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The three-dimensional organization of telomeres in the nucleus of mammalian cells.

Chuang TC, Moshir S, Garini Y, Chuang AY, Young IT, Vermolen B, van den Doel R, Mougey V, Perrin M, Braun M, Kerr PD, Fest T, Boukamp P, Mai S - BMC Biol. (2004)

Bottom Line: In tumor cells, the 3D telomere organization is distorted and aggregates are formed.The results emphasize a non-random and dynamic 3D nuclear telomeric organization and its importance to genomic stability.Based on our findings, it appears possible to examine telomeric aggregates suggestive of genomic instability in individual interphase nuclei and tissues without the need to examine metaphases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, 675 McDermot Avenue, Winnipeg, MB, R3E 0V9, Canada. tcychuang@hotmail.com

ABSTRACT

Background: The observation of multiple genetic markers in situ by optical microscopy and their relevance to the study of three-dimensional (3D) chromosomal organization in the nucleus have been greatly developed in the last decade. These methods are important in cancer research because cancer is characterized by multiple alterations that affect the modulation of gene expression and the stability of the genome. It is, therefore, essential to analyze the 3D genome organization of the interphase nucleus in both normal and cancer cells.

Results: We describe a novel approach to study the distribution of all telomeres inside the nucleus of mammalian cells throughout the cell cycle. It is based on 3D telomere fluorescence in situ hybridization followed by quantitative analysis that determines the telomeres' distribution in the nucleus throughout the cell cycle. This method enables us to determine, for the first time, that telomere organization is cell-cycle dependent, with assembly of telomeres into a telomeric disk in the G2 phase. In tumor cells, the 3D telomere organization is distorted and aggregates are formed.

Conclusions: The results emphasize a non-random and dynamic 3D nuclear telomeric organization and its importance to genomic stability. Based on our findings, it appears possible to examine telomeric aggregates suggestive of genomic instability in individual interphase nuclei and tissues without the need to examine metaphases. Such new avenues of monitoring genomic instability could potentially impact on cancer biology, genetics, diagnostic innovations and surveillance of treatment response in medicine.

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BrdU-positive cells were live sorted and synchronized in the S phase. They were harvested from a culture at time intervals of 3.5–9 hours. The cells were then fixed for 3D analysis. For each time point we have measured: 1. the fraction of nuclei with a telomeric disk; 2. the fraction of cells in mitosis; and 3. the fraction of cells with interphase nuclei but without a telomeric disk. Ninety percent of the cells formed a telomeric disk 3.5 hours after BrdU incorporation and were therefore interpreted as cells in the late G2 phase (black line and circles). Cells entering mitosis (dashed line and squares) peaked at 7.5 hours (65%) and cells in G1 (dotted line and triangles) peaked after 8.5 hours (57%). The increase in the number of metaphases at 9.5 hours cannot be explained and probably lies within the limits of experimental errors.
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Figure 6: BrdU-positive cells were live sorted and synchronized in the S phase. They were harvested from a culture at time intervals of 3.5–9 hours. The cells were then fixed for 3D analysis. For each time point we have measured: 1. the fraction of nuclei with a telomeric disk; 2. the fraction of cells in mitosis; and 3. the fraction of cells with interphase nuclei but without a telomeric disk. Ninety percent of the cells formed a telomeric disk 3.5 hours after BrdU incorporation and were therefore interpreted as cells in the late G2 phase (black line and circles). Cells entering mitosis (dashed line and squares) peaked at 7.5 hours (65%) and cells in G1 (dotted line and triangles) peaked after 8.5 hours (57%). The increase in the number of metaphases at 9.5 hours cannot be explained and probably lies within the limits of experimental errors.

Mentions: To further study the phase transition timing along the cell cycle we used the synchronous bromodeoxyuridine (BrdU) sorting method. The cell population was pulse-labelled with BrdU in the S phase and flow sorted. Cells were placed back into culture and sub-populations harvested at 3.5, 4, 5, 6, 7, 8, 8.5, 9 and 10 hours after labelling and sorting. The cells were then fixed for 3D analysis. A minimum of 20 cells from each of these sub-populations were measured, analyzed and divided into the following three categories: 1) nuclei with a telomeric disk; 2) cells in mitosis; 3) cells in interphase without telomeric disk and mitotic figures (evaluated as G1 cells). The cell fractions as a function of time are shown in Fig. 6. Most cells (90%) form a telomeric disk 3.5 hours after BrdU incorporation. These cells are, therefore, interpreted as cells in the G2 phase. The fraction of metaphase cells peaks at 7.5 hours (65%) and the cell fraction of interphase cells that does not have a telomeric disk (and is interpreted as being in the G1 phase) peaks at 8.5 hours (57%).


