Eimeria tenella ROP kinase EtROP1 induces G0/G1 cell cycle arrest and inhibits host cell apoptosis

Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the aetiologic agent of caecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterised. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N‐terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co‐immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterisation confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its noncanonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.     

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G₀/G₁ phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G₀ phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G₁ and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21^Cip1, p27^Kip1, and p16^INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G₁ cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G₁ cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G₀/G₁ phase.     

Valeric acid induces cell cycle arrest at G1 phase in CHO cell cultures and improves recombinant antibody productivity

To find a more effective chemical reagent for improved monoclonal antibody (mAb) production, eight chemical reagents (curcumin, quercein, DL‐sulforaphane, thymidine, valeric acid, phenyl butyrate, valproic acid, and lithium chloride) known to induce cell cycle arrest were examined individually as chemical additives to recombinant CHO (rCHO) cell cultures producing mAb. Among these chemical additives, valeric acid showed the best production performance. Valeric acid decreased specific growth rate (μ), but increased culture longevity and specific mAb productivity (q_mAb) in a dose‐dependent manner. The beneficial effect of valeric acid on culture longevity and q_mAb outweighed its detrimental effect on μ, resulting in 2.9‐fold increase in the maximum mAb concentration when 1.5 mM valeric acid was added to the cultures. Furthermore, valeric acid did not negatively affect the mAb quality attributes with regard to aggregation, charge variation, and galactosylation. Unexpectedly, galactosylation of the mAb increased by the 1.5 mM valeric acid addition. Taken together, the results obtained here demonstrate that valeric acid is an effective chemical reagent to increase mAb production in rCHO cells.     

RNA interference-mediated silencing of iASPP induces cell proliferation inhibition and G0/G1 cell cycle arrest in U251 human glioblastoma cells

iASPP is an evolutionally conserved inhibitory member of the ASPP (apoptosis-stimulating protein of p53) protein family. Overexpression of iASPP was observed in several types of human tumors, however, its role in tumorigenesis has not been fully clarified. To investigate the role of iASPP in human glioblastoma multiforme (GMB) progression, the authors employed lentivirus-mediated shRNA to silence endogenous iASPP expression and elucidated iASPP function by analysis of viability, colony formation, DNA synthesis, and cell cycle in p53-mutant glioblastoma cell line U251. iASPP was significantly and sustainably knocked down by iASPP-specific shRNA in U251 cells. Stable down-regulation of iASPP expression-induced cell proliferation inhibition and G0/G1 cell cycle arrest by down-regulation of cyclin D1 and up-regulation of p21(Waf1/Cip1). Thus, the findings not only provide a molecular basis for the role of iASPP in cell cycle progression of glioblastoma cells but also suggest a novel therapeutic target for the treatment of GBM.     

Inhibition of cell proliferation by mechanical agitation involves transient cell cycle arrest at G1 phase in dinoflagellates

Summary.  Cell proliferation of dinoflagellates is negatively affected by mechanical agitation and red tides caused by members of the group have been correlated with periods of calm sea conditions. The mechanism involved in the mechanically transduced inhibition of cell proliferation is thought to involve the disruption of the cell division apparatus. In this study, we used highly synchronized cells and flow cytometry to study the effects of mechanical agitation on cell cycle progression. We observed that mechanical agitation induced transient cell cycle arrest at G₁ phase, in both the heterotrophic dinoflagellate Crypthecodinium cohnii and the photosynthetic dinoflagellate Heteroscapsa triquetra. Received March 12, 2002; accepted July 20, 2002; published online November 29, 2002     

Escherichia coli cyclomodulin Cif induces G2 arrest of the host cell cycle without activation of the DNA-damage checkpoint-signalling pathway

