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

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that aberrant spindle formation increases the chance of obtaining erroneous kinetochore attachments and that proper bipolar spindle assembly is essential to obtain normal, amphitelic attachments. In line with this, it was shown that washout of drugs that affect spindle formation in prophase also increased the number of merotelic attachments and induced chromosome segregation errors 345 . Interestingly, multipolar spindles are often observed in CIN tumors 349-352 , which indicates that aberrant spindle assembly might be responsible for merotelic attachments and CIN induction in vivo as well. These multipolar spindles are often the result of centrosome overduplication, a common trait of cancer cells 353 . Persistence of multipolar spindles in anaphase results in severe chromosome missegregations and the severely aneuploid daughter cells often die in the next G1 phase 354 . Tumor cells that harbor multiple centrosomes have also been shown to sometimes progress through a transient multipolar state, after which supernumerary centrosomes cluster during mitosis and eventually form a seemingly normal bipolar spindle 355,356 (Fig.7A). Mitotic entry in the presence of a multipolar spindle results in attachments of single kinetochores to multiple spindle poles 347,348,355,356 . Subsequent centrosome clustering converts these attachments to merotelic attachments, thus providing a mechanism that links multipolarity to lagging chromosomes 355,356 (Fig.7A). Supernumerary centrosomes can arise through a number of different mechanisms, such as centrosome overduplication, cytokinesis failure or virus-induced cell fusion, of which the last two also result in increased ploidy. Interestingly, tetraploidy is also frequently observed in tumor cells and has been shown to promote tumorigenesis in mice 357 and transformation in vitro and in vivo 358 . One remarkable mechanism by which tetraploidy could arise is ‘entosis’: the invasion of a cell into another cell, a phenotype often observed in tumors that increases with increasing tumor grade 359-361 . The mere presence of an invading cell in the host cell’s cleavage plane inhibits cytokinesis and as a result causes tetraploidy and multipolarity in the next mitosis, resulting in aneuploid daughter cells 361,362 . This illustrates that one single cytokinesis failure could initiate chromosome segregation errors in the tumor cell’s off-spring, ultimately leading to CIN. Increased stability of kinetochore MT attachments The proper control of kinetochore-microtubule stability is another crucial factor that determines the fidelity of chromosome segregation (Fig.7B). Increasing the stability of microtubules bound to kinetochores through depletion of MCAK or Kif2b, two kinetochore-localized microtubule depolymerases, results in an increased occurrence of merotelic attachments and severe chromosome missegregations in human tumor cells 363 . These results stress the importance of the error-correction machinery, because a slight decrease in kinetochore-MT turnover upon Kif2b or MCAK depletion can clearly affect Aurora B-dependent correction of misattached kinetochores 363 . A follow-up study in which the kinetochore- MT turnover was compared between an untransformed cell line and several CIN lines, revealed that CIN tumor cell lines often display an increased stability of kinetochore MTs, which could result in persistent misattachments until anaphase 364 . Importantly, increasing kinetochore-MT turnover in these CIN cell lines by overexpressing Kif2b or MCAK could partially revert the chromosomal instability phenotype 364 . A recent report revealed an unexpected link between MAD2 overexpression, often found in CIN tumors, and increased kinetochore-MT stability. Aurora B centromere localization and activity was significantly decreased upon MAD2 overexpression, which could explain the increased occurrence of chromosome segregation errors in these cells 365 . The authors observed that the effect on Aurora B localization is independent of the checkpoint function of MAD2, but the exact underlying mechanism is still unknown. This effect on Aurora B localization and activity is in line with another study, which suggested that the Aurora B-dependent error correction machinery works more efficiently in healthy cells when compared to tumor cells 366 , thus providing an 23 General Introduction 1
