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

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- Loop out BamHI site with site-directed mutagenesis Primers used: #2-2_#2-3-(BamHI) loopF: GCGTCACCACAGTTCCAGAACTGTACAGTGGTCAG #2-2_#2-3-(BamHI) loopR: CTGACCACTGTACAGTTCTGGAACTGTGGTGACGC - Ligate #2-123 from pCDNA into pAC16 (HindIII/PacI) (pAC16-#2-123) 2) Ligate 5’ recombination arm (fragment#1) into PmeI-AscI (upstream of 5’ loxPsite): Fragment#1=PmeI-(lastpartof) intron14-exon15-intron15-exon16-(firstpartof)intron16-ApaI-AscI (2kb): GTTTAAACTCGAAGGCCTCAACCTCACAGAGATCTTTCTGCTTCTGCCTCTCTCCTGAGTGCTGAGATTAAAGGTGTGTG- CAGCCATGCCTAGCTGTGTGTGGCTTTCCTGTTACCACATTCTGTGGATAGCTTATCTGTTGTTTGTGCCCCACCTTGTACT- TAATATCAAGATGAGATGTTTGGCTGTTCGGTGATACTAGCTCTGGTGACTAAAACTGAATAGGACCTTTATTCCTTTGCT- GAGTGTTCCTCCTTGGCTATCAGGCTGTTTTATGTTATGGTGATACATGAACAGAGATAAAGGGCTTATTTTAAGTTTTACTG- TAATTCTCTAAGCCAGCTAGCTGTAATTAGACTTGCTTCTGGCTATTTTATTAACTTATAGCAAGTAATTGGAAAGCATCCCAT- CAGACCCATTTGTATAATTCCTGCACACAGTACGCTGAGGCGGGATGATTGCCTGGGCTACATACATGCACTGTATAG- GCTGCTGCTATGGCTTTGGTGCAGGGCCCTGGCTTTAATCTCTTAACATAAAAACCATAAGCAAAAGACAAAATAGGTAG- GAGTGTATATTTCCACATGGAGCATGTCTTCCCATAAATATTTTCCTTTTCACGCTCCCCTTATTAGATTTTCAGTTATGAG- CATAAGGAAGAAGGTGGGGTAAGAGTGATTGAACAAGAGTGACAGGGAAGAGGATTCGTGCAGGGGAGGGAAGT- GCATGCATGTTCTAGGCACTGAGTGATGGGTGTGCTGGAAGCTGTAAACTGCGTGGGGGCCTCTTCCTCAGTGCTT- TAAGAAATTGATTCATAGGAACATCATTGCTCCTGCCAACCTAACTCAACTGTGACTTGCGCTGCTTTCCACAAATGAAT- GTAGTGATGGCTTACAATTACTGTGATTTTTAAAAATATTCCCTATCAGAGAAATGAATTGGTTGATAGTAGGCACAAT- GAAAAGGTGGGAGTTGGTGGGGAGAGGGGTCTAGGAAGTGAACTGTCAAATCACAGCCTTAGTCATCCTTATCGTCTTC- GAATGTCTTTTCCTTGATGTTTTCTCCCTGGATAAGAAAGGCATCCCTAGAATTTTGTGGATATAGCAACATCATATT- TAAGTTGGTTTTCTTAGACACTGATGTAGAAAACCTTTGAATTATTTGAATGTCCATTGTTATAGGGGCTGGAAATG- GATACTTAGCTTCTCATGTTGGTATTTCTTTAGGAATATCCTCAGCCTGAGACTGTTAGTGTTAAATGGAAAGGTACTGCTC- CAGTTTTCAGAGGGAGACATGTCCTAAGCTCTTTCTCCACTTTTTATGTAGGTGTTTCAGGTATTGAATGAGAAAAAA- CAGATAAACGCTATCAAATATGTGAACCTAGAAGACGCCGATAGCCAAACTATTGAGAGCTACCGCAACGAGATAGC- GTTTTTGAACAAACTACAGCAACACAGTGATAAGATCATCCGCCTCTATGATTAGTATGAATTCATTTTTATTTTAAAAATA- AAAGTTTGTTCTTGCCATAATTCTTAGGCAAAGAGTAAATCCTTAATGACATAATGTGGGCATTTATTGTTTTGTTGTGTCTGTT- TATCTTTAATTGCAGTGAAATCACCGAGCAGTACATCTACATGGTAATGGAATGTGGAAACATTGACCTAAATAGTTGGCT- TAAAAAGAAAAAATCCATCAATCCATGGGAACGCAAGAGCTACTGGAAAAACATGTTGGAGGCAGTACACATAATCCAT- CAGCATGGTATTTTCATATCTCTTCATACACGTAAAGTTAAAATAGTTGTTAATTGTGCCATTTTAGAAACATACCCTTAACTG- GAAGTTCATTAGAGGTGAAGGCACTCTTAAGAGTGGTTATACACAGGCTACAGAACACAAACAAGCACAGGATGTAGAA- CAGAAATGGCCACATGTACAATGTAAACTTACCCTCCTCTGGTACCTGGGGATTCCTATCTTCAAGTCCTGAGGATTTGGA- CATCCTACAGATAGGAGGAGAGGGCCCGGCGCGCC - PCR fragment#1 from genomic DNA (from 129/Ola-derived IB10 ES cells, (kind gift from Dr. Hans Clevers)) (Italic is overhang, in Capitals/Italic the restriction sites): Primers used: #1Forward-Short2kb2: ctagcgGTTTAAACTCGAAGGCCTCAACCTCACAGAGATCTTTC #1 Reverse: atcttaGGCGCGCCGGGCCCTCTCCTCCTATCTGTAGGATG - Ligate into pAC16 (>>pAC16-fragment#1) (PmeI&AscI) - Ligate into pAC16-#2-123 (>>pAC16- #1-#2-123) (PmeI&AscI) 3) Ligate 3’ recombination arm, including point mutation (*) (fragment#3) into SbfI-NotI (downstream of 3’ loxP site) Fragment#3=SbfI-(lastpartof) intron16-exon17*-intron17-NotI (2.2kb): (*GCT* =D637A, *GCA* =T649A) CCTGCAGGATGGCCAGTTTTTCAGTCTCCAAAGGATTTTCTCTTTGGAGCCCGGTGTGAGAGTTGGTCTGTTCTTGCT- GTTTTCTGTTTCATTATTTGTTTGTTCTTGTTCTCTCCAGTTTCCTCTGCCTTTCCCTTGTCTTTTAAACAGATGTGTTCAATCT- CATGCGCTTCCTCTGCTTCTCCTTTTAGAAGCAGTAACTAAGCAGAGGATAGGACTCTTTGGGGGTGAGGGAGGCCTTGC- GATCTTATTGTCTGACAGTTCTTGGTGGTGCTTTCCTGGCCTGTGCATGGCAGTGTGCAGCACACACTGACAGGCTAGGCCTG- GAATCCTGAACAGTTCACTTCAACTCAAATCACATTGAGTGTCCTCACCCTGAAAATGATGATGATAATGGGACTTATTTGCTAT- GCATTGCTATAGAATGGTACCTGGCCCACAACATTTGCTAAGAAAGTGTAGAATGTATAATAACTAAATATTAATGTGTTGCCTA- ATTGAAAGGATAAGCCTGACCTAAAGATTATGAAATGCTTTTTTGTCCCCTGGCCATAGAATGTTTTTCTTCATCTAACAGAAC- 65 Cre-inducible Mps1 knock-in mouse model (CiMKi) 3
3 GGATTTTATTTTGAAGGTATTGTTCATAGTGATCTGAAGCCTGCTAACTTTGTGATAGTGGATGGAATGCTAAAGCTAATT *GCT*TTTGGGATTGCAAACCAAATGCAGCCAGACACA*GCA*AGCATTGTTAAAGATTCTCAGGTAGGAGTTTTGCT- GTCTTGGTTGTATTTTAGTGTTTTGAACCAGGGTTTTGCATCAGGGTTTTGCATAACCTAGAATGCTCTTGACTTTGATCAGTG- GCCTTCAGCTCCTGATCCTGCTGCCTGTGCATCCCAGGTGTGGGCTTATAGGTGTCAGCCCCGACACCCGACTTCAGGTAG- GATTTTAATGATGGCTGGTTACTACAAGGCTTAGTTCATTTTTATCTGTTAAATATGTTGCCAATATTATATTTTTACCAACCATGT- TATTCCAAAAATTTGAAGTCTTTTTAAAGAATAGAAACTATGTTTATAAAAGACCATGGTCAAAGCCATGGTCAATTTGATT- TATAAAAGCAGTTCAAGATCAGACAAGTATATTTATGAATTTTGGATGATTTTCTCATAGCTGAGGCAGGGCAGAGAGTAATTG- CACCTTCATGTTCTCCACTGTCCTGTTTCTTTTTCTTACTGCTTAAATTTGGGAGAAAGTTTTAAGAGAGCCTTATTGGGAATACT- GAAGCGTTCCACTCAGCTACGCGTTAAAAAGGAAATATTTTACTTACTGTTTGGGGGGCGGTGTGCGGATTCCAGATGTA- AGTGTGCCATTGTGGAGGTGTGGGGATGGCTTCGGTAAACCACTTCTCTCCTACTGTGGGCCCTGGGAGCCAAAGTCA- GATTGTGTGTGCAGCAAGCTCTACAGCCCAGCTGTGCTTTGTAGTAACATTTGCTGTGGTAAATCTCATGAAGCTGAAG- TAGTGAGGGGAAAACAGAGCTGAAAGGTGATGTCGACTGCACCTCGCAGGCTGTGTCCAGGGATGGAGATAAATCAGAA- GATAAATTACCATGCACGTAGAAAGTCATTCTTCTTGACAGCCATTCATTGTTTTTTGTTTCAGAAGTACAGATGATGAACAGT- GAGTGTAGATGAGACTGAAGTTTTCTATGGCAAGGTCTTAGCAGGCCGACATTTTGTTACCTTAGAACTAAAGGATTTT- GCGTATTATCTCCATGCCCAGCTAGAGAAGCTCTTCTCTGATATAGGTTTCCAACCCATCTTGATCTGCACATGGAGC- CGAAGAATATTGGGAGATAAAGCTAGCTGGTTCCTTTTATTCATGTATTAATTTGTTGCTTGGTTTATTGAGGGAAGAATAT- GTTGAATTTATTGGAACATGAAAGTGAATGAAAGGCCAAGTTCAGAATCCGCCTACGCAGTTGTAAAGACTTAGTACTTAG- TACTTAGTACTTAGCACTAGCTCTCCAGCACAGCTGCAGACAGCACAGTGCTCCCTGTGCTCCAGACGGAGCCCGTTCATTCT- CAGCCCAGCTCATCTGATTGTACCTGGGATGGGATAGTACATACATTCTTATATTGTTAGCAGTTATTTGAATTTTTCAAGTCTGT- CATTTAAATCATTAGTTATTCAAATTTCCAAGAATCTGACATTTACATATTTACAAATCTAGAAAGATATTCTCATTGATTTCTTTGT- GATTTGCAAATAGGCGGCCGC - PCR fragment#3 from genomic DNA (from 129/Ola-derived IB10 ES cells, (kind gift from Dr. Hans Clevers)) (Italic is overhang, in Capitals/Italic the restriction sites): Primers used: #3 Forward: ccgccCCTGCAGGATGGCCAGTTTTTCAG #3 Reverse: ctagcgGCGGCCGCCTATTTGCAAATCACAAAG - Ligate into pAC16 (>>pAC16-#3) (PmeI&AscI) - Introduce point mutations with site-directed mutagenesis Primers used: mMps1-T649A-F: CAAATGCAGCCAGACACA*gca*AGCATTGTTAAAGATTC mMps1-T649A-R: GAATCTTTAACAATGCTTGCTGTGTCTGGCTGCATTTG mMps1-D637A (KD)-F: GAATGCTAAAGCTAATT*gct*TTTGGGATTGCAAAC mMps1-D637A (KD)-R: GTTTGCAATCCCAAAAGCAATTAGCTTTAGCATTC - Ligate into pAC16- #1-#2-123 (>>pAC16-#1-#2-123-#3) (PmeI&AscI): (=CIMKI-D637A & CIMKI-T649A) Cell culture & reagents ES cells were grown in G-MEM supplemented with L-Glutamine, Sodium Pyruvate, NonEssential Amino Acids (all from GIBCO Invitrogen), 10% Foetal Calf Serum (Greiner), β-Mercaptoethanol and LIF (CHEMICON international). ES cells were grown on either 0.1% gelatin coated dishes or a layer of irradiated MEFS and transfected with the Not1 linearized targeting construct pAC16-D637A or T649A using electroporation with a Biorad gene pulser. Electroporated ES cells were selected with 1mg/ml puromycin (Sigma) to obtain stable transfectants. Single colonies were subsequently picked and grown in 96-wells plates before genotype analysis. MEFs were cultured in DMEM (4,5 g/L glucose) (Lonza) supplemented with antibiotics, ultraglutamine (Lonza) and 15% FCS. Ad-Cre-GFP and Ad-GFP (both from Vector Biolabs) were used to infect MEF cells at 100 MOI. Nocodazole (250ng/ml), MG132 (2mM) and DAPI (1mg/ml) were all from Sigma. PCR analysis ES clones ES cell clones were grown in 96 wells plates, lysed using Hotshot lysis buffer 456 and analyzed using PCR. DNA from targeted ES cells yielded a band of ~2.8 kb. 66
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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
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 24 and 25: that aberrant spindle formation inc
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 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 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
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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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