• Early chromatin unfolding by RUNX1: a molecular explanation for differential requirements during specification versus maintenance of the hematopoietic gene expression program.

      Hoogenkamp, Maarten; Lichtinger, Monika; Krysinska, Hanna; Lancrin, Christophe; Clarke, Deborah; Williamson, Andrew J K; Mazzarella, Luca; Ingram, Richard; Jorgensen, Helle; Fisher, Amanda; et al. (2009-07-09)
      At the cellular level, development progresses through successive regulatory states, each characterized by their specific gene expression profile. However, the molecular mechanisms regulating first the priming and then maintenance of gene expression within one developmental pathway are essentially unknown. The hematopoietic system represents a powerful experimental model to address these questions and here we have focused on a regulatory circuit playing a central role in myelopoiesis: the transcription factor PU.1, its target gene colony-stimulating-factor 1 receptor (Csf1r), and key upstream regulators such as RUNX1. We find that during ontogeny, chromatin unfolding precedes the establishment of active histone marks and the formation of stable transcription factor complexes at the Pu.1 locus and we show that chromatin remodeling is mediated by the transient binding of RUNX1 to Pu.1 cis-elements. By contrast, chromatin reorganization of Csf1r requires prior expression of PU.1 together with RUNX1 binding. Once the full hematopoietic program is established, stable transcription factor complexes and active chromatin can be maintained without RUNX1. Our experiments therefore demonstrate how individual transcription factors function in a differentiation stage-specific manner to differentially affect the initiation versus maintenance of a developmental program.
    • Eight-channel iTRAQ enables comparison of the activity of six leukemogenic tyrosine kinases.

      Pierce, Andrew; Unwin, Richard D; Evans, Caroline A; Griffiths, Stephen D; Carney, Louise; Zhang, Liqun; Jaworska, Ewa; Lee, Chia-Fang; Blinco, David; Okoniewski, Michal J; et al. (2008-05)
      There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL, TEL/PDGFRbeta, FIP1/PDGFRalpha, D816V KIT, NPM/ALK, and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRalpha. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin, although this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes, illustrating the utility of such a combined approach.
    • Endoglin expression in blood and endothelium is differentially regulated by modular assembly of the Ets/Gata hemangioblast code.

      Pimanda, John E; Chan, Wan Y I; Wilson, Nicola K; Smith, Aileen M; Kinston, Sarah; Knezevic, Kathy; Janes, Mary E; Landry, Josette-Renee; Kolb-Kokocinski, Anja; Frampton, Jonathan; et al. (2008-12-01)
      Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast, early hematopoietic, and vascular development. We have previously shown that an upstream enhancer, Eng -8, together with the promoter region, mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements, we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8, +7+9 enhancers in both blood and endothelial cells. By contrast Pu.1, an Ets factor specific to the blood lineage, and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence.
    • Evaluation of circulating tumor cells and serological cell death biomarkers in small cell lung cancer patients undergoing chemotherapy.

      Hou, Jian-Mei; Greystoke, Alastair; Lancashire, Lee J; Cummings, Jeffrey; Ward, Timothy H; Board, Ruth E; Amir, Eitan; Hughes, Sarah; Krebs, Matthew G; Hughes, Andrew; et al. (2009-08)
      Serological cell death biomarkers and circulating tumor cells (CTCs) have potential uses as tools for pharmacodynamic blood-based assays and their subsequent application to early clinical trials. In this study, we evaluated both the expression and clinical significance of CTCs and serological cell death biomarkers in patients with small cell lung cancer. Blood samples from 88 patients were assayed using enzyme-linked immunosorbent assays for various cytokeratin 18 products (eg, M65, cell death, M30, and apoptosis) as well as nucleosomal DNA. CTCs (per 7.5 ml of blood) were quantified using Veridex CellSearch technology. Before therapeutic treatment, cell death biomarkers were elevated in patients compared with controls. CTCs were detected in 86% of patients; additionally, CD56 was detectable in CTCs, confirming their neoplastic origin. M30 levels correlated with the percentage of apoptotic CTCs. M30, M65, lactate dehydrogenase, and CTC number were prognostic for patient survival as determined by univariate analysis. Using multivariate analysis, both lactate dehydrogenase and M65 levels remained significant. CTC number fell following chemotherapy, whereas levels of serological cell death biomarkers peaked at 48 hours and fell by day 22, mirroring the tumor response. A 48-hour rise in nucleosomal DNA and M30 levels was associated with early response and severe toxicity, respectively. Our results provide a rationale to include the use of serological biomarkers and CTCs in early clinical trials of new agents for small cell lung cancer.
    • Exon level integration of proteomics and microarray data.

      Bitton, Danny A; Okoniewski, Michal J; Connolly, Yvonne; Miller, Crispin J; Cancer Research UK, Applied Computational Biology and Bioinformatics Group, Paterson Institute for Cancer Research, The University of Manchester, Christie Hospital Site, Wilmslow Road, Manchester, M20 4BX, UK. dbitton@picr.man.ac.uk (2008)
      BACKGROUND: Previous studies comparing quantitative proteomics and microarray data have generally found poor correspondence between the two. We hypothesised that this might in part be because the different assays were targeting different parts of the expressed genome and might therefore be subjected to confounding effects from processes such as alternative splicing. RESULTS: Using a genome database as a platform for integration, we combined quantitative protein mass spectrometry with Affymetrix Exon array data at the level of individual exons. We found significantly higher degrees of correlation than have been previously observed (r = 0.808). The study was performed using cell lines in equilibrium in order to reduce a major potential source of biological variation, thus allowing the analysis to focus on the data integration methods in order to establish their performance. CONCLUSION: We conclude that part of the variation observed when integrating microarray and proteomics data may occur as a consequence both of the data analysis and of the high granularity to which studies have until recently been limited. The approach opens up the possibility for the first time of considering combined microarray and proteomics datasets at the level of individual exons and isoforms, important given the high proportion of alternative splicing observed in the human genome.
    • Expression of the leukemia oncogene Lmo2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors.

      Landry, Josette-Renee; Bonadies, Nicolas; Kinston, Sarah; Knezevic, Kathy; Wilson, Nicola K; Oram, S Helen; Janes, Mary E; Piltz, Sandie; Hammett, Michelle; Carter, Jacinta; et al. (2009-06-04)
      The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.
    • Fission yeast MAP kinase Sty1 is recruited to stress-induced genes.

      Reiter, Wolfgang; Watt, Stephen; Dawson, Keren; Lawrence, Clare L; Bähler, Jürg; Jones, Nic; Wilkinson, Caroline R M; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, UK. (2008-04-11)
      The stress-induced expression of many fission yeast genes is dependent upon the Sty1 mitogen-activated protein kinase (MAPK) and Atf1 transcription factor. Atf1 is phosphorylated by Sty1 yet this phosphorylation is not required for stress-induced gene expression, suggesting another mechanism exists whereby Sty1 activates transcription. Here we show that Sty1 associates with Atf1-dependent genes and is recruited to both their promoters and coding regions. This occurs in response to various stress conditions coincident with the kinetics of the activation of Sty1. Association with promoters is not a consequence of increased nuclear accumulation of Sty1 nor does it require the phosphorylation of Atf1. However, recruitment is completely abolished in a mutant lacking Sty1 kinase activity. Both Atf1 and its binding partner Pcr1 are required for association of Sty1 with Atf1-dependent promoters, suggesting that this heterodimer must be intact for optimal recruitment of the MAPK. However, many Atf1-dependent genes are still expressed in a pcr1Delta mutant but with significantly delayed kinetics, thus providing an explanation for the relatively mild stress sensitivity displayed by pcr1Delta. Consistent with this delay, Sty1 and Atf1 cannot be detected at these promoters in this condition, suggesting that their association with chromatin is weak or transient in the absence of Pcr1.
    • 'Fit-for-purpose' validation of SearchLight multiplex ELISAs of angiogenesis for clinical trial use.

