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

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Materials and Methods Cloning targeting vector (D637A=kinase-dead and T649A=Hm676) Targeting vector; pAC16 (based on pFlexible 455 ) (kind gift from Dr. Jos Jonkers): F=FRT site L=loxP site 1) Ligate conditional part (fragment#2) into HindIII-PacI (between loxP sites) 2) Ligate 5’ recombination arm (fragment#1) into PmeI-AscI (upstream of 5’ loxPsite) 3) Ligate 3’ recombination arm, including point mutation (fragment#3) into SbfI-NotI (downstream of 3’ loxP site) Fragment#1 Fragment#2 Fragment#3 63 Cre-inducible Mps1 knock-in mouse model (CiMKi) 3
3 1) Ligate conditional part (fragment#2) into HindIII-PacI (between loxP sites) Fragment#2 was assembled from 3 parts: #2-1=HindIII-(lastpartof)intron16-XhoI (600 bp) #2-2=XhoI-exon17-exon18-exon19-exon20-exon21-exon22-BamHI (1000 bp) #2-3=BamHI-intron22(polyAsignal)-PacI (600 bp) AAGCTTATGGCCAGTTTTTCAGTCTCCAAAGGATTTTCTCTTTGGAGCCCGGTGTGAGAGTTGGTCTGTTCTTGCTGTTTTCT- GTTTCATTATTTGTTTGTTCTTGTTCTCTCCAGTTTCCTCTGCCTTTCCCTTGTCTTTTAAACAGATGTGTTCAATCTCATGC- GCTTCCTCTGCTTCTCCTTTTAGAAGCAGTAACTAAGCAGAGGATAGGACTCTTTGGGGGTGAGGGAGGCCTTGCGATCT- TATTGTCTGACAGTTCTTGGTGGTGCTTTCCTGGCCTGTGCATGGCAGTGTGCAGCACACACTGACAGGCTAGGCCTG- GAATCCTGAACAGTTCACTTCAACTCAAATCACATTGAGTGTCCTCACCCTGAAAATGATGATGATAATGGGACTTATTTGC- TATGCATTGCTATAGAATGGTACCTGGCCCACAACATTTGCTAAGAAAGTGTAGAATGTATAATAACTAAATATTAATGTGTT- GCCTAATTGAAAGGATAAGCCTGACCTAAAGATTATGAAATGCTTTTTTGTCCCCTGGCCATAGAATGTTTTTCTTCATCTAA- CAGAACGGATTTTATTTTGAAGGTATTGTTCATAGTGATCTGAAGCCTGCTAACTTTGTGATAGTGGATGGAATGCTAAAGC- TAATTGATTTTGGGATTGCAAACCAAATGCAGCCAGACACAACAAGCATTGTTAAAGATTCTCAGGTTGGCACAGTTAAC- TATATGGCCCCAGAAGCAATCAGAGACATGTCTTCTTCAAGAGAAAATTCGAAAATCAGAACCAAGGTAAGTCCCAGAAGT- GATGTCTGGTCCTTGGGGTGCATTTTGTACTACATGACTTATGGGAGGACGCCATTTCAGCACATCATCAATCAGGTCTCTA- AACTGCACGCCATAATCAACCCTGCTCATGAGATTGAATTTCCCGAGATTTCGGAAAAAGATCTTCGAGACGTGCTAAAGT- GCTGTTTAGTGAGGAACCCTAAAGAGAGGATATCTATCCCTGAGCTCCTCACACATCCGTATGTTCAAATTCAGCCCCATC- CAGGCAGCCAAATGGCTAGGGGAGCCACTGATGAAATGAAATATGTGTTGGGTCAACTTGTTGGTCTGAATTCTCCTA- ACTCCATCTTGAAAACTGCAAAAACTTTGTATGAACGTTATAATTGTGGTGAAGGTCAAGATTCTTCGTCATCCAAGACTTTT- GACAAAAAGAGAGAAAGAAAGTGATGCACAGCTACGTACAAACCAAGAACACTAGATTGTTTCCTCTGCCATACTCTT- GAATCTCTGAGGAAATCTACCAGTTGGAAACAACCTCACCTGGATTTTATCAGTTAAAAAAACAAACAAACAAAACTTCAG- TAGATTATCCTCAAAAGAAAGCTGTAAAGTTAACCACTCATAGCACTGTGTATATTAAATTATAGAGTTGTGCTTTTCTTT- TATGCTTTTCTGTAAATCTGCTAATGTTTTACGTTTGGAACAGTGAATGATAGCTGGAATGTTGAAGAGCTCTGTAAATA- AAGCGTCACCACAGTTCCAGAACTGTACAGTGGTCAGTTTCTTCAATCAAATGTGTTCTTGGCATGATAGCAAAATTTT- TAGAAAAACGGGATTAAGAATAGACCGTAGTAAATAAAGTTTAACAATTAAATTTCCAAAGGATTTAGGACTGTAAA- CAGGCTCACACCTTTAGTCCCAGCACTTGGGAGGCAGAGGCAGGCAGATTTCTGAGTTCGAGGCCAGCCTGGTCTA- CAGAGTGAGTTCCAGGACAGCCAGGGCTACACAGAGAAACCCTGTCTTGGCGGGGAGGGGCCGGGGAGGGGGGGA- GATCCAAAGGATGTAGGAGGCAGAGACAGGTGGATCTCTGTGATTCTGAAGCCAGCCTGGTCTACAGAACTAGTTC- TAGGATAGCCAAGGCTATACAGACACTTTCTCCCCACCACCATCCTGCCCTCAAAAAAATTGTAAATAAATTTCCTA- ATTGTGTACAGCCATGATACCATCTATAGTATTTGGTCTGCAAGTGGCTTTTTCAGTTCTCCCTTTGACTCTTCAAAGTA- CATATGGGGTTTGGTGTTCTAAATATTGTGCTGGAGTTTGTGATTTAATGTCTATAGTTAATACATGCCATTATTGAGTTAATTAA PCRs performed to obtain fragment #2-1, #2-2, #2-3 (Italic is overhang, in Capitals/Italic the restriction sites): - PCR #2-1 from genomic DNA (from 129/Ola-derived IB10 ES cells, (kind gift from Dr. Hans Clevers)) Primers used: #2-1Forward:cggcgAAGCTTATGGCCAGTTTTTCAG #2-1 Reverse: cgccgCTCGAGCTTCAAAATAAAATCCGTC - PCR #2-2 from mouse cDNA (ordered from Imagenes, Germany, BC 058851, ID: 30023533) Primers used: #2-2long Forward: ccggcCTCGAGgcgctactctggtgGTATTGTTCATAGTG #2-2long Reverse: ccgcgGGATCCgcgctactctggtgCTGGAACTGTGGTGAC - PCR #2-3 from genomic DNA (from 129/Ola-derived IB10 ES cells, Clevers lab) Primers used: #2-3 Forward: ccgccGGATCCAACTGTACAGTGGTC #2-3 Reverse: ccgccgTTAATTAACTCAATAATGGCATG Ligation steps to assemble 3 fragments: - Ligate #2-1 & #2-2 simultaneously into pCDNA3 (>>pCDNA-#2-12) - Loop out XhoI site with site-directed mutagenesis: Primers used: #2-1_#2-2-(XhoI) loopF: GACGGATTTTATTTTGAAGGTATTGTTCATAGTGATC #2-1_#2-2-(XhoI) loopR: GATCACTATGAACAATACCTTCAAAATAAAATCCGTC - Ligate #2-3 (from pGEM-Teasy) into pCDNA-#2-12 (>>pCDNA-#2-123) 64
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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
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 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 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 110 and 111: clear accumulation of cells in mito
Page 112 and 113: 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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