4N DNA Research Articles

V-Src-induced nuclear localization of YAP is involved in multipolar spindle formation in tetraploid cells

The protein-tyrosine kinase, c-Src, is involved in a variety of signaling events, including cell division. We have reported that v-Src, which is a mutant variant of the cellular proto-oncogene, c-Src, causes delocalization of Aurora B kinase, resulting in a furrow regression in cytokinesis and the generation of multinucleated cells. However, the effect of v-Src on mitotic spindle formation is unknown. Here we show that v-Src-expressing HCT116 and NIH3T3 cells undergo abnormal cell division, in which cells separate into more than two cells. Upon v-Src expression, the proportion of multinucleated cells is increased in a time-dependent manner. Flow cytometry analysis revealed that v-Src increases the number of cells having a ≥4N DNA content. Microscopic analysis showed that v-Src induces the formation of multipolar spindles with excess centrosomes. These results suggest that v-Src induces multipolar spindle formation by generating multinucleated cells. Tetraploidy activates the tetraploidy checkpoint, leading to a cell cycle arrest of tetraploid cells at the G1 phase, in which the nuclear exclusion of the transcription co-activator YAP plays a critical role. In multinucleated cells that are induced by cytochalasin B and the Plk1 inhibitor, YAP is excluded from the nucleus. However, v-Src prevents this nuclear exclusion of YAP through a decrease in the phosphorylation of YAP at Ser127 in multinucleated cells. Furthermore, v-Src decreases the expression level of p53, which also plays a critical role in the cell cycle arrest of tetraploid cells. These results suggest that v-Src promotes abnormal spindle formation in at least two ways: generation of multinucleated cells and a weakening of the tetraploidy checkpoint.

Preclinical activity of MBM-5 in gastrointestinal cancer by inhibiting NEK2 kinase activity.

NEK2 is a conserved mitotic regulator critical for cell cycle progression. Aberrant expression of NEK2 has been found in a variety of human cancers, making it an attractive molecular target for the design of novel anticancer therapeutics. In the present study, we have identified a novel compound MBM-5, which was found to bind to NEK2 with high affinity by docking simulations study. MBM-5 potently inhibited NEK2 kinase activity in vitro in a concentration-dependent manner. MBM-5 also suppressed cellular NEK2 kinase activity, as evidenced by the decreased phosphorylation of its substrate Hec1 on S165 in a concentration- and time-dependent manner. This inhibition impeded mitotic progression by inducing chromosome segregation defects and cytokinesis failure; therefore leading to accumulation of cells with ≥4N DNA content, which finally underwent apoptosis. More importantly, MBM-5 treatment effectively suppressed the tumor growth of human gastric and colorectal cancer cells xenografts. Taken together, we demonstrated that MBM-5 effectively inhibited the kinase activity of NEK2 and showed a potential application in anti-cancer treatment regimens.

Cell Cycle-Dependent Mechanisms Underlie Vincristine-Induced Death of Primary Acute Lymphoblastic Leukemia Cells.

Microtubule-targeting agents (MTA), such as the taxanes and vinca alkaloids, are used to treat a variety of cancers due to their ability to perturb microtubule dynamics. In cell culture, MTAs exert their anticancer effects primarily by causing mitotic arrest and cell death. However, accumulating indirect evidence suggests that MTAs may exert their cytotoxicity in human tumors by interfering with interphase microtubules. In this study, we sought to develop and characterize an experimental system in which to test the hypothesis that MTAs induce cell death during interphase. Primary adult acute lymphoblastic leukemia (ALL) cells treated with vincristine only weakly exhibited colocalization between mitotic and apoptotic markers and major characteristics of mitotic death, such as an increase in cells with 4N DNA content before the appearance of cells with <2N DNA content, suggesting a mixed response. Therefore, we separated ALL cells into distinct phases of the cell cycle by centrifugal elutriation, labeled cells with 5-ethynyl-2'-deoxyuridine (EdU), and then treated each population with vincristine. Cells isolated during G1 underwent cell death without evidence of EdU uptake, indicating that the cytotoxic effects of vincristine took place during G1 Conversely, cells isolated during S or G2-M phases underwent death following mitotic arrest. Thus, vincristine induces distinct death programs in primary ALL cells depending on cell-cycle phase, and cells in G1 are particularly susceptible to perturbation of interphase microtubules. Primary ALL cells may therefore provide a powerful model system in which to study the multimodal mechanisms underlying MTA-induced cell death. Cancer Res; 76(12); 3553-61. ©2016 AACR.

