GENES, CHROMOSOMES & CANCER 53:934–950 (2014)

Different TP53 Mutations are Associated with Specific Chromosomal Rearrangements, Telomere Length Changes, and Remodeling of the Nuclear Architecture of Telomeres Oumar Samassekou,1,2 Nathalie Bastien,1 Daniel Lichtensztejn,2 Ju Yan,1* Sabine Mai,2* and R egen Drouin1* 1 Division of Genetics,Department of Pediatrics,Faculty of Medicine and Health Sciences,Universite de Sherbrooke, Sherbrooke,QC, Canada 2 Manitoba Institute of Cell Biology, CancerCare Manitoba, Department of Physiology, Faculty of Medicine,University of Manitoba, Winnipeg, MB, Canada

TP53 mutations are the most common mutations in human cancers, and TP53-R175H and TP53-R273H are the most frequent. The impact of these mutations on genomic instability after tumor initiation is still uncovered. To gain insight into this, we studied the effects of three specific TP53 mutants (TP53-V143A, TP53-R175H, and TP53-R273H) on genomic instability using four isogenic lines of LoVo cells. Multicolor fluorescence in situ hybridization (FISH), three-dimensional (3D) quantitative FISH (Q-FISH) on interphase and Q-FISH on metaphases were used to investigate genomic instability. We found that LoVo cells expressing mutant TP53-R175H displayed the highest level of chromosomal instability among the LoVo cell lines. Furthermore, we observed that mutant TP53-R175H and TP53-V143A showed more alterations in their 3D nuclear architecture of telomeres than the mutant TP53-R273H and the wild type. Moreover, we noted an association between some chromosomal abnormalities and telomere elongation in the mutant TP53-R175H. Taken together, our results indicate that the mutation TP53-R175H is more likely to cause higher levels of genomic instability than the other TP53 mutations. We proposed that the type of TP53 mutations and the genetic background of a cancer cell are major C 2014 Wiley Periodicals, Inc. V determinants of the TP53-dependent genomic instability.

INTRODUCTION

Telomere dysfunctions and chromosomal instability (CIN) are two of the main hallmarks of cancer cells. Telomere dysfunctions are characterized by their shortening, loss of their protection, and remodeling of their nuclear architecture (Artandi and DePinho, 2010; Mai, 2010). These changes in telomere length and structure can lead to the appearance of dicentric chromosomes resulting in breakage–fusion–bridge cycles, nonreciprocal translocations, and finally CIN (Louis et al., 2005), which is characterized by the presence of structural and numerical aberrations of chromosomes (Bayani et al., 2007). Structural aberrations are generally caused by telomere dysfunctions or remodeling of nuclear architecture of chromosomes (Mai, 2010) while numerical aberrations are usually due to missegregation of chromosomes and/or tetraploidy (Pino and Chung, 2010). Four major mechanisms have been proposed to explain the presence of tetraploid cells in cancer: (1) cell fusion, (2) failure to complete mitosis (mitotic slippage), (3) failure to C 2014 Wiley Periodicals, Inc. V

complete cytokinesis, and (4) loss of telomere protection associated with deletion of TP53 (Ganem et al., 2007; Davoli et al., 2010). Additional Supporting Information may be found in the online version of this article. Supported by: The Cancer Research Society Inc. (J.Y.); The CancerCare Manitoba Foundation (CCMF) (S.M.); The Canada Research Chair Program (R.D.); Canadian Foundation for Innovation (CFI) and CCMF (S. M.). The Canadian Foundation for  Innovation and from the Centre de Recherche Clinique EtienneLe Bel (The Cell Imaging Facility of the Faculty of Medicine and Health Sciences, Universit e de Sherbrooke). Disclosure: R.D. held a Canada Research Chair in Genetics, Mutagenesis, and Cancer. S.M. is the Director of the Genomic Centre for Cancer Research and Diagnosis. R.D. is member of  the FRQS-funded Centre de recherche clinique Etienne-Le Bel. *Correspondence to: Ju Yan; Division of Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Universit e de Sherbrooke, 3001 12th Avenue North, Sherbrooke, QC J1H 5N4, Canada. E-mail: [email protected] or Sabine Mai; Manitoba Institute of Cell Biology, 675 McDermot Avenue, Winnipeg, MB R3E 0V9, Canada. E-mail: [email protected] or R egen Drouin; Division of Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Universit e de Sherbrooke, 3001 12th Avenue North, Sherbrooke, QC J1H 5N4, Canada. E-mail: [email protected] Received 11 June 2014; Accepted 2 July 2014 DOI 10.1002/gcc.22205 Published online 25 July 2014 in Wiley Online Library (wileyonlinelibrary.com).

