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

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death is not induced in the short term in those analyses because the deleterious effects of severe chromosome missegregations are not expected to become apparent before 2-6 divisions have occurred 292 . In support of this, the present study determined the effect on cell viability after 4 to 11 days and we find that high doses of taxol are equally toxic to parental as well as Mps1- or BubR1depleted cells (Fig.2, 1000nM taxol). The apparent resistance of CIN cells to high taxol treatment 280 and chromosome missegregations 289 has led to the hypothesis that these cells have obtained a survival mechanism that protects them against cell death induced by aneuploidy. In contrast, stable diploid (tumor) cells, which have become aneuploid, are sensitive to the genome unbalances and are removed from the cell population 289 . To address the question whether such aneuploidy-tolerance mechanisms would allow CIN cells to also tolerate simultaneous Mps1 depletion and taxol treatment, we tested the effect of this combined treatment on the CIN cell line SW480. As expected 270 , these cells displayed frequent chromosome missegregations in the absence of any treatment (Fig.S8G untreated). Similar to what was observed with the other four tumor cell lines (Fig.2), simultaneous treatment of SW480 cells with low taxol (10nM) and partial depletion of Mps1 (Fig.S8D) enhanced the frequency and severity of chromosome segregation errors (Fig.S8G) and caused cell death (Fig.S8E,F). It is important to note that the level of chromosome missegregations induced by this combined treatment reached far beyond the level of missegregations found in CIN tumor cells ( 270 and Fig. S8G). In all likelihood, therefore, the tolerance to aneuploidy of CIN tumor cells is insufficient to cope with the severity of chromosome segregation errors induced by our treatments. Together, our data show that substantial reduction of Mps1 or BubR1 leads to an increase in mild chromosome segregation errors but fails to induce massive tumor cell death. However, combining reduction in Mps1 or BubR1 with low, non-lethal doses of taxol increases the level and severity of segregation errors and severely compromises viability in all tumor cell lines tested. Synergy in cell killing with low taxol depends on inefficient chromosome alignment in Mps1- or BubR1depleted cells In addition to their function in the mitotic checkpoint, both Mps1 and BubR1 have been shown to control chromosome alignment by influencing the stability of the kinetochore-microtubule interaction or error-correction 116,425,442 . In contrast, Mad2 is a protein which is critical for the mitotic checkpoint but has no apparent role in efficient chromosome alignment 73,442 . To examine if the mitotic checkpoint deficiency caused by reduction in Mps1 or BubR1 levels is sufficient to cause cell killing with low doses of taxol, we created an inducible monoclonal U2OS-TetRMad2 cell line in which the mitotic checkpoint was compromised to a similar extent after doxycycline addition as compared to the U2OS cells in which Mps1 or BubR1 were partially reduced (Fig.3A and S9A, C). Assessment of the time spent in mitosis in the presence or absence of low doses of taxol did also not reveal any difference between U2OS cells with partial Mps1, BubR1 or Mad2 knockdown (Fig.S9B). Importantly, partial Mad2 depletion did not lead to synergistic lethality with low doses of taxol (Fig.3B), which contrasts the effects seen with partial reduction of Mps1 or BubR1 (Fig.2A,K). This lack of synergy in cell killing correlated with a lack of synergy in severe chromosome missegregations (Fig.3C) and aneuploidy (Fig.S4B) in these cells. The rate of severe segregation errors was significantly lower than in cells with reduced Mps1 or BubR1 (40%, 85% and 65% respectively) (Fig.3C, compare with Figs.2B and 2L). In addition, in the partial Mps1- or BubR1depleted U2OS cells almost all of the remaining cell divisions display mild missegregations, while 50% of all divisions in cells with partial Mad2 knockdown and 5nM taxol show no segregation errors at all. The previous observations indicated that inducing chromosome missegregations to a level sufficient to cause cell killing was substantially more efficient when chromosome alignment was disturbed 93 Chromosome missegregations kill tumor cells 5