The effect of amifostine on differentiation of the human megakaryoblastic Dami cell line.

Amifostine is a cytoprotective drug that was initially used to control and treat nuclear radiation injury and is currently used to provide organ protection in cancer patients receiving chemotherapy. Clinical studies have also found that amifostine has some efficacy in the treatment of cytopenia caused by conditions such as myelodysplastic syndrome and immune thrombocytopenia, both of which involve megakaryocyte maturation defects. We hypothesized that amifostine induced the differentiation of megakaryocytes and investigated this by exposing the human Dami megakaryocyte leukemia cell line to amifostine (1 mmol/L). After 12 days of amifostine exposure, optical microscopy showed that the proportion of Dami cells with diameters >20 μm had increased to 24.63%. Transmission electron microscopy identified the development of a platelet demarcation membrane system, while flow cytometry detected increased CD41a expression and decreased CD33 expression on the Dami cell surface. Ploidy analysis found that the number of polyploid cells with >4N DNA content increased to 27.96%. We did not detect any elevation in the mRNA or protein levels of megakaryocytic differentiation‐associated transcription factors GATA‐binding factor 1 (GATA‐1) and nuclear factor, erythroid 2 (NF‐E2), but nuclear import assay revealed an increased nuclear translocation of these proteins. These findings indicate that amifostine induced the differentiation of Dami cells into mature megakaryocytes via a mechanism involving increased nuclear translocation of the transcription factors, NF‐E2 and GATA‐1.

Abstract B48: Intratumor ploidy heterogeneity as a chemoresistance mechanism in triple-negative breastcancer

Abstract Background: Triple negative breast cancers (TNBC) are aggressive, heterogeneous tumors, and currently lack targeted therapies. Chemotherapy is the mainstay treatment, but tumor recurrence is a major cause of mortality and morbidity. Chemotherapy may select for resistant subclones that have a survival advantage over the bulk population of cancer cells and cause tumor relapse. DNA aneuploidy (abnormal number of chromosomes) is a common genetic feature in breast cancer and has been frequently associated with poor prognosis. Although, aneuploid or polyploid cancer cells can contribute to intratumor ploidy heterogeneity, their functional role in tumorigenesis and chemoresistance is largely undefined. Traditionally, polyploid cells were thought to arise due to defects in cell division and were considered to be senescent. However, recent reports have shown that polyploid cells represent a viable and proliferating subpopulation within a tumor and may have a direct role in regulating chemoresistance. Methods: In this study, we isolated and functionally characterized the diploid and polyploid sub-populations derived from TNBC cell lines. DNA ploidy was assessed in Hoechst 33342 stained cells using flow cytometry. Cells with greater than 4N DNA content were considered as polyploid. Next, we analyzed cell proliferation in HCC1937 cells using CFSE and DNA labeling. Live-cell imaging was performed on flow-sorted diploid and polyploid cells using IncuCyte Zoom to monitor cell proliferation in the absence and presence of mitotic inhibitor (docetaxel). Results: DNA ploidy analysis identified presence of polyploid cells in HCC1395 and HCC1937 cell lines at a frequency of 6.7% and 5.8% respectively. Polyploid cells have higher forward-scatter than diploid cells suggesting that they are larger in size. In addition, nuclear staining with Hoechst 33342 showed that giant polyploid cells contain enlarged and/or multiple nuclei. Time-lapse microscopy in polyploid cells revealed mitotic structures suggestive of multipolar cell division. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labeling showed that polyploid cycle slower than the predominant diploid population. Further, the IncuCyte cell proliferation assay also confirmed that sorted polyploid cells divide slower than the diploid cells and displayed resistance to docetaxel. Concusions: These findings indicate that there are functional differences between diploid and polyploid sub-populations. In our future studies, we will examine the isolated diploid and polyploid cells for differences in their tumorigenic potential and role in tumor recurrence. Citation Format: Rekha Gyanchandani, Adrian Lee. Intratumor ploidy heterogeneity as a chemoresistance mechanism in triple-negative breastcancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B48.

