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Epidemiological studies have shown that the regular use of non-steroidal anti-inflammatory (NSAIDs) drugs is associated with a reduced risk of various cancers. In addition, in vitro and experiments in mouse models have demonstrated that NSAIDs decrease tumor initiation and/or progression of several cancers. However, there are limited preclinical studies investigating the effects of NSAIDs in ovarian cancer. Here, we have studied the effects of two NSAIDs, diclofenac and indomethacin, in ovarian cancer cell lines and in a xenograft mouse model. Diclofenac and indomethacin treatment decreased cell growth by inducing cell cycle arrest and apoptosis. In addition, diclofenac and indomethacin reduced tumor volume in a xenograft model of ovarian cancer. To identify possible molecular pathways mediating the effects of NSAID treatment in ovarian cancer, we performed microarray analysis of ovarian cancer cells treated with indomethacin or diclofenac. Interestingly, several of the genes found downregulated following diclofenac or indomethacin treatment are transcriptional target genes of E2F1. E2F1 was downregulated at the mRNA and protein level upon treatment with diclofenac and indomethacin, and overexpression of E2F1 rescued cells from the growth inhibitory effects of diclofenac and indomethacin. In conclusion, NSAIDs diclofenac and indomethacin exert an anti-proliferative effect in ovarian cancer in vitro and in vivo and the effects of NSAIDs may be mediated, in part, by downregulation of E2F1. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs) are pluripotent and have unlimited self- renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co- regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B- MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity. [ABSTRACT FROM AUTHOR]

Krüppel-like factor 4 (KLF4), a transcription factor, plays a key role in the pluripotency of stem cells. We sought to determine the function of KLF4 in T-cell development and differentiation by using T-cell-specific Klf4-knockout (KO) mice. We found that KLF4 was highly expressed in thymocytes and mature T cells and was rapidly down-regulated in mature T cells after activation. In Klf4-KO mice, we observed a modest reduction of thymocytes (27%) due to the reduced proliferation of double-negative (DN) thymocytes. We demonstrated that a direct repression of Cdkn1b by KLF4 was a cause of decreased DN proliferation. During in vitro T-cell differentiation, we observed significant reduction of IL-17-expressing CD44 T cells (Th17; 24%) but not in other types of Th differentiation. The reduction of Th17 cells resulted in a significant attenuation of the severity (35%) of experimental autoimmune encephalomyelitis in vivo in Klf4-KO mice as compared with the Klf4 wild-type littermates. Finally, we demonstrated that KLF4 directly binds to the promoter of Il17a and positively regulates its expression. In summary, these findings identify KLF4 as a critical regulator in T-cell development and Th17 differentiation. [ABSTRACT FROM AUTHOR]

Exceptional genomic stability is one of the hallmarks of mouse embryonic stem (ES) cells. However, the genes contributing to this stability remain obscure. We previously identified Zscan4 as a specific marker for two-cell embryo and ES cells. Here we show that Zscan4 is involved in telomere maintenance and long-term genomic stability in ES cells. Only 5% of ES cells express Zscan4 at a given time, but nearly all ES cells activate Zscan4 at least once during nine passages. The transient Zscan4-positive state is associated with rapid telomere extension by telomere recombination and upregulation of meiosis-specific homologous recombination genes, which encode proteins that are colocalized with ZSCAN4 on telomeres. Furthermore, Zscan4 knockdown shortens telomeres, increases karyotype abnormalities and spontaneous sister chromatid exchange, and slows down cell proliferation until reaching crisis by passage eight. Together, our data show a unique mode of genome maintenance in ES cells. [ABSTRACT FROM AUTHOR]

Background: Cardiomyocyte (CM) cell cycle analysis has been impeded because of a reliance on primary neonatal cultures of poorly proliferating cells or chronic transgenic animal models with innate compensatory mechanisms. Methodology/Principal Findings: We describe an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CM. Following induction with ascorbate and selection with puromycin, early CM cultures are .98% pure, and at least 85% of the cells actively proliferate. During the proliferative stage, cells express high levels of E2F3a, B-Myb and phosphorylated forms of retinoblastoma (Rb), but with continued cultivation, cells stop dividing and mature functionally. This developmental transition is characterized by a switch from slow skeletal to cardiac TnI, an increase in binucleation, cardiac calsequestrin and hypophosphorylated Rb, a decrease in E2F3, B-Myb and atrial natriuretic factor, and the establishment of a more negative resting membrane potential. Although previous publications suggested that Rb was not necessary for cell cycle control in heart, we find following acute knockdown of Rb that this factor actively regulates progression through the G1 checkpoint and that its loss promotes proliferation at the expense of CM maturation. Conclusions/Significance: We have established a unique model system for studying cardiac cell cycle progression, and show in contrast to previous reports that Rb actively regulates both cell cycle progression through the G1 checkpoint and maturation of heart cells. We conclude that this in vitro model will facilitate the analysis of cell cycle control mechanisms of CMs. [ABSTRACT FROM AUTHOR]

