• A casein kinase 1 and PAR proteins regulate asymmetry of a PIP(2) synthesis enzyme for asymmetric spindle positioning.

      Panbianco, Costanza; Weinkove, David; Zanin, Esther; Jones, David R; Divecha, Nullin; Gotta, Monica; Ahringer, Julie; The Gurdon Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB21QN, UK. (2008-08)
      Spindle positioning is an essential feature of asymmetric cell division. The conserved PAR proteins together with heterotrimeric G proteins control spindle positioning in animal cells, but how these are linked is not known. In C. elegans, PAR protein activity leads to asymmetric spindle placement through cortical asymmetry of Galpha regulators GPR-1/2. Here, we establish that the casein kinase 1 gamma CSNK-1 and a PIP(2) synthesis enzyme (PPK-1) transduce PAR polarity to asymmetric Galpha regulation. PPK-1 is posteriorly enriched in the one-celled embryo through PAR and CSNK-1 activities. Loss of CSNK-1 causes uniformly high PPK-1 levels, high symmetric cortical levels of GPR-1/2 and LIN-5, and increased spindle pulling forces. In contrast, knockdown of ppk-1 leads to low GPR-1/2 levels and decreased spindle forces. Furthermore, loss of CSNK-1 leads to increased levels of PIP(2). We propose that asymmetric generation of PIP(2) by PPK-1 directs the posterior enrichment of GPR-1/2 and LIN-5, leading to posterior spindle displacement.
    • CD4+ T-cell recognition of human 5T4 oncofoetal antigen: implications for initial depletion of CD25+ T cells.

      Elkord, Eyad; Burt, Deborah J; Drijfhout, Jan W; Hawkins, Robert E; Stern, Peter L; Department of Immunology, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. eelkord@picr.man.ac.uk (2008-06)
      BACKGROUND: The human 5T4 (h5T4) oncofoetal antigen is expressed by a wide variety of human carcinomas including colorectal, ovarian, gastric and renal, but rarely on normal tissues. Its restricted expression on tumour tissues as well as its association with tumour progression and bad prognosis has driven the development of a MVA-based vaccine (TroVax) which has been tested in several early phase clinical trials and these studies have led to the start of a phase III trial in renal cell carcinoma patients. We have recently shown that CD8(+) T cells recognizing h5T4 can be generated in the absence of CD4(+) T cells from peripheral blood lymphocytes of human healthy individuals. RESULTS: We report the existence and expansion of human CD4(+) T cells against h5T4 by stimulation with autologous monocyte-derived dendritic cells infected with a replication defective adenovirus encoding the h5T4 cDNA (Ad-h5T4). The h5T4-specific T-cell responses in normal individuals are enhanced by initial depletion of CD25(+) cells (putative T regulatory cells) prior to the in vitro stimulation. We have identified a novel h5T4-derived 15-mer peptide recognized by CD4(+) T cells in HLA-DR4 positive healthy individuals. Interestingly, CD4(+) T cells spontaneously recognizing a different 5T4 epitope restricted by HLA-DR were identified in tumour-infiltrating lymphocytes isolated from a regressing renal cell carcinoma lung metastasis. CONCLUSION: Our data show that CD4(+) T cells recognizing h5T4 can be expanded and detected in healthy individuals and a renal cell carcinoma patient. Such h5T4-specific CD4(+) T cells boosted or induced by vaccination could act to modulate both cell or antibody mediated anti-tumour responses.
    • The cellular adaptations to hypoxia as novel therapeutic targets in childhood cancer.