The three-dimensional organization of telomeres in the nucleus of mammalian cells.

Chuang TC, Moshir S, Garini Y, Chuang AY, Young IT, Vermolen B, van den Doel R, Mougey V, Perrin M, Braun M, Kerr PD, Fest T, Boukamp P, Mai S - BMC Biol. (2004)

BrdU-positive cells were live sorted and synchronized in the S phase. They were harvested from a culture at time intervals of 3.5–9 hours. The cells were then fixed for 3D analysis. For each time point we have measured: 1. the fraction of nuclei with a telomeric disk; 2. the fraction of cells in mitosis; and 3. the fraction of cells with interphase nuclei but without a telomeric disk. Ninety percent of the cells formed a telomeric disk 3.5 hours after BrdU incorporation and were therefore interpreted as cells in the late G2 phase (black line and circles). Cells entering mitosis (dashed line and squares) peaked at 7.5 hours (65%) and cells in G1 (dotted line and triangles) peaked after 8.5 hours (57%). The increase in the number of metaphases at 9.5 hours cannot be explained and probably lies within the limits of experimental errors.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC425602&req=5

Figure 6: BrdU-positive cells were live sorted and synchronized in the S phase. They were harvested from a culture at time intervals of 3.5–9 hours. The cells were then fixed for 3D analysis. For each time point we have measured: 1. the fraction of nuclei with a telomeric disk; 2. the fraction of cells in mitosis; and 3. the fraction of cells with interphase nuclei but without a telomeric disk. Ninety percent of the cells formed a telomeric disk 3.5 hours after BrdU incorporation and were therefore interpreted as cells in the late G2 phase (black line and circles). Cells entering mitosis (dashed line and squares) peaked at 7.5 hours (65%) and cells in G1 (dotted line and triangles) peaked after 8.5 hours (57%). The increase in the number of metaphases at 9.5 hours cannot be explained and probably lies within the limits of experimental errors.
Mentions: To further study the phase transition timing along the cell cycle we used the synchronous bromodeoxyuridine (BrdU) sorting method. The cell population was pulse-labelled with BrdU in the S phase and flow sorted. Cells were placed back into culture and sub-populations harvested at 3.5, 4, 5, 6, 7, 8, 8.5, 9 and 10 hours after labelling and sorting. The cells were then fixed for 3D analysis. A minimum of 20 cells from each of these sub-populations were measured, analyzed and divided into the following three categories: 1) nuclei with a telomeric disk; 2) cells in mitosis; 3) cells in interphase without telomeric disk and mitotic figures (evaluated as G1 cells). The cell fractions as a function of time are shown in Fig. 6. Most cells (90%) form a telomeric disk 3.5 hours after BrdU incorporation. These cells are, therefore, interpreted as cells in the G2 phase. The fraction of metaphase cells peaks at 7.5 hours (65%) and the cell fraction of interphase cells that does not have a telomeric disk (and is interpreted as being in the G1 phase) peaks at 8.5 hours (57%).

Bottom Line: In tumor cells, the 3D telomere organization is distorted and aggregates are formed.The results emphasize a non-random and dynamic 3D nuclear telomeric organization and its importance to genomic stability.Based on our findings, it appears possible to examine telomeric aggregates suggestive of genomic instability in individual interphase nuclei and tissues without the need to examine metaphases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, 675 McDermot Avenue, Winnipeg, MB, R3E 0V9, Canada. tcychuang@hotmail.com

ABSTRACT

Background: The observation of multiple genetic markers in situ by optical microscopy and their relevance to the study of three-dimensional (3D) chromosomal organization in the nucleus have been greatly developed in the last decade. These methods are important in cancer research because cancer is characterized by multiple alterations that affect the modulation of gene expression and the stability of the genome. It is, therefore, essential to analyze the 3D genome organization of the interphase nucleus in both normal and cancer cells.

Results: We describe a novel approach to study the distribution of all telomeres inside the nucleus of mammalian cells throughout the cell cycle. It is based on 3D telomere fluorescence in situ hybridization followed by quantitative analysis that determines the telomeres' distribution in the nucleus throughout the cell cycle. This method enables us to determine, for the first time, that telomere organization is cell-cycle dependent, with assembly of telomeres into a telomeric disk in the G2 phase. In tumor cells, the 3D telomere organization is distorted and aggregates are formed.

Conclusions: The results emphasize a non-random and dynamic 3D nuclear telomeric organization and its importance to genomic stability. Based on our findings, it appears possible to examine telomeric aggregates suggestive of genomic instability in individual interphase nuclei and tissues without the need to examine metaphases. Such new avenues of monitoring genomic instability could potentially impact on cancer biology, genetics, diagnostic innovations and surveillance of treatment response in medicine.

Show MeSH
Related in: MedlinePlus