Cell Cycle Phenotype Research Articles

Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which controls programs regulating cell proliferation, differentiation, and apoptosis. We have identified an unexpected role for GR in mitosis. We discovered that specifically modified GR species accumulate at the mitotic spindle during mitosis in a distribution that overlaps with Aurora kinases. We found that Aurora A was required to mediate mitosis-driven GR phosphorylation, but not recruitment of GR to the spindle. GR was necessary for mitotic progression, with increased time to complete mitosis, frequency of mitotic aberrations, and death in mitosis observed following GR knockdown. Complementation studies revealed an essential role for the GR ligand-binding domain, but no clear requirement for ligand binding in regulating chromosome segregation. The GR N-terminal domain, and specifically phosphosites S203 and S211, were not required. Reduced GR expression results in a cell cycle phenotype, with isolated cells from mouse and human subjects showing changes in chromosome content over prolonged passage. Furthermore, GR haploinsufficient mice have an increased incidence of tumor formation, and, strikingly, these tumors are further depleted for GR, implying additional GR loss as a consequence of cell transformation. We identified reduced GR expression in a panel of human liver, lung, prostate, colon, and breast cancers. We therefore reveal an unexpected role for the GR in promoting accurate chromosome segregation during mitosis, which is causally linked to tumorigenesis, making GR an authentic tumor suppressor gene.

Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes.

The eukaryotic cell division cycle is a highly regulated process that consists of a complex series of events and involves thousands of proteins. Researchers have studied the regulation of the cell cycle in several organisms, employing a wide range of high-throughput technologies, such as microarray-based mRNA expression profiling and quantitative proteomics. Due to its complexity, the cell cycle can also fail or otherwise change in many different ways if important genes are knocked out, which has been studied in several microscopy-based knockdown screens. The data from these many large-scale efforts are not easily accessed, analyzed and combined due to their inherent heterogeneity. To address this, we have created Cyclebase—available at http://www.cyclebase.org—an online database that allows users to easily visualize and download results from genome-wide cell-cycle-related experiments. In Cyclebase version 3.0, we have updated the content of the database to reflect changes to genome annotation, added new mRNA and protein expression data, and integrated cell-cycle phenotype information from high-content screens and model-organism databases. The new version of Cyclebase also features a new web interface, designed around an overview figure that summarizes all the cell-cycle-related data for a gene.

Abstract C39: Docetaxel-induced growth and cell cycle phenotypes in prostate stromal cells

Abstract Background: Docetaxel (D) is a clinically used front-line chemotherapeutic agent for castration resistant advanced prostate cancer. However, its long-term benefits are limited with most patients eventually progressing because of inherent or acquired drug resistance. The treatment of advanced prostate cancer is a significant challenge and there are no effective treatments that stably suppress the disease. The mechanisms behind resistance to Docetaxel are not fully understood. Since in vivo Docetaxel cytotoxicity is directed not only to cancer cells, we hypothesize that the response of prostate stromal cells exposed to chemotherapeutic agents could modify drug response or therapeutic outcome in prostate cancer. One of cellular responses to chemotherapy is NF-kB activation and inflammatory signaling. NF-κB activation has been proposed as an important event in PCa development, castration-resistant progression and resistance to chemotherapy. Objective: In this study, we examined the response of prostate stromal (WPMY- 1) cells to Docetaxel. Current Results: Cellular proliferation was analyzed by MTT assay. The IC50 of Docetaxel for WPMY-1 cells was 10nM. Cell cycle profiles were assessed by flow cytometry. The analysis showed that Docetaxel (10-100nM) induced prominent G2-M arrest in these stromal cells within 24 hours, especially in the higher dose ranges. Interestingly lower doses of Docetaxel (10nM), in contrast to higher doses, consistently resulted in accumulation of sub-G1 population (28% 10nM vs 6% in 100nM) of cells suggestive of fragmented DNA associated with apoptotic process. We show that Docetaxel exhibits strong anti-proliferative effects on prostate stromal WPMY-1 cells. Conclusions: These preliminary results show that stromal cells are sensitive to Docetaxel. Why lower doses of the drug induce sub-G1 (apoptotic) accumulation while higher doses preferentially induce G2-M arrest is currently unknown. Future Steps: We will compare responses to D between prostate cancer cells and stromal cells in individual and co-culture models. NF-kB activity following D treatment or radiation, and the expression of genes in NF-kB pathway in D treated cells will be examined. Citation Format: Dominique Gales, Brionna McMcmeans, Temesgen Samuel. Docetaxel-induced growth and cell cycle phenotypes in prostate stromal cells. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C39. doi:10.1158/1538-7755.DISP13-C39