      Backen, Alison C; Cummings, Jeffrey; Mitchell, Claire L; Jayson, Gordon C; Ward, Timothy H; Dive, Caroline; CR-UK Translational Angiogenesis Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK. (2009-03-15)
      Validated assays of circulating biomarkers of angiogenesis to predict and determine the efficacy of vascular-targeted anticancer drugs would facilitate successful drug development. Multiple biomarker candidates exist and a multiplex approach was sought to minimise the requisite patient blood volume and to aid selection of those biomarkers with greatest potential clinical utility. Validation of the SearchLight multiplex ELISA platform comprising two multiplex assays of nine potential angiogenesis biomarkers was conducted (plex 1; VEGF R1 and R2, IL-8, KGF, PlGF; plex 2; PDGFbb, HGF, FGFb and VEGF). The study focused on instrument qualification, analyte specificity within the multiplex format, assay precision and reproducibility. No evidence was found within the multiplex that signals output from one analyte impinged on another or that antibody cross-reactivity occurred. Spike recovery for 5 between-experiment repeats was within +/-15% of input values for 7 of the 9 multiplexed analytes, with a coefficient of variation (CV) of <20% for 6 of the 9 analytes. Plasma samples from 8 ovarian cancer patients (who were not receiving therapy) were assessed using the two multiplexes on this platform to explore the likely baseline variability in this disease context. This study suggests that the platform and the multiplex approach will be useful to evaluate pharmacodynamic responses to vascular targeted therapy in early clinical trials.
    • Flipping of alkylated DNA damage bridges base and nucleotide excision repair.

      Tubbs, Julie L; Latypov, Vitaly F; Kanugula, Sreenivas; Butt, Amna; Melikishvili, Manana; Kraehenbuehl, Rolf; Fleck, Oliver; Marriott, Andrew S; Watson, Amanda J; Verbeek, Barbara; et al. (2009-06-11)
      Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O(6)-alkylguanine-DNA alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the reactive cysteine and alkyltransferase activity of AGT. Here we determine Schizosaccharomyces pombe ATL structures without and with damaged DNA containing the endogenous lesion O(6)-methylguanine or cigarette-smoke-derived O(6)-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating that ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to mammalian XPG (also known as ERCC5) and ERCC1 in S. pombe homologues Rad13 and Swi10 and biochemical interactions with Escherichia coli UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair.
    • Frequency of human T regulatory cells in peripheral blood is significantly reduced by cryopreservation.

      Elkord, Eyad; Clinical Immunotherapy Laboratory, Department of Medical Oncology, University of Manchester, Wilmslow Road, Manchester M204BX, UK. eelkord@picr.man.ac.uk (2009-08-15)
      Cryopreservation of peripheral blood mononuclear cells (PBMC) is essential for many clinical and research assays. Some studies reported consistent changes in PBMC phenotype following cryopreservation. We hypothesized that PBMC freezing may have a negative impact on estimation of the frequency of T regulatory cell (Treg). Treg levels were measured in 6 fresh PBMC samples isolated from 6 healthy donors and these levels were re-measured after freezing for three weeks. Herein, we report a significant reduction in Treg frequency in all samples following cryopreservation.
    • GSTM1 copy number and lung cancer risk.

      Crosbie, Philip A J; Barber, Philip V; Harrison, Kathryn L; Gibbs, Alan R; Agius, Raymond M; Margison, Geoffrey P; Povey, Andrew C; Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom. (2009-05-12)
      The GSTM1 null genotype is associated with a small increased lung cancer risk when compared to controls with at least one copy of the GSTM1 gene. As two copies of the GSTM1 gene might provide more protection than a single copy, we have determined GSTM1 copy number in a lung cancer case-control study. Cases with incident lung cancer were identified through a Bronchoscopy Unit and two separate hospital based control groups with non-malignant disease were selected with one from the same Bronchoscopy Unit and the other from a chest clinic at the same hospital. Subjects with at least one GSTM1 copy had a decreased lung cancer risk whatever the control group: the odds ratio (95% CI), after adjustment for age, gender and smoking duration, was 0.64 (0.41-0.98) and 0.54 (0.32-0.91) with bronchoscopy and chest clinic controls, respectively. Lung cancer risk varied with GSTM1 copy number with chest clinic controls only: the OR was 0.56 (0.32-0.97) for one copy of the GSTM1 gene and with two copies 0.43 (0.15-1.22), a trend that was significant (p=0.02): with bronchoscopy controls the trend was not significant (p=0.07). Results then confirm that the presence of GSTM1 provides protection against the risk of lung cancer. In addition there is equivocal evidence that this protection varies with the number of gene copies.
    • The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage.

      Lancrin, Christophe; Sroczynska, Patrycja; Stephenson, Catherine; Allen, Terence D; Kouskoff, Valerie; Lacaud, Georges; Cancer Research UK Stem Cell Biology Group. (2009-02-12)
      It has been proposed that during embryonic development haematopoietic cells arise from a mesodermal progenitor with both endothelial and haematopoietic potential called the haemangioblast. A conflicting theory instead associates the first haematopoietic cells with a phenotypically differentiated endothelial cell that has haematopoietic potential (that is, a haemogenic endothelium). Support for the haemangioblast concept was initially provided by the identification during mouse embryonic stem cell differentiation of a clonal precursor, the blast colony-forming cell (BL-CFC), which gives rise to blast colonies with both endothelial and haematopoietic components. Although recent studies have now provided evidence for the presence of this bipotential precursor in vivo, the precise mechanism for generation of haematopoietic cells from the haemangioblast still remains completely unknown. Here we demonstrate that the haemangioblast generates haematopoietic cells through the formation of a haemogenic endothelium intermediate, providing the first direct link between these two precursor populations. The cell population containing the haemogenic endothelium is transiently generated during BL-CFC development. This cell population is also present in gastrulating mouse embryos and generates haematopoietic cells on further culture. At the molecular level, we demonstrate that the transcription factor Tal1 (also known as Scl; ref. 10) is indispensable for the establishment of this haemogenic endothelium population whereas the core binding factor Runx1 (also known as AML1; ref. 11) is critical for generation of definitive haematopoietic cells from haemogenic endothelium. Together our results merge the two a priori conflicting theories on the origin of haematopoietic development into a single linear developmental process.
    • Hierarchical maintenance of MLL myeloid leukemia stem cells employs a transcriptional program shared with embryonic rather than adult stem cells.

      Somervaille, Tim C P; Matheny, Christina J; Spencer, Gary J; Iwasaki, Masayuki; Rinn, John L; Witten, Daniela M; Chang, Howard Y; Shurtleff, Sheila A; Downing, James R; Cleary, Michael L; et al. (2009-02-06)
      The genetic programs that promote retention of self-renewing leukemia stem cells (LSCs) at the apex of cellular hierarchies in acute myeloid leukemia (AML) are not known. In a mouse model of human AML, LSCs exhibit variable frequencies that correlate with the initiating MLL oncogene and are maintained in a self-renewing state by a transcriptional subprogram more akin to that of embryonic stem cells (ESCs) than to that of adult stem cells. The transcription/chromatin regulatory factors Myb, Hmgb3, and Cbx5 are critical components of the program and suffice for Hoxa/Meis-independent immortalization of myeloid progenitors when coexpressed, establishing the cooperative and essential role of an ESC-like LSC maintenance program ancillary to the leukemia-initiating MLL/Hox/Meis program. Enriched expression of LSC maintenance and ESC-like program genes in normal myeloid progenitors and poor-prognosis human malignancies links the frequency of aberrantly self-renewing progenitor-like cancer stem cells (CSCs) to prognosis in human cancer.
    • The histone acetyl transferase activity of monocytic leukemia zinc finger is critical for the proliferation of hematopoietic precursors.

      Perez-Campo, Flor-Maria; Borrow, Julian; Kouskoff, Valerie; Lacaud, Georges; Paterson Institute for Cancer Research, University of Manchester, USA. (2009-05-14)
      The monocytic leukemia zinc finger (MOZ) gene encodes a large multidomain protein that contains, besides other domains, 2 coactivation domains for the transcription factor Runx1/acute myeloid leukemia 1 and a histone acetyl transferase (HAT) catalytic domain. Recent studies have demonstrated the critical requirement for the complete MOZ protein in hematopoietic stem cell development and maintenance. However, the specific function of the HAT activity of MOZ remains unknown, as it has been shown that MOZ HAT activity is not required either for its role as Runx1 coactivator or for the leukemic transformation induced by MOZ transcriptional intermediary factor 2 (TIF2). To assess the specific requirement for this HAT activity during hematopoietic development, we have generated embryonic stem cells and mouse lines carrying a point mutation that renders the protein catalytically inactive. We report in this study that mice exclusively lacking the HAT activity of MOZ exhibit significant defects in the number of hematopoietic stem cells and hematopoietic committed precursors as well as a defect in B-cell development. Furthermore, we demonstrate that the failure to maintain a normal number of hematopoietic precursors is caused by the inability of HAT(-/-) cells to expand. These results indicate a specific role of MOZ-driven acetylation in controlling a desirable balance between proliferation and differentiation during hematopoiesis.
    • Identification of gene transcript signatures predictive for estrogen receptor and lymph node status using a stepwise forward selection artificial neural network modelling approach.