Background: The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Methodology/Principal Findings: In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. Conclusions/Significance: Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiationassociated cell death. [ABSTRACT FROM AUTHOR]

Hypermutation in immunoglobulin genes produces a high frequency of substitutions of all four bases, which are likely generated by low-fidelity DNA polymerases. Indeed, humans deficient for DNA polymerase (pol) η have decreased substitutions of A·T base pairs in variable and switch regions. To study the role of pol η in a genetically tractable system, we created mice lacking pol η. B cells from Polh-/- mice produced normal amounts of lgG, indicating that pol η does not affect class switch recombination. Similar to their human counterparts, variable and switch regions from PoIh-/- mice had fewer substitutions of A·T base pairs and correspondingly more mutations of CG base pairs, which firmly establishes a central role for pol η in hypermutation. Notably, the location and types of substitutions differ markedly from those in Msh6-/- clones, which also have fewer A·T mutations. The data suggest that pol η preferentially synthesizes a repair patch on the nontranscribed strand, whereas MSH6 functions to generate the patch. [ABSTRACT FROM AUTHOR]

The balance of activities between the proto-oncogene phosphoinositide 3-kinase (PI3K) and the tumour suppressor gene PTEN has been shown to affect cellular growth and proliferation, as well as tumorigenesis. Previously, PTEN expression in the PTEN-null Jurkat T cell leukaemia line was shown to cause reduced proliferation without cell cycle arrest. Here, we further these investigations by determining the basis for this phenomenon. By BrdU pulse-chase and cell cycle arrest and release assays, we find that PTEN expression reduced proliferation by slowing progression through all phases of the cell cycle. This was associated with reduced levels of cyclins A, B1 and B2, cdk4, and cdc25A and increased p27KIP1 expression. Apoptosis played no role in the antiproliferative effect of PTEN, since only marginal increases in the rate of apoptosis were detected upon PTEN expression, and inhibitors of effector caspases did not restore proliferative capacity. Active Akt blocked the antiproliferative effects of PTEN, indicating that PTEN mediates its effects through conventional PI3K-linked signalling pathways. Similar results were obtained from a different PTEN-null leukaemia T cell line, CEM. Together, these results show that PTEN expression in leukaemic T cells leads to reduced proliferation via an apoptosis-independent mechanism involving slower passage through the cell cycle.Oncogene (2003) 22, 8195-8204. doi:10.1038/sj.onc.1206872 [ABSTRACT FROM AUTHOR]

Studies the use of green fluorescent proteins as markers to identify and track genetically modified hematopoietic stem cells. Vectors containing cell-surface markers; Identification of hematopoietic cells expressing the marker gene; Detection of gene-modified human and non-human hematopoietic cells.

The cellular abundance of topoisomerase IIa (TOP2A) critically maintains DNA topology after replication and determines the efficacy of TOP2 inhibitors in chemotherapy. Here, we report that the RNA-binding protein HuR, commonly overexpressed in cancers, binds to the TOP2A 3'-untranslated region (3'UTR) and increases TOP2A translation. Reducing HuR levels triggered the recruitment of TOP2A transcripts to RNA-induced silencing complex (RISC) components and to cytoplasmic processing bodies. Using a novel MS2-tagged RNA precipitation method, we identified microRNA miR-548c-3p as a mediator of these effects and further uncovered that the interaction of miR-548c-3p with the TOP2A 3'UTR repressed TOP2A translation by antagonizing the action of HuR. Lowering TOP2A by silencing HuR or by overexpressing miR-548c-3p selectively decreased DNA damage after treatment with the chemotherapeutic agent doxorubicin. In sum, HuR enhances TOP2A translation by competing with miR-548c-3p; their combined actions control TOP2A expression levels and determine the effectiveness of doxorubicin. [ABSTRACT FROM AUTHOR]