Galectin-3 Inhibition As a Novel Therapeutic Approach in Acute Lymphoblastic Leukemia (ALL)

Pre-B acute lymphoblastic leukemia (ALL) is one of the leading causes of cancer-related death, largely due to the high risk of relapse. Relapse often originates in the bone marrow microenvironment where stroma protects ALL cells against drug treatment. Galectin-3 (Gal3) is a carbohydrate-binding protein which recognizes cell surface receptors by binding LacNAc branches of N-glycans. We recently found that the stromal cells of the tumor microenvironment are the main source of Gal3 in ALL cells. In co-culture of ALL cells with supportive stroma (OP9), stroma-secreted Gal3 is actively internalized by ALL cells. Expression of Gal3 in ALL cells leads to significant protection of these cells against drug-induced apoptosis. The application and therapeutic benefits of targeting Gal3 and the lectin-mediated communication between the tumor microenvironment and leukemic cells in ALL treatment have not been explored. Here, we tested the hypothesis that Gal3 conveys important signals between the tumor microenvironment and ALL cells and that it represents a viable therapeutic target.We tested the preclinical activities of a first in class Gal3 inhibitor "KB1019.7" by studying its antileukemic effects on ALL cells in vitro. KB1019.7 is a novel carbohydrate-based compound with a micro Molar binding affinity for Gal3 as well as significant selectivity for Gal3 over Gal1. KB1019.7 acts by interfering with carbohydrate recognition activities of Gal3.Treatment of different ALL cells with KB1019.7 resulted in a dose-dependent reduction in cell viability. ALL cells which were cultured on a protective stroma (OP9) layer were plated alone or together with OP9 cells prior to drug treatment. The IC50 values (concentration of the inhibitor at which 50% cell death occurred) did not vary significantly among different ALL subgroups and were within mid micromolar range (average ~470 μM). The high tolerance of the cells to KB1019.7 may be explained by the fact that this compound is an optimized monovalent carbohydrate and not a classic Galectin inhibitor which are usually in the form of peptide or carbohydrate ligands. Gal3 is involved in several cellular activities including cell growth, cell differentiation and apoptosis. We evaluated the effects of KB1019.7 on modulating the biological functions of Gal3 in US7 (Ph-negative) and TXL2 (Ph-positive) B-cell leukemic cells. To this end, analysis of ALL cells exposed to 250 and 500 μM of KB1019.7 revealed a dose-dependent increase in the number of cells with 4N DNA content after 48 hours, suggesting that Gal3 inhibition may lead to accumulation of ALL cells in G2/M phase. More importantly, KB1019.7 had adverse effect on the acquired resistance to Vincristine in ALL cells. We treated US7 cells with vehicle control, KB1019.7 (500 μM), Vincristine (2.5 nM) or a combination of KB1019.7 and Vincristine in the presence of protective stroma (OP9) cells. Vincristine was chosen as the model for standard cytotoxic therapy in pre-B ALL. US7 cells developed resistance to Vincristine after continued exposure (~12 days) as evident by recovery and increase in cell viability observed after a time-dependent decline in cell number. Interestingly, introduction of KB1019.7 in combination with Vincristine effectively inhibited resistance to Vincristine and led to efficient cell death. Furthermore, KB1019.7 treatment promoted a significant downregulation in pERK1/2 protein levels which is often upregulated upon emergence of environmentally mediated drug resistance in ALL cells.Together our data demonstrate a role for Gal3 inhibition with KB1019.7 in inducing antileukemic responses in ALL cells including induction of cell death and cell cycle arrest. Furthermore, our results highlight the value of combining Gal3 inhibition with conventional chemotherapy as a tool to efficiently circumvent resistance to such therapies. Gal3 pro-tumor activities are documented in other hematologic malignancies and further studies to initiate and expand Gal3-targeted therapies in leukemia are highly warranted. DisclosuresNo relevant conflicts of interest to declare.