TP53 MUTATIONS, CHROMOSOME INSTABILITY, AND TELOMERES

The protein TP53 and telomeres can jointly induce genomic instability when TP53 is absent and telomeres are uncapped or short. Indeed, in the absence of TP53, a deletion of POT1 leads to dysfunction of telomere structure and a persistent DNA damage response, involving ATM and ATR kinases. Then, an endoreplication occurs, followed by a tetraploidization, which can subsequently lead to aneuploidy (Davoli et al., 2010; Davoli and de Lange, 2012). Moreover, the dual action of TP53 and short telomeres to generate CIN has been highlighted in mice deficient in Tp53 and Terc, the ARN component of telomerase. Terc2/2 and Tp532/2 mice present greater CIN, manifested by fusions of chromosome ends and nonreciprocal translocations (Artandi et al., 2000). These data indicate that telomere dysfunctions and the absence of TP53 lead to genomic instability and promote tumorigenesis. TP53, a key player in the maintenance of genome stability and integrity, is mutated in more than 50% of cancers, and most of these mutations are missense mutations located in the DNAbinding domain (Hainaut and Hollstein, 2000). The mutant TP53 protein can form a heterotetramer with the wild-type protein, which exerts a dominant negative effect. This leads to the loss of DNA-binding ability of the tetramer and the reduction of its transactivation activity (Goh et al., 2011). The mutations R175H and R273H are among the most frequent TP53 mutations in human cancers and occur in most cancers after tumor initiation (Goh et al., 2011). In mouse model, the mutant TP53-R172H (equivalent to human TP53R175H) leads to increased formation of skin tumors, accelerated tumor growth, and induction of metastasis. In addition, the resulting tumors exhibit aneuploidy that is associated with centrosome amplification (Caulin et al., 2007). These results suggest an involvement of TP53 mutants in the generation of genomic instability in cancers. However, the impact of TP53 mutations in the genesis of genomic instability when these mutations occur at late stages of the neoplastic process is still not well-deciphered. We hypothesized that specific TP53 mutations lead to different degrees of genomic instability when they occur after tumor initiation. To test this hypothesis, we studied in the LoVo cell lines, derived from colon carcinoma, the impact of the mutant TP53-V143A, TP53R175H, and TP53-R273H on genomic instability by assessing the level of CIN, the length of individual telomeres, the remodeling of the nuclear architecture of telomeres, and the expression pro-

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file of shelterin genes. Telomere dysfunctions have been associated with genomic instability and their study in this context can help to better evaluate the extent of genomic instability in mutant TP53 and to understand their impact on various elements of the telomere biology. We used the LoVo cell line because it is chromosomally stable in culture, present few chromosomal abnormalities (Dridi et al., 2003; Stewenius et al., 2005), and thus, allows one to make a representative model to study the impact of a TP53 mutation, as a secondary event in neoplastic process of inducing genomic instability. MATERIALS AND METHODS Cell Lines and Culture

Four isogenic LoVo cell lines were used for this study, one TP53 wild type and three mutant TP53 lines (LoVo TP53-wt, LoVo TP53-V143A, LoVo TP53-R175H, and LoVo TP53-R273H). They were generated from the LoVo colon adenocarcinoma cell line and kindly given by Dr. MarieFrance Poupon (Institut Marie-Curie, Paris, France; Pocard et al., 1996). The transfection technique, its efficiency, as well as the transfectant selection were described in Pocard et al. (1996). The normal fibroblasts and the SW480 cell line, purchased from ATCC (Manassas, VA), were only used for the relative quantification of mRNA. All the LoVo cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) Nut-mix F-12w/Gut-1 supