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

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A B U2OS + H2B-YFP percentage of cells 0’ 10’ 15’ 20’ 25’ 30’ 35’ 100 0’ 5’ 10’ 15’ 20’ 25’ 30’ 80 60 40 20 0 n=34 n=55 - + RPE-1 n=46 - + U2OS n=72 n=52 n=61 - + Hela no errors mild errors (1-5) severe errors (>5) 100nM Cpd-5 Control Compound-5 (100nM) Mps1, we treated U2OS, Hela and RPE-1 cells with low doses of compound-5 and simultaneously treated them with sub-lethal doses of taxol. Indeed, combined inhibition of Mps1 and treatment with taxol resulted in a synergistic effect on cell viability in both Hela and U2OS cells (Fig.S2A, B) whereas such synergy was not evident in the untransformed cell line RPE-1 (Fig.S2A). Moreover, when we treated various tumor and normal, untransformed cell lines with increasing doses of the Mps1 inhibitor compound-5, we also observed selectivity towards tumor cells with low doses of compound-5 in the absence of taxol (Fig.4A). All tumor cell lines lost the capacity to form colonies upon treatment with 20-30 nM compound-5, whereas viability of both RPE-1 and BJ-Tert cells was not affected in the presence of 50 nM compound-5 (Fig.4A). The effect on cell viability was specifically due to Mps1 inhibition, since the Mps1-602Q mutant could overcome the lethal effects of compound-5 in U2OS cells (Fig.S2C). The difference in sensitivity between cell lines is not due to differences in compound-5 uptake, since treatment of RPE-1 cells with compound-5 affected the ability to delay mitosis in the presence of nocodazole as potently as in U2OS and Hela cells (Fig.2A). We have hypothesized before that untransformed, healthy cells could be less prone to missegregate their chromosomes upon Mps1 depletion or inhibition when compared to aneuploid tumor cells 299,447 . Indeed, when we followed chromosome segregation using time-lapse imaging of various cell lines stably expressing tagged H2B, we observed that tumor cell lines were more prone to missegregate their chromosomes upon low-dose compound-5 treatment (30 nM) when compared to untransformed RPE-1 cells (Fig.4B). In unperturbed anaphases, tumor cell lines showed a chromosome missegregation rate of 20-50% (including both mild and severe segregation errors), which increased to 80-95% when 30 nM compound-5 was added (Fig.4B). In contrast to the tumor cell lines, RPE-1 cells showed mild missegregation events in only 20% of anaphases when treated with low dose compound-5 versus 10% in controls and almost never displayed percentage of cells 100 80 60 40 20 0 U2OS: n=87 n=38 - + - n=25 aligned mild misalignment (1-5) severe misalignment (>5) n=20 - + Mps1-602Q 30nM Cpd-5 Figure 3. Selective inhibition of Mps1 results in the induction of chromosome misalignments and segregation errors A) (top) Representative images of U2OS cells stably expressing H2B-GFP undergoing division in the absence or presence of missegregating chromosomes. (bottom) Quantification of time-lapse movies of RPE-1, U2OS and Hela cells stably expressing tagged H2B. Three phenotypes were scored; anaphase without missegregations, mild missegregations (1-5 chromosomes) and severe missegregations (>5 chromosomes). Time indicates minutes after chromosome condensation. N indicates number of cells analyzed. Compound-5 was added 10 minutes prior to filming. B) (top) Representative images of fixed U2OS cells showing no, mild (1-5 misaligned chromosomes) and severe misalignments (>5 misaligned chromosomes). DNA was visualized using DAPI. (bottom) Quantification of chromosome alignment of indicated U2OS cell lines after 30 minutes of treatment with MG132 in the presence or absence of 30nM compound-5. 121 Mps1 inhibition kills aneuploid cells 6
6 severe missegregations in either treated or control anaphases (2% and 1%, respectively) (Fig.4B). Chromosome segregation errors result in aneuploidy and DNA damage 290,389 . To determine whether an increase in DNA damage could be observed specifically in tumor cells treated with the Mps1 inhibitor, we imaged the tumor cell lines MCF-7 and SW480 and the untransformed cell line RPE-1 all stably expressing H2B-RFP and 53BP1-GFP, a marker for DNA damage (Fig.4C). This analysis revealed a clear increase in G1 daughter cells with DNA damage foci after treatment of MCF-7 and SW480 cells with 30 nM compound-5 (18% and 42% respectively with more than 3 foci versus 5% and 21% in controls), whereas RPE-1 cells hardly showed any DNA damage foci formation following mitotic exit (5% versus 0.5% in controls) (Fig.4C). Overall, these data suggest that the deleterious effects of selective Mps1 inhibition specifically on tumor cells are due to the induction of severe chromosome segregation defects, eventually resulting in massive aneuploidy and DNA damage. A 0 nM compound-5 10 20 30 50 B RPE-1 C Figure 4. Pharmacological inhibition of Mps1 specifically kills tumor cells A) Colony formations of indicated cell lines treated with increasing concentrations of compound-5. Cells were harvested 6 days after addition of compound-5. B) Quantification of chromosome segregation performed as in Fig.3A of indicated cell lines expressing tagged-H2B. N indicates number of cells. C) (left) Representative images of MCF-7 cells stably expressing H2B-RFP (red) and 53BP1- GFP (green) undergoing division in the absence or presence of missegregating chromosomes. (right) Graph depicts quantification of percentage of G1 daughter cells having more than three 53BP1 foci within 300 minutes after mitotic exit (3 experiments + SEM, >50 cells/experiment). Alterations in mitotic progression sensitize tumor cells to Mps1 inhibition One explanation for the different effects that Mps1 inhibition has on chromosome segregation in transformed versus non-transformed cells could be that aneuploid tumor cells have more difficulties aligning their chromosomes when compared to healthy diploid cells 539 and are therefore more dependent on mitotic checkpoint function to prevent segregation errors. Inhibition of the mitotic checkpoint in combination with problems in chromosome alignment, due to partial Mps1 inhibition, could then lead to a more pronounced defect in the fidelity of chromosome segregation in aneuploid 122 BJ-Tert MCF-7 SW480 U2OS HeLa LS174T HCT116 HCT116 p53-/- No error H2B-RFP 53BP1-GFP Segregation error percentage of cells 100 80 60 40 20 0 n=134 n=139 - + 0’ 13’ 26’ 39’ 52’ 65’ 170’ 210’ RPE-1 n=203 n=185 n=24 n=54 No errors Mild errors Severe errors - + - + - + - + 30nM Comp.5 MCF-7 SW480 percentage of cells n=115 60 40 20 0 U2OS n=138 n=74 - + RPE Hela n=73 >3 53BP1 foci (within 300 minutes after Mitosis) - + - + 30nM Comp.5 MCF-7 SW480
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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
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 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 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
Page 157 and 158: 8 tumorformation using intravital i
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
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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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