Original Article

Oncogene (2007) 26, 1398–1406. doi:10.1038/sj.onc.1209928; published online 4 September 2006

Telomeric aggregates and end-to-end chromosomal fusions require myc box II

A Caporali1,2,4, L Wark2,4, B J Vermolen3, Y Garini3 and S Mai2

  1. 1Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Biochimica Clinica e Biochimica dell'Esercizio Fisico, Università degli Studi di Parma, Parma, Italy
  2. 2Manitoba Institute of Cell Biology and The Genomic Center for Cancer Research and Diagnosis, CancerCare Manitoba, University of Manitoba, Winnipeg, MB, Canada
  3. 3Department of Imaging Science and Technology, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands

Correspondence: Dr S Mai, Manitoba Institute of Cell Biology, Physiology; Biochemistry and Medical Genetics, 675 McDermot, Winnipeg, MB, Canada R3E 0V9. E-mail: smai@cc.umanitoba.ca

4These two authors contributed equally to this work.

Received 21 March 2006; Revised 26 June 2006; Accepted 7 July 2006; Published online 4 September 2006.

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Abstract

Telomeres of tumor cells form telomeric aggregates (TAs) within the three-dimensional (3D) interphase nucleus. Some of these TAs represent end-to-end chromosomal fusions and may subsequently initiate breakage–bridge–fusion cycles. Wild-type (wt) and myc box II mutant (mt) Myc induce different types of genomic instability when conditionally expressed in mouse proB cells (Ba/F3). Only wt Myc overexpressing Ba/F3 cells are capable of tumor formation in severe combined immunodeficient mice. In this study, we investigated whether telomere dysfunction leading to TA formation is linked to the genetic changes that permit wt c-Myc-dependent transformation of Ba/F3 cells. To this end, we examined the 3D organization of telomeres after the deregulated expression of deletion myc boxII mutant (Delta106) or wt Myc. Delta106-Myc overexpression did not induce TAs, whereas wt-Myc deregulation did. Instead, Delta106-Myc remodelled the 3D telomeric organization such that telomeres aligned in the center of the 3D interphase nucleus forming a telomeric disk owing to a Delta106-induced G1/S cell cycle arrest. In contrast, wt-Myc overexpression led to distorted telomere distribution and TA formation. Analysis of chromosomal alterations using spectral karyotyping confirmed Delta106-Myc and wt-Myc-associated genomic instability. A significant number of chromosomal end-to-end fusions indicative of telomere dysfunction were noted in wt-Myc-expressing cells only. This study suggests that TAs may play a fundamental role in Myc-induced tumorigenesis and provides a novel way to dissect tumor initiation.

Keywords:

three-dimensional nuclear organization, telomere aggregates, c-Myc, genomic instability, telomeric fusions

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Introduction

Chromosomes are organized into discrete territories in the interphase nucleus. This nonrandom organization of chromosomes, conserved during evolution, suggests a role for a spatial organization in the control of gene expression and replication (for reviews, see Cremer and Cremer, 2001; Parada et al., 2002). A number of reports describe an architectural stability of the chromosomal positions in the nucleus (Gerlich et al., 2003), whereas other studies described considerable changes in chromosomal positions during the cell cycle (Gasser, 2002; Essers et al., 2005). In particular, chromosome motility increases during mitosis and early-stage G1, but it is limited to local diffusion during the rest of cell cycle (Walter et al., 2003). The spatial organization of chromosomes might contribute to chromosomal translocations found in many tumors (Kozubek et al., 1999; Neves et al., 1999).

Three-dimensional (3D) fluorescent in situ hybridization (FISH) is an innovative approach to study the nuclear architecture in fixed cultured cells during cell cycle, cell differentiation and malignant transformation (Solovei et al., 2002; Cremer et al., 2003).