The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins and effector proteins, the cyclomodulins, that deregulate the host cell cycle. Upon injection into HeLa cells by the enteropathogenic Escherichia coli (EPEC) type III secretion system, Cif induces a cytopathic effect characterized by the recruitment of focal adhesion plates and the formation of stress fibres, an irreversible cell cycle arrest at the G(2)/M transition, and sustained inhibitory phosphorylation of mitosis inducer, CDK1. Here, we report that the reference typical EPEC strain B171 produces a functional Cif and that lipid-mediated delivery of purified Cif into HeLa cells induces cell cycle arrest and actin stress fibres, implying that Cif is necessary and sufficient for these effects. EPEC infection of intestinal epithelial cells (Caco-2, IEC-6) also induces cell cycle arrest and CDK1 inhibition. The effect of Cif is strikingly similar to that of cytolethal distending toxin (CDT), which inhibits the G(2)/M transition by activating the DNA-damage checkpoint pathway. However, in contrast to CDT, Cif does not cause phosphorylation of histone H2AX, which is associated with DNA double-stranded breaks. Following EPEC infection, the checkpoint effectors ATM/ATR, Chk1 and Chk2 are not activated, the levels of the CDK-activating phosphatases Cdc25B and Cdc25C are not affected, and Cdc25C is not sequestered in host cell cytoplasm. Hence, Cif activates a DNA damage-independent signalling pathway that leads to inhibition of the G(2)/M transition.     

A novel nucleolar protein, PAPA-1, induces growth arrest as a result of cell cycle arrest at the G1 phase

We have identified a novel nucleolar protein, PAP-1-associated protein-1 (PAPA-1), after screening the interacting proteins with Pim-1-associated protein-1 (PAP-1), a protein that is a phosphorylation target of Pim-1 kinase. PAPA-1 comprises 345 amino acids with a basic amino-acid cluster. PAPA-1 was found to be localized in the nucleolus in transfected HeLa cells, and the lysine/histidine cluster was essential for nucleolar localization of PAPA-1. PAPA-1 protein and mRNA expression decreased upon serum restimulation of starvation-synchronized cells, which displayed maximum level of PAPA-1 expression at G0 and early G1 phase of the cell cycle. Ectopic expression of PAPA-1 induced growth suppression of cells, and the effect was dependent on its nucleolar localization in established HeLa cell lines that inducibly express PAPA-1 or its deletion mutant under the control of a tetracycline-inducible promoter. Furthermore, when PAPA-1-inducible HeLa cells were synchronized by thymidine, colcemid or mimosine, and then PAPA-1 was expressed, the proportion of cells at the G1 phase was obviously increased. These results suggest that PAPA-1 induces growth and cell cycle arrests at the G1 phase of the cell cycle.     

Interactions between Toxoplasma gondii and its mammalian host cells

Toxoplasma is a protozoan parasite that is uniquely adapted for invading and surviving within a wide range of host cells. The parasite actively invades the cell, forming a novel vacuole that originates from the host cell plasma membrane. The vacuole membrane is rapidly modified to remove host cell proteins and this compartment subsequently resists fusion with all other host cell endocytic compartments. Shortly after invasion, the parasite secretes a variety of proteins by a process of regulation exocytosis and elaborates an extensive array of membranous tubules that form a network connecting with the vacuolar membrane. Understanding the formation and modification of this unique vacuole may reveal novel mechanisms for subverting host cell endocytic pathways that lead to intracellular survival.     

Toxoplasma gondii manipulates host cell signaling pathways via its secreted effector molecules

The obligate intracellular parasite Toxoplasma gondii secretes a vast variety of effector molecules from organelles known as rhoptries (ROPs) and dense granules (GRAs). ROP proteins are released into the cytosol of the host cell where they are directed to the cell nucleus or to the parasitophorous vacuole (PV) membrane. ROPs secrete proteins that enable host cell penetration and vacuole formation by the parasites, as well as hijacking host-immune responses. After invading host cells, T. gondii multiplies within a PV that is maintained by the parasite proteins secreted from GRAs. Most GRA proteins remain within the PV, but some are known to access the host cytosol across the PV membrane, and a few are able to traffic into the host-cell nucleus. These effectors bind to host cell proteins and affect host cell signaling pathways to favor the parasite. Studies on host–pathogen interactions have identified many infection-altered host signal transductions. Notably, the relationship between individual parasite effector molecules and the specific targeting of host-signaling pathways is being elucidated through the advent of forward and reverse genetic strategies. Understanding the complex nature of the host–pathogen interactions underlying how the host-signaling pathway is manipulated by parasite effectors may lead to new molecular biological knowledge and novel therapeutic methods for toxoplasmosis. In this review, we discuss how T. gondii modulates cell signaling pathways in the host to favor its survival.     