Abstract A38: In vitro and in vivo characterization of a novel NEK2 inhibitor MBM5 for the treatment of gastrointestinal cancer

Abstract NEK2 is a conserved mitotic regulator critical for centrosome splitting and correct chromosome segregation. Aberrant expression of NEK2 has been found in a variety of human cancers, making it an attractive molecular target for the design of novel anticancer therapeutics. Here we report the discovery of a novel small molecule NEK2 inhibitor, MBM5, for the treatment of gastrointestinal cancer cells. A series of compounds have been synthesized to determine their potency against NEK2. Through in vitro kinase assay, we found that compound MBM5 exhibited a significant inhibitory effect on the kinase activity of NEK2. MBM5 also demonstrated strong inhibition on proliferation of a broad spectrum of tumor cells, especially gastrointestinal cancer cells (IC50&amp;lt;1 μM). Treating cells with MBM5 triggered a significant accumulation of cells at G2/M phase and a modest accumulation of cells with &amp;gt;4N DNA content. By releasing cells from the G1/S boundary, we found that the progression from G2/M to G1 phases were completely abrogated in the presence of MBM5. In consistence, we further observed that MBM5 induced chromosomal misalignment in metaphase and slightly increased the level of multinucleated cells, confirming the perturbed mitotic progression. And these was correlated with MBM5-inhibited phosphorylation of Hec1 and Histone 3. Over time, the accumulation of chromosomal abnormalities led to apoptosis, as evidenced by the increased population of Annexin-V/PI positive cells, the up-regulated expression of cleaved-caspase 3 and the cleavage of PARP. In vivo daily treatment with MBM5 (ip, 20 mg/kg, 3 weeks) significantly inhibited the growth of MGC-803 gastric tumor and HCT-116 colonal tumor xenografts. Altogether, this study demonstrates that MBM5 interfered with NEK2 functions both in cells and tumor xenografts, thus showing its potential in anti-cancer treatment regimens. Citation Format: Yanfen Fang, Xiongwen Zhang, Wenhao Hu, Yannan Kong, Hong Wang, Mengli Zhu. In vitro and in vivo characterization of a novel NEK2 inhibitor MBM5 for the treatment of gastrointestinal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A38.

The EGF receptor ligand amphiregulin controls cell division via FoxM1.

Epidermal growth factor receptor (EGFR) is central to epithelial cell physiology, and deregulated EGFR signaling has an important role in a variety of human carcinomas. Here we show that silencing of the EGF-related factor amphiregulin (AREG) markedly inhibits the expansion of human keratinocytes through mitotic failure and accumulation of cells with ⩾ 4n DNA content. RNA-sequencing-based transcriptome analysis revealed that tetracycline-mediated AREG silencing significantly altered the expression of 2331 genes, 623 of which were not normalized by treatment with EGF. Interestingly, genes irreversibly upregulated by suppression of AREG overlapped with genes involved in keratinocyte differentiation. Moreover, a significant proportion of the irreversibly downregulated genes featured upstream binding sites recognized by forkhead box protein M1 (FoxM1), a key transcription factor in the control of mitosis that is widely dysregulated in cancer. The downregulation of FoxM1 and its target genes preceded mitotic arrest. Constitutive expression of FoxM1 in AREG knockdown cells normalized cell proliferation, reduced the number of cells with ⩾ 4n DNA content and rescued expression of FoxM1 target genes. These results demonstrate that AREG controls G2/M progression and cytokinesis in keratinocytes via activation of a FoxM1-dependent transcriptional program, suggesting new avenues for treatment of epithelial cancer.

Abstract 3554: A short ASPM splicing variant but not the full length variant as a molecular target in hepatocellular carcinoma