The Society of Biomolecular Imaging and Informatics First Annual Conference

The Society of Biomolecular Imaging and Informatics First Annual Conference

Multiple metabolic requirements for size homeostasis and initiation of division in Saccharomyces cerevisiae.

Most cells must grow before they can divide, but it is not known how cells determine when they have grown enough so they can commit to a new round of cell division. Several parameters affect the timing of initiation of division: cell size at birth, the size cells have to reach when they commit to division, and how fast they reach that size. We report that Saccharomyces cerevisiae mutants in metabolic and biosynthetic pathways differ in these variables, controlling the timing of initiation of cell division in various ways. Some mutants affect the size at birth, size at initiation of division, the rate of increase in size, or any combination of the above. Furthermore, we show that adenylate kinase, encoded by ADK1, is a significant determinant of the efficiency of size control mechanisms. Finally, our data argue strongly that the cell size at division is not necessarily a function of the rate cells increase in size in the G1 phase of the cell cycle. Taken together, these findings reveal an unexpected diversity in the G1 cell cycle phenotypes of metabolic and biosynthetic mutants, suggesting that growth requirements for cell division are multiple, distinct and imposed throughout the G1 phase of the cell cycle.

In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

A transgenic zebrafish model expressing KIT-D816V recapitulates features of aggressive systemic mastocytosis.

Systemic mastocytosis (SM) is a rare myeloproliferative disease without curative therapy. Despite clinical variability, the majority of patients harbour a KIT-D816V mutation, but efforts to inhibit mutant KIT with tyrosine kinase inhibitors have been unsatisfactory, indicating a need for new preclinical approaches to identify alternative targets and novel therapies in this disease. Murine models to date have been limited and do not fully recapitulate the most aggressive forms of SM. We describe the generation of a transgenic zebrafish model expressing the human KIT-D816V mutation. Adult fish demonstrate a myeloproliferative disease phenotype, including features of aggressive SM in haematopoeitic tissues and high expression levels of endopeptidases, consistent with SM patients. Transgenic embryos demonstrate a cell-cycle phenotype with corresponding expression changes in genes associated with DNA maintenance and repair, such as reduced dnmt1. In addition, epcam was consistently downregulated in both transgenic adults and embryos. Decreased embryonic epcam expression was associated with reduced neuromast numbers, providing a robust in vivo phenotypic readout for chemical screening in KIT-D816V-induced disease. This study represents the first zebrafish model of a mast cell disease with an aggressive adult phenotype and embryonic markers that could be exploited to screen for novel agents in SM.

Abstract P6-05-18: A53, cell cycle phenotype and Cdc7 kinase: Identifying patients likely to benefit from targeted Cdc7 therapy