      Lancashire, Lee J; Rees, Robert C; Ball, Graham R; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. LLancashire@picr.man.ac.uk (2008-06)
      OBJECTIVE: The advent of microarrays has attracted considerable interest from biologists due to the potential for high throughput analysis of hundreds of thousands of gene transcripts. Subsequent analysis of the data may identify specific features which correspond to characteristics of interest within the population, for example, analysis of gene expression profiles in cancer patients to identify molecular signatures corresponding with prognostic outcome. These high throughput technologies have resulted in an unprecedented rate of data generation, often of high complexity, highlighting the need for novel data analysis methodologies that will cope with data of this nature. METHODS: Stepwise methods using artificial neural networks (ANNs) have been developed to identify an optimal subset of predictive gene transcripts from highly dimensional microarray data. Here these methods have been applied to a gene microarray dataset to identify and validate gene signatures corresponding with estrogen receptor and lymph node status in breast cancer. RESULTS: Many gene transcripts were identified whose expression could differentiate patients to very high accuracies based upon firstly whether they were positive or negative for estrogen receptor, and secondly whether metastasis to the axillary lymph node had occurred. A number of these genes had been previously reported to have a role in cancer. Significantly fewer genes were used compared to other previous studies. The models using the optimal gene subsets were internally validated using an extensive random sample cross-validation procedure and externally validated using a follow up dataset from a different cohort of patients on a newer array chip containing the same and additional probe sets. Here, the models retained high accuracies, emphasising the potential power of this approach in analysing complex systems. These findings show how the proposed method allows for the rapid analysis and subsequent detailed interrogation of gene expression signatures to provide a further understanding of the underlying molecular mechanisms that could be important in determining novel prognostic markers associated with cancer.
    • Immune evasion mechanisms in colorectal cancer liver metastasis patients vaccinated with TroVax (MVA-5T4).

      Elkord, Eyad; Dangoor, Adam; Burt, Deborah J; Southgate, Thomas D; Daayana, Sai; Harrop, Richard; Drijfhout, Jan W; Sherlock, David J; Hawkins, Robert E; Stern, Peter L; et al. (2009-02-17)
      We have recently reported the results of a phase II trial in which two TroVax [modified vaccinia ankara (MVA) encoding the tumour antigen 5T4] vaccinations were given to patients both pre- and post-surgical resection of liver metastases secondary to colorectal cancer (CRC). 5T4-specific cellular responses were assessed at the entry and 2 weeks after each vaccination by proliferation of fresh lymphocytes and ELISA for antibody responses; 18 from the 19 CRC patients mounted a 5T4-specific cellular and/or humoral response. Here, we present a comparison of individual and between patient responses over the course of the treatments using cryopreserved peripheral blood mononuclear cells (PBMC) samples from the baseline until after the fourth vaccination at 14 weeks. Assays used were proliferation assay with 5T4-Fc fusion protein, overlapping 32mer 5T4 peptides, MVA-LacZ and MVA-5T4 infected autologous monocytes. Responses to 5T4 protein or one or more peptide pools were pre-existing in 12/20 patients and subsequently 10 and 12 patients showed boosted and/or de novo responses, respectively. Cumulatively, 13/20 patients showed proliferative responses by week 14. We also assessed the levels of systemic T regulatory cells, plasma cytokine levels, phenotype of tumour-infiltrating lymphocytes including T regulatory cells and tumour HLA class I loss of expression. More than half of the patients showed phenotypes consistent with relative immune suppression and/or escape highlighting the complexity of positive and negative factors challenging any simple correlation with clinical outcome.
    • In silico screening and biological evaluation of inhibitors of Src-SH3 domain interaction with a proline-rich ligand.

      Atatreh, Noor; Stojkoski, Cvetan; Smith, Phillippa; Booker, Grant W; Dive, Caroline; Frenkel, A David; Freeman, Sally; Bryce, Richard A; School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PT, UK. (2008-02-01)
      Src signalling and transduction are directly involved in cell growth, cell cycle, malignant transformation and cell migration, providing therapeutic opportunities through inhibition of Src. Here we report virtual screening for novel compounds that inhibit the Src-SH3 protein-protein interaction with a proline-rich peptide ligand. Computational docking of the ZINC compound database was performed using GOLD. Top-scoring compounds were assayed using a fluorescence polarization-based assay. A benzoquinoline derivative showed micromolar inhibition of binding between Src-SH3 and the proline-rich peptide. Several analogues were subsequently assayed showing the requirement of a linker between the benzoquinoline and phenyl rings, and electron donating substituents on the phenyl ring.
    • Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast.