Using 3D FISH experiments with peptide-nucleic-acid (PNA)-telomeric probes, we demonstrated that telomeres of normal cells are organized in a nonoverlapping manner in the 3D interphase nucleus (Chuang et al., 2004; Louis et al., 2005). In contrast, tumor cells display an aberrant organization of telomeres and form clusters of telomeres, the so-called telomeric aggregates (TAs) (Chuang et al., 2004; Mai and Garini, 2005, 2006).

The position of telomeres during the cell cycle is an important indicator of the stage at which these fusions may occur. It has been shown previously that the 3D telomere organization varies during different phases of the cell cycle and displays a highly ordered, dynamic assembly in the interphase nucleus. During G0/G1 and S phases, telomeres are widely distributed throughout the nucleus, whereas in late G2 phase, they align in the middle of the nucleus forming a telomeric disk (Chuang et al., 2004; Vermolen et al., 2005).

The deregulation of Myc protein is found in a wide range of human cancers and is associated with disease progression. The deregulated expression of Myc can drive cells into proliferation (Deb-Basu et al., 2006), reduce cell adhesion (Frye et al., 2003), promote metastasis (Pelengaris et al., 2002) and genomic instability (for a review see Mai and Mushinski 2003; Kuttler and Mai, 2005).

The N-terminus of Myc has three highly conserved elements, known as Myc boxes. Of these, Myc box I has been implicated in Myc turnover (Bahram et al., 2000). Myc box I is essential for Myc function in vivo and is required for full transactivation and repression of many target genes (Oster et al., 2003). Myc box II is required for all the known biological functions of Myc (Stone et al., 1987) but not all Myc target genes require the integrity of this box for activation, which shows that there are other mechanisms of Myc-dependent activation (Nikiforov et al., 2002)

The conditional expression of wild-type (wt) Myc and deleted box II mutant-Myc (Delta106-Myc) in spontaneously immortalized mouse Ba/F3 pro-lymphocytes was previously characterized (Fest et al., 2002). Mutant Myc protein induced lower level of apoptosis but higher level of genomic instability than its wt counterpart. It is of note that in these cells, genomic instability and tumorigenesis are two separable events: Only wt-Myc but not Delta106-Myc-expressing cells induced tumor formation in the severe combined immunodeficient mouse model (Fest et al., 2005).

We have recently reported that c-Myc deregulation induces cycles of TA formation and remodels the interphase nucleus by changing the organization of telomeres and chromosomes (Louis et al., 2005, for reviews, see Mai and Garini, 2005, 2006). In addition, the presence of TAs in cells constitutively expressing Myc contributed to genomic instability by forcing abnormal chromosome segregation during mitosis (Ermler et al., 2004).

Telomere function is essential for the preservation of chromosomal integrity (for a review see Feldser et al., 2003). Loss of various telomere-capping proteins or critical shortening of the telomeric repeats led to dysfunctional telomeres. The formation of dicentric chromosomes that led to specific rearrangements was observed more than 60 years ago by Barbara McClintock. Dicentric chromosomes can initiate ongoing chromosomal instability via breakage–bridge–fusion (BBF) cycles (McClintock, 1941). During mitotic segregation, the two centromeres of a dicentric chromosome are pulled to opposite poles and chromosomes can break. These breaks generate telomere-free ends and new chromosome fusions, nonreciprocal translocations and overall genetic changes that contribute to genomic instability. Our previous study showed that c-Myc is one key factor that initiates chromosomal rearrangements through BBF cycles (Louis et al., 2005).

In the present study, we investigated whether Myc box II is required for TA formation in Ba/F3 cells. In order to evaluate the difference in initiating and promoting tumorigenesis between wt-Myc and Delta106-Myc-expressing cells, we analysed the organization of telomeres in the interphase nucleus and the presence of chromosomal rearrangements resulting from BBF cycles.

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Results

Telomere disk after cell cycle synchronization

It has been shown previously that varying telomere organization is observed during different phases of the cell cycle with telomeric disks forming in the G2 phase of the cell cycle (Chuang et al. 2004). Telomere positions in the 3D nucleus were calculated by using a program (Teloview) and algorithms that we have developed for this purpose (Chuang et al. 2004; Vermolen et al., 2005). Briefly, using an adequate threshold, the program calculates the center of gravity, the volume and intensity for each telomere. Using the quick-hull algorithm (Barber et al. 1996), the distribution of the telomeres in the nucleus volume is found by fitting the smallest set of polygons that contains all the telomeres. In general, this volume is an ellipsoid with two similar radii (a=b) and one dissimilar radius (c) (i.e. spheroid). Therefore, the level of flatness of the volume occupied by the telomeres can be described by an a/c ratio. The larger the ratio, the more disk-like is the shape of the volume occupied by the telomeres.

In order to confirm the position of telomeres during different phases of the cell cycle and the presence of telomeric disk, mouse diploid immortalized Pre-B lymphocytes were synchronized in late G2 with 0.5 mug/ml of nocodazole. Synchronized cells were reintroduced into culture and harvested again after 8 h at G1 phase (Figure 1a).

Figure 1.
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FACS analysis for Pre-B cells. Control group proliferated normally with no detectable sign of cell cycle arrest (a). Cells were harvested 0 h after having been synchronized at G2/M with nocodazole and 8 h after release from synchronization with nocodazole. (b) For G1/S synchronization, Pre-B cells were incubated for 42 h in RPMI media that had been depleted of the amino acids methionine, cysteine and L-glutamine, released from G1/S synchronization and placed in normal media. Cells were then harvested at 0 h after synchronization and once every hour for 8 h. The cell cycle profile was expressed as the percentage (plusminuss.d.) of cells in each phase (G0/G1; S; G2/M). These values were calculated from data collected from three independent experiments.

Full figure and legend (65K)

Using PNA-FISH hybridization in 3D fixed Pre-B lymphocytes, we confirmed the formation of a telomeric disk at the time of synchronization (Figure 2b). Eight hours after release from synchronization, telomeres returned to a wide distribution throughout the interphase nucleus (Figure 2c). The calculated a/c ratios in the cells arrested in G2/M phase and in the cells in G1 phase were 11.8plusminus2.9 and 8.1plusminus1.7, respectively (Table 1).

Figure 2.
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Three-dimensional analysis of telomere position after G2/M and G1/S synchronization. (a) Fixed control cell untreated. (b) Fixed cells harvested 0 h after having been synchronized at G2/M with nocodazole. (c) Fixed cells harvested 8 h after release from synchronization with nocodazole. (d) Fixed cells harvested 0 h after synchronization at G1/S with depleted medium/mimosine. (e) Fixed cells harvested 8 h after release from synchronization with depleted medium/mimosine. Telomeres are shown in red; nuclei were stained with DAPI (blue). 3DF (3D front view), 3DS (3D side view).

Full figure and legend (153K)


To detect telomere positions after G1/S synchronization, Pre-B cells were incubated for 42 h in RPMI 1640 that had been depleted of the amino acids methionine, cysteine and L-glutamine, and then returned to complete RPMI 1640 with mimosine at a concentration of 0.4 mug/ml 8 h (Kuschak et al., 2002). The G1/S block was confirmed by fluorescent-activated cell sorter (FACS) analysis (Figure 1a). Eight hours after release from the G1/S block, Pre-B cells returned to normal cycling conditions (Figure 1b). Under conditions of G1/S synchronization, the telomeres aligned in the center of 3D interphase nucleus and formed a telomere disk (Figure 2d). The high value of the a/c ratio (9.6plusminus2.9 time 0), calculated for G1/S synchronized cells, confirmed the flatness of the volume occupied by the telomeres. Eight hours after release from the G1/S arrest, the telomeres were widely distributed in 3D interphase nucleus with a calculated a/c ratio of 7.7plusminus1.4 (Table 2, Figure 2e).


The 3D organization of telomeres in wt-Myc and Delta106-Myc-induced Ba/F3 cells

To study the organization of telomeres in the nucleus after conditional wt-Myc and Delta106-Myc induction in immortalized mouse pro-B lymphocytes (Ba/F3) stably transfected with MycER (Fest et al., 2002), we performed PNA-telomere FISH hybridization. After addition of a single dose of 4-hydroxytamoxifen (4HT), nuclear c-Myc signal was quantified by quantitative fluorescent immunostaining (Kuschak et al., 1999). In nontreated control cells, MycER was found in the cytoplasm. The nuclear signal of both wt and Delta106-Myc proteins increased threefold over a 2–4-h period and decreased to the levels of nontreated cells after 6 h (data not shown).

Consistent with our previous results (Louis et al., 2005), Ba/F3 cells without MycER activation showed nonoverlapping telomere positions (Figure 3a). At 24 h after wt-Myc activation, Ba/F3 cells displayed a wide spatial telomere distribution and the presence of TAs (Figure 3b). At the same time point, in Delta106-myc-induced Ba/F3 cells, telomeres were aligned in the center of the 3D interphase nucleus (Figure 3c).

Figure 3.
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Telomere distribution in 3D interphase nuclei of Ba/F3 cells at 24 h after wt-Myc and Delta106-Myc activation. (a) Control (non-4HT treated) Ba/F3 cells; (b) wt-Myc-activated Ba/F3 cells with a wide distribution of telomeres and with TAs formation (yellow arrow); (c) in Delta106-Myc expressing cells, telomeres are aligned in the center of the interphase nucleus, forming a disk-like structure without TAs. Telomeres are shown in red; nuclei were stained with DAPI (blue). 3DF (3D front view), 3DS (3D side view).

Full figure and legend (94K)

To better describe telomere distribution in 3D nucleus, we measured a/c ratios after wt and Delta106-Myc activation (Table 3). Telomeres were widely distributed throughout the nucleus after wt-Myc induction with a calculated a/c ratio of 4.1plusminus1.1 at 24 h and 4.9plusminus0.7 at 48 h, which means a spherical-like volume of distribution. However, after Delta106-Myc induction, the a/c ratio was 10.1plusminus4.3 at 24 h and 9.2plusminus2.9 at 48 h.


The possible relationship between telomere distribution and cell cycle in this model was investigated by flow cytometry. Propidium iodide staining of cellular DNA indicated that non-4HT-treated Ba/F3 cells proliferated normally with no detectable sign of cell cycle arrest (Figure 4, 0 h). As expected, the overexpression of wt-Myc increased G1/S transition after 24 and 48 h (Figure 4a). In contrast, the Delta106-Myc-expressing cells accumulated at G1/S phase of the cell cycle. By 24 h, over 80% of the cells were arrested in G1/S (Figure 4b).

Figure 4.
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Effect of wt-Myc and Delta106-Myc expression on cell cycle profiles. Cell cycle analysis of Ba/F3 cells at 24 and 48 h after wt-MycER (a) and Delta106-MycER (b) activation. The cells were collected, permeablized and DNA was stained with propidium iodide. The overexpression of wt-Myc increased G1/S transition after 24 and 48 h. The Delta106-Myc-expressing cells accumulated at G1/S phase of the cell cycle. The cell cycle profile was expressed as the percentage (plusminuss.d.) of cells in each phase (G0/G1, S, G2/M). These values were calculated from data collected from three independent experiments.

Full figure and legend (51K)

Delta106-Myc expression does not induce TA formation in interphase nuclei

In tumor cells, the ordered and nonoverlapping 3D nuclear space that telomeres normally occupy is compromised and telomeres can form aggregates that may fuse their respective chromosomes, favoring structural chromosomal aberrations. In order to investigate whether the mutation in Myc box II impacted on the formation of TAs, we analysed wt and Delta106-Myc-expressing cells. To this end, cells were harvested every 6 h over a time period of 48 h. Measurement of TAs was performed after 3D image acquisition and constrained iterative deconvolution (Louis et al., 2005).

This time course experiment confirmed that only wt-Myc expression in Ba/F3 cells induced TAs. Representative images showed that TAs varied in number in wt-Myc-expressing cells at 24 h (Figure 5B, b). In contrast, TAs were not detectable in the 3D nucleus at 24 h, with control Ba/F3 cells (non-4HT treated cells) and Delta106-Myc-expressing cells (Figure 5B, a and c, respectively). A single dose of 4HT induced the highest levels of TA formation after 24 h in wt-Myc-expressing cells. Thus, only a single TA cycle is observed in Ba/F3 after wt-Myc activation (Figure 5A). In Ba/F3 cells, the nuclear localization of Myc completely disappeared 6 h after 4HT-induced MycER activation (data not shown). As reported previously (Louis et al., 2005), the number of TA cycles was directly linked to the duration of wt-Myc deregulation. As wt-Myc but not Delta106-Myc-expressing cells induced tumorigenesis in vivo (Fest et al., 2005), the presence of TAs seems to be linked to the initiation and/or progression of tumorigenic potential seen in Ba/F3 cells with deregulated wt-Myc expression.

Figure 5.
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Overview of TA formation in nuclei of Ba/F3 cells expressing wt-Myc or Delta106-Myc. (A) Fold increase in TAs over control level during a period of 48 h. The highest levels of TAs were observed at 24 after wt-Myc activation. Error bars represent a 95% confidence interval of binomial distribution. (B) Representative individual images showing TA formation over the time frame shown in (A). (B, a) Control (non-4HT treated) Ba/F3 cells display nonoverlapping telomeres. (B, b) wt-Myc activated Ba/F3 show the formation of TAs (yellow arrow). (B, c) Delta106-Myc-expressing cells do not show TAs.

Full figure and legend (105K)

A significant number of chromosomal fusions were noted only in wt-Myc-expressing cells

To determine whether the formation of TA was associated with BBF events, spectral karyotyping (SKY) analysis on metaphase chromosomes was performed at different times: prior (6 h), during (24 h) and after (42 h) the peak of TA formation in both wt and Delta106-Myc-expressing cells. Table 4 summarizes the genomic aberrations detected in wt-Myc-expressing Ba/F3 cells. As expected for an immortalized cell line (Fest et al., 2005), control Ba/F3 cells (non-4HT treated) showed some chromosomal alterations. At 24 h, six out of 20 metaphases in wt-Myc-expressing cells showed a significant increase in end-to-end chromosomal fusions and nonreciprocal translocations over control levels. At 42 h after wt-Myc activation, the percentage of fusions and translocation decreased. The karyotype of spontaneously immortalized, tetraploid (Ba/F3) cells is shown in Figure 6a. Representative images (Figure 6b), 24 h after wt-Myc activation, show fusions at telomeric ends between two chromosomes X and between chromosomes 10 and 9. Nonreciprocal translocation between chromosomes 15 and 9 and broken chromosomes (4, 16 and 9) were observed. Other aberrations included the insertion of chromosome X material into chromosome 1 and of chromosome 19 material into chromosome 4.

Figure 6.
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SKY of Ba/F3 wt-MycER and Delta106-MycER cells. Representative images of Ba/F3 cells control (non-4HT treated) (a), 24 h after MycER activation (+4HT) for (b) wt-Myc and Delta106-Myc (c) are shown. Notice the presence of fusion between two chromosomes X and between chromosome 10 and 9 (see yellow arrows) after wt-Myc activation. No fusions were detected by SKY after Delta106-Myc activation. For a detailed overview of the aberration detected by SKY, see Tables 4 (wt-Myc) and 5 (Delta106-Myc). Each panel of the figure shows the following order of images, the raw spectral image of the metaphase (top left corner), the classified image of the identical metaphase (top middle panel), the inverted DAPI image of the identical metaphase (top right corner) and the classified karyotype of the identical metaphase is displayed in the bottom panel. A minimum of 20 metaphases was analysed per time point.