Abstract We previously reported multiparameter analysis of cell cycle regulatory proteins in breast cancer and identified three prognostic ‘cell cycle phenotypes’. Phenotype iii represents cells traversing through the mitotic cell division cycle and is associated with poor prognosis and high Cdc7 levels1. Cdc7 kinase initiates DNA synthesis at chromosomal replication origins and is rate-limiting for S phase entry. In cell-based studies we discovered a new, p53-dependent cell cycle checkpoint which reversible arrests normal cells in G1 phase if DNA replication initiation is perturbed. In cancer cells treated with Cdc7 inhibitors this checkpoint function is lost, resulting in aberrant S phase entry and subsequent apoptotic cell death2,3. Cdc7 kinase is therefore emerging as a novel therapeutic target in cancers bearing mutant p53. In this study we investigated whether the inclusion of p53 mutation status adds further prognostic power to the cell cycle phenotype. We also questioned whether p53 mutant breast cancers express high Cdc7 levels and are thus more likely to be of cell cycle phenotype iii (susceptible to Cdc7-targeted therapy). Our results show that Cdc7 levels are inversely correlated with p53 mutation status (n = 173) and are highest in cell cycle phenotype iii and harder to treat Her-2 and triple negative breast cancer subgroups. Cell cycle phenotype is the strongest predictor of early relapse compared to p53, Ki67 and Cdc7 on univariate analysis and remains so on multivariate analysis after adjusting for p53 status (HR = 3.80, p = 0.002). These findings support the supposition that p53 negatively regulates Cdc7 expression. Combined testing for p53 status, cell cycle phenotype and Cdc7 expression identifies those patients with breast cancer most likely to benefit from targeted therapy with Cdc7 inhibitors. 1 Br J Cancer. 2009 Mar 24;100(6):959-70 2 EMBO J. 2010 Oct 6;29(19):3381-94 3 Am J Pathol. 2010 Oct;177(4):2034-45. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-05-18.

Control analysis of the eukaryotic cell cycle using gene copy-number series in yeast tetraploids

BackgroundIn the model eukaryote, Saccharomyces cerevisiae, previous experiments have identified those genes that exert the most significant control over cell growth rate. These genes are termed HFC for high flux control. Such genes are overrepresented within pathways controlling the mitotic cell cycle.ResultsWe postulated that the increase/decrease in growth rate is due to a change in the rate of progression through specific cell cycle steps. We extended and further developed an existing logical model of the yeast cell cycle in order elucidate how the HFC genes modulated progress through the cycle. This model can simulate gene dosage-variation and calculate the cycle time, determine the order and relative speed at which events occur, and predict arrests and failures to correctly execute a step. To experimentally test our model’s predictions, we constructed a tetraploid series of deletion mutants for a set of eight genes that control the G2/M transition. This system allowed us to vary gene copy number through more intermediate levels than previous studies and examine the impact of copy-number variation on growth, cell-cycle phenotype, and response to different cellular stresses.ConclusionsFor the majority of strains, the predictions agreed with experimental observations, validating our model and its use for further predictions. Where simulation and experiment diverged, we uncovered both novel tetraploid-specific phenotypes and a switch in the determinative execution point of a key cell-cycle regulator, the Cdc28 kinase, from the G1/S to the S/G2 boundaries.

Human corneal epithelial subpopulations: oxygen-dependentex vivoexpansion and transcriptional profiling

Corneal epithelium is being regenerated throughout life by limbal epithelial stem cells (LESCs) believed to be located in histologically defined stem cell niches in corneal limbus. Defect or dysfunctional LESCs result in limbal stem cell deficiency (LSCD) causing pain and decreased visual acuity. Since the first successful treatment of LSCD by transplantation of ex vivo expanded LESCs in 1997, many attempts have been carried out to optimize culture conditions to improve outcome of surgery. To date, progress in this field of bioengineering is substantially hindered not only by the lack of specific biomarkers of LESCs, but also by the lack of a precise molecular characterization of in situ epithelial subpopulations. The aim of this dissertation (Bath 2013a) was to optimize culture systems with regard to the environmental oxygen concentration for selective ex vivo expansion of LESCs and to analyse in situ subpopulations in human corneal epithelium using a combination of laser capture microdissection and RNA sequencing for global transcriptomic profiling. We compared dissociation cultures, using either expansion on γ-irradiated NIH/3T3 feeder cells in serum-rich medium or expansion directly on plastic in serum-free EpiLife medium, using a range of physiologically relevant oxygen concentrations (2%, 5%, 10%, 15% and 20%). Using immunocytochemistry and advanced fluorescence microscopy, cells were characterized according to growth, cell cycle distribution, colony-forming efficiency, phenotypes, and cytomorphometry. Limbal epithelial cells expanded in 2% O2 exhibited slow growth, low fraction of cells in S/G2, high CFE, high expression of stem cell markers ABCG2 and p63α, and low fraction of differentiation marker CK3 resembling an LESC phenotype. The effect of hypoxia to maintain LESCs in culture was not dependent on the system used for propagation (Bath et al. 2013b). Laser capture microdissection was used to isolate cellular subpopulations in situ from the spatially defined differentiation pathway in human corneal epithelium according to an optimized protocol for maintenance of expression profiles. Isolated total RNA from basal limbal crypts (BLCs), superficial limbal crypts (SLCs), paracentral/central cornea and limbal stroma was amplified and converted to fragmented cDNA libraries for use in deep paired-end next-generation sequencing. Global transcriptional profiling was carried out using bioinformatics. The location of primitive cells in BLCs, migratory and activated cells in SLCs, and differentiated cells in paracentral/central cornea was evident from mapping of significantly upregulated genes in each compartment to the Gene Ontology (GO). Interestingly, many GO terms in BLCs were also involved in neurogenic processes, whereas many GO terms in SLCs were related to vasculature. Mapping upregulated genes in BLCs to pathway annotations in Kyoto Encyclopaedia of Genes and Genomes described many active pathways as signalling and cancer-associated pathways. We supply extensive information on possible novel biomarkers, reveal insight into both active pathways and novel regulators of LESCs such as Lrig1 and SOX9, and provide an immense amount of data for the future exploration (Bath et al. 2013c). Selective ex vivo expansion of LESCs in hypoxia and the comprehensive molecular characterization of corneal epithelial subpopulations in situ are expected to be beneficial for the future treatment for LSCD by cultured limbal epithelial transplantation.

Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes

The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.

Pax6 is required for normal cell-cycle exit and the differentiation kinetics of retinal progenitor cells.

The coupling between cell-cycle exit and onset of differentiation is a common feature throughout the developing nervous system, but the mechanisms that link these processes are mostly unknown. Although the transcription factor Pax6 has been implicated in both proliferation and differentiation of multiple regions within the central nervous system (CNS), its contribution to the transition between these successive states remains elusive. To gain insight into the role of Pax6 during the transition from proliferating progenitors to differentiating precursors, we investigated cell-cycle and transcriptomic changes occurring in Pax6 - retinal progenitor cells (RPCs). Our analyses revealed a unique cell-cycle phenotype of the Pax6-deficient RPCs, which included a reduced number of cells in the S phase, an increased number of cells exiting the cell cycle, and delayed differentiation kinetics of Pax6 - precursors. These alterations were accompanied by coexpression of factors that promote (Ccnd1, Ccnd2, Ccnd3) and inhibit (P27 kip1 and P27 kip2) the cell cycle. Further characterization of the changes in transcription profile of the Pax6-deficient RPCs revealed abrogated expression of multiple factors which are known to be involved in regulating proliferation of RPCs, including the transcription factors Vsx2, Nr2e1, Plagl1 and Hedgehog signaling. These findings provide novel insight into the molecular mechanism mediating the pleiotropic activity of Pax6 in RPCs. The results further suggest that rather than conveying a linear effect on RPCs, such as promoting their proliferation and inhibiting their differentiation, Pax6 regulates multiple transcriptional networks that function simultaneously, thereby conferring the capacity to proliferate, assume multiple cell fates and execute the differentiation program into retinal lineages.

