Influenza A Virus NS1 Induces G0/G1 Cell Cycle Arrest by Inhibiting the Expression and Activity of RhoA Protein

Influenza A virus is an important pathogenic virus known to induce host cell cycle arrest in G₀/G₁ phase and create beneficial conditions for viral replication. However, how the virus achieves arrest remains unclear. We investigated the mechanisms underlying this process and found that the nonstructural protein 1 (NS1) is required. Based on this finding, we generated a viable influenza A virus (H1N1) lacking the entire NS1 gene to study the function of this protein in cell cycle regulation. In addition to some cell cycle regulators that were changed, the concentration and activity of RhoA protein, which is thought to be pivotal for G₁/S phase transition, were also decreased with overexpressing NS1. And in the meantime, the phosphorylation level of cell cycle regulator pRb, downstream of RhoA kinase, was decreased in an NS1-dependent manner. These findings indicate that the NS1 protein induces G₀/G₁ cell cycle arrest mainly through interfering with the RhoA/pRb signaling cascade, thus providing favorable conditions for viral protein accumulation and replication. We further investigated the NS1 protein of avian influenza virus (H5N1) and found that it can also decrease the expression and activity of RhoA, suggesting that the H5N1 virus may affect the cell cycle through the same mechanism. The NS1/RhoA/pRb cascade, which can induce the G₀/G₁ cell cycle arrest identified here, provides a unified explanation for the seemingly different NS1 functions involved in viral replication events. Our findings shed light on the mechanism of influenza virus replication and open new avenues for understanding the interaction between pathogens and hosts.     

Absence of Inhibin Alpha and Retinoblastoma Protein Leads to Early Sertoli Cell Dysfunction

Sertoli cells, the support cells of mammalian spermatogenesis, are regulated by a number of nuclear factors and express retinoblastoma (RB) tumor suppressor protein. We hypothesized that RB is an important mediator of Sertoli cell tumorigenesis in inhibin α knockout (Inha KO) mice. In our previous mouse studies, we found that conditional knockout (cKO) of Rb in Sertoli cells caused progressive Sertoli cell dysfunction. Initially, loss of RB had no gross effect on Sertoli cell function as the mice were fertile with normal testis weights at 6 weeks of age, but by 10–14 weeks of age, mutant mice demonstrated severe Sertoli cell dysfunction and infertility. Although double knockout (dKO) of Rb and Inha did not result in exacerbation of the tumorigenic phenotype of Inha-null mice, we found that the dKO mice demonstrate an acceleration of Sertoli cell dysfunction compared to Rb cKO mice. Specifically, in contrast to Rb cKO mice, Inha/Rb dKO mice showed signs of Sertoli cell dysfunction as early as 4 weeks of age. These results demonstrate that RB is not essential for Sertoli cell tumorigenesis in Inha KO mice but that loss of Inha accelerates the infertility phenotype of Rb cKO mice.     

Depletion of the SR-Related Protein TbRRM1 Leads to Cell Cycle Arrest and Apoptosis-Like Death in Trypanosoma brucei

Arginine-Serine (RS) domain-containing proteins are RNA binding proteins with multiple functions in RNA metabolism. In mammalian cells this group of proteins is also implicated in regulation and coordination of cell cycle and apoptosis. In trypanosomes, an early branching group within the eukaryotic lineage, this group of proteins is represented by 3 members, two of them are SR proteins and have been recently shown to be involved in rRNA processing as well as in pre-mRNA splicing and stability. Here we report our findings on the 3rd member, the SR-related protein TbRRM1. In the present study, we showed that TbRRM1 ablation by RNA-interference in T. brucei procyclic cells leads to cell-cycle block, abnormal cell elongation compatible with the nozzle phenotype and cell death by an apoptosis-like mechanism. Our results expand the role of the trypanosomal RS-domain containing proteins in key cellular processes such as cell cycle and apoptosis-like death, roles also carried out by the mammalian SR proteins, and thus suggesting a conserved function in this phylogenetically conserved protein family.     

Preservation of Dendritic Cell Function during Vesicular Stomatitis Virus Infection Reflects both Intrinsic and Acquired Mechanisms of Resistance to Suppression of Host Gene Expression by Viral M Protein

Inhibition of host-directed gene expression by the matrix (M) protein of vesicular stomatitis virus (VSV) effectively blocks host antiviral responses, promotes virus replication, and disables the host cell. However, dendritic cells (DC) have the capacity to resist these effects and remain functional during VSV infection. Here, the mechanisms of DC resistance to M protein and their subsequent maturation were addressed. Flt3L-derived murine bone marrow dendritic cells (FDC), which phenotypically resemble resident splenic DC, continued to synthesize cellular proteins and matured during single-cycle (high-multiplicity) and multicycle (low-multiplicity) infection with VSV. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived myeloid DC (GDC), which are susceptible to M protein effects, were nevertheless capable of maturing, but the response was delayed and occurred only during multicycle infection. FDC resistance was manifested early and was type I interferon (IFN) receptor (IFNAR) and MyD88 independent, but sustained resistance required IFNAR. MyD88-dependent signaling contributed to FDC maturation during single-cycle infection but was dispensable during multicycle infection. Similar to FDC, splenic DC were capable of maturing in vivo during the first 24 h of infection with VSV, and neither Toll-like receptor 7 (TLR7) nor MyD88 was required. We conclude that FDC resistance to M protein is controlled by an intrinsic, MyD88-independent mechanism that operates early in infection and is augmented later in infection by type I IFN. In contrast, while GDC are not intrinsically resistant, they can acquire resistance during multicycle infection. In vivo, splenic DC resist the inhibitory effects of VSV, and as in multicycle FDC infection, MyD88-independent signaling events control their maturation.     

Induction of Retinoblastoma Gene Expression during Terminal Growth Arrest of a Conditionally Immortalized Fetal Rat Lung Epithelial Cell Line and during Fetal Lung Maturation

The process by which fetal lung epithelial cells differentiate into type 1 and type 2 cell is largely unknown. In order to study lung epithelial cell proliferation and differentiation we have infected 20-day fetal lung epithelial cells with a retrovirus carrying a temperature-sensitive SV40 T antigen (T Ag) and isolated several immortalized fetal epithelial cell lines. Cell line 20-3 has characteristics of lung epithelial cells including the presence of distinct lamellar bodies, tight junctions, keratin 8 and 18 mRNA, HFH8, and T1α mRNA and low levels of surfactant protein A mRNA. At 33°C 20-3 grows with a doubling time of 21 h. At 40°C the majority of cells cease to proliferate. Growth arrest is accompanied by significant morphological changes including an increase in cell size, transition to a squamous phenotype that resembles type 1 cells, and an increase in the number of multinucleated cells within the population. Greater than 95% of the cells incorporate [³H]thymidine into DNA at 33°C whereas at 40°C label incorporation drops to less than 20%. When shifted down to 33°C 40% of the cells remain terminally growth arrested. In addition, cells plated at 40°C have a reduced ability to form colonies when replated at 33°C. Treatment with TGF-β increases the percentage of cells that terminally growth arrest to greater than 80%. Growth arrest is accompanied by an increase in the levels of c-jun, jun D, cyclin D1, C/EBP-β, transglutaminase type II, and retinoblastoma (Rb) mRNA and an induction of p105, the hypophosphorylated, growth regulatory form of Rb. Evaluation of Rb mRNA in fetal lung indicates that it is induced 2.5-fold between 17 and 21 days of gestation. These studies indicate that 20-3 terminally growth arrests in culture at the nonpermissive temperature and that it may be useful in studying changes in gene expression that accompany terminal growth arrest during lung development.     

Disrupting the Interaction between Retinoblastoma Protein and Raf-1 Leads to Defects in Progenitor Cell Proliferation and Survival during Early Inner Ear Development

The retinoblastoma protein (pRb) is required for cell-cycle exit of embryonic mammalian hair cells but is not required for hair cell fate determination and early differentiation, and this provides a strategy for hair cell regeneration by manipulating the pRb pathway. To reveal the mechanism of pRb functional modification in the inner ear, we compared the effects of attenuated pRb phosphorylation by an inhibitor of the Mitogen-Activated Protein (MAP) kinase pathway and an inhibitor of the Rb–Raf-1 interaction on cultured chicken otocysts. We demonstrated that the activity of pRb is correlated with its phosphorylation state, which is regulated by a newly established cell cycle-independent pathway mediated by the physical interaction between Raf-1 and pRb. The phosphorylation of pRb plays an important role during the early stage of inner ear development, and attenuated phosphorylation in progenitor cells leads to cell cycle arrest and increased apoptosis along with a global down-regulation of the genes involved in cell cycle progression. Our study provides novel routes to modulate pRb function for hair cell regeneration.     

Reevesioside A,a Cardenolide Glycoside,Induces Anticancer Activity against Human Hormone-Refractory Prostate Cancers through Suppression of c-myc Expression and Induction of G1 Arrest of the Cell Cycle

In the past decade, there has been a profound increase in the number of studies revealing that cardenolide glycosides display inhibitory activity on the growth of human cancer cells. The use of potential cardenolide glycosides may be a worthwhile approach in anticancer research. Reevesioside A, a cardenolide glycoside isolated from the root of Reevesia formosana, displayed potent anti-proliferative activity against human hormone-refractory prostate cancers. A good correlation (r² = 0.98) between the expression of Na⁺/K⁺-ATPase α₃ subunit and anti-proliferative activity suggested the critical role of the α₃ subunit. Reevesioside A induced G1 arrest of the cell cycle and subsequent apoptosis in a thymidine block-mediated synchronization model. The data were supported by the down-regulation of several related cell cycle regulators, including cyclin D1, cyclin E and CDC25A. Reevesioside A also caused a profound decrease of RB phosphorylation, leading to an increased association between RB and E2F1 and the subsequent suppression of E2F1 activity. The protein and mRNA levels of c-myc, which can activate expression of many downstream cell cycle regulators, were dramatically inhibited by reevesioside A. Transient transfection of c-myc inhibited the down-regulation of both cyclin D1 and cyclin E protein expression to reevesioside A action, suggesting that c-myc functioned as an upstream regulator. Flow cytometric analysis of JC-1 staining demonstrated that reevesioside A also induced the significant loss of mitochondrial membrane potential. In summary, the data suggest that reevesioside A inhibits c-myc expression and down-regulates the expression of CDC25A, cyclin D1 and cyclin E, leading to a profound decrease of RB phosphorylation. G1 arrest is, therefore, induced through E2F1 suppression. Consequently, reevesioside A causes mitochondrial damage and an ultimate apoptosis in human hormone-refractory prostate cancer cells.     

