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

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C % abnormal nuclei D E % of cells 60 40 20 0 In vivo 60 50 40 30 20 10 0 In vitro C26 Before After Dtx n=277 C26 n=115 Before n=491 n=175 n=53 m n=30 Day1 Day2 50 µm After Dtx % abnormal nuclei % of cells 60 50 40 30 20 10 0 SW480 n=486 n=131 Before Before Day 1 Day 2 60 40 20 0 SW480 Before After Dtx After Dtx Normal Abnormal Figure 4. Docetaxel induces apoptosis in vivo independent of mitotic defects A) Representative stills of SW480 cells in vitro entering mitosis in the presence of docetaxel resulting in a mitotic delay (top) and eventually apoptosis (bottom). Quantification of CFP-YFP ratios in representative images is shown. Each line represents one cell (black-alive, grey-dying). B) Top: Representative images of mitotic C26 cells in vivo. Bottom: Quantification of number of mitotic cells found at indicated time points in C26 tumors. Mitotic cells were determined according to nuclear morphology. C) Quantification of the number of abnormal nuclei in vitro in C26 (left) and SW480 cells (right) before or after treatment with 1nM docetaxel (low dose). D) Left: Representative images of SW480 cells in vivo with normal or abnormal nuclear morphology visualized using H2B-D. Right: Quantification of the number of cells with abnormal nuclei (black) and number of apoptotic cells (grey) in SW480 and C26 tumors at indicated time points after docetaxel treatment. Apoptotic cells were determined according to CFP-YFP ratios: 30% more than the average CFP-YFP ratio was used as a cut-off. N indicates number of cells analyzed. E) Left: representative images of photo-marked SW480 cells in vivo switched before treatment and imaged on day 1 and 2 after treatment. Right: Quantification of the number of photo-marked (red) H2B-D cells in SW480 tumors in vivo. Relative ratio compared to day 0 (before treatment) is n=359 n=143 n=182 n=77 Day1 Day2 After Dtx relative number of cells In vivo 10 µm % abnormal nuclei % apoptotic cells 1,0 0,8 0,6 0,4 0,2 0 Before Photomarked cells SW480 in vivo Day1 Day2 After Dtx 141 Intravital imaging of Docetaxel response in tumors 7
7 chromosome unstable, which means that they frequently lose and gain whole chromosomes during cell division, already in the absence of any treatment 270,289 . Importantly, neither the number of photomarked SW480 cells in vivo (Fig.4E), nor the number of nuclei with an aberrant morphology (Fig.4D) increased over time upon docetaxel treatment (34% and 25% of cells had an abnormal nucleus 20 and 48 hours after treatment respectively, compared to 27% before treatment) , indicating that these cells have not divided following docetaxel treatment, whereas the percentage of apoptotic cells increased substantially in the same tumor fields (15%, 48% and 57% before, 20 and 48 hours after docetaxel treatment respectively) (Fig.4D). Taken together, these data show that the onset of docetaxel-induced apoptosis correlates with the number of mitotic defects in vitro, but not in vivo. From this we conclude that docetaxel, in contrast to its effect in vitro, induces apoptosis in vivo independent of its effect on mitotic progression. Discussion Although Taxanes have been used in the clinic already for over a decade, a discrepancy between in vitro and in vivo data exists on this drug’s therapeutic action. In this report, we have used two colorectal tumor models in which we analyze the effect of docetaxel on both mitotic progression and apoptosis induction in in vitro and in vivo models. For this, we have developed intravital imaging techniques that enabled us to track individual cells in multiple imaging sessions, and simultaneously determine mitotic progression and the induction of apoptosis. Our comparison shows that taxanes, as shown before342,486,553,596, affect mitotic progression in vitro, but our in vivo data indicate that other deleterious effects are more likely to be causative in tumor regression. Since we have used the same cell lines in our in vitro and in vivo study, the diverse effects cannot be explained by genetic differences. How can such a striking difference in drug response be explained? To start, tumor cells grown in vitro lack the complex microenvironment provided by neighboring cells, soluble secreted factors, and non-cellular matrix components 602. Surrounding cells include tumor-, stromal-, immune- and endothelial cells, which mutually influence each other. Given that the constantly changing microenvironment controls many signaling pathways in tumor cells, it is not surprising that many in vitro observations do not correlate with in vivo observations 611. For example, cells in culture divide once every day, while cells in primary human breast tumors divide less frequent (40-300 days) 612. Importantly, the cell cycle is well controlled by many signaling pathways and therefore the heterogeneous microenvironment may potentially affect the action of taxanes on tumor cells. Indeed, immunohistological analysis and intravital imaging of mouse and human tumor tissues only revealed small increases in mitotic index following paclitaxel treatment 597-600, supporting our finding that mitotic perturbations are not causative for taxane-induced tumor regression 599,600. Interesting recent data suggest that the microenvironment contributes to drug responses via regulation of vascular permeability and innate immune cell infiltration613. It has indeed been hypothesized that immune cells might elicit an anti-tumor effect upon taxane administration 614 and macrophages can be directly activated by paclitaxel resulting in pro-inflammatory effects 615. Although effects of the innate immune system cannot be excluded, the clear apoptotic effects of docetaxel administration in our human xenograft model using immune-compromised SCID mice indicates that the adaptive immune system is not the main contributor to taxane-dependent cell killing in vivo. The ability of docetaxel to kill tumor cells independent of its effects on mitosis could have important implications for the use and development of mitosis-specific drugs, such as Eg5 or Aurora A inhibitors 142
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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
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control treated MEFs (unpaired t te
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Materials and Methods Cloning targe
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- Loop out BamHI site with site-dir
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Primers: pGH_pA_FW#2: TCTATGGCTTCTG
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was prepared using standard procedu
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71 Cre-inducible Mps1 knock-in mous
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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
Page 92 and 93: Partial depletion of Mps1 or BubR1
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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
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Page 139 and 140: 7 A Low CFP lifetime High CFP lifet
Page 141: 7 docetaxel treatment in vitro resu
Page 145 and 146: 7 Cells (~50.000/well) were plated
Page 147 and 148: 7 Supplemental data Supplemental fi
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Page 151 and 152: 8 Thesis summary Unequal separation
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Page 155 and 156: 8 1.4 NoCut: preventing the inducti
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Page 160 and 161: Addendum References Nederlandse sam
Page 162 and 163: 41. McEwen, B.F., et al., CENP-E is
Page 164 and 165: 2010. 6(5): p. 359-68. 124. Liu, S.
Page 166 and 167: 210. Stucki, M., et al., MDC1 direc
Page 168 and 169: tumors in mice. Cancer Res, 2007. 6
Page 170 and 171: adiotherapy in breast cancer. Breas
Page 172 and 173: 470. Marcus, A.I., et al., Mitotic
Page 174 and 175: 559. Kienitz, A., et al., Partial d
Page 176 and 177: Nederlandse Samenvatting Celdeling,
Page 178 and 179: verdeling van tumorcellen compleet
Page 180 and 181: Publications A. Janssen, R.H.Medema
Page 182 and 183: En dan zijn er nu nog een aantal li
Page 184 and 185: promotie-stukjes! Je bent een voorb
Page 186: edankt voor al jullie hulp bij de i
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