estimates for family history were based on small numbers of case patients with an affected sibling (n = 4) or parent (n = 10). Pooled studies with larger sample sizes will be required to further assess the potential role of gene–environmental exposures for family history of NHL. In contrast to Crump et al. (7), a large meta-analysis showed no statistically significant association between high or low birth weight and NHL (12), and the authors concluded that birth weight might not be a good metric for an association between fetal growth and lymphoma (12). Advancing maternal age appears to be a risk factor for many childhood cancers possibly due to differential gene expression in cell cycle control and DNA damage repair mechanisms among older women, age-related mutations in oocyte genes, increasing use of fertility technology by older mothers, or changes in maternal hormonal exposures with advancing age (13). The association of low birth order after adjustment for maternal age is consistent with the hypothesis of “reduced or delayed exposure” to first viral or bacterial infection in infancy due to the absence of other siblings (14); however, others have found little support for an association between NHL and birth order or family size (15,16). Finally, though male predominance in the incidence of cancer is well established (17), the age interaction or effect modification by sex before age 15 years is not easily explained.

Mitotic exit kinase Dbf2 directly phosphorylates chitin synthase Chs2 to regulate cytokinesis in budding yeast

How cell cycle machinery regulates extracellular matrix (ECM) remodeling during cytokinesis remains poorly understood. In the budding yeast Saccharomyces cerevisiae, the primary septum (PS), a functional equivalent of animal ECM, is synthesized during cytokinesis by the chitin synthase Chs2. Here, we report that Dbf2, a conserved mitotic exit kinase, localizes to the division site after Chs2 and directly phosphorylates Chs2 on several residues, including Ser-217. Both phosphodeficient (chs2-S217A) and phosphomimic (chs2-S217D) mutations cause defects in cytokinesis, suggesting that dynamic phosphorylation-dephosphorylation of Ser-217 is critical for Chs2 function. It is striking that Chs2-S217A constricts asymmetrically with the actomyosin ring (AMR), whereas Chs2-S217D displays little or no constriction and remains highly mobile at the division site. These data suggest that Chs2 phosphorylation by Dbf2 triggers its dissociation from the AMR during the late stage of cytokinesis. Of interest, both chs2-S217A and chs2-S217D mutants are robustly suppressed by increased dosage of Cyk3, a cytokinesis protein that displays Dbf2-dependent localization and also stimulates Chs2-mediated chitin synthesis. Thus Dbf2 regulates PS formation through at least two independent pathways: direct phosphorylation and Cyk3-mediated activation of Chs2. Our study establishes a mechanism for direct cell cycle control of ECM remodeling during cytokinesis.