Chromosome segregation errors: a double-edged sword - TI Pharma

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A B C percentage of cells % PI positive cells control cenp-e siRNA 0 5 0 5 0 5 0 5 nM taxol 20 15 10 5 0 n=71 - doxycycline 0 5 0 5 0 5 0 5 + doxycycline (Mad2 depleted) nM taxol control cenp-e control cenp-e siRNA - doxycycline + doxycycline (Mad2 depleted) time-lapse (H2B-GFP) n=63 n=38 n=29 n=69 n=65 n=36 n=34 control cenp-e D E relative signal intensity CENP-E/ CREST F time-lapse (DIC) G U2OS-TetR-Mad2 VH10-TetR Mps1 600 500 100 100 400 80 80 300 60 60 200 40 40 100 20 0 0 5 0 5 0 5 0 5 nM taxol 20 0 0 2 0 2 nM taxol 0 control cenp-e control cenp-e siRNA - + doxycycline - doxycycline no segregation defects + doxycycline (Mad2 depleted) mild segregation defects (1-5) severe segregation defects (>5) percentage of cells n=27 n=56 n=39 n=28 x1000 cells 1,2 0,8 0,4 0 Mad2 actin - + doxycycline control cenp-e control cenp-e control cenp-e control cenp-e siRNA siRNA CREST CENP-E merge w/DNA - + doxycycline VH10-TetR Mps1 day 0 day 2 day 4 day 7 day 9 - dox + dox - dox + 2nM tax + dox + 2nM tax control siRNA cenp-e siRNA Figure S10. A) Representative pictures of colony formations of U2OS-TetRMad2 cells on day 6 treated with or without doxycycline (day 0), control or CENP-E siRNA (day 2) and 0 or 5nM taxol (day 3). Luciferase or GAPDH siRNA were used as a control. B) Automated analysis of the percentage of PI positive over Hoechst positive U2OS-TetRMad2 cells on day 6 after treatment as in (A). Bars represent the average of 4 independent experiments (+SEM). C) Quantification of time-lapse analysis as in Fig.S1C. siRNA transfections were performed 36 hours prior to filming. Indicated taxol concentrations were added 1 hour prior to filming. n represents amount of cells filmed. D) Immunoblots of Mad2 and actin of U2OS-TetRMad2 cells treated with and without doxycycline and control or CENP-E siRNA. E) Top: Localization of CENP-E (green) and CREST (red) in mitotic U2OS-TetRMad2 cells transfected with control or CENP-E siRNA. Bottom: Quantification of intensity of CENP-E staining over CREST. Average is shown of 35 kinetochores +SD of 7 analyzed cells per condition. F) Quantification of time-lapse analysis (DIC) of chromosome segregation in anaphase in VH10 cells. n indicates amount of cells filmed. G) Growth curve of VH10-TetRMps1 cells after indicated treatments. On day 0 doxycycline was added, on day 2 2nM of taxol was added where indicated. 105 Chromosome missegregations kill tumor cells 5
5 106 Addendum Targeting the mitotic checkpoint to kill tumor cells. Aniek Janssen 1,2 , Miranda van Amersfoort 1 , Geert J. Kops 1,3 and René H. Medema 1,2 . 1 Department of Medical Oncology and Cancer Genomics Center, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, the Netherlands 2 Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, the Netherlands 3 Department of Molecular Cancer Research, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, the Netherlands Adapted from: Horm Cancer. 2011 Apr;2(2):113-6
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Chromosome segregation errors: a do
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Chromosome segregation errors: a do
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Get up, stand up, stand up for your
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Chapter 1 General Introduction Anie
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cytoplasms and pinches off the memb
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which sister-chromatids are not pro
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Non-MCC members, but important mito
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completely separated. Following cle
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Two striking features of cancer cel
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predisposition at a very young age,
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that aberrant spindle formation inc
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(Fig.7C). As discussed in section 4
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Thesis outline Chromosome segregati
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286,395,407,408 Increase (thymus) I
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2 Abstract Various types of chromos
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2 34 A C D E % of cells Control 100
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2 assess whether cytokinesis induce
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2 A Asynchronous Monastrol release