Phthalocyanine-mediated photodynamic therapy induces cell death and a G0/G1 cell cycle arrest in cervical cancer cells

We have developed a series of novel photosensitizers which have potential for anticancer photodynamic therapy (PDT). Photosensitizers include zinc phthalocyanine tetra-sulphonic acid and a family of derivatives with amino acid substituents of varying alkyl chain length and degree of branching. Subcellular localization of these photosensitizers at the phototoxic IC(50) concentration in human cervical carcinoma cells (SiHa Cells) was similar to that of the lysosomal dye Lucifer Yellow. Subsequent nuclear relocalization was observed following irradiation with 665nm laser light. The PDT response was characterized using the Sulforhodamine B cytotoxicity assay. Flow cytometry was used for both DNA cell cycle and dual Annexin V-FITC/propidium iodide analysis. Phototoxicity of the derivatives was of the same order of magnitude as for tetrasulphonated phthalocyanine but with an overall trend of increased phototoxicity with increasing amino acid chain length. Our results demonstrate cell death, inhibition of cell growth, and G(0)/G(1) cell cycle arrest during the phthalocyanine PDT-mediated response.     

Human parvovirus B19 nonstructural protein (NS1) induces cell cycle arrest at G(1) phase

Human parvovirus B19 infects predominantly erythroid precursor cells, leading to inhibition of erythropoiesis. This erythroid cell damage is mediated by the viral nonstructural protein 1 (NS1) through an apoptotic mechanism. We previously demonstrated that B19 virus infection induces G(2) arrest in erythroid UT7/Epo-S1 cells; however, the role of NS1 in regulating cell cycle arrest is unknown. In this report, by using paclitaxel, a mitotic inhibitor, we show that B19 virus infection induces not only G(2) arrest but also G(1) arrest. Interestingly, UV-irradiated B19 virus, which has inactivated the expression of NS1, still harbors the ability to induce G(2) arrest but not G(1) arrest. Furthermore, treatment with caffeine, a G(2) checkpoint inhibitor, abrogated the B19 virus-induced G(2) arrest despite expression of NS1. These results suggest that the B19 virus-induced G(2) arrest is not mediated by NS1 expression. We also found that NS1-transfected UT7/Epo-S1 and 293T cells induced cell cycle arrest at the G(1) phase. These results indicate that NS1 expression plays a critical role in G(1) arrest induced by B19 virus. Furthermore, NS1 expression significantly increased p21/WAF1 expression, a cyclin-dependent kinase inhibitor that induces G(1) arrest. Thus, G(1) arrest mediated by NS1 may be a prerequisite for the apoptotic damage of erythroid progenitor cells upon B19 virus infection.     

Ambient particulate matter induces alveolar epithelial cell cycle arrest: role of G1 cyclins

We hypothesized that the ambient air pollution particles (particulate matter; PM) induce cell cycle arrest in alveolar epithelial cells (AEC). Exposure of PM (25microg/cm(2)) to AEC induced cells cycle arrest in G1 phase, inhibited DNA synthesis, blocked cell proliferation and caused decrease in cyclin E, A, D1 and Cyclin E- cyclin-dependent kinase (CDK)-2 kinase activity after 4h. PM induced upregulation of CDK inhibitor, p21 protein and p21 activity in AEC. SiRNAp21 blocked PM-induced downregulation of cyclins and AEC G1 arrest. Accordingly, we provide the evidence that PM induces AEC G1 arrest by altered regulation of G1 cyclins and CDKs.     

Releasing latent Toxoplasma gondii cysts from host cells to the extracellular environment induces excystation

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects a wide variety of mammals and birds. Although T. gondii infects the brain and muscles in its latent cyst form containing bradyzoite stage parasites during chronic infection, when a chronically infected host becomes immunodeficient or is preyed upon by a predator, the latent cyst undergoes excystation. However, it is not yet known how T. gondii recognises the triggers of excystation in the microenvironment surrounding the cyst. In this study, we incubated T. gondii cysts from host cells in several solutions containing a variety of ionic compositions. Excystation occurred in a solution with an ionic composition which mimicked that of the extracellular environment. However, excystation did not occur in a solution that mimicked the intracellular environment. We also found that the specific Na⁺/K⁺ ratio and the presence of Ca²⁺, mimicking the extracellular environment, are required to trigger excystation. To examine whether the stage conversion of bradyzoite to tachyzoite occurs prior to egress, we constructed a gene-modified T. gondii strain expressing a green fluorescent protein specifically in the tachyzoite stage. During the process of cyst reactivation of this strain, green fluorescence was detected prior to excystation. This suggests that stage conversion from bradyzoite to tachyzoite occurs prior to cyst disruption. These results indicate that T. gondii bradyzoites monitor the ionic composition of their surroundings to recognise their expulsion from host cells, to effectively time their excystation and stage conversion.     