      Sanchez-Diaz, Alberto; Marchesi, Vanessa; Murray, Stephen M; Jones, Richard C; Pereira, Gislene; Edmondson, Ricky D; Allen, Terence D; Labib, Karim; Cancer Research U.K., Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK. (2008-04)
      By rapidly depleting each of the essential budding yeast proteins of unknown function, we identified a novel factor that we call Inn1, which associates with the contractile actomyosin ring at the end of mitosis and is needed for cytokinesis. We show that Inn1 has a C2 domain at the amino terminus of the protein that is required for ingression of the plasma membrane, whereas the remainder of the protein recruits Inn1 to the actomyosin ring. The lethal effects of deleting the INN1 gene can be suppressed by artificial fusion of the C2 domain to other components of the actomyosin ring, restoring membrane ingression on contraction of the actomyosin ring. Our data indicate that recruitment of the C2 domain of Inn1 to the contractile actomyosin ring is crucial for ingression of the plasma membrane during cytokinesis in budding yeast.
    • Int6/eIF3e promotes general translation and Atf1 abundance to modulate Sty1 MAPK-dependent stress response in fission yeast.

      Udagawa, T; Nemoto, N; Wilkinson, Caroline R M; Narashimhan, J; Jiang, L; Watt, S; Zook, A; Jones, Nic; Wek, R; Bähler, J; et al. (2008-08-08)
      int-6 is one of the frequent integration sites for mouse mammary tumor viruses. Although its product is the e-subunit of translation initiation factor eIF3, other evidence indicates that it interacts with proteasomes or other proteins to regulate protein stability. Here we report that the fission yeast int6(+) is required for overcoming stress imposed by histidine starvation, using the drug 3-aminotriazole (3AT). Microarray and complementary Northern studies using wild-type, int6Delta or gcn2Delta mutants indicate that 3AT-treated wild-type yeast induces core environmental stress response (CESR) genes in addition to typical general amino acid control (GAAC) genes whose transcription depends on the eIF2 kinase, Gcn2. In agreement with this, Sty1 MAPK and its target transcription factor Atf1, which signal the CESR, are required for overcoming 3AT-induced starvation. We find that Int6 is required for maintaining the basal level of Atf1 and for rapid transcriptional activation of the CESR on 3AT-insult. Pulse labeling experiments indicate that int6Delta significantly slows down de novo protein synthesis. Moreover, Atf1 protein half-life was reduced in int6Delta cells. These effects would account for the compromised Atf1 activity on 3AT-induced stress. Thus, the robust protein synthesis promoted by intact eIF3 appears to be a part of the requisites for sound Sty1 MAPK-dependent signaling governed by the activity of the Atf1 transcription factor.
    • An introduction to artificial neural networks in bioinformatics--application to complex microarray and mass spectrometry datasets in cancer studies.

      Lancashire, Lee J; Lemetre, Christophe; Ball, Graham R; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK. llancashire@picr.man.ac.uk (2009-05)
      Applications of genomic and proteomic technologies have seen a major increase, resulting in an explosion in the amount of highly dimensional and complex data being generated. Subsequently this has increased the effort by the bioinformatics community to develop novel computational approaches that allow for meaningful information to be extracted. This information must be of biological relevance and thus correlate to disease phenotypes of interest. Artificial neural networks are a form of machine learning from the field of artificial intelligence with proven pattern recognition capabilities and have been utilized in many areas of bioinformatics. This is due to their ability to cope with highly dimensional complex datasets such as those developed by protein mass spectrometry and DNA microarray experiments. As such, neural networks have been applied to problems such as disease classification and identification of biomarkers. This review introduces and describes the concepts related to neural networks, the advantages and caveats to their use, examples of their applications in mass spectrometry and microarray research (with a particular focus on cancer studies), and illustrations from recent literature showing where neural networks have performed well in comparison to other machine learning methods. This should form the necessary background knowledge and information enabling researchers with an interest in these methodologies, but not necessarily from a machine learning background, to apply the concepts to their own datasets, thus maximizing the information gain from these complex biological systems.