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

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Simultaneously imaging mitotic progression and the onset of apoptosis To unequivocally prove that docetaxel-induced apoptosis is mediated through defects in mitotic progression, both processes should be visualized simultaneously in the same cells in vitro and in vivo before and after docetaxel treatment. Here, we have developed an imaging method to achieve this (Fig 2A, B). Activation of the protease caspase-3 is crucial for apoptosis induction and catalyzes the cleavage of several key cellular proteins. Caspase-3 activation precedes chromosome condensation and cell fragmentation, the typical morphological changes associated with apoptotic cell death 602 . We and others have recently used a caspase-3 Fluorescent Resonance Energy Transfer (FRET) probe to visualize the onset of apoptosis in vitro and in vivo 601,603-605 . The caspase-3 FRET sensor consists of a CFP and YFP moiety separated by the caspase-3 DEVD cleavage motif and is targeted to the plasma membrane by a C-terminal CAAX sequence 606 (Fig.2A,C). Under normal conditions, caspase-3 is inactive and CFP and YFP are in close proximity, so that CFP can transfer its energy to YFP. As a result of this energy transfer, CFP fluorescence will drop and YFP fluorescence will increase, leading to a low CFP to YFP ratio (referred to as CFP/YFP ratio). Caspase-3-dependent cleavage of the DEVD motif will perturb energy transfer from CFP to YFP and result in a decreased CFP/YFP ratio. We created both C26 and SW480 cell lines stably expressing this apoptosis sensor and, as expected, we were able to visualize apoptosis induction both in vitro and in vivo using this caspase-3 FRET sensor (Fig 2C). To follow both caspase-3 activation and mitotic progression before and after docetaxel treatment, we, in addition to the caspase-3 biosensor, also stably introduced Histone 2B tagged to photo-switchable Dendra2 607 (H2B-D) in C26 and SW480 cell lines (Fig.2B,D). In prior studies, we have used Dendra2 to specifically photo-mark and trace individual cells 608,609 . By switching the color of H2B-Dendra2 (H2B-D) from green to red using a violet laser 607 (Fig.2B, D), we were able to track the same cells both in vitro and in vivo over multiple imaging sessions (Fig.2D, E). Photo-switching of Dendra2 did not affect caspase-3 FRET measurements and more importantly, FRET levels did not change during mitotic progression, excluding possible effects of the mitotic state on FRET efficiency (Fig.S1A, B). These data show that by imaging the caspase-3 FRET probe and H2B-Dendra2 simultaneously, we are able to visualize mitotic progression and the onset of apoptosis in the same cells both in vitro and in vivo. Docetaxel treatment induces caspase-3 activation both in vitro and in vivo Next, we determined the onset of apoptosis in our cell lines after docetaxel administration in vitro. The CFP/YFP ratio clearly increased in mitotic cells after prolonged docetaxel treatment (Fig.3A). Importantly, cells that were morphologically clearly apoptotic showed an even higher CFP/YFP ratio (5.6) when compared to the rest of the mitotic cells treated with docetaxel (3.7). This indicates that we can observe the docetaxel-induced increase in caspase-3 activity well before cells undergo the typical morphological changes associated with apoptosis (Fig.2A,C), which is in line with a recently proposed model in which caspase-3 activity slowly increases during a mitotic delay imposed by treatments with anti-mitotic drugs 342 . To evaluate the effects of docetaxel in vivo, H2B-D-expressing cells in the tumor were photo-marked and CFP/YFP ratios of single cells were subsequently determined before, 20 and 48 hours after intravenous docetaxel administration (25mg/kg) (Fig.3B). This analysis revealed a significant increase in the CFP/YFP ratios of both C26 and SW480 tumor cells after docetaxel treatment when compared to the control situation, which indicates that there is an increase in caspase-3 activity (Fig.3B). Before docetaxel treatment, SW480 cells displayed a CFP/YFP ratio of 2.1 on average, which increased to 2.7 and 3.3 after 20 and 48 hours of docetaxel 137 Intravital imaging of Docetaxel response in tumors 7
7 A Low CFP lifetime High CFP lifetime Increase CFP/YFP ratio 138 860 nm 480nm CFP 860 nm 480nm CFP DEVD Caspase3 DEVD DEV 530nm eYFP eYFP membrane CAAX CAAX C In vitro In vivo D E In vivo Before H2B-D switching After Alive Early apoptotic Late apoptotic H2B-D C3-CAAX B Photomarking Mitotic progression in vitro 50 µm H2B-D Switch Dendra2 Dendra2 Cell cycles progression H2B-D C3-CAAX Day1 Day2 Figure 2. Simultaneous imaging of apoptosis and mitosis A) Schematic representation of the caspase-3 FRET probe. In the absence of caspase-3 activity, CFP excitation results in the excitation of YFP bound to the membrane (FRET) and a low CFP fluorescence lifetime. A rise in caspase-3 activity, indicative of apoptosis induction, results in the cleavage of the DEVD motif in between the CFP and YFP moieties, which will result in loss of FRET, an increase in CFP-YFP ratio and an increased CFP fluorescence lifetime. B) Schematic representation of photo-marking of H2B-D cells. Switching of H2B-D from green to red enables the tracking of single cells and visualization of mitotic progression. C) Color scheme and representative cells (alive, early apoptotic, late apoptotic) in vitro and in vivo showing CFP/YFP ratio changes. D) In vitro cells representative of H2B-Dendra2 (H2B-D) switching and mitotic progression. E) Left: Representative image of H2B-D photo-switching of SW480 tumor cells in vivo. Right: Stills of individual SW480 cell in vivo followed for 2 consecutive days.
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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
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Introduction Aneuploidy is a common
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activity by ~80% 170-172 . With the
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A WT/CiMKi T649A Embryo’s: Figure
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control treated MEFs (unpaired t te
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Materials and Methods Cloning targe
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- Loop out BamHI site with site-dir
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Primers: pGH_pA_FW#2: TCTATGGCTTCTG
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was prepared using standard procedu
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71 Cre-inducible Mps1 knock-in mous
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4 Abstract Most of the current drug
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4 Two other interesting mitotic tar
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4 inhibition of the anti-apoptotic
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4 cluster their centrosomes. This
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4 of checkpoint function can be ach
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4 4.5 Disadvantages of exploiting m
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Page 90 and 91: Introduction Error-free chromosome
Page 92 and 93: Partial depletion of Mps1 or BubR1
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Page 96 and 97: had survived growth for 7 days in t
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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 106 and 107: A B C percentage of cells % PI posi
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
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Page 151 and 152: 8 Thesis summary Unequal separation
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Page 155 and 156: 8 1.4 NoCut: preventing the inducti
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Page 160 and 161: Addendum References Nederlandse sam
Page 162 and 163: 41. McEwen, B.F., et al., CENP-E is
Page 164 and 165: 2010. 6(5): p. 359-68. 124. Liu, S.
Page 166 and 167: 210. Stucki, M., et al., MDC1 direc
Page 168 and 169: tumors in mice. Cancer Res, 2007. 6
Page 170 and 171: adiotherapy in breast cancer. Breas
Page 172 and 173: 470. Marcus, A.I., et al., Mitotic
Page 174 and 175: 559. Kienitz, A., et al., Partial d
Page 176 and 177: Nederlandse Samenvatting Celdeling,
Page 178 and 179: verdeling van tumorcellen compleet
Page 180 and 181: Publications A. Janssen, R.H.Medema
Page 182 and 183: En dan zijn er nu nog een aantal li
Page 184 and 185: promotie-stukjes! Je bent een voorb
Page 186: edankt voor al jullie hulp bij de i
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