QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 15 August 2006
doi: 10.1242/jcs.03145

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Girdler, F. Articles by Taylor, S. S. Search for Related Content PubMed PubMed Citation Articles by Girdler, F. Articles by Taylor, S. S. Social Bookmarking                         What's this?

Validating Aurora B as an anti-cancer drug target

¹ Faculty of Life Sciences, Michael Smith Building, Oxford Road, University of Manchester, Manchester, M13 9PT, UK
² Cancer and Infection Research Area, AstraZeneca Pharmaceuticals, Mereside, Alderley Park, Cheshire, SK10 4TG, UK

View larger version (49K): [in a new window]   Fig. 1. Characterisation of model system. HEK293 cell lines stably transfected with Aurora transgenes were induced with tetracycline then analysed by immunoblot, immunofluorescence and immunoprecipitation kinase assays. (A) Immunoblot showing that the anti-Aurora A, B and C antibodies are monospecific for Aurora (Ar) A, B and C respectively. DN, Aurora kinase D-N mutant; KR, Aurora kinase K-R mutant; WT, wild type. (B-D) Immunoblots probed with antibodies against Aurora proteins, the Myc-epitope tag and phosphorylated Histone H3 (S10), showing tetracycline induced expression of Aurora transgenes and effects on H3 phosphorylation. (E) Immunofluorescence images showing localisation of exogenous Aurora proteins. Bar, 5 µm. (F,G) Immunoprecipitation kinase assays showing that the Aurora kinase mutants are catalytically inactive.

View larger version (23K): [in a new window]   Fig. 2. Suppression of Aurora B kinase activity inhibits cell division. Aurora transgenic lines were induced with tetracycline (tet), harvested at various time points and analysed by flow cytometry to determine DNA content. (A) Histograms 32 hours post induction showing that cells expressing the Aurora B and C mutants accumulate DNA contents 4N. (B) Line graphs quantifying cells with DNA contents >4N over a 40-hour time course. At t=0 tetracycline was added to the Aurora transgenic lines indicated or, alternatively, ZM1 was added to uninduced HEK293 cells. The values shown are representative of multiple independent experiments. DN, D-N mutant; KR; K-R mutant; WT, wild type; ZM, ZM1.

View larger version (28K): [in a new window]   Fig. 3. Suppression of Aurora B kinase activity compromises the spindle checkpoint. Aurora transgenic lines were induced with tetracycline for 4 hours, exposed to spindle toxins then analysed by flow cytometry to determine mitotic index, or time-lapse to measure mitotic timing. In parallel, cells were exposed to 2 µM ZM1. (A,B) Bar graphs measuring mitotic index 16 hours post addition of spindle toxins nocodazole (A) or taxol (B) showing that the Aurora B and C mutants mimic the effect of ZM1. The values represent the mean ± s.e.m. derived from three independent experiments. (C,D) Box plots measuring time spent in mitosis in the absence (C) or presence (D) of taxol, showing that the Aurora B D-N mutant mimics the effect of ZM1.

View larger version (73K): [in a new window]   Fig. 4. Suppression of Aurora B kinase activity compromises cell proliferation and viability. (A) Line graphs plotting relative cell number over an 8-day time course in the continuous presence of tetracycline, showing that like exposure to ZM1, induction of the Aurora B kinase mutants inhibits cell proliferation. Results are from a representative experiment in which each value represents the mean of three assay wells. (B,C) Transgenic lines were induced with tetracycline for 24 hours, replated in the absence of tetracycline, then fixed 17 days later and stained with crystal violet to determine colony number. Images of culture plates (B) and bar graph quantifying cell number (C), both showing that the Aurora B D-N mutant mimics the effect of ZM1. The data are derived from a representative experiment in which each value represents the mean of two assay plates.

View larger version (18K): [in a new window]   Fig. 5. ZM2 and ZM3: novel Aurora kinase inhibitors. (A) Chemical structures of ZM1, ZM2 and ZM3. Note that ZM1 is ZM447439 as described (Ditchfield et al., 2003). (B) Table summarising results from in vitro kinase assays to determine the effects of ZM compounds on Aurora kinase activity. Shown are the IC50 values; the relative potency of ZM2 and 3 with respect to ZM1; and the selectivity of each ZM compound towards Aurora B relative to Aurora A.

View larger version (69K): [in a new window]   Fig. 6. Aurora A kinase activity is required for spindle bipolarity. Transgenic lines encoding RNAi-resistant Aurora A transgenes were transfected with siRNAs designed to repress Aurora A or Lamin B1, then exposed to tetracycline as indicated, to induce transgene expression. (A) Immunoblot showing simultaneous repression of endogenous Aurora A and induction of Myc-tagged Aurora A D-N. The arrow indicates the Myc-tagged exogenous Aurora A, whereas the asterisk indicates endogenous protein. (B) Immunofluorescence images showing monopolar spindles in Aurora A RNAi cells expressing the Aurora A transgenes. In panels i and iii, the horizontal arrows indicate bipolar Aurora-A-positive spindles in untransfected cells and the arrowheads indicate prometaphase-like Aurora-A-deficient cells. In panels ii and iv, the vertical arrows indicate bipolar or monopolar spindles, respectively, in cells expressing the Aurora A transgene. Bars, 10 µm. (C) Bar graph quantifying monopolar spindles showing that although the wild-type Aurora A rescues the RNAi phenotype, the Aurora A D-N kinase mutant does not. The values represent the mean ± s.e.m. derived from three independent experiments in which at least 100 mitotic cells were scored.

View larger version (23K): [in a new window]   Fig. 7. ZM3 inhibits spindle bipolarity. DLD-1 cells were exposed to MG132 plus either 2 µM ZM1, 2 µM ZM3 or monastrol (Mon) for 2 hours, then analysed by immunofluorescence. (A) Images showing examples of monopolar spindles in the presence of monastrol and ZM3. Bar, 5 µm. (B) Bar graph quantifying monopolar spindles. The values represent the mean ± s.e.m. derived from three independent experiments in which at least 100 mitotic cells were scored. (C) Dot plot showing interpolar distances. (D) Line graph showing proportion of monopolar spindles over a range of ZM concentrations. The data are derived from a single representative experiment in which at least 100 mitotic cells were scored per concentration.

View larger version (54K): [in a new window]   Fig. 8. Expression of a ZM3-resistant Aurora A mutant restores spindle bipolarity. (A) Immunoblots and immunofluorescence images of DLD-1 cell lines showing that the tetracycline-induced wild-type and W277A Aurora A proteins localise to the spindle poles. The arrow indicates exogenous Myc-tagged Aurora A and the asterisk indicates endogenous protein. (B) Following tetracycline (Tet) induction, cells were exposed to 2 µM ZM3 and MG132 for 2 hours then analysed by immunofluorescence. Images show examples of monopolar spindles in the absence of W277A expression. Bar, 5 µm. (C) Bar graph quantifying monopolar spindles. The values represent the mean ± s.e.m. derived from three independent experiments in which at least 100 mitotic cells were scored. (D) Line graph showing proportion of monopolar spindles over a range of ZM3 concentrations. The data is derived from a single representative experiment in which at least 100 mitotic cells were scored per concentration.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter