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

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Partial depletion of Mps1 or BubR1 sensitizes tumor cells to clinically relevant doses of taxol. The observed correlation between the level of Mps1 or BubR1 depletion, the severity of chromosome missegregations and the extent of cell death pressed us to investigate if increasing the amount of segregation errors beyond that obtained by partial depletion of Mps1 or BubR1 would now impact tumor cell viability. To this end we introduced low, clinically relevant doses of taxol (1-10nM) that are known to cause aneuploidy 554,555 without causing the severe mitotic delay that occurs at high doses of taxol (50nM-10uM) 342,484,553 . We found that in U2OS-, HCT-116-, LS174- and Hela-TetRMps1 cells with normal Mps1 levels, viability as well as the fidelity of chromosome segregation was only marginally compromised at 2nM taxol (Fig.2A-H –doxycycline, 2nM taxol). However, viability was drastically reduced by this low dose of taxol when Mps1 expression was additionally partially suppressed (Fig.2A,C,E,G + doxycycline, 2nM taxol): The number of colonies was reduced 2- to 10-fold when compared to doxycycline treatment alone in U2OS-, HCT-116-, LS174T and Hela-TetRMps1 cells (Fig.2A,C,E,G). This lethality correlated strongly with an increase in the frequency and severity of segregation errors (Fig.2B, D, F, H +doxycycline, 2nM taxol). A slight increase in the dosage of taxol in U2OS-TetRMps1 cells (from 2 to 5 nM) or in LS174-T-TetRMps1 cells (from 2 to 3 nM) induced a moderate increase in chromosome missegregations when Mps1 levels were kept high, but only a partial reduction in colony formation was observed (Fig.2A,B,E,F). Nonetheless, additional partial depletion of Mps1 at these slightly higher taxol concentrations completely blocked colony formation (Fig.2A, E and S2D), which correlated with an even further increase in the amount and severity of segregation errors (Fig.2B, F). We confirmed that the observed reduction in colony formation capacity was due to cell death, and not due to a post-mitotic arrest, by filming U2OS-TetRMps1 cells in the presence of PI on day 4, 5 and 6 after treatment (Fig.S2A, B). The frequency of severe segregation errors correlated well with increased aneuploidy after 4 divisions as determined by chromosome spreads (Fig.S4A). LS174T-TetRMps1 cells were near-diploid in the absence of any treatment. After treatment with doxycycline or 3nM taxol the karyotype resembled that of control cells. This is in line with previous observations in which aneuploid cells in (near- ) diploid cell lines are removed from the population 289 . Nevertheless, upon combined treatment of LS174T-TetRMps1 cells with taxol and doxycycline, the observed chromosome segregation errors (Fig.2F, 3nM taxol) induced a clear increase in aneuploidy and multinucleated cells (Fig.S4A, C). Although untreated U2OS-TetRMps1 cells were already significantly aneuploid, a clear increase in the breadth of distribution in the karyotypes was observed upon combining low doses of taxol and partial Mps1 knockdown (Fig.S4A), a combination that leads to the demise of the population (Fig.2A). The synergistic effect with low doses of taxol was not specific for partial Mps1 depletion, since reduced BubR1 levels also sensitized Hela and U2OS cells to low doses of taxol (Fig.2I, K, S2C and S3). This correlated with an increase in severe chromosome missegregations and enhanced aneuploidy as revealed by the karyotypes of the Hela-TetRBubR1 cells (Fig.2J, L and S4A). To rule out the possibility that the synergy in cell killing by low taxol and reduction of Mps1 or BubR1 was due to clonal variations within one cell type, we used titration of doxycycline in the U2OS clone in which high doxycycline addition (1mg/ml) causes almost full depletion of Mps1 (U2OS- TetRMps1-clone #2, see Fig.S1A). Such titrations enabled us to reach a reduction of Mps1 levels to 22% by adding only 2ng/ml doxycycline (Fig.S5A, B). In agreement with results obtained with the ‘partial reduction’ clone U2OS-TetRMps1 (Fig.1A), the reduced levels of Mps1 achieved by 2ng/ml doxycycline addition did not cause lethality on its own, whereas additional administration of 5nM taxol did (Fig.S5A, D). This again correlated with the amount of chromosome segregation errors (Fig. S5C). Unexpected side-effects of doxycycline administration on cell viability were also ruled out, as we observed no toxicity of simultaneous addition of doxycycline and low doses of taxol in the different founder cell lines (Fig.S6). Finally, the sensitization to low taxol we observed was not due to 91 Chromosome missegregations kill tumor cells 5
A U2OS-TetR Mps1 B relative colony formation 5 a general sensitization to cytotoxic drugs, since partial depletion of Mps1 or BubR1 did not synergize with low doses of doxorubicin, a drug resulting in induction of double-strand DNA breaks (Fig.S7). To further address our hypothesis that cell death in our experiments was caused by inducing a certain threshold level of chromosome missegregations and not, for instance, by cytotoxic effects of taxol, we introduced siRNA's against CENP-E, KIF18A or HEC-1, all known to cause chromosome congression defects upon knockdown in human cells 125,562 . Knockdown of these proteins mimicked the effects of low taxol: Simultaneous reduction of Mps1 and RNAi of CENP-E, KIF18A or HEC-1 in U2OS-TetRMps1 cells increased cell death compared to siRNA transfected controls (Fig.S8A). Importantly, cell death in CENP-E RNAi cells partially depleted of Mps1 correlated with an increase in severe segregation errors (Fig.S8B, C). The differential sensitivity to low taxol when reducing Mps1 or BubR1 was no longer observed when using very high doses of taxol (Fig.2, 1000nM taxol). Although this is in apparent contradiction with reports suggesting that depletion of mitotic checkpoint components can rescue the lethality induced by high doses of taxol (50nM -10uM) 557-559 , in those studies cell death was analyzed within 48 hours after taxol treatment. Indeed we have previously also observed that compromising the mitotic checkpoint causes resistance to high doses of spindle poisons in the short term292 Figure 2. Low doses of taxol reduce the viability of Mps1 or BubR1 depleted tumor cells. A, C, E, G, I, K). Quantification of colony formations of indicated cell lines treated with and without dox for 11 days. Indicated taxol concentrations were added 1 day after dox administration. Colony formation capacity of control cells (no taxol treatment) was set at 1. Average of three independent experiments is shown. B, D, F, H, J, L) Quantification of time-lapse movies performed as in Fig. S1C. Indicated taxol concentrations were added 1 hour prior to filming. n=amount of cells filmed. , most likely because mitotic checkpoint inhibition prevents mitotic catastrophe in the presence of those high doses. Cell 92 1,0 0,8 0,6 0,4 0,2 0 0 2 5 10 20 1000 nM taxol - doxycycline + doxycycline percentage of cells 100 80 60 40 20 C HCT-116-TetR Mps1 D 1,0 100 0,8 80 0,6 60 0,4 40 0,2 20 0 0 1 2 3 4 1000 0 nM taxol relative colony formation percentage of cells E LS174-TetR Mps1 F relative colony formation 1,0 0,8 0,6 0,4 0,2 0 0 1 2 3 4 1000 nM taxol percentage of cells time-lapse (H2B-GFP) 0 100 80 60 40 20 0 n=77 n=82 n=95 n=53 n=70 n=72 0 2 5 0 2 5 nM taxol - + doxycycline n=113 n=107 n=101 n=83 n=81 n=84 0 1 2 0 1 2 nM taxol - + doxycycline n=31 n=44 n=54 n=39 n=47 n=40 no chrom. segregation defects mild chrom. segregation defects (1-5) severe chrom. segregation defects (>5) G 0 2 3 0 2 3 nM taxol - + doxycycline relative colony formation HeLa-TetR Mps1 1,0 0,8 0,6 0,4 0,2 0 0 1 2 3 4 1000 nM taxol H percentage of cells I J HeLa-TetR BubR1 relative colony formation 1,0 0,8 0,6 0,4 0,2 0 0 1 2 3 4 1000 nM taxol percentage of cells K U2OS-TetR BubR1 L relative colony formation 1,0 0,8 0,6 0,4 0,2 0 0 2 5 10 201000 nM taxol percentage of cells 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 time-lapse (H2B-GFP) 0 n=115 n=95 n=102 n=142 n=103 n=73 0 1 2 0 1 2 nM taxol - + doxycycline n=54 n=63 n=40 n=43 0 1 0 1 nM taxol - + doxycycline n=91 n=98 n=84 n=72 0 5 0 5 nM taxol - + doxycycline
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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
Page 41 and 42: 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 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 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
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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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