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

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the mice did not receive any doxycycline, one third of the mice were given doxycycline in their drinking water from day 0 onwards, and one third of the mice received doxycycline in their drinking water from day 7 onwards. Clearly, tumor growth was reduced in the mice that received doxycycline in their drinking water (Fig.3A). Tumor growth was initially most efficiently inhibited in the group that received doxycycline on day 0, but almost all of the mice displayed progressive tumor growth (Fig.3A). Thus, the conditional inactivation of Mps1 that is achieved in this system is capable of slowing down tumor growth, but it is unable to fully arrest tumor growth. Whether this is due to incomplete inactivation of Mps1 in vivo is currently unclear. Alternatively, tumor cells in vivo might be less dependent on Mps1 function for proper chromosome segregation than tumor cells grown in vitro. To analyze if tumors in vivo had lost the ability to shut down Mps1 expression in response to doxycycline, we isolated tumors from the mice that received doxycycline in their drinking water and analyzed their growth in vitro. Importantly, their viability and mitotic checkpoint function was again severely compromised when doxycycline was added to the culture medium, indicating that these cells had not simply lost conditional expression of the Mps1 shRNA (Fig.3B). However, this observation does not discriminate between the possibility that doxycycline does not efficiently reach the tumors in situ, or the possibility that in vivo tumor growth is less dependent on Mps1 function. In order to resolve this we will have to examine the effect of doxycycline on chromosome segregation in vivo. For this purpose we wish to perform intravital imaging of cell division of the xenografted tumor cells. Nonetheless, our data show that inactivation of Mps1 can partially inhibit tumor growth and suggest that targeting Mps1 function might be a useful anti-cancer strategy. Materials and Methods Tissue Culture, Transfections and Treatments LS174T cells were grown in DMEM (Lonza) with 10% Tet-approved FCS (Clontech), supplemented with pen/strep (Invitrogen) and ultraglutamine (Lonza). Taxol, MG132 and doxycycline (used at 1mg/ml) were from Sigma. LS174T cells expressing TetR were infected with retrovirus carrying pSuperior-retro-puro- Mps1 and selected with 2µg/ml puromycin. Single colonies were selected after replating 1-2 cells/well. Immunofluorescence Microscopy Cells plated on 12mm coverslips were harvested after 90 minutes MG132 treatment. Fixation was done using 4% PFA in PEM buffer. CREST was incubated O/N in PBS 3% BSA. Anti-human Alexafluor647 and DAPI were incubated in PBS 0, 1% Tween. Stained coverslips were mounted with Vectashield Mounting Medium (Vector). Images were acquired on a Zeiss 510 Meta confocal laser scanning microscope with a 63X/1.4NA Plan-ApoChromat objective using the Zeiss LSM software. Chromosome spreads Nocodazole was added for 4 hours to the medium to enrich for mitotic cells. Cells were treated with 0.75 M KCl at 37ºC for 10 minutes, centrifuged at 2000 rpm and fixed for 20 minutes with Methanol: Acetic Acid (3:1). Fixation procedure was repeated 3 times. Samples were collected in Methanol and DAPI to stain for DNA. Chromosome spreads were created by allowing the drops to fall from 30 cm height onto glass slides. Images were acquired as described above for Immunofluorescence. 111 Chromosome missegregations kill tumor cells 5
5 Flow Cytometry Flow cytometry samples were harvested and fixed using 70% ethanol. α-MPM2 was incubated for 1 hour in PBS-2% BSA-0,1% Tween and α-Mouse Cy5 for 1 hour in PBS-0,1% Tween. Stained cells were collected in PBS containing RNAse and Propidium Iodide. Fluorescence was measured on the FACSCalibur and analyzed with Cell Quest Pro software (BD Biosciences). Immuno Blotting and antibodies Cells were lysed in Laemmli buffer. Samples were separated by SDS-page and transferred to PVDF (Immobilin FL, Millipore). The membranes were cut in half and blotted with anti-Mps1 and anti-αtubulin. The following antibodies have been used for Western Blot, Immunofluoresence and FACS analysis: Mps1 (Upstate), α-tubulin (Sigma), CREST (Cortex Biochem), MPM2 (Upstate), anti-human Alexafluor647, anti-mouse cy-5 (Jackson) Colony formation assays Cells (+/- 50.000/well) were plated on 6-wells plates (Costar). Doxycycline was added at day 0 to allow knockdown of the proteins. At day 11, plates were washed with PBS, fixed 5 minutes with 96% Methanol and stained with 0,1% crystal violet. Xenografting 0,5 x 10 6 LS174T cells were injected subcutaneously on day 0. Doxycycline (1mg/ml) was added to the drinking water of the mice on indicated days until the end of the experiment. Drinking water was supplemented with 2% sucrose. Tumor growth of LS174T cells was assessed every 3 days for a period of 30 days. 112
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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
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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 110 and 111: clear accumulation of cells in mito
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
Page 160 and 161: 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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