HIV-1初始传播病毒Vpr基因遗传变异对诱导G2期阻滞及细胞凋亡的影响

人免疫缺陷病毒(HIV-1)急性感染过程中,病毒的遗传多样性显著减少,往往只有一株或几株病毒可以建立有效感染,这种病毒被称为初始传播病毒(Transmitted/Founder virus)。病毒蛋白R(Vpr)是HIV-1的辅助蛋白之一,在病毒复制过程中起重要作用。研究初始传播病毒Vpr基因遗传变异与生物学特征对于阐明病毒建立感染的关键环节具有重要意义。文章利用流式细胞术分析了C亚型HIV-1初始传播病毒株与慢性感染株MJ4的 Vpr蛋白诱导细胞G₂期阻滞和细胞凋亡的能力。结果显示,初始传播病毒ZM246和ZM247的Vpr诱导细胞G₂期阻滞和细胞凋亡的能力显著高于慢性感染株MJ4 Vpr。氨基酸序列分析表明,初始传播病毒Vpr在第77、85和94位上存在高频突变。研究结果提示初始传播病毒可能在病毒感染早期,通过Vpr基因的遗传突变,提升病毒诱导细胞停滞G₂期和细胞凋亡的能力,进而促进病毒在宿主体内的复制和传播。     

Caudatin induces cell cycle arrest and caspase-dependent apoptosis in HepG2 cell

In the present study, we investigate the anti-cancer activity and mechanism of caudatin, the C-21 steroidal glycosides, on human hepatoma cell line HepG2. The MTT assay and flow cytometry were used to evaluate HepG2 cell proliferation and cell cycle. Annexin-V/PI and DAPI staining were used to investigate cell apoptosis. Western blotting analysis was used to evaluate the expression levels of proteins. It is found that caudatin inhibits HepG2 cell growth and induces of G₀/G₁ phase arrest in a dose dependent manner, which is associated with a decreased in the expression of cyclinD₁ and increased the levels of p21 and p53. HepG2 cells dealing with caudatin showed typical characteristics of apoptosis. Western blotting analysis indicated that the levels of Bcl-2 were down-regulated after caudatin treatment, whereas the expression of Bax was up-regulated. Furthermore, caudatin-induced apoptosis was accompanied by activation of caspase-3, -9, and poly(ADP-Ribose) Polymerase (PARP). Treatment with caudatin also induced phosphorylation of extracellular-signal regulating kinase (ERK) and c-Jun N-terminal kinase (JNK). These results demonstrate that caudatin inhibits cell proliferation via DNA synthesis reduction and induces caspase-dependent apoptosis in HepG2 cell. Activation of ERK and JNK may be involved in caudatin-induced hepatoma cell apoptosis.     

Smad7 induces G0/G1 cell cycle arrest in mesenchymal cells by inhibiting the expression of G1 cyclins

The major Smad pathways serve in regulating the expression of genes downstream of TGFbeta signals. In this study, we examined the effects of sustained Smad7 expression in cultured cells. Interestingly, Smad7 caused various mesenchymal cells, including NIH3T3 fibroblast and ST2 bone-marrow stromal cells, to undergo a marked morphological alteration into a flattened cell shape, but kept them alive for as long as 60 days. Furthermore, Smad7 arrested the proliferation of the cells even before they reached confluence. These cells became quiescent in G0/G1 phase and accumulated a hypophosphorylated form of retinoblastoma. The cytostatic effect of Smad7 was closely associated with a preceding decrease in the levels of G1 cyclins, such as cyclin D1 and cyclin E. Accordingly, ectopic cyclin E was able to overcome the Smad7-induced arrest of proliferation. These results indicate that Smad7 functions upstream of G1 cyclins and suggest a novel role for Smad7 as an antiproliferative factor. In contrast to the growth of mesenchymal cells, that of epithelial cells was little susceptible to Smad7. The present findings raise the possibility that a link between Smad7 and the G1 to S phase transition may also contribute to the cell cycle control by certain Smad7-inducing stimuli in a cell-type-dependent fashion.