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2 for 14 hours (unless indicated ot
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2 Immuno Blotting Cells were lysed
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2 Supplemental figures 44 A B C % o
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2 A MCF7 B C 53BP1 foci/cell 46 % o
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2 A siLuc siATM C 48 p-S1981 ATM me
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2 50 A B C % of EdU positive cells
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Chapter 3 Studying Chromosomal inst
Page 56 and 57: Introduction Aneuploidy is a common
Page 58 and 59: activity by ~80% 170-172 . With the
Page 60 and 61: A WT/CiMKi T649A Embryo’s: Figure
Page 62 and 63: control treated MEFs (unpaired t te
Page 64 and 65: Materials and Methods Cloning targe
Page 66 and 67: - Loop out BamHI site with site-dir
Page 68 and 69: Primers: pGH_pA_FW#2: TCTATGGCTTCTG
Page 70 and 71: was prepared using standard procedu
Page 72: 71 Cre-inducible Mps1 knock-in mous
Page 75 and 76: 4 Abstract Most of the current drug
Page 77 and 78: 4 Two other interesting mitotic tar
Page 79 and 80: 4 inhibition of the anti-apoptotic
Page 81 and 82: 4 cluster their centrosomes. This
Page 83 and 84: 4 of checkpoint function can be ach
Page 85 and 86: 4 4.5 Disadvantages of exploiting m
Page 88 and 89: Chapter 5 Elevating the frequency o
Page 90 and 91: Introduction Error-free chromosome
Page 92 and 93: Partial depletion of Mps1 or BubR1
Page 94 and 95: death is not induced in the short t
Page 96 and 97: had survived growth for 7 days in t
Page 98 and 99: The following antibodies have been
Page 100 and 101: Supplemental data Supplemental figu
Page 102 and 103: A BubR1 α-tubulin Hela-TetRBubR1 -
Page 104 and 105: - doxycycline + doxycycline - doxyc
Page 108 and 109: Abstract One of the most common hal
Page 110 and 111: clear accumulation of cells in mito
Page 112 and 113: the mice did not receive any doxycy
Page 114 and 115: 113 Chromosome missegregations kill
Page 116 and 117: Chapter 6 Aneuploid tumor cells are
Page 118 and 119: Introduction Equal segregation of t
Page 120 and 121: S1A, data not shown). Based on thes
Page 122 and 123: A B U2OS + H2B-YFP percentage of ce
Page 124 and 125: tumor cells, leaving diploid cells
Page 126 and 127: A B p-S10-Histone H3 DAPI C µmol/l
Page 128 and 129: Supplemental Figure-1. Protein bioc
Page 130 and 131: Bioavailability of compound-5 after
Page 132: A B U2OS (7.5nM) Hela (10nM) RPE-1
Page 135 and 136: 7 Abstract Taxanes, such as docetax
Page 137 and 138: 7 of mitotic aberrations. These dat
Page 139 and 140: 7 A Low CFP lifetime High CFP lifet
Page 141 and 142: 7 docetaxel treatment in vitro resu
Page 143 and 144: 7 chromosome unstable, which means
Page 145 and 146: 7 Cells (~50.000/well) were plated
Page 147 and 148: 7 Supplemental data Supplemental fi
Page 149 and 150: 7 148
Page 151 and 152: 8 Thesis summary Unequal separation
Page 153 and 154: 8 exerted through contraction of th
Page 155 and 156: 8 1.4 NoCut: preventing the inducti
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8 tumorformation using intravital i
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Addendum References Nederlandse sam
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41. McEwen, B.F., et al., CENP-E is
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2010. 6(5): p. 359-68. 124. Liu, S.
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210. Stucki, M., et al., MDC1 direc
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tumors in mice. Cancer Res, 2007. 6
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adiotherapy in breast cancer. Breas
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470. Marcus, A.I., et al., Mitotic
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559. Kienitz, A., et al., Partial d
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Nederlandse Samenvatting Celdeling,
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verdeling van tumorcellen compleet
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Publications A. Janssen, R.H.Medema
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En dan zijn er nu nog een aantal li
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promotie-stukjes! Je bent een voorb
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edankt voor al jullie hulp bij de i
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Chromosome segregation errors: a double-edged sword - TI Pharma

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