丙型肝炎病毒NS5A蛋白对Huh7细胞周期的影响

应用PCR技术从含有丙型肝炎病毒（HCV）全长开放阅读框的质粒pBRTM／HCV1～3011中获得NS5A全长基因片段,利用基因重组技术将其克隆至真核表达载体pcDNA3．1（-）中。通过酶切、PCR及测序鉴定证实,NS5A基因已正确插入到pcDNA3．1（-）中。再利用脂质体介导转染Huh7细胞,30h后收获细胞,经Western blot验证,证实HCV的NS5A基因在Huh7细胞中已经获得表达。在培养条件完全一致的条件下,表达NS5A基因的Huh7细胞与pcDNA3．1（-）转染的细胞在转染30h后被收集起来,乙醇固定,PI染色后利用流式细胞仪检测细胞周期变化。G0／G1期由60．6％下降到49．7％,S期由23．9％上升到32．7％,而转染pcDNA3．1（-）细胞的细胞周期与正常的Huh7细胞则差别不大。从而证明HCV NS5A蛋白对Huh7细胞周期具有调节作用。     

Human Immunodeficiency Virus Type 1 Vpr Induces Cell Cycle G2 Arrest through Srk1/MK2-Mediated Phosphorylation of Cdc25

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G₂ arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G₂ arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G₂/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G₂ arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G₂ delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G₂/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G₂ arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G₂ arrest at least in part through a Srk1/MK2-mediated mechanism.     

SD-208, a Novel Protein Kinase D Inhibitor,Blocks Prostate Cancer Cell Proliferation and Tumor Growth In Vivo by Inducing G2/M Cell Cycle Arrest

Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.     

The Accumulation of Cyclin-Dependent Kinase Inhibitor p27Is a Primary Response to Staurosporine and Independent of G1 Cell Cycle Arrest

The staurosporine-induced G1 cell cycle arrest was analyzed in a variety of cell lines which includes human tumor cell lines and oncogene-transformed NIH3T3 cell lines. All the cell lines which were sensitive to staurosporine-induced G1 arrest contained a functional retinoblastoma protein (pRB). However, when pRB-lacking fibroblast cells derived from pRB knockout mice were tested they were also sensitive to G1 arrest by staurosporine, indicating that the inactivation of pRB alone is not sufficient for the abrogation of staurosporine-induced G1 arrest. In searching for a common event caused by staurosporine, the cyclin-dependent kinase (CDK) inhibitor protein p27^kip1but not p21^cip1was found to accumulate after staurosporine treatment in all the cell lines examined. This accumulation occurred regardless of the induction of the G1 arrest. The result indicates that the accumulation of p27^kip1is the cell's primary response to staurosporine and that the capability of staurosporine to induce G1 arrest depends on the integrity of cell cycle regulatory components which are downstream of p27^kip1.     

Phosphorylation of Cdc25C by pp90Rsk Contributes to a G2 Cell Cycle Arrest in Xenopus Cycling Egg Extracts

In unfertilized Xenopus eggs, the p42 mitogen activated protein kinase (p42MAPK) pathway isknown to maintain cell cycle arrest at metaphase of meiosis II. However, constitutive activation ofp42MAPK in post-meiotic, cycling Xenopus egg extracts can lead to either a G2 or M-phase arrestof the cell cycle, depending on the timing of p42MAPK activation. Here, we examined themolecular mechanism by which activation of the p42MAPK pathway during interphase leads to cellcycle arrest in G2. When either a recombinant wild type Cdc25C(WT) or a mutated form ofCdc25C, in which serine 287 was replaced by an alanine (S287A), was added to cycling eggextracts, S287A accelerated entry into M-phase. Furthermore, the addition of S287A overcame theG2 arrest caused by p42MAPK, driving the extract into M-phase. p90Rsk, a kinase that is the targetof p42MAPK, was phosphorylated and activated (pp90Rsk) in the G2-arrested egg extracts, and wasable to phosphorylate WT but not S287A in vitro. 14-3-3 proteins were associated with endogenousCdc25C in G2-arrested extracts. Cdc25C(WT) that had been phosphorylated by pp90Rsk bound 14-3-3?, whereas S287A could not. These data suggest that the link between the p42MAPK signalingpathway and Cdc25C involves the activation of pp90Rsk and its phosphorylation of Cdc25C at S287,causing the binding of 14-3-3 proteins. We propose that the binding of 14-3-3 proteins to pp90Rskphosphorylated-Cdc25C results in a G2 arrest in a manner similar to the cell cycle delays inducedby differentiation signals that occur later in embryonic development.