      Adamski, J K; Estlin, Edward J; Makin, Guy W J; School of Cancer and Imaging Studies, Faculty of Medical and Human Studies, University of Manchester, United Kingdom. JAdamski@picr.man.ac.uk (2008-05)
      Exposure of tumour cells to reduced levels of oxygen (hypoxia) is a common finding in adult tumours. Hypoxia induces a myriad of adaptive changes within tumour cells which result in increased anaerobic glycolysis, new blood vessel formation, genetic instability and a decreased responsiveness to both radio and chemotherapy. Hypoxia correlates with disease stage and outcome in adult epithelial tumours and increasingly it is becoming apparent that hypoxia is also important in paediatric tumours. Despite its adverse effects upon tumour response to treatment hypoxia offers several avenues for new drug development. Bioreductive agents already exist, which are preferentially activated in areas of hypoxia, and thus have less toxicity for normal tissue. Additionally the adaptive cellular response to hypoxia offers several novel targets, including vascular endothelial growth factor (VEGF), carbonic anhydrase, and the central regulator of the cellular response to hypoxia, hypoxia inducible factor-1 (HIF-1). Novel agents have emerged against all of these targets and are at various stages of clinical and pre-clinical development. Hypoxia offers an exciting opportunity for new drug development that can include paediatric tumours at an early stage.
    • Circulating biomarkers of cell death after treatment with the BH-3 mimetic ABT-737 in a preclinical model of small-cell lung cancer.

      Micha, Dimitra; Cummings, Jeffrey; Shoemaker, Alex; Elmore, Steven; Foster, Kelly; Greaves, Martin J; Ward, Timothy H; Rosenberg, Saul; Dive, Caroline; Simpson, Kathryn L; et al. (2008-11-15)
      PURPOSE: This study evaluated epithelial cell death ELISAs that measure circulating cytokeratin 18 in mice bearing small-cell lung cancer xenografts treated with a proapoptotic dose of the BH-3 mimetic ABT-737. EXPERIMENTAL DESIGN: H146 tumor-bearing and non-H146 tumor-bearing severe combined immunodeficient (SCID)/bg mice were treated with ABT-737 or vehicle control. Plasma collected before and 2 to 360 hours after treatment was analyzed by M30 (caspase-cleaved cytokeratin 18) and M65 (intact and cleaved cytokeratin 18) ELISA. In parallel, tumors were interrogated for cleaved caspase-3 and cleaved cytokeratin 18 as biomarkers of apoptosis. RESULTS: ABT-737-treated tumors regressed by 48 hours (P < 0.01) compared with controls, correlating with increased cleaved cytokeratin 18 (P < 0.01; 6 and 24 hours) and increased intact cytokeratin 18 (P < 0.01; 24 hours). Cleaved cytokeratin 18 levels decreased below baseline between 72 and 360 hours for ABT-737-treated and control mice whereas intact cytokeratin 18 decreased below the level of detection at 8 and 15 days in ABT-737-treated mice only. Apoptosis in tumors reflected changes in circulating cytokeratin 18 (cleaved caspase-3, P < 0.05 at 2 hours and P < 0.001 at 6, 12, and 24 hours; caspase-cleaved cytokeratin 18, P < 0.05 at 15 days, for drug treated versus controls). CONCLUSIONS: ABT-737 caused tumor regression by apoptosis in H146 xenografts that mapped to a drug-specific, early increase in circulating cleaved cytokeratin 18 that subsequently declined. Circulating, intact cytokeratin 18 levels correlated with tumor burden. Cleaved caspase-3 and caspase-cleaved cytokeratin 18 in tumor correlated with treatment (P < 0.05, 2 hours; P < 0.001, 6, 12, and 24 hours; cleaved caspase-3, P < 0.05, 15 days; caspase-cleaved cytokeratin 18), indicating that events in plasma were tumor derived. These circulating biomarker data will be translated to clinical trials wherein serial tumor biopsies are rarely obtained.
    • Contribution of HIF-1 and drug penetrance to oxaliplatin resistance in hypoxic colorectal cancer cells.

