Normal Cell Division Research Articles

Abstract 4106: Cool-1-mediated inhibition of c-Cbl as a therapuetic target, which modulates multiple critical properties of glioblastomas.

Abstract Glioblastomas (GBMs), the most malignant glial tumors, differ from normal glial progenitor cells in many ways, but little is known about the molecular circuitry underlying these differences. We have discovered a novel means by which GBMs become chemo-resistant, based on inhibition of the redox/Fyn/c-Cbl pathway by overexpression of Cool-1(Beta-Pix). The protein c-Cbl, an E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of multiple receptor tyrosine kinases critical for cell division and cell survival. In normal glial progenitor cells of the CNS, exposure to chemotherapy oxidizes cells, leading to sequential activation of Fyn and c-Cbl. However in GBM cells, exposure to BCNU or other chemical pro-oxidants does not lead to c-Cbl activation. Further studies demonstrated that these agents do cause Fyn activation, suggesting that GBMs inhibit c-Cbl phosphorylation leading to decreased degradation of EGFR, accounting for increased EGFR signaling. We next discovered that the decrease in c-Cbl activation via the redox/Fyn/c-Cbl pathway was due to c-Cbl sequestration by Cool-1, which is overexpressed in GBM cells and samples of tumors. We found that oxidant-associated c-Cbl activation can be restored by shRNA-mediated inhibition of Cool-1 expression, leading to restoration of normal c-Cbl-mediated degradation of RTKs (including EGFR). We further found that restoration of Cool-1 activity modulated multiple other hallmarks of GBM, including changes in migration, cell cycle parameters, and altering the cancer stem cell phenotype of our cells. In contrast, Cool-1/c-Cbl complexes were not found in normal brain or normal glial progenitors and Cool-1 knockdown did not reduce progenitor cell division. The translation of these findings into an in vivo intracranial xenograft model of GBM showed that Cool-1 is essential for tumorigenesis and decreasing Cool-1 levels leads to decreased tumor take, decreased tumor growth, and increased survival. In sum, the ability of Cool-1/c-Cbl interactions to modulate a variety of tumor cell properties suggests that these interactions offer an attractive target for modulating GBM growth, particularly due to the absence of Cool-1/c-Cbl complexes in normal brain tissue and to the lack of effects of Cool-1 knockdown on division of normal glial progenitor cells. The activation of c-Cbl activity through pharmacological agents is a potentially viable strategy for the specific eradication of GBM cells. Citation Format: Brett M. Stevens, Christopher J. Folts, Warner Chen, Addie L. Bardin, Mark Noble. Cool-1-mediated inhibition of c-Cbl as a therapuetic target, which modulates multiple critical properties of glioblastomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4106. doi:10.1158/1538-7445.AM2013-4106

The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line

Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.

Rab24 is Required for Normal Cell Division

Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co-localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i.e., chromatin bridges). Furthermore, in CHO cells stably transfected with GFP-Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.

Cell-Cycle Perturbations Suppress the Slow-Growth Defect ofspt10ΔMutants inSaccharomyces cerevisiae

Spt10 is a putative acetyltransferase of Saccharomyces cerevisiae that directly activates the transcription of histone genes. Deletion of SPT10 causes a severe slow growth phenotype, showing that Spt10 is critical for normal cell division. To gain insight into the function of Spt10, we identified mutations that impair or improve the growth of spt10 null (spt10Δ) mutants. Mutations that cause lethality in combination with spt10Δ include particular components of the SAGA complex as well as asf1Δ and hir1Δ. Partial suppressors of the spt10Δ growth defect include mutations that perturb cell-cycle progression through the G1/S transition, S phase, and G2/M. Consistent with these results, slowing of cell-cycle progression by treatment with hydroxyurea or growth on medium containing glycerol as the carbon source also partially suppresses the spt10Δ slow-growth defect. In addition, mutations that impair the Lsm1-7−Pat1 complex, which regulates decapping of polyadenylated mRNAs, also partially suppress the spt10Δ growth defect. Interestingly, suppression of the spt10Δ growth defect is not accompanied by a restoration of normal histone mRNA levels. These findings suggest that Spt10 has multiple roles during cell division.