Systems biology of Ewing sarcoma: a network model of EWS-FLI1 effect on proliferation and apoptosis

Ewing sarcoma is the second most frequent pediatric bone tumor. In most of the patients, a chromosomal translocation leads to the expression of the EWS-FLI1 chimeric transcription factor that is the major oncogene in this pathology. Relative genetic simplicity of Ewing sarcoma makes it particularly attractive for studying cancer in a systemic manner. Silencing EWS-FLI1 induces cell cycle alteration and ultimately leads to apoptosis, but the exact molecular mechanisms underlying this phenotype are unclear. In this study, a network linking EWS-FLI1 to cell cycle and apoptosis phenotypes was constructed through an original method of network reconstruction. Transcriptome time-series after EWS-FLI1 silencing were used to identify core modulated genes by an original scoring method based on fitting expression profile dynamics curves. Literature data mining was then used to connect these modulated genes into a network. The validity of a subpart of this network was assessed by siRNA/RT-QPCR experiments on four additional Ewing cell lines and confirmed most of the links. Based on the network and the transcriptome data, CUL1 was identified as a new potential target of EWS-FLI1. Altogether, using an original methodology of data integration, we provide the first version of EWS-FLI1 network model of cell cycle and apoptosis regulation.

Functional Characterization of Fission Yeast Transcription Factors by Overexpression Analysis

In Schizosaccharomyces pombe, over 90% of transcription factor genes are nonessential. Moreover, the majority do not exhibit significant growth defects under optimal conditions when deleted, complicating their functional characterization and target gene identification. Here, we systematically overexpressed 99 transcription factor genes with the nmt1 promoter and found that 64 transcription factor genes exhibited reduced fitness when ectopically expressed. Cell cycle defects were also often observed. We further investigated three uncharacterized transcription factor genes (toe1+–toe3+) that displayed cell elongation when overexpressed. Ectopic expression of toe1+ resulted in a G1 delay while toe2+ and toe3+ overexpression produced an accumulation of septated cells with abnormalities in septum formation and nuclear segregation, respectively. Transcriptome profiling and ChIP-chip analysis of the transcription factor overexpression strains indicated that Toe1 activates target genes of the pyrimidine-salvage pathway, while Toe3 regulates target genes involved in polyamine synthesis. We also found that ectopic expression of the putative target genes SPBC3H7.05c, and dad5+ and SPAC11D3.06 could recapitulate the cell cycle phenotypes of toe2+ and toe3+ overexpression, respectively. Furthermore, single deletions of the putative target genes urg2+ and SPAC1399.04c, and SPBC3H7.05c, SPACUNK4.15, and rds1+, could suppress the phenotypes of toe1+ and toe2+ overexpression, respectively. This study implicates new transcription factors and metabolism genes in cell cycle regulation and demonstrates the potential of systematic overexpression analysis to elucidate the function and target genes of transcription factors in S. pombe.

Isolation and biological characteristics of mesenchymal stem cells derived from human placenta decidua basalis

Comparing to bone marrow mesenchymal stem cells (MSCs), placenta-derived MSCs have the advantages of adequate sources, low immunogenicity, little risk of viral contamination, and no ethical controversy, and thus possess a better prospect for clinical application. Placental tissue not only includes chorionic and amniotic, but also contains decidua basalis which locate in the maternal placenta surface. The biological characteristics of MSCs isolated from decidua basalis have not been well studied. This study was aimed to investigate the biologic characteristics of placenta decidua basalis-derived MSC from placenta decidua basalis (DB) by enzymatic digestion. Short tandem repeats (STR) test was used to identify the cells derived from the maternal placenta surface. Growth rate of decidua basalis mesenchymal stem cells (DB-MSC) was measured by MTT. Cell cycle and cell phenotype were detected by flow cytometry. Inducing differentiation was used to evaluate multipotency of DB-MSC. For testing the immunosuppression of DB-MSC, they were co-cultured with peripheral blood mononuclear cells (PBMNC) stimulated by phytohemagglutinin (PHA) and then IFN-γ in the co-cultured media was quantified by ELISA. The results showed that the cells were derived from the maternal placenta by STR analysis. DB-MSC showed typical fibroblast morphology in the culture and were positive for the MSC surface markers: CD90, CD73, CD105, CD44 and negative for CD45, CD11b, and CD34. DB-MSC underwent osteogenic, adipogenic and chondrogenic differentiation in inducing medium. DB-MSC could inhibit the secretion of IFN-γ by PBMNC. It is concluded that the cells are isolated from placenta decidua basalis and possess the basic characteristics of MSC. DB-MSC can be an important maternal autologous MSC and may be a safe and effective treatment for immune system diseases, which makes the DB-MSC as an important source of autologous MSC from mother. DB-MSC can be safely for the treatment of the mother's immune system diseases.