      Roberts, Darren L; Williams, Kaye J; Cowen, Rachel L; Barathova, M; Eustace, A J; Brittain-Dissont, S; Tilby, Michael J; Pearson, D Graham; Ottley, Christopher J; Stratford, Ian J; et al. (2009-10-20)
      BACKGROUND: Hypoxia is as an indicator of poor treatment outcome. Consistently, hypoxic HCT116 colorectal cancer cells are resistant to oxaliplatin, although the mechanistic basis is unclear. This study sought to investigate the relative contribution of HIF-1 (hypoxia-inducible factor-1)-mediated gene expression and drug penetrance to oxaliplatin resistance using three-dimensional spheroids. METHODS: Hypoxia-inducible factor-1alpha function was suppressed by the stable expression of a dominant-negative form in HCT116 cells (DN). Cells were drug exposed as monolayer or multicellular spheroid cultures. Cells residing at differing oxygenation status were isolated from Hoechst 33342-treated spheroids using flow cytometry. Sub-populations were subjected to clonogenic survival assays and to Inductively-Coupled Plasma Mass Spectroscopy to determine oxaliplatin uptake. RESULTS: In spheroids, a sensitivity gradient (hypoxic
    • Detection of BRAF mutations in the tumour and serum of patients enrolled in the AZD6244 (ARRY-142886) advanced melanoma phase II study.

      Board, Ruth E; Ellison, G; Orr, M C M; Kemsley, K R; McWalter, G; Blockley, L Y; Dearden, S P; Morris, C; Ranson, Malcolm R; Cantarini, M V; et al. (2009-11-17)
      BACKGROUND: This study investigated the potential clinical utility of circulating free DNA (cfDNA) as a source of BRAF mutation detection in patients enrolled into a phase II study of AZD6244, a specific MEK1/2 inhibitor, in patients with advanced melanoma. METHODS: BRAF mutations were detected using Amplification Refractory Mutation System allele-specific PCR. BRAF mutation status was assessed in serum-derived cfDNA from 126 patients enrolled into the study and from 94 matched tumour samples. RESULTS: Of 94 tumour samples, 45 (47.9%) were found to be BRAF mutation positive (BRAF+). Serum-derived cfDNA was BRAF+ in 33 of 126 (26.2%) samples, including in five samples for which tumour data were unavailable. Of BRAF+ tumours, 25 of 45 (55.6%) were BRAF+ in cfDNA. In three cases in which the tumour was negative, cfDNA was BRAF+. Progression-free survival (PFS) of patients with BRAF+ tumour and cfDNA was not significantly different compared with tumour BRAF+ but cfDNA BRAF-negative patients, indicating that cfDNA BRAF detection is not associated with poorer prognosis on PFS in stage III/IV advanced melanoma. CONCLUSIONS: These data demonstrate the feasibility of BRAF mutation detection in cfDNA of patients with advanced melanoma. Future studies should aim to incorporate BRAF mutation testing in cfDNA to further validate this biomarker for patient selection.
    • A developmentally regulated heparan sulfate epitope defines a subpopulation with increased blood potential during mesodermal differentiation.

      Baldwin, Rebecca J; Ten Dam, Gerdy; Van Kuppevelt, Toin H; Lacaud, Georges; Gallagher, John T; Kouskoff, Valerie; Merry, Catherine L R; University of Manchester and Cancer Research UK Department of Medical Oncology, Manchester, United Kingdom. (2008-12)
      Heparan sulfate (HS) is a mandatory coreceptor for many growth factors and morphogens involved in embryonic development; its bioactivity is dictated by complex sulfation motifs embedded within the polymer chain. Using a panel of HS-specific antibodies we have identified a unique HS epitope recognized by antibody HS4C3 that is selectively expressed during differentiation of embryonic stem (ES) cells along the mesodermal lineage to the hemangioblast stage. The appearance of this high-affinity HS4C3-binding (HS4C3(high)) epitope is transient; the epitope is specifically expressed within the emerging Brachyury(+) (Bry(+)) population and marks those cells that will become fetal liver kinase 1 (Flk1)(+). Fluorescence-activated cell sorting (FACS) separation and colony forming assays revealed that HS4C3(high)/Flk1(+) cells have a dramatically increased potential to form both blast and endothelial colonies, both of which depend upon the HS-binding growth factor vascular endothelial growth factor. Critically, expression of this HS epitope is tightly regulated, disappearing from the cell surface as the resultant hematopoietic lineages mature, in a similar manner to protein markers Bry and Flk1. In vivo studies showed a remarkable correlation with in vitro findings, with expression of HS4C3-binding epitopes restricted to newly formed mesodermal tissues during gastrulation. We believe this is the first time a defined HS epitope has been implicated in a specific developmental pathway and that this provides, in addition, a novel enrichment technique for the isolation of hemangioblasts from mixed differentiated ES cell cultures.
    • Dietary variables associated with DNA N7-methylguanine levels and O6-alkylguanine DNA-alkyltransferase activity in human colorectal mucosa.