Protein Complexes and Proteolytic Activation of the Cell Wall Hydrolase RipA Regulate Septal Resolution in Mycobacteria

Peptidoglycan hydrolases are a double-edged sword. They are required for normal cell division, but when dysregulated can become autolysins lethal to bacteria. How bacteria ensure that peptidoglycan hydrolases function only in the correct spatial and temporal context remains largely unknown. Here, we demonstrate that dysregulation converts the essential mycobacterial peptidoglycan hydrolase RipA to an autolysin that compromises cellular structural integrity. We find that mycobacteria control RipA activity through two interconnected levels of regulation in vivo—protein interactions coordinate PG hydrolysis, while proteolysis is necessary for RipA enzymatic activity. Dysregulation of RipA protein complexes by treatment with a peptidoglycan synthase inhibitor leads to excessive RipA activity and impairment of correct morphology. Furthermore, expression of a RipA dominant negative mutant or of differentially processed RipA homologues reveals that RipA is produced as a zymogen, requiring proteolytic processing for activity. The amount of RipA processing differs between fast-growing and slow-growing mycobacteria and correlates with the requirement for peptidoglycan hydrolase activity in these species. Together, the complex picture of RipA regulation is a part of a growing paradigm for careful control of cell wall hydrolysis by bacteria during growth, and may represent a novel target for chemotherapy development.

Mouse Versus Human Early Embryogenesis: What Can We Learn from Time-Lapse Microscopy (TLM)?

Early embryogenesis relies on key mitotic events for normal cell division and differentiation. Proper division timing has been correlated with an embryo’s success in developing to the blastocyst stage.

The Holliday junction resolvase RecU is required for chromosome segregation and DNA damage repair in Staphylococcus aureus

BackgroundThe Staphylococcus aureus RecU protein is homologous to a Bacillus subtilis Holliday junction resolvase. Interestingly, RecU is encoded in the same operon as PBP2, a penicillin-binding protein required for cell wall synthesis and essential for the full expression of resistance in Methicillin Resistant S. aureus strains. In this work we have studied the role of RecU in the clinical pathogen S. aureus.ResultsDepletion of RecU in S. aureus results in the appearance of cells with compact nucleoids, septa formed over the DNA and anucleate cells. RecU-depleted cells also show increased septal recruitment of the DNA translocase SpoIIIE, presumably to resolve chromosome segregation defects. Additionally cells are more sensitive to DNA damaging agents such as mitomycin C or UV radiation. Expression of RecU from the ectopic chromosomal spa locus showed that co-expression of RecU and PBP2 was not necessary to ensure correct cell division, a process that requires tight coordination between chromosome segregation and septal cell wall synthesis.ConclusionsRecU is required for correct chromosome segregation and DNA damage repair in S. aureus. Co-expression of recU and pbp2 from the same operon is not required for normal cell division.

Direct Measurements of Human Colon Crypt Stem Cell Niche Genetic Fidelity: The Role of Chance in Non-Darwinian Mutation Selection

Perfect human stem cell genetic fidelity would prevent aging and cancer. However, perfection would be difficult to achieve, and aging is universal and cancers common. A hypothesis is that because mutations are inevitable over a human lifetime, downstream mechanisms have evolved to manage the deleterious effects of beneficial and lethal mutations. In the colon, a crypt stem cell architecture reduces the number of mitotic cells at risk for mutation accumulation, and multiple niche stem cells ensure that a lethal mutation within any single stem cell does not lead to crypt death. In addition, the architecture of the colon crypt stem cell niche may harness probability or chance to randomly discard many beneficial mutations that might lead to cancer. An analysis of somatic chromosome copy number alterations (CNAs) reveals a lack of perfect fidelity in individual normal human crypts, with age-related increases and higher frequencies in ulcerative colitis, a proliferative, inflammatory disease. The age-related increase in somatic CNAs appears consistent with relatively normal replication error and cell division rates. Surprisingly, and similar to point mutations in cancer genomes, the types of crypt mutations were more consistent with random fixation rather than selection. In theory, a simple “non-Darwinian” way to nullify selection is to reduce the size of the reproducing population. Fates are more determined by chance rather than selection in very small populations, and therefore selection may be minimized within small crypt niches. The desired effect is that many beneficial mutations that might lead to cancer are randomly lost by drift rather than fixed by selection. The subdivision of the colon into multiple very small stem cell niches may trade Darwinian evolution for non-Darwinian somatic cell evolution, capitulating to aging but reducing cancer risks.

MeCP2 modulates gene expression pathways in astrocytes

BackgroundMutations in MECP2 encoding methyl-CpG-binding protein 2 (MeCP2) cause the X-linked neurodevelopmental disorder Rett syndrome. Rett syndrome patients exhibit neurological symptoms that include irregular breathing, impaired mobility, stereotypic hand movements, and loss of speech. MeCP2 protein epigenetically modulates gene expression through genome-wide binding to methylated CpG dinucleotides. While neurons have the highest level of MeCP2 expression, astrocytes and other cell types also express detectable levels of MeCP2. Recent studies suggest that astrocytes likely control the progression of Rett syndrome. Thus, the object of these studies was to identify gene targets that are affected by loss of MeCP2 binding in astrocytes.MethodsTo identify gene targets of MeCP2 in astrocytes, combined approaches of expression microarray and chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) were compared between wild-type and MeCP2-deficient astrocytes. MeCP2 gene targets were compared with genes in the top 10% of MeCP2 binding levels in gene windows either within 2 kb upstream of the transcription start site, or the ‘gene body’ that extended from transcription start to end site, or 2 kb downstream of the transcription end site.ResultsA total of 118 gene transcripts surpassed the highly significant threshold (P < 0.005, fold change > 1.2) in expression microarray analysis from triplicate cultures. The top 10% of genes with the highest levels of MeCP2 binding were identified in two independent ChIP-seq experiments. Together this integrated, genome-wide screen for MeCP2 target genes provided an overlapping list of 19 high-confidence MeCP2-responsive gene transcripts in astrocytes. Validation of candidate target gene transcripts by RT-PCR revealed that expression of Apoc2, Cdon, Csrp and Nrep were consistently responsive to MeCP2 deficiency in astrocytes.ConclusionsThe first MeCP2 ChIP-seq and gene expression microarray analysis in astrocytes reveals a set of potential MeCP2 target genes that may contribute to normal astrocyte signaling, cell division and neuronal support functions, the loss of which may contribute to the Rett syndrome phenotype.

Antitumor activity of L-asparaginase from Erwinia Carotovora from against different leukemic and solid tumours cell lines

We have studied dose- and time-dependent antitumor and cytotoxic effects of Erwinia carotovora L-asparaginase (ECAR LANS) and Escherichia coli L-asparaginase (MEDAC) on human leukemic cells and human and animal solid tumor cells. We determined the sensitivity of tumor cells to L-asparaginases, as well the effect L-asparaginases on cell growth rate, protein and DNA synthesis per se and with addition of different cytostatics. The data obtained demonstrated that ECAR LANS L-asparaginase suppressed growth of all tested solid tumor cells. Evaluation of leukemic cell number after treatment with L-asparaginases for 24, 48 and 72 h demonstrated that asparagine deficiency did not kill cells but stopped normal cell division and had no effect on protein and DNA synthesis. Cytofluorometric study of solid and leukemic cells demonstrated that the treatment with L-asparaginase for 72 h did not change cell cycle phase distribution and did not increase the number of apoptotic cells. The HL-60 cell line was only exemption. At the same time, cells treatment with L-asparaginase and doxorubicin combination leaded to increase of apoptotypical cell number to 60% for MCF7 cells, to 40% for Jurkat cells and to 99% for HL-60 cells. We have excluded apoptosis as main reason for tumor cell death after asparaginase treatment because multi resistant Jurkat/A4 cells have been asparaginase sensitive. We have not found ECAR LANS L-asparaginase effect on normal human fibroblasts growth ability and we had come to conclusion that enzyme cytotoxcisity related only with asparagine deficiency.

ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress

Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease (MNase) that is normalized for nucleosome density, the NCAM (normalized chromatin accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress, revealing novel functions for the kinase in replication stress response. These results suggest a possibility that Mec1 may facilitate DNA replication fork progression during replication stress by increasing chromatin accessibility around replication forks.

Plant Vascular Cell Division Is Maintained by an Interaction between PXY and Ethylene Signalling

The procambium and cambium are meristematic tissues from which vascular tissue is derived. Vascular initials differentiate into phloem towards the outside of the stem and xylem towards the inside. A small peptide derived from CLV-3/ESR1-LIKE 41 (CLE41) is thought to promote cell divisions in vascular meristems by signalling through the PHLOEM INTERCALLATED WITH XYLEM (PXY) receptor kinase. pxy mutants, however, display only small reductions in vascular cell number, suggesting a mechanism exists that allows plants to compensate for the absence of PXY. Consistent with this idea, we identify a large number of genes specifically upregulated in pxy mutants, including several AP2/ERF transcription factors. These transcription factors are required for normal cell division in the cambium and procambium. These same transcription factors are also upregulated by ethylene and in ethylene-overproducing eto1 mutants. eto1 mutants also exhibit an increase in vascular cell division that is dependent upon the function of at least 2 of these ERF genes. Furthermore, blocking ethylene signalling using a variety of ethylene insensitive mutants such as ein2 enhances the cell division defect of pxy. Our results suggest that these factors define a novel pathway that acts in parallel to PXY/CLE41 to regulate cell division in developing vascular tissue. We propose a model whereby vascular cell division is regulated both by PXY signalling and ethylene/ERF signalling. Under normal circumstances, however, PXY signalling acts to repress the ethylene/ERF pathway.

Influenza Virus A/Beijing/501/2009(H1N1) NS1 Interacts with β-Tubulin and Induces Disruption of the Microtubule Network and Apoptosis on A549 Cells

NS1 of influenza A virus is a key multifunctional protein that plays various roles in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. These functions rely on its ability to participate in a multitude of protein-protein and protein-RNA interactions. To gain further insight into the role of NS1, a tandem affinity purification (TAP) method was utilized to find unknown interaction partner of NS1. The protein complexes of NS1 and its interacting partner were purified from A549 cell using TAP-tagged NS1 as bait, and co-purified cellular factors were identified by mass spectrometry (MS). We identified cellular β-tubulin as a novel interaction partner of NS1. The RNA-binding domain of NS1 interacts with β-tubulin through its RNA-binding domain, as judged by a glutathione S-transferase (GST) pull-down assay with the GST-fused functional domains of NS1. Immunofluorescence analysis further revealed that NS1 with β-tubulin co-localized in the nucleus. In addition, the disruption of the microtubule network and apoptosis were also observed on NS1-transfected A549 cells. Our findings suggest that influenza A virus may utilize its NS1 protein to interact with cellular β-tubulin to further disrupt normal cell division and induce apoptosis. Future work will illustrate whether this interaction is uniquely specific to the 2009 pandemic H1N1 virus.

Loss of expression of the double strand break repair protein ATM is associated with worse prognosis in colorectal cancer and loss of Ku70 expression is associated with CIN

Repair of double strand DNA breaks (DSBs) is pivotal in maintaining normal cell division and disruption of this system has been shown to be a key factor in carcinogenesis. Loss of expression of the DSB repair proteins have previously been shown to be associated with poorer survival in colorectal cancer. We wished to ascertain the relationship of altered expression of the DSB repair proteins γ-H2AX (gamma-H2AX), ATM and Ku70 with biological and clinico-pathological features of colorectal cancer. 908 tumours from the VICTOR clinical trial of stage II/III colorectal cancer were analysed for expression of γ-H2AX, ATM and Ku70 using immunohistochemistry. Expression levels were correlated with CIN and with disease-free survival, correcting for microsatellite instability, BRAF/KRAS mutation status, Dukes stage, chemo/radiotherapy, age, gender and tumour location. Down-regulated Ku70 expression was associated with chromosomal instability (p=0.029) in colorectal cancer. Reduced ATM expression was an independent marker of poor disease-free survival (HR=1.67, 95% CI 1.11-2.50, p=0.015). For Ku70, further studies are required to investigate the potential relationship of non-homologous end joining with chromosomal instability. Loss of ATM expression might serve as a biomarker of poor prognosis in colorectal cancer.

Antitumor activity of L-asparaginase from Erwinia carotovora against different human and animal leukemic and solid tumor cell lines

The dose- and time-dependent antitumor and cytotoxic effects of L-asparaginases from Erwinia carotovora (ECAR LANS) and Escherichia coli (MEDAC) have been investigated using human leukemic cells and human and animal solid tumor cells. These included human T-cell acute lymphoblastic leukemia cell lines (Jurkat, Jurkat/A4, Molt-4), human chronic myeloid leukemia K562 cells, human promyelocytic leukemia HL-60, and also human solid tumor cells (prostate carcinoma LnCap, breast adenocarcinoma MCF7, ovarian adenocarcinoma SCOV-3 and carcinoma CaOV, hepatocarcinoma Hep G2, fibrosarcoma HT-1080) and animal solid tumor cells (rat Gasser’s ganglion neurinoma cells GGNC-1, mouse glioblastoma EPNT-5). We investigated sensitivity of tumor cells (seeded at different density) to L-asparaginases, as well the effect of L-asparaginases on cell growth rate, protein and DNA synthesis in the presence of various cytostatics. Cell cycle analysis by flow cytofluorimetry and detection of apoptotic cells before and after treatment with L-asparaginases indicate that ECAR LANS L-asparaginase suppressed growth of all tested solid tumor cells. Evaluation of leukemic cell number after treatment with L-asparaginases for 24, 48 and 72 h demonstrated that asparagine deficiency did not kill cells but stopped normal cell division. The cytofluorometric study of solid and leukemic cells revealed that except HL-60 cells the treatment with L-asparaginase for 72 h did not change cell cycle phase distribution and did not increase the number of apoptotic cells. Combined treatment of cells using a combination of L-asparaginase and doxorubicin significantly increased the number of apoptotic cells up to 60% (MCF-7 cells), 40% (Jurkat cells) and even 99% (HL-60). High levels of DNA and protein synthesis rates in asparaginase-treated tumor cells suggest lack of massive entry of tumor cells to apoptosis. This conclusion is based on the fact of sensitivity of multi-resistant Jurkat/A4 cells to L-asparaginases (it is nearly impossible to induce apoptosis in these cells). Since ECAR LANS did not influence growth of normal human fibroblasts it appears that the enzyme cytotoxicity is associated only asparagine deficiency.