Human Corneal Epithelial Subpopulations: Oxygen Dependent Ex Vivo Expansion and Transcriptional Profiling

Corneal epithelium is being regenerated throughout life by limbal epithelial stem cells (LESCs) believed to be located in histologically defined stem cell niches in corneal limbus. Defective or dysfunctional LESCs result in limbal stem cell deficiency (LSCD) causing pain and decreased visual acuity. Since the first successful treatment of LSCD by transplantation of ex vivo expanded LESCs in 1997, many attempts have been carried out to optimize culture conditions to improve the outcome of surgery. To date, progress in this field of bioengineering is substantially hindered by both the lack of specific biomarkers of LESCs and the lack of a precise molecular characterization of in situ epithelial subpopulations. The aim of this dissertation was to optimize culture systems with regard to the environmental oxygen concentration for selective ex vivo expansion of LESCs and to analyse in situ subpopulations in human corneal epithelium using a combination of laser capture microdissection and RNA sequencing for global transcriptomic profiling. We compared dissociation cultures, using either expansion on γ-irradiated NIH/3T3 feeder cells in serum-rich medium or expansion directly on plastic in serum-free EpiLife medium, using a range of physiologically relevant oxygen concentrations (2%, 5%, 10%, 15% and 20%). Using immunocytochemistry and advanced fluorescence microscopy, cells were characterized regarding growth, cell cycle distribution, colony-forming efficiency (CFE), phenotypes and cytomorphometry. Limbal epithelial cells expanded in 2% O2 exhibited slow growth, low fraction of cells in S/G2 , high CFE, high expression of stem cell markers ABCG2 and p63α, and low fraction of differentiation marker CK3 resembling a LESC phenotype. The effect of hypoxia to maintain LESCs in culture was not dependent on the system used for propagation (Bath etal. 2013a). Laser capture microdissection was used to isolate cellular subpopulations in situ from the spatially defined differentiation pathway in human corneal epithelium according to an optimized protocol for maintenance of expression profiles. Isolated total RNA from basal limbal crypts (BLCs), superficial limbal crypts (SLCs), paracentral/central cornea and limbal stroma was amplified and converted to fragmented cDNA libraries for use in deep paired-end next-generation sequencing. Global transcriptional profiling was carried out using bioinformatics. The location of primitive cells in BLCs, migratory and activated cells in SLCs and differentiated cells in paracentral/central cornea was evident from mapping of significantly upregulated genes in each compartment to the gene ontology (GO). Interestingly, many GO terms in BLCs were also involved in neurogenic processes, whereas many GO terms in SLCs were related to vasculature. Mapping upregulated genes in BLCs to pathway annotations in Kyoto Encyclopedia of Genes and Genomes described many active pathways as signalling and cancer-associated pathways. We supply extensive information on possible novel biomarkers, reveal insight into both active pathways and novel regulators of LESCs such as Lrig1 and SOX9 and provide an immense amount of data for future exploration (Bath etal. 2013b). Selective ex vivo expansion of LESCs in hypoxia and the comprehensive molecular characterization of corneal epithelial subpopulations in situ are expected to be beneficial for the future treatment of LSCD by cultured limbal epithelial transplantation.