5 A Mad2 U2OS-TetR MAD2 100 30 B U2OS-TetR MAD2 C time-lapse (H2B-GFP) U2OS-TetR MAD2 α-tubulin - + 10 µM STLC doxycycline 1,0 0,8 0,6 - doxycycline + doxycycline 100 80 no segregation defects 20 0,4 0,2 60 40 mild segregation defects (1-5) severe segregation defects (>5) 15 10 5 0 - doxycycline + doxycycline 0 0 2 5 10 201000 nM taxol 20 0 0 5 - 0 5 nM taxol + doxycycline D in addition to the mitotic checkpoint. In support of this, when cells were allowed to align their chromosomes for 90 minutes in the presence of the proteasome inhibitor MG132, we found that severe chromosome misalignments were more prevalent in low-taxol-treated populations of cells with diminished BubR1 or Mps1 levels than those with diminished Mad2 (Fig.3D,E). This suggests that reducing Mad2 levels, when weakening mitotic checkpoint activity to a similar extent as partial Mps1 or BubR1 depletion, does not cause sufficient segregation errors in the presence of low doses of taxol to compromise tumor cell viability. These results show that chromosome congression defects play an important role in the synergistic lethality with low doses of taxol. Although congression defects per se are not expected to lead to chromosome missegregations when the mitotic checkpoint is working efficiently, it is formally possible that mere alignment defects are sufficient to cause lethality and that mitotic checkpoint weakening is not required. To address this, we introduced CENP-E siRNA in U2OS-TetRMad2 cells to enhance congression defects in a checkpointweakened cell system and thus recapitulate the effects of reducing Mps1 or BubR1 (Fig.S10D, E). The chromosome alignment defects induced by CENP-E siRNA and 5nM taxol treatment led to an increase in segregation errors when compared to control siRNA-treated cells (Fig.S10C -doxycycline), but caused only a minor increase in cell killing (Fig.S10A, B). Importantly, introducing a checkpoint deficiency under these conditions, by partial knockdown of Mad2 (Fig.S10D), clearly increased the level of severe segregation errors and resulted in a significant increase in cell death (Fig.S10A-C +doxycycline). These results support the hypothesis that the synergy in cell killing by low doses of taxol and partial depletion of Mps1 or BubR1 requires significant congression defects combined with a weakened mitotic checkpoint. To rule out that the failure to compromise tumor cell viability by partial depletion of Mad2 was due to selection for cells with higher Mad2 expression during growth in low taxol, we analyzed the ability to delay mitosis in respect to spindle poison (1mM taxol) in U2OS-TetRMad2 cells that 94 mitotic duration (hours) n=61 n=64 crest dapi merge relative colony formation capacity alignment mild misalignment (1-5 chromosomes) severe misalignment (>5 chromosomes) E 100 percentage of cells 80 60 40 20 percentage of cells percentage of cells 0 0 5 0 5 - + U2OS-TetRMps1 100 n=60 n=65 + MG132 (fixed) 80 60 40 20 n=60 n=75 0 0 5 0 5 - + U2OS-TetRBubR1 percentage of cells 100 80 60 40 20 0 0 5 0 5 nM taxol - + doxycycline U2OS-TetRMad2 Figure 3. Chromosome alignment dysfunction in Mps1- or BubR1-depleted cells enhances cell death upon taxol [low] treatment. A) Top: U2OS-TetRMad2 cells that were treated with and without dox were immuno-blotted for Mad2 and α-tubulin. Values indicate relative percentage of Mad2 levels. Bottom: Live cell analysis of the mitotic duration of U2OS-TetRMad2 cells treated and measured as in Fig.1B. B) Quantification of colony formations analyzed as in Fig. 2. C) Quantification of anaphase progression as in Fig. 1E. n=amount of cells filmed. D) Immunofluorescence images of cells with different chromosome alignment phenotypes after 90 minutes addition of MG132. Centromeres (CREST) are in green and DNA (DAPI) is in blue. ‘Alignment’, ‘Mild misalignment’ or ‘Severe misalignment’ indicate cells with 0, 1-5 or more than 5 chromosomes not aligned on the metaphase plate, respectively. E) The indicated cell lines were treated with or without dox for 3 days and 1 hour prior to MG132 addition indicated taxol concentrations were added. n=amount of cells analyzed per condition.
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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
Page 43 and 44: 2 Immuno Blotting Cells were lysed
Page 45 and 46: 2 Supplemental figures 44 A B C % o
Page 47 and 48: 2 A MCF7 B C 53BP1 foci/cell 46 % o
Page 49 and 50: 2 A siLuc siATM C 48 p-S1981 ATM me
Page 51 and 52: 2 50 A B C % of EdU positive cells
Page 54 and 55: 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 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 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
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7 Cells (~50.000/well) were plated
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7 Supplemental data Supplemental fi
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7 148
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8 Thesis summary Unequal separation
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8 exerted through contraction of th
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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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