      Billson, H A; Harrison, Kathryn L; Lees, Nicholas P; Hall, C Nicholas; Margison, Geoffrey P; Povey, Andrew C; Occupational and Environmental Health Research Group, School of Translational Medicine, University of Manchester, Manchester M13 9PL, UK. (2009-04)
      Components of human diets may influence the incidence of colorectal adenomas, by modifying exposure or susceptibility to DNA-damaging alkylating agents. To examine this hypothesis, a food frequency questionnaire was used to assess the diet of patients recruited for a case-referent study where biopsies of normal colorectal mucosa were collected during colonoscopy and subsequently analysed for DNA N7-methylguanine (N7-MeG) levels, as an indicator of exposure, and activity of the DNA repair protein O6-alkylguanine DNA-alkyltransferase (MGMT), as an indicator of potential susceptibility. Cases with histologically proven colorectal adenomas (n = 38) were compared with referents (n = 35) free of gastrointestinal neoplasia. The case group consumed significantly more red meat (4.5 versus 3.4 servings/week, P < 0.05), processed meats, (4.7 versus 3.2 servings/week, P < 0.05) and % food energy as fat (34.9 versus 30.7%, P < 0.001). N7-MeG [mean: 95% confidence interval (CI)] levels were significantly lower in the group that consumed the highest proportion of dietary fibre/1000 kcal in comparison with the group with the lowest intake (0.61; 0.35-0.86 versus 1.88; 0.88-2.64 micromol/mol dG, P < 0.05). N7-MeG levels were also inversely associated with folate consumption (P < 0.05). MGMT activity (mean; 95% CI) was significantly higher in the group with the lowest consumption of vegetables than in the group with the greatest vegetable consumption (7.02; 5.70-8.33 versus 4.93; 3.95-5.91 fmol/microg DNA, P < 0.05). Our results are consistent with the hypothesis that dietary factors may modify exposure or susceptibility, respectively, to DNA damage by alkylating agents.
    • The differential activities of Runx1 promoters define milestones during embryonic hematopoiesis.

      Sroczynska, Patrycja; Lancrin, Christophe; Kouskoff, Valerie; Lacaud, Georges; Cancer Research UK Stem Cell Biology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom. (2009-12-17)
      The transcription factor RUNX1/AML1 is a master regulator of hematopoietic development. Its spatiotemporal expression is tightly regulated during embryonic development and is under the control of 2 alternative promoters, distal and proximal. Despite the functional significance of Runx1, the relative and specific activities of these 2 promoters remain largely uncharacterized. To investigate these activities, we introduced 2 reporter genes under the control of the proximal and distal promoters in embryonic stem cell and transgenic mouse lines. Our study reveals that both in vitro and in vivo the proximal Runx1 isoform marks a hemogenic endothelium cell population, whereas the subsequent expression of distal Runx1 defines fully committed definitive hematopoietic progenitors. Interestingly, hematopoietic commitment in distal Runx1 knockout embryos appears normal. Altogether, our data demonstrate that the differential activities of the 2 Runx1 promoters define milestones of hematopoietic development and suggest that the proximal isoform plays a critical role in the generation of hematopoietic cells from hemogenic endothelium. Identification and access to the discrete stages of hematopoietic development defined by the activities of the Runx1 promoters will provide the opportunity to further explore the cellular and molecular mechanisms of hematopoietic development.
    • 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.