Abstract Most solid tumor cells are aneuploidy and various cancer cell lines show “chromosomal instability” (CIN), meaning that they frequently lose and gain whole chromosomes during divisions. The cause of these observed chromosome imbalances is unknown, but will probably be found in defects in the processes that control chromosome segregation during mitosis. The clinical importance of CIN is underscored by its association with poor patient outcome in multiple cancer types. However, despite the prevalence and the clinical relevance of CIN, a consistent basis for how it is generated at the molecular level is poorly understood. ASPM gene is the human orthologue of the Drosophila abnormal spindle (asp) protein associates with centrosomes and the most commonly mutated gene of autosomal recessive primary microcephaly. The homozygous semi-lethal mutation of Drosophila asp causes abnormal spindles, frequent polyploid cells and cytokinesis failure, leading to arrest of neuroblasts in metaphase and larval-pupal lethality. Centrosome defects cause chromosome misaggregation and aneuploidy, leading to genetic instability, a major driving force for malignant transformation and tumor progression. In our previous work, ASPM was shown often overexpressed in human HCC and associated with high serum AFP, bigger tumor, high-grade, higher- vascular invasion stage, and lower 5-year survival, early tumor recurrence and poor prognosis. We assessed the role of ASPM in ensuring the balance of genomic instability and cancer invasion in liver cancer. Using the in vitro invasion-selection cell model, increased ASPM mRNA was corresponded to the invasive potential. Moreover, knock down of ASPM, using RNAi oligos, caused in tested cell lines a rising impaired chromosomal instability resulting in multi-nucleation, bi-nucleus and micronucleus formation of liver cancer cells. After ASPM RNAi treatment, an image-cytometry analysis revealed a correlation between the expression of genes related to chromosomal segregation such as CEP55 in mRNA, TPX2, Aurora A, and cyclin B1 in protein level and increasing 4N DNA content fraction of testing cells, indicating mitotic phase progression failure and loss the balance of genomic instability. In addition, Knock down of ASPM resulted in reduction of tumor cell growth, cell cycle progression and invasion in HCC cell lines. Surprisingly, theses cell functions inhibition were only come with downregulation of ASPM short splicing variant (variant #1) after a particular ASPM RNAi oligos transfection. In conclusion, downregulation of ASPM short splicing variant suppressed tumor cell growth, delayed cell cycle progression and the formation of tumors in vitro, and led to chromosomal instability in liver cancer cells. Mechanistic analysis showed that the knockdown of ASPM short splicing variant reduced the expression of genes related to chromosomal segregation. Citation Format: Hung-Wei Pan, Yu-Chia Chen. A short ASPM splicing variant but not the full length variant as a molecular target in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3554. doi:10.1158/1538-7445.AM2015-3554

Midostaurin preferentially attenuates proliferation of triple-negative breast cancer cell lines through inhibition of Aurora kinase family.

BackgroundBreast cancer is classified into three subtypes by the expression of biomarker receptors such as hormone receptors and human epidermal growth factor receptor 2. Triple-negative breast cancer (TNBC) expresses none of these receptors and has an aggressive phenotype with a poor prognosis, which is insensitive to the drugs that target the hormone receptors and human epidermal growth factor receptor 2. It is, thus, required to develop an effective therapeutic reagent to treat TNBC.ResultsThe study using a panel of 19 breast cancer cell lines revealed that midostaurin, a multi-target protein kinase inhibitor, suppresses preferentially the growth of TNBC cells comparing with non-TNBC cells. Clustering analysis of the drug activity data for the panel of cancer cell lines predicted that midostaurin shares the target with Aurora kinase inhibitors. Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells.ConclusionMidostaurin suppresses the proliferation of TNBC cells among the breast cancer cell lines presumably through the inhibition of the Aurora kinase family. The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0150-2) contains supplementary material, which is available to authorized users.

Distinct Pathways of Vincristine Induced Cell Death in Primary Acute Lymphoblastic Leukemia Cells

Microtubule-targeting agents (MTAs), such as the taxanes and vinca alkaloids, are anti-cancer agents that perturb microtubule function. In cell culture, these drugs exert their cytotoxicity primarily by causing mitotic arrest and cell death. However, accumulated indirect evidence suggests that MTAs may exert their cytotoxicity in tumors by interfering with interphase microtubules. We therefore sought to develop and characterize a cell system in which MTAs induced interphase death. In primary adult acute lymphoblastic leukemia (ALL) cell cultures treated with vincristine, the major characteristics of mitotic death, which include an increase in cells with 4N DNA content prior to the appearance of cells with sub-G1 DNA content, as well as the co-existence of mitotic and apoptotic markers, were only weakly represented, suggesting a mixed response. The ALL cells were therefore separated into distinct phases of the cell cycle by centrifugal elutriation and labeled with 5-ethynyl-2'-deoxyuridine (EdU) prior to treatment with vincristine. A proportion of cells in G1 phase underwent vincristine-induced cell death without evidence of EdU staining, indicating that they had died without entering S phase and had thus died directly from G1 phase. In contrast, cells in S or G2/M phases underwent death following mitotic arrest. Thus, vincristine induces distinct, cell cycle-dictated pathways of death in primary ALL cells, with cells in G1 phase evidently susceptible to perturbation of interphase microtubules. Hence, primary ALL cells provide a powerful model system to study clinically relevant mechanisms of MTA-induced cell death. Supported by NIH CA109821

Ex Vivo Induction of Megakaryocytic Differentiation of Umbilical Cord Blood CD34+ Cells

