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

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clear accumulation of cells in mitosis of up to 75% of all cells (Fig.1B). In contrast, depletion of Mps1 by the addition of doxycycline leads to clear inactivation of the mitotic checkpoint, since less than 5% of the cells is mitotic in these cultures (Fig.1B). Addition of doxycycline to the parental LS174 cells did not result in a checkpoint override, indicating that this effect was due to specific depletion of Mps1 (data not shown). These results indicate that conditional depletion of Mps1 from LS174-T tumor cells leads to inactivation of the mitotic checkpoint and a concomitant reduction in cell viability. A B Mps1 α-tubulin doxycycline - + - doxycycline + doxycycline % of MPM2-positive cells in 4N population 80 60 40 20 0 + 1µM taxol - doxycycline + doxycycline Figure 1: Conditional inactivation of Mps1 causes inactivation of the mitotic checkpoint and tumor cell death. A) Left: LS174T-TetRMps1, treated without (-) and with (+) doxycycline (dox) for 3 days, were immuno- blotted for Mps1 and α-tubulin. Right: Colony formations of LS174T-TetRMps1 cells treated with and without dox for 11 days. B) FACS analysis of LS174T- TetRMps1 cells treated with or without dox for 3 days and 1mM taxol for 18 hours. The percentage of mitotic cells was measured as the fraction of cells with 4N DNA content that were positive for MPM2. Mps1 depletion causes severe chromosome alignment defects and enhanced aneuploidy. Inactivation of Mps1 is expected to compromise a cell’s ability to achieve proper chromosome alignment on the mitotic spindle 116 . Therefore we analyzed if conditional depletion of Mps1 compromised the fidelity of chromosome alignment in LS174-T tumor cells. To this end, LS174-T cells grown in the presence or absence of doxycycline for 3 days were incubated with the proteasome inhibitor MG132 for 90 minutes to arrest cells at the metaphase-to-anaphase transition. In control cells grown in the absence of doxycycline, we found that 90% of all mitotic cells had fully aligned their chromosomes (Fig.2A). In contrast, depletion of Mps1 by the addition of doxycycline to the culture medium resulted in a severe reduction, as less than 25% of the cells managed to align their chromosomes in this time (Fig.2A). This increase in segregation errors coincides with a more severe aneuploidy, as evidence by the karyotyping shown in figure 2B (Fig.2B). Taken together, these data show that conditional inactivation of Mps1 compromises the mitotic checkpoint, and results in a severe defect in chromosome alignment. The combination of these two effects results in enhanced segregation errors and as a consequence the karyotype of the tumor cells becomes highly unstable. Conditional inactivation of Mps1 inhibits tumor growth in vivo. Our data thus far demonstrate that conditional inactivation of Mps1 inactivates the mitotic checkpoint, increases chromosome alignment defects and compromises cell viability in vitro. We next asked if conditional inactivation of Mps1 could inhibit tumor growth in vivo. For this, we subcutaneously injected LS174-T tumor cells in Balb/c nude mice and monitored tumor growth over time. One third of 109 Chromosome missegregations kill tumor cells 5
5 110 A B percentage of cells 100 80 60 40 20 0 + MG132 (fixed) - + doxycycline no alignment defects mild alignment defects (1-5 chromosomes) severe alignment defects (>5 chromosomes) % of cells 50 40 30 20 10 0 55
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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
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 106 and 107: A B C percentage of cells % PI posi
Page 108 and 109: Abstract One of the most common hal
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
Page 157 and 158: 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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