Full figure and legend (270K)


The analysis of 20 metaphases from Delta106-Myc-expressing cells revealed an increased number of nonreciprocal translocations over 42 h in comparison with control (non-4HT treated) Ba/F3 cells. In contrast to wt-Myc-overexpressing Ba/F3 cells, Delta106-Myc-overexpressing cells did not show chromosomal end-to-end fusions. Nonreciprocal translocations were found in wt-Myc-expressing cells at 24 h in 30% of the metaphases (P=0.0101). As time progressed, translocations increased (in 40% of metaphases P<0.001) (Table 5). Figure 6c summarizes the most common aberrations seen in Delta106-Myc-expressing cells at 24 h. SKY analysis confirmed the presence of unbalanced translocation involving chromosomes 2, 3, 4 and 15.


Both wt and Delta106-Myc proteins showed high levels of significant karyotypic instability. The main difference between Delta106-Myc and wt-Myc-expressing cells were that the latter exhibited a significant number of chromosome fusions related to telomere dysfunction.

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Discussion

There are at least two types of telomere dysfunction in tumor cells. One type involves critically short telomeres (DePinho and Polyak, 2004). The other one involves the formation of TAs and is independent of telomere size and telomerase activity (Louis et al., 2005). Telomeres of normal cells are organized in a nonoverlapping manner in the 3D interphase nucleus. In contrast, tumor cells display an aberrant organization of telomeres that impact on the numbers of TAs (Chuang et al., 2004).

Telomere dynamics have been observed and measured by live cell imaging approaches in different cell lines. Long and short ranging movements were observed over a time period of 20 min (Molenaar et al., 2003). 3D imaging has permitted to determine that the telomere organization in the nucleus is cell cycle dependent. As such the position of telomeres during the cell cycle is an important indicator of the stage at which these TAs may occur (Chuang et al., 2004).

In the present study, we have investigated a possible correlation between telomere positions during the cell cycle and the formation of TAs and the different tumorigenic potential of wt and mutant Myc proteins (Fest et al., 2005). At the G1/S boundary of a synchronized cell cycle, the telomeres of Pre-B cells have a tendency to align in the center of the nucleus, in a structure we had termed earlier a telomeric disk (Chuang et al., 2004). This is the first time such a telomere organization has been found during an induced G1/S block. This alignment of telomeres dissociates and telomeres are observed throughout the nucleus when cells re-enter into the cycle after release from the synchronization event.

The telomeric disk naturally reforms at the G2/M transition of the cell cycle (Chuang et al., 2004). Thereafter, the telomeres resume their distribution throughout the nucleus. This feature of a dynamic telomeric organization throughout the cell cycle is mimicked by an experimental G2/M synchronization. Eight hours after release from the G2/M block, telomeres will assume their normal cell cycle-dependent organization.

The expression of wt-Myc stimulates the G1/S transition by regulating the levels and the activity of the cyclins (Trumpp et al., 2001). In Ba/F3 cells, wt-Myc activation promotes G1/S transition and is accompanied by a distorted telomere distribution that results from the presence of TAs (Figure 3, yellow arrow). In contrast, Myc box II mutants expressing cells were blocked in G1/S and the telomeres were aligned in the center of the 3D interphase nucleus, forming a telomere disk. These results agree with the data obtained in synchronized Pre-B cells in G1/S phase of the cell cycle.

Previous work had shown in Ba/F3 cells that wt and mutant Myc proteins induced genomic instability but only wt-Myc protein had the potential of initiating and promoting tumorigenesis in vivo (Fest et al., 2005). Using PNA-telomere FISH hybridization in 3D-fixed cells, we demonstrated that the formation of TAs takes part in MYC-induced tumorigenesis.

TAs were detectable only in wt-Myc expressing and tumorigenic Ba/F3 cells (Fest et al., 2005) reaching the highest peak after 24 h after Myc activation, whereas Delta106-Myc expressing and nontumorigenic Ba/F3 cells (Fest et al., 2005) did not show a significant number of TAs. The presence of TAs in malignant cells is supported by data from different cell lines and human tumors (Chuang et al., 2004).

SKY data show that conditional wt-Myc protein expression (Table 4) led to a higher level of chromosomes end-to-end fusions than conditional Delta106-Myc protein expression (Table 5). These results are in agreement with the absence of TAs in the mutant Myc-expressing cells.

TAs are not just a transient aberration in the 3D organization of the nucleus, but these events precede the formation of BBF cycles. As reported in our previous work, once aggregates form and chromosome fusions occur, BBF cycles result and the genetic information of the chromosomes will be remodelled (Louis et al., 2005).

Nothing is known about the mechanisms that cause TA formation in the context of c-Myc deregulation. Shelterin is a protein complex with DNA remodelling activity that, together with several DNA repair protein, such as WRN, the Mre1 complex and DNA-PK, protects the integrity of the chromosome ends (De Lange, 2005). In cells constitutively expressing Myc and characterized by the presence of TAs (Ermler et al., 2004), the level of TRF2 protein, a shelterin subunit protein, was reduced. These data lend support to the hypothesis that Myc may somehow interact with proteins of the shelterin or DNA repair complexes to mediate TAs formation. This capacity is lost in myc box II mutant. Understanding whether TAs may be the earliest events in tumor development and which genetic background is more susceptible to TA formation will provide a novel way to dissect the benign-to-malignant transition in cancer.

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Materials and methods

Cell cultures and treatments

Mouse Pre-B lymphocytes (Mai et al., 1999) were grown in RPMI 1640 supplemented with 0.1% beta-mercaptoethanol, 1% L-glutamine, 1% sodium-pyruvate, 1% penicillin/streptomycin and 10% fetal bovine serum (FBS; Gibco, Burlington ON, Canada) at 37°C, in a humidified atmosphere and in the presence of 5%CO2.

For G2/M synchronization, Pre-B cells were incubated for 8 h in standard RPMI 1640 medium (Gibco, Burlington ON, Canada) with nocodazole (Sigma-Aldrich, Oakville ON, Canada) at a concentration of 1 mug/ml. Upon completion of synchronization, cells were removed from nocodazole and returned to nocodazole-free media and harvested at 0 and 8 h.

For G1/S synchronization, Pre-B cells were incubated for 42 h in RPMI 1640 that had been depleted of the amino acids methionine, cysteine and L-glutamine, and then returned to complete RPMI 1640 with mimosine at a concentration of 0.4 mug/ml for 8 h (Kuschak et al., 2002). Cells were then harvested at 0 h and once every hour for 8 h.

The two Ba/F3 cell lines with conditional wt-MycER (Littlewood et al., 1995) and Delta106-MycER used in this study have been previously described (Fest et al., 2002). Cells were grown in RPMI 1640 containing 10% FBS, 1% WEHI cells supernatant (mouse myelomonocytic leukemia macrophage-like cells derived from a BALB/c mouse; the cells produce IL3 supernatant) and 0.21% of plasmocin (Cayla, Toulouse, France).

Wt-Myc and Delta106-Myc cells were induced with 4-hydroxytamoxifen (4HT) (Sigma-Aldrich, Oakville ON, Canada) to a final concentration of 100 nM in 105 cells/ml to activate the c-Myc protein. Cells were split 24 h prior induction and every 48 h after 4HT induction.

Cells were grown and maintained at a density of 105–106 cells/ml. Cell viability was determined by hemocytometer counts using trypan blue (Sigma-Aldrich, Oakville ON, Canada).

Immunohistochemistry

Immunohistochemistry was performed as described previously (Fukasawa et al., 1997). The primary antibody used was a rabbit polyclonal anti-c-Myc (N262) at a dilution of 1:100 (Santa Cruz, Santa Cruz, California, USA) visualized by using a secondary goat anti-rabbit IgG fluorescein isothiocyanate antibody at a dilution of 1:100 (Sigma-Aldrich, Oakville ON, Canada). Images were acquired using a Hamamatsu CCD SensiCam Camera and the Northern Eclipse v 6.0 software.

FACS analysis

For FACS analysis, Pre-B and Ba/F3 cells were fixed in 70% cold ethanol and stained with propidium iodide (Sigma-Aldrich, Oakville ON, Canada) (1 mug/ml) following RNAse digestion (Sigma-Aldrich, Oakville ON, Canada) (20 mug/ml). The stained cells were analysed for DNA content by flow cytometry in an EPICS Altra cytometer (Beckman-Coulter, Mississauga, ON, Canada).

Telomere FISH and 3D image analysis

Cells were fixed using 3:1 methanol/acetic acid fixative (Fluka, Oakville, ON, Canada) and then placed on 26 times 76 mm2 microscope slides. Telomeres were stained using quantitative fluorescent FISH with a telomere-specific CY3-labeled PNA probe (DAKO, Mississauga, ON, Canada). Nuclear volumes did not significantly change during the denaturation protocol used for 3D telomere FISH. Counterstaining was performed with 4'-6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Oakville ON, Canada). Three independent experiments were performed and at least 30 nuclei were examined per time point.

Cells were imaged on a Zeiss Axioplan 2 microscope with a Zeiss Axiocam HRm and deconvolution module, including Axiovision (Zeiss, North York, ON, Canada) software v3.1. Images were deconvolved using a constrained iterative algorithm (Schaefer et al., 2001). Analysis was performed with TeloView (Chuang et al., 2004; Vermolen et al., 2005).

By choosing a simple threshold for the telomeres, the volume, intensity and center of gravity are calculated. The integrated intensity of each telomere is calculated because it is proportional to the telomere length (Poon et al., 1999). The integration region is determined by growing a sphere on top of the found coordinate. After every step of growth (iteration), the sum under this volume (the telomere) is subtracted by the sum immediately surrounding it (background level). When the process of the growth of the sphere does not contribute to an integrated intensity increase, the algorithm stops and the integrated intensity of the telomere with an automatic background correction is obtained.

The telomeric distribution inside the nucleus is described by fitting an ellipsoid to the volume occupied by the telomeres. The distributions were found to be either oblate or spherical. It is therefore convenient to describe the distribution volume as a spheroid (i.e. an ellipsoid having two axes of equal length). As such, it is simpler to describe the spheroid degree of variation from a perfect sphere by the ratio a/c where a and b are the similar semiaxes and c is the third dissimilar axis. Such a description reflects the degree to which the telomere's volume is oblate. Nuclear flattening that may affect a/c ratios was considered in this study. All cells (samples) in an experiment were processed at the same time. If nuclei on one slide consistently showed high a/c ratios, whereas nuclei on parallel processed slides did not, we assumed that the a/c ratio reflected the flatness of telomere distribution of nuclei within that specific sample. All experiments were performed three times.

SKY analysis

SKY was performed by using the ASI (Applied Spectral Imaging, Vista, CA, USA) kit for mouse and Spectra Cube on a Carl Zeiss Axioplan 2 microscope. At least 20 metaphases were examined per time points.

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Acknowledgements

We thank Dr Ludger Klewes and Dr Ed Rector for assistance with FACS analysis, and Mary Cheang (Biostatistics Unit) for statistical analyses. We acknowledge support from CancerCare Manitoba Foundation, the National Cancer Institute of Canada (NCIC), and the Canadian Institutes of Health Research (CIHR). AC and LW were recipients of a Strategic Training Program stipend from CIHR.

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