Background and Objectives: Due to platelet shortage, megakaryocytes have been regarded as an effective substitute to the alleviation of frequent thrombocytopenia after stem cell transplantation. However, ex vivo expansion of megakaryoblasts and their subsequent differentiation into mature megakaryocytes for clinical applications remains a challenge. Here, we describe the development of a two-stage culture system for producing megakaryocytes from cord blood CD34+ cells.Design and Methods: Firstly, we expanded CD34+ hematopoietic progenitor/stem cells for 6 days in a serum-free culture system (IMDM basal medium with the addition of biotin, putrescine, insulin, human serum albumin, selenium, and some other nutrients) supplemented with stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), low density lipoprotein (LDL), StemRegenin 1 (SR1), and DMSO. CD34+ cells expansion was monitored by flow cytometric analysis of cell surface markers coupled with cell counting. Subsequently, these expanded cells were induced toward the megakaryocytic lineage for additional 7 days in the same serum-free medium as above supplemented with SCF, TPO, IL-3, IL-6, IL-11, granulocyte-macrophage colony-stimulating factor (GM-CSF), and LDL. Megakaryocytes were detected by flow cytometry using antibodies against specific cell surface markers including CD41a and CD42b. Differentiated megakaryocytes were also confirmed by morphological criteria such as cell size and DNA polyploidy. To functionally evaluate induced megakaryocytes, these cells were transplanted into sublethally irradiated NOD/SCID mice. Viability and cell being of these mice were monitored after injection. Mice of the negative control group (n=3) were injected with saline. In the experimental group (n=6), each mouse was injected with 1.0×107 cells from the second stage of culture.Results: After the first stage culture, proliferation folds of total cells and CD34+ cells were 85.65±7.03 and 62.91±4.36, respectively. The calculated yield from each CD34+ cell was between 1.0×104 to 1.5×104 CD41+ megakaryocytes with a purity of CD41+ and CD42+ cells reaching 93.7%±2.8% and 80.3%±5.8%, respectively. Differentiated cells were morphologically discernible as they were much larger than starting CD34+ cells with apparent lobular nuclei and numerous α-granules. In addition, about 32.67%±7.43% of induced megakaryocytes exhibited 4N or larger DNA content (4N 17.6%±4.12%; 8N 10.93%±2.48%; >8N 3.23%±1.34%). In mouse studies, samples collected from the negative control group contained no cells positive for human CD41a and CD42b markers. In the experimental group, human platelets were detected in mouse peripheral blood 3 days post-transplantation. At day 14 post-transplantation, the percentage of platelets derived from injected human megakaryocytes reached 13.6%±6.2%. Human megakaryocytes were also detected in mouse bone marrow 7 days post-transplantation, peaking at day 14 (~2.38% of total bone marrow megakaryocytes).Conclusions: We have established a stem cell expansion and differentiation platform that can be adapted to large-scale production of mature megakaryocytes from umbilical cord blood cells. Significantly, induced megakaryocytes are capable of engrafting in mouse bone marrow and producing platelets after transplantation into irradiated NOD/SCID mice. Therefore, our experimental platform is capable of producing a sufficient number of functional megakaryocytes for various clinical applications in the future. DisclosuresQin:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment. Ren:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.

Apc and p53 interaction in DNA damage and genomic instability in hepatocytes.

Disruption of Apc within hepatocytes activates Wnt signaling, perturb differentiation and ultimately lead to neoplasia. Apc negatively regulates Wnt signaling but is also involved in organizing the cytoskeleton and may play a role in chromosome segregation. In vitro studies have implicated Apc in the control of genomic stability. However, the relevance of this data has been questioned in vivo as Apc is lost earlier than the onset of genomic instability. Here, we analyse the relationship between immediate loss of Apc and the acquisition of genomic instability in hepatocytes. We used Cre-lox technology to inactivate Apc and in combination with p53 in vivo, to define the consequences of gene loss upon cell-cycle regulation, proliferation, death and aneuploidy. We show that whilst Apc loss leads to increased proliferation, it also leads to increased apoptosis, the accumulation of p53, p21 and markers of DSBs and DNA repair. Flow cytometry revealed an increased 4N DNA content, consistent with a G2 arrest. Levels of anaphase bridges were also elevated, implicating failed chromosome segregation. This was accompanied by an increase in centrosome number which demonstrates a role for Apc in maintaining euploidy.To address the role of p53 in these processes, we analyzed combined loss of Apc and p53, which led to a further increase in proliferation, cell death, DNA damages and repair and a bypass of G2 arrest than was observed with Apc loss. However we observed only a marginal effect on anaphase bridges and centrosome number which could be due to increased cell death. Our data therefore establishes, in an in vivo setting, that APC loss leads to a DNA damage signature and genomic instability in the liver and that additional loss of p53 leads to an increase in the DNA damage signal but not to an immediate increase in the genomic instability phenotype.

A small molecule, MTBT, prevents cancer cell growth by activating p38 MAPK

Cancer is a disease of unscheduled cell division and many anticancer drugs target the cell cycle to inhibit the proliferation of cancer cells. We conducted a screen for new anticancer drugs that induce cell cycle arrest using a small compound library. From this screen, we identified 2-(3-methyl-thiophen-2-yl)-4-(3,4-dioxybenzene) thiazole (MTBT), which causes accumulation of cancer cells with 4N DNA content and inhibits colony formation of several cancer cell lines. We further showed that the treatment of cancer cells with this compound for a longer time period leads to apoptosis, as indicated by the presence of cells with a sub-G1 peak and the appearance apoptotic markers. The increased phosphorylation of serine 10 on histone H3 in MTBT-treated cancer cells suggests cell cycle arrest in the M-phase. Strikingly, MTBT-induced cell cycle arrest and enhanced H3 (Ser10) phosphorylation are abrogated by the pretreatment with SB203580, a specific inhibitor of mitogen-activated protein kinase p38. Moreover, treatment of cancer cells with MTBT induces the phosphorylation of p38, indicative of p38 activation. Together, we have identified a new compound that inhibits cancer cell proliferation, which is likely a consequence of p38 activation.

Heteropathogenic virulence and phylogeny reveal phased pathogenic metamorphosis in Escherichia coli O2:H6

Extraintestinal pathogenic and intestinal pathogenic (diarrheagenic) Escherichia coli differ phylogenetically and by virulence profiles. Classic theory teaches simple linear descent in this species, where non-pathogens acquire virulence traits and emerge as pathogens. However, diarrheagenic Shiga toxin-producing E. coli (STEC) O2:H6 not only possess and express virulence factors associated with diarrheagenic and uropathogenic E. coli but also cause diarrhea and urinary tract infections. These organisms are phylogenetically positioned between members of an intestinal pathogenic group (STEC) and extraintestinal pathogenic E. coli. STEC O2:H6 is, therefore, a ‘heteropathogen,’ and the first such hybrid virulent E. coli identified. The phylogeny of these E. coli and the repertoire of virulence traits they possess compel consideration of an alternate view of pathogen emergence, whereby one pathogroup of E. coli undergoes phased metamorphosis into another. By understanding the evolutionary mechanisms of bacterial pathogens, rational strategies for counteracting their detrimental effects on humans can be developed.Subject Categories Microbiology, Virology & Host Pathogen Interaction

Senescence affects endothelial cells susceptibility to dengue virus infection.

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.

The Janus kinases inhibitor AZD1480 attenuates growth of small cell lung cancers in vitro and in vivo.

The prognosis of small cell lung cancer (SCLC) is poor, and there has been very little progress in the medical treatment of SCLC in the past two decades. We investigated the potential of Janus-activated kinases (JAK) inhibitor, AZD1480, for treatment of SCLC in vitro and in vivo. JAK1 and JAK2 were inhibited by AZD1480 or siRNAs, and the effect of inhibition of JAK gene family on SCLC cell viability was evaluated. The effect of AZD1480 on cell-cycle distribution and apoptosis induction was studied. Antitumor effects of AZD1480 in tumor xenografts were assessed. AZD1480 significantly inhibited growth of six out of 13 SCLC cells with IC50s ranging from 0.73 to 3.08 μmol/L. Knocking down of JAK2 and JAK1 inhibited proliferation of Jak2-positive/Jak1-negative H82 cells and Jak1-positive/Jak2-negative GLC4 cells, respectively. Treatment of SCLC cells with AZD1480 for 24 hours resulted in an increase of 4N DNA content and histone 3 serine 10 phosphorylation, indicative of G2-M phase arrest. Moreover, SCLCs underwent apoptosis after AZD1480 treatment as exemplified by the downregulation of MCL1, the accumulation of cleaved caspase 3, cleaved PARP, and increase of annexin-V-positive cells. Finally, xenograft experiments showed that AZD1480 attenuated the growth of H82 and GLC4 tumors in mice, and we observed stronger apoptosis as well as decreased CD31-positive endothelial cells in H82 and GLC4 xenografts upon AZD1480 treatment. JAK inhibitor AZD1480 attenuated growth of SCLC cells in vitro and in vivo. Clinical development of anti-JAKs therapies in SCLC warrants further investigation.

Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template

Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks.

AMG 900, pan-Aurora kinase inhibitor, preferentially inhibits the proliferation of breast cancer cell lines with dysfunctional p53

Aurora kinases play important roles in cell division and are frequently overexpressed in human cancer. AMG 900 is a novel pan-Aurora kinase inhibitor currently being tested in Phase I clinical trials. We aimed to evaluate the in vitro activity of AMG 900 in a panel of 44 human breast cancer and immortalized cell lines and identify predictors of response. AMG 900 inhibited proliferation at low nanomolar concentrations in all cell lines tested. Response was further classified based on the induction of lethality. 25 cell lines were classified as highly sensitive (lethality at 10nM of AMG 900>10%), 19 cell lines as less sensitive to AMG 900 (lethality at 10nM of AMG 900<10%). Traditional molecular subtypes of breast cancer did not predict for this differential response. There was a weak association between AURKA amplification and response to AMG 900 (response ratio=2.53, p=0.09). mRNA expression levels of AURKA, AURKB, and AURKC and baseline protein levels of Aurora kinases A and B did not significantly associate with response. Cell lines with TP53 loss of function mutations (RR=1.86, p=0.004) and low baseline p21 protein levels (RR=2.28, p=0.0004) were far more likely to be classified as highly sensitive to AMG 900. AMG 900 induced p53 and p21 protein expression in cell lines with wt TP53. AMG 900 caused the accumulation of cells with >4N DNA content in a majority of cell lines independently of sensitivity and p53 status. AMG 900 induced more pronounced apoptosis in highly sensitive p53-dysfunctional cell lines. We have found that AMG 900 is highly active in breast cancer cell lines and that TP53 loss of function mutations as well as low baseline expression of p21 protein predict strongly for increased sensitivity to this compound in vitro.

Contribution of BubR1 to oxidative stress‐induced aneuploidy in p53‐deficient cells

DNA aneuploidy is observed in various human tumors and is associated with the abnormal expression of spindle assembly checkpoint (SAC) proteins. Oxidative stress (OS) causes DNA damage and chromosome instability that may lead to carcinogenesis. OS is also suggested to contribute to an increase in aneuploid cells. However, it is not clear how OS is involved in the regulation of SAC and contributes to carcinogenesis associated with aneuploidy. Here we show that an oxidant (KBrO3) activated the p53 signaling pathway and suppressed the expression of SAC factors, BubR1, and Mad2, in human diploid fibroblast MRC5 cells. This suppression was dependent on functional p53 and reactive oxygen species. In p53 knockdown cells, KBrO3 did not suppress BubR1 and Mad2 expression and increased both binucleated cells and cells with >4N DNA content. BubR1 and not Mad2 downregulation suppressed KBrO3-induced binucleated cells and cells with >4N DNA content in p53 knockdown cells, suggesting that BubR1 contributes to enhanced polyploidization by a mechanism other than its SAC function. In analysis of 182 gastric cancer specimens, we found that BubR1 expression was significantly high when p53 was positively stained, which indicates loss of p53 function (P = 0.0019). Moreover, positive staining of p53 and high expression of BubR1 in tumors were significantly correlated with DNA aneuploidy (P = 0.0065). These observations suggest that p53 deficiency may lead to the failure of BubR1 downregulation by OS and that p53 deficiency and BubR1 accumulation could contribute to gastric carcinogenesis associated with aneuploidy.We found that OS could contribute to the emergence of polyploid cells when p53 was deficient in normal human fibroblast cells. Importantly, this polyploidization could be suppressed by downregulating the expression of one spindle assembly checkpoint factor, BubR1. We also found that p53 dysfunction and BubR1 accumulation strongly correlate with the extent of aneuploidy in gastric cancer specimen and our data suggest that p53 deficiency and BubR1 accumulation could contribute to gastric carcinogenesis associated with aneuploidy.