Aurora A kinase (AURKA) in normal and pathological cell division

Temporally and spatially controlled activation of the Aurora A kinase (AURKA) regulates centrosome maturation, entry into mitosis, formation and function of the bipolar spindle, and cytokinesis. Genetic amplification and mRNA and protein overexpression of Aurora A are common in many types of solid tumor, and associated with aneuploidy, supernumerary centrosomes, defective mitotic spindles, and resistance to apoptosis. These properties have led Aurora A to be considered a high-value target for development of cancer therapeutics, with multiple agents currently in early-phase clinical trials. More recently, identification of additional, non-mitotic functions and means of activation of Aurora A during interphase neurite elongation and ciliary resorption have significantly expanded our understanding of its function, and may offer insights into the clinical performance of Aurora A inhibitors. Here we review the mitotic and non-mitotic functions of Aurora A, discuss Aurora A regulation in the context of protein structural information, and evaluate progress in understanding and inhibiting Aurora A in cancer.

Folic acid inhibits endothelial cell proliferation through activating the cSrc/ERK 2/NF-κB/p53 pathway mediated by folic acid receptor

Folate is important for normal cell division. Folate deficiency has been implicated in various diseases, including atherosclerosis, neural tube defects, and cancer. However, the effect of folate on angiogenesis was unclear. The aim of this study was to investigate the anti-angiogenic action of folic acid (FA). FA (0-10 μmol/L) concentration-dependently decreased DNA synthesis and proliferation in cultured human umbilical venous endothelial cells (HUVEC). Western blot analyses demonstrated that the levels of p21, p27 and p53 protein in HUVEC were increased by FA. The FA-inhibited [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were knocked-down together. Knock-down of p53 prevented the FA-induced increases in p21 and p27 protein level. The levels of phosphorylated Src (p-Src) and p-Src-FA receptor (FR) complex in HUVEC were increased by FA. Knock-down of FR reduced the FA-induced increases of p-Src and p53. The FA-induced increases of p21, p27 and p53 protein levels were abolished when cSrc was knocked-down. FA also increased NF-κB nuclear translocation and binding onto the p53 promoter. The FA-induced up-regulation of the p53 promoter activity was prevented by knocked-down of ERK. Matrigel angiogenesis assay in mice demonstrate the anti-angiogenic effect of FA in vivo. In conclusion, our data indicate that FA bound to FR in HUVEC, subsequently activated the cSrc/ERK 2/NF-κB/p53 signaling pathway, which in turn up-regulated the expression of p21 and p27, and finally resulted in cell cycle arrest at the G0/G1 phase. In the present study, we uncover a completely novel role of FA for anti-angiogenesis.

Chryseolinea serpens gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from soil

An aerobic chemoheterotrophic gliding bacterium, designated RYG(T), was isolated from a soil in Germany. Cells were Gram-stain-negative, thin rods (0.4-0.6 µm in width and 2.0-5.5 µm in length). Cells multiplied by normal cell division and no resting stages were observed. Colonies were yellow and displayed swarming edges. Gliding motility was observed in wet mounts. Strain RYG(T) grew at pH 5.6-7.7 (optimum pH 6.6-7.0), at 13-37 °C (optimum 25-30 °C) and with 0-1.0 % NaCl (optimum 0-0.1 %). The isolate was incapable of atmospheric nitrogen fixation and grew on most mono- and disaccharides as well as a few polysaccharides and organic acids. The predominant menaquinone was MK-7, the major cellular fatty acids were C(16 : 1)ω5c and iso-C(15 : 0) and the major intact polar lipids were composed of phosphatidylethanolamine derivatives and two unknown series. The DNA G+C content was 49.9 mol%. Based on 16S rRNA gene sequence analysis, the isolate belonged to the phylum Bacteroidetes, class Cytophagia, order Cytophagales, but was only distantly related to any cultured bacteria. The closest relatives were Ohtaekwangia koreensis 3B-2(T) and Ohtaekwangia kribbensis 10AO(T) (both 93 % 16S rRNA gene sequence similarity). We propose a novel genus and species, Chryseolinea serpens gen. nov., sp. nov.. Strain RYG(T) ( = DSM 24574(T) = ATCC BAA-2075(T)) is the type strain.

Small proteins link coat and cortex assembly during sporulation in Bacillus subtilis

Mature spores of the bacterium Bacillus subtilis are encased by two concentric shells: an inner shell (the 'cortex'), made of peptidoglycan; and an outer proteinaceous shell (the 'coat'), whose basement layer is anchored to the surface of the developing spore via a 26-amino-acid-long protein called SpoVM. During sporulation, initiation of cortex assembly depends on the successful initiation of coat assembly, but the mechanisms that co-ordinate the morphogenesis of both structures are largely unknown. Here, we describe a sporulation pathway involving SpoVM and a 37-amino-acid-long protein named 'CmpA' that is encoded by a previously un-annotated gene and is expressed under control of two sporulation-specific transcription factors (σ(E) and SpoIIID). CmpA localized to the surface of the developing spore and deletion of cmpA resulted in cells progressing through the sporulation programme more quickly. Overproduction of CmpA did not affect normal growth or cell division, but delayed entry into sporulation and abrogated cortex assembly. In those cells that had successfully initiated coat assembly, CmpA was removed by a post-translational mechanism, presumably in order to overcome the sporulation inhibition it imposed. We propose a model in which CmpA participates in a developmental checkpoint that ensures the proper orchestration of coat and cortex morphogenesis by repressing cortex assembly until coat assembly successfully initiates.

Abstract 2074: Cool-1-mediated inhibition of c-Cbl modulates multiple critical properties of glioblastomas, including the ability to generate tumors in vivo

Abstract Glioblastomas (GBMs), the most malignant glial tumors, differ from normal glial progenitor cells in many ways, but little is known about the molecular circuitry underlying these differences. We have discovered a novel means by which GBMs become chemo-resistant, based on inhibition of the redox/Fyn/c-Cbl pathway by overexpression of Cool-1. The protein c-Cbl, an E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of multiple receptor tyrosine kinases critical for cell division and cell survival. In normal glial progenitor cells of the CNS, exposure to chemotherapy oxidizes cells, leading to sequential activation of Fyn and c-Cbl. However in GBM cells, exposure to BCNU or other chemical pro-oxidants does not lead to c-Cbl activation. Further studies have demonstrated that these agents do cause Fyn activation, suggesting that GBMs inhibit c-Cbl phosphorylation leading to decreased degradation of EGFR, accounting for increased EGFR signaling. We therefore set out to investigate the factors involved in the decreased c-Cbl activity specific to GBMs. In this work we discovered that oxidant-associated c-Cbl activation can be restored by suppressing expression of the protein Cool-1, found to be over-expressed in GBM. Our data further demonstrate that Cool-1 and c-Cbl formed complexes, that genetic reduction in Cool-1 levels restored normal c-Cbl-mediated degradation of RTKs (including EGFR) in response to exposure to pro-oxidants, and that restoration of normal c-Cbl activation has multiple effects on hallmarks of GBM. Cool-1 decreases lead to decreased migration and decreased numbers of cells in the cell cycle. Cool-1 inhibition leads to decreased number of cells that exhibit cancer stem cells characteristics including decreased antigen positivity and decreased sphere forming ability. In contrast, Cool-1/c-Cbl complexes were not found in normal brain or normal glial progenitors and Cool-1 knockdown did not reduce progenitor division. The translation of these findings into an in vivo intracranial xenograft model of GBM shows that Cool-1 is essential for tumorigenesis and decreasing Cool-1 levels leads to decreased tumor take, decreased tumor growth, and increased survival. In sum, the ability of Cool-1/c-Cbl interactions, to modulate a variety of tumor cell properties suggests that these interactions offer an attractive target for modulating GBM growth, particularly due to the absence of Cool-1/c-Cbl complexes in normal brain tissue and to the lack of effects of Cool-1 knockdown on division of normal glial progenitor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2074. doi:1538-7445.AM2012-2074