1 explanation for the occurrence of CIN in those tumor cells. Since MAD2 overexpression results in chromosome segregation errors and tumorigenesis in mice 331,332 (Table I), aberrant Aurora B localization and activation could very well explain the CIN observed in MAD2-overexpressing tumors. One other interesting possibility which could explain the increased stability of kinetochore-MTs observed in CIN cells 363 , is the mutation or loss of the tumor-suppressor gene Adenomatous Polyposis Coli (APC), which frequently occurs in colon carcinomas 367 . These APC mutations are thought to be an initiating event in colon tumorigenesis through loss of APC’s inhibitory role in Wnt signaling 368 . APC loss and CIN are both found in early cancer lesions 369 and APC loss-of-function has been shown to result in genetic instability 370,371 , which is thought to, at least partially, be dependent on its function in kinetochore-MT stability 372-374 . APC localizes to centrosomes, kinetochores and MT plus-ends 369 and depletion of APC results in reduced inter-kinetochore tension, which is thought to be due to decreased kinetochore-MT dynamics 372,373 . Although a very interesting hypothesis, future research will have to assess the direct effects of APC loss on CIN in vivo, since defects in APC function also result in activation of Wnt signaling and its downstream targets, which might explain the induction of CIN as well 375,376 . 24 A) Centrosome clustering B) Increased KT-MT stability C) Multiple attachment sites Aurora B/ MCAK/ Kif2b Aurora B/ MCAK/ Kif2b Figure 7. Schematic representation of underlying causes of merotelic attachments A) Centrosome coalescence creates merotelic attachments by allowing clustering of centrosomes, which had already established microtubule interactions with kinetochores. B) Impaired kinetochore microtubule dynamics inhibits the release of erroneous attachments, whereas the presence of multiple centromeres (C) increases the chance of creating faulty attachments. Multiple attachment sites The presence of extra centromeres on chromosomes could be another cause of lagging chromosomes
Page 2 and 3: Chromosome segregation errors: a do
Page 4 and 5: Chromosome segregation errors: a do
Page 6: Get up, stand up, stand up for your
Page 10 and 11: Chapter 1 General Introduction Anie
Page 12 and 13: cytoplasms and pinches off the memb
Page 14 and 15: which sister-chromatids are not pro
Page 16 and 17: Non-MCC members, but important mito
Page 18 and 19: completely separated. Following cle
Page 20 and 21: Two striking features of cancer cel
Page 22 and 23: predisposition at a very young age,
Page 26 and 27: (Fig.7C). As discussed in section 4
Page 28 and 29: Thesis outline Chromosome segregati
Page 30: 286,395,407,408 Increase (thymus) I
Page 33 and 34: 2 Abstract Various types of chromos
Page 35 and 36: 2 34 A C D E % of cells Control 100
Page 37 and 38: 2 assess whether cytokinesis induce
Page 39 and 40: 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
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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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Chapter 5 Elevating the frequency o
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Introduction Error-free chromosome
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Partial depletion of Mps1 or BubR1
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death is not induced in the short t
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had survived growth for 7 days in t
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The following antibodies have been
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Supplemental data Supplemental figu
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A BubR1 α-tubulin Hela-TetRBubR1 -
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- doxycycline + doxycycline - doxyc
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A B C percentage of cells % PI posi
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Abstract One of the most common hal
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clear accumulation of cells in mito
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the mice did not receive any doxycy
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113 Chromosome missegregations kill
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Chapter 6 Aneuploid tumor cells are
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Introduction Equal segregation of t
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S1A, data not shown). Based on thes
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A B U2OS + H2B-YFP percentage of ce
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tumor cells, leaving diploid cells
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A B p-S10-Histone H3 DAPI C µmol/l
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Supplemental Figure-1. Protein bioc
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Bioavailability of compound-5 after
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A B U2OS (7.5nM) Hela (10nM) RPE-1
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7 Abstract Taxanes, such as docetax
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7 of mitotic aberrations. These dat
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7 A Low CFP lifetime High CFP lifet
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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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