MiR-29a modulates the angiogenic properties of human endothelial cells

Although extensive investigation has been made on miR-29a in relation to malignancies, only a little information has been provided about the angiogenic property of this miRNA so far. Herein, we sought to investigate the role of miR-29a in regulating cell cycle and angiogenic phenotype of endothelial cells. The results showed that miR-29a is highly expressed and upregulated by hypoxia-mimicking reagents in human umbilical vein endothelial cells (HUVEC). Consistent with this preliminary finding, introduction of exogenous agomiR-29a, or Antagomir-29a altered cell cycle progression and promoted, or repressed the proliferation and tube formation of HUVEC, respectively. Furthermore, by using luciferase reporter assay, the expression of HBP1, a suppressor transcription factor was directly regulated by miR-29a through 3′-UTR. Increased or decreased HBP1 protein level was associated with the inhibition or overexpression of miR-29a, respectively. We conclude that miR-29a has a significant role in regulating cell cycle, proliferation and angiogenic properties of HUVEC, and this function is likely mediated through HBP1 protein at the post-transcriptional level. As a novel molecular target, miR-29a may have a potential value for the treatment of angiogenesis-associated diseases such as cardiovascular diseases and cancers.

Functional interplay between the cell cycle and cell phenotypes.

Cell cycle distribution of adherent cells is typically assessed using flow cytometry, which precludes the measurements of many cell properties and their cycle phase in the same environment. Here we develop and validate a microscopy system to quantitatively analyze the cell-cycle phase of thousands of adherent cells and their associated cell properties simultaneously. This assay demonstrates that population-averaged cell phenotypes can be written as a linear combination of cell-cycle fractions and phase-dependent phenotypes. By perturbing the cell cycle through inhibition of cell-cycle regulators or changing nuclear morphology by depletion of structural proteins, our results reveal that cell cycle regulators and structural proteins can significantly interfere with each other's prima facie functions. This study introduces a high-throughput method to simultaneously measure the cell cycle and phenotypes at single-cell resolution, which reveals a complex functional interplay between the cell cycle and cell phenotypes.

LQB-118, an orally active pterocarpanquinone, induces selective oxidative stress and apoptosis in Leishmania amazonensis

The pterocarpanquinone LQB-118, previously demonstrated to be effective in vivo via oral delivery, was investigated for its mechanism in selective parasite killing. Oxidative stress in Leishmania amazonensis was analysed by evaluating reactive oxygen species (ROS) production (2',7'-dichlorodihydrofluorescein diacetate) and the loss of mitochondrial membrane potential (ΔΨm) using rhodamine, JC-1 and MitoCapture. Ultrastructural analysis was performed using transmission electron microscopy (TEM). DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Treatment with LQB-118 induced ROS production in the promastigotes of L. amazonensis in a concentration-dependent manner for the first 4 h and was sustained for 24 h. TEM analysis revealed several alterations typical of apoptosis. Promastigotes presented a reduction of ΔΨm after 24 h of incubation with 2.5 μM (18.7%), 5 μM (63.7%) or 10 μM (70.7%) LQB-118. A sub-G0/G1 cell cycle phenotype was observed in 21%-83% of the promastigotes incubated with 1.25-10 μM LQB-118. Concentration-dependent DNA fragmentation was observed in promastigotes treated with 2.5-10 μM LQB-118, and selective DNA fragmentation was observed in intracellular amastigotes after 72 h with 2.5 μM treatment. Our results suggest that LQB-118 selectively induces ROS-triggered and mitochondria-dependent apoptosis in this parasite.

Transcriptional Networks Controlling the Cell Cycle

In this work, we map the transcriptional targets of 107 previously identified Drosophila genes whose loss caused the strongest cell-cycle phenotypes in a genome-wide RNA interference screen and mine the resulting data computationally. Besides confirming existing knowledge, the analysis revealed several regulatory systems, among which were two highly-specific and interconnected feedback circuits, one between the ribosome and the proteasome that controls overall protein homeostasis, and the other between the ribosome and Myc/Max that regulates the protein synthesis capacity of cells. We also identified a set of genes that alter the timing of mitosis without affecting gene expression, indicating that the cyclic transcriptional program that produces the components required for cell division can be partially uncoupled from the cell division process itself. These genes all have a function in a pathway that regulates the phosphorylation state of Cdk1. We provide evidence showing that this pathway is involved in regulation of cell size, indicating that a Cdk1-regulated cell size checkpoint exists in metazoans.