• The stepwise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to Bmp4, activin A, bFGF and VEGF.

      Pearson, Stella; Sroczynska, Patrycja; Lacaud, Georges; Kouskoff, Valerie; Cancer Research UK, Paterson Institute for Cancer Research, Manchester University, Wilmslow Road, M20 4BX, Manchester, UK. (2008-04)
      The differentiation of embryonic stem (ES) cells offers a powerful approach to study mechanisms implicated in cell fate decision. A major hurdle, however, is to promote the directed and efficient differentiation of ES cells toward a specific lineage. Here, we define in serum-free media the minimal factor requirement controlling each step of the differentiation process, resulting in the production of highly enriched hematopoietic progenitors. Four factors - Bmp4, activin A, bFGF (Fgf2) and VEGF (VegfA) - are sufficient to drive the selective and efficient differentiation of mouse ES cells to hematopoiesis. Each of these factors appears to regulate a step of the process: Bmp4 promotes the very efficient formation of mesoderm; bFGF and activin A induce the differentiation of these mesodermal precursors to the hemangioblast fate; and VEGF is required for the production of fully committed hematopoietic progenitors. The stimulation of mesodermal precursors by bFGF and activin A switches on very rapidly the hematopoietic program, allowing us to dissect the molecular events leading to the formation of the hemangioblast. Runx1, Scl (Tal1) and Hhex expression is upregulated within 3 hours of stimulation, whereas upregulation of Lmo2 and Fli1 is observed later. Interestingly, increased expression levels of genes such as cMyb, Pu.1 (Sfpi1), Gata1 and Gata2 are not observed at the onset of hemangioblast commitment. This stepwise control of differentiation is extremely efficient, giving rise to a very high frequency of hematopoietic precursors, and provides an optimal system for understanding the molecular machineries involved in blood progenitor commitment.
    • Stress-regulated kinase pathways in the recovery of tip growth and microtubule dynamics following osmotic stress in S. pombe.

      Robertson, Alasdair M; Hagan, Iain M; CRUK Cell Division Laboratory, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008-12-15)
      The cell-integrity and stress-response MAP kinase pathways (CIP and SRP, respectively) are stimulated by various environmental stresses. Ssp1 kinase modulates actin dynamics and is rapidly recruited to the plasma membrane following osmotic stress. Here, we show that osmotic stress arrested tip growth, induced the deposition of abnormal cell-wall deposits at tips and led to disassociation of F-actin foci from cell tips together with a reduction in the amount of F-actin in these foci. Osmotic stress also ;froze' the dynamics of interphase microtubule bundles, with microtubules remaining static for approximately 38 minutes (at 30 degrees C) before fragmenting upon return to dynamic behaviour. The timing with which microtubules resumed dynamic behaviour relied upon SRP activation of Atf1-mediated transcription, but not on either CIP or Ssp1 signalling. Analysis of the recovery of tip growth showed that: (1) the timing of recovery was controlled by SRP-stimulated Atf1 transcription; (2) re-establishment of polarized tip growth was absolutely dependent upon SRP and partially dependent upon Ssp1 signalling; and (3) selection of the site for polarized tip extension required Ssp1 and the SRP-associated polarity factor Wsh3 (also known as Tea4). CIP signalling did not impact upon any aspect of recovery. The normal kinetics of tip growth following osmotic stress of plo1.S402A/E mutants established that SRP control over the resumption of tip growth after osmotic stress is distinct from its control of tip growth following heat or gravitational stresses.
    • Suppressor role of activating transcription factor 2 (ATF2) in skin cancer.

      Bhoumik, Anindita; Fichtman, Boris; Derossi, Charles; Breitwieser, Wolfgang; Kluger, Harriet M; Davis, Sean; Subtil, Antonio; Meltzer, Paul; Krajewski, Stan; Jones, Nic; et al. (2008-02-05)
      Activating transcription factor 2 (ATF2) regulates transcription in response to stress and growth factor stimuli. Here, we use a mouse model in which ATF2 was selectively deleted in keratinocytes. Crossing the conditionally expressed ATF2 mutant with K14-Cre mice (K14.ATF2(f/f)) resulted in selective expression of mutant ATF2 within the basal layer of the epidermis. When subjected to a two-stage skin carcinogenesis protocol [7,12-dimethylbenz[a]anthracene/phorbol 12-tetradecanoate 13-acetate (DMBA/TPA)], K14.ATF2(f/f) mice showed significant increases in both the incidence and prevalence of papilloma development compared with the WT ATF2 mice. Consistent with these findings, keratinocytes of K14.ATF2(f/f) mice exhibit greater anchorage-independent growth compared with ATF2 WT keratinocytes. Papillomas of K14.ATF2(f/f) mice exhibit reduced expression of presenilin1, which is associated with enhanced beta-catenin and cyclin D1, and reduced Notch1 expression. Significantly, a reduction of nuclear ATF2 and increased beta-catenin expression were seen in samples of squamous and basal cell carcinoma, as opposed to normal skin. Our data reveal that loss of ATF2 transcriptional activity serves to promote skin tumor formation, thereby indicating a suppressor activity of ATF2 in skin tumor formation.
    • To determine the cytotoxicity of chlorambucil and one of its nitro-derivatives, conjugated to prasterone and pregnenolone, towards eight human cancer cell-lines.

      Shervington, Leroy A; Smith, Nigel K; Norman, Emma; Ward, Timothy H; Phillips, Roger M; Shervington, Amal; School of Pharmacy and Pharmaceutical Sciences, University of Central Lancashire, Preston, UK. lashervington@uclan.ac.uk (2009-07)
      Four ester prodrugs derived from the bifunctional alkylating agent chlorambucil, and one of its nitro-derivatives, 3-nitrochlorambucil conjugated to prasterone and pregnenolone, were synthesized and tested for their cytotoxic activity against eight human cell lines, using the standard MTT assay. A comparison between the esters and the controls, namely chlorambucil and 3-nitrochlorambucil would suggest that all four esters possess to varying degrees, specificity towards the breast adenocarcinoma cell line (MDA-mb468) than the other seven cells' lines tested. The overall findings are encouraging since it infers that these lipophilic esters not only have the ability to traverse specific cell membranes but also exhibit cytotoxicity towards most of the cell lines tested.
    • The transcription factor Atf1 binds and activates the APC/C ubiquitin ligase in fission yeast.

      Ors, Aslihan; Grimaldi, Margaret; Kimata, Yuu; Wilkinson, Caroline R M; Jones, Nic; Yamano, Hiroyuki; Cell Cycle Control Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom. (2009-09-04)
      Fission yeast Atf1 is a member of the ATF/CREB basic leucine zipper (bZIP) family of transcription factors with strong homology to mammalian ATF2. Atf1 regulates transcription in response to stress stimuli and also plays a role in controlling heterochromatin formation and recombination. However, its DNA binding independent role is poorly studied. Here, we report that Atf1 has a distinct role in regulating the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. We have identified atf1(+) as a dose-dependent suppressor of apc5-1, a mutation causing mitotic arrest. Remarkably, the suppression is not dependent upon the bZIP domain and is therefore independent of the ability of Atf1 to bind DNA. Interestingly, Atf1 physically binds the APC/C in vivo. Furthermore, we show that addition of purified Atf1 proteins into a cell-free system stimulates ubiquitylation of cyclin B and securin by the APC/C. These results reveal a novel role for Atf1 in cell cycle control through protein-protein interaction.
    • Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity.

      Karanam, Balasubramanyam; Gambhira, Ratish; Peng, Shiwen; Jagu, Subhashini; Kim, Dae-Jin; Ketner, Gary W; Stern, Peter L; Adams, Robert J; Roden, Richard B S; Department of Pathology, The Johns Hopkins University, Baltimore, MD 21231, USA. (2009-02-11)
      A vaccine comprising human papillomavirus type 16 (HPV16) L2, E6 and E7 in a single tandem fusion protein (termed TA-CIN) has the potential advantages of both broad cross-protection against HPV transmission through induction of L2 antibodies able to cross neutralize different HPV types and of therapy by stimulating T cell responses targeting HPV16 early proteins. However, patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. Here we test TA-CIN formulated along with the adjuvant GPI-0100, a semi-synthetic quillaja saponin analog that was developed to promote both humoral and cellular immune responses. Subcutaneous administration to mice of TA-CIN (20 microg) with 50microg GPI-0100, three times at biweekly intervals, elicited high titer HPV16 neutralizing serum antibody, robust neutralizing titers for other HPV16-related types, including HPV31 and HPV58, and neutralized to a lesser extent other genital mucosatropic papillomaviruses like HPV18, HPV45, HPV6 and HPV11. Notably, vaccination with TA-CIN in GPI-0100 protected mice from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP without adjuvant. Formulation of TA-CIN with GPI-0100 enhanced the production of E7-specific, interferon gamma producing CD8(+) T cell precursors by 20-fold. Vaccination with TA-CIN in GPI-0100 also completely prevented tumor growth after challenge with 5x10(4) HPV16-transformed TC-1 tumor cells, whereas vaccination with TA-CIN alone delayed tumor growth. Furthermore, three monthly vaccinations with 125 microg of TA-CIN and 1000 microg GPI-0100 were well tolerated by pigtail macaques and induced both HPV16 E6/E7-specific T cell responses and serum antibodies that neutralized all HPV types tested.
    • A validated gene expression profile for detecting clinical outcome in breast cancer using artificial neural networks.

      Lancashire, Lee J; Powe, D G; Reis-Filho, J S; Rakha, E; Lemetre, Christophe; Weigelt, B; Abdel-Fatah, T M; Green, Anthony R; Mukta, R; Blamey, R; et al. (2009-04-04)
      Gene expression microarrays allow for the high throughput analysis of huge numbers of gene transcripts and this technology has been widely applied to the molecular and biological classification of cancer patients and in predicting clinical outcome. A potential handicap of such data intensive molecular technologies is the translation to clinical application in routine practice. In using an artificial neural network bioinformatic approach, we have reduced a 70 gene signature to just 9 genes capable of accurately predicting distant metastases in the original dataset. Upon validation in a follow-up cohort, this signature was an independent predictor of metastases free and overall survival in the presence of the 70 gene signature and other factors. Interestingly, the ANN signature and CA9 expression also split the groups defined by the 70 gene signature into prognostically distinct groups. Subsequently, the presence of protein for the principal prognosticator gene was categorically assessed in breast cancer tissue of an experimental and independent validation patient cohort, using immunohistochemistry. Importantly our principal prognosticator, CA9, showed that it is capable of selecting an aggressive subgroup of patients who are known to have poor prognosis.
    • X:Map: annotation and visualization of genome structure for Affymetrix exon array analysis.

      Yates, Tim; Okoniewski, Michal J; Miller, Crispin J; Cancer Research UK, Bioinformatics Group, Paterson Institute for Cancer Research, The University of Manchester, Christie Hospital Site, Wilmslow Road, Withington, Manchester, M20 4BX, UK. (2008-01)
      Affymetrix exon arrays aim to target every known and predicted exon in the human, mouse or rat genomes, and have reporters that extend beyond protein coding regions to other areas of the transcribed genome. This combination of increased coverage and precision is important because a substantial proportion of protein coding genes are predicted to be alternatively spliced, and because many non-coding genes are known also to be of biological significance. In order to fully exploit these arrays, it is necessary to associate each reporter on the array with the features of the genome it is targeting, and to relate these to gene and genome structure. X:Map is a genome annotation database that provides this information. Data can be browsed using a novel Google-maps based interface, and analysed and further visualized through an associated BioConductor package. The database can be found at http://xmap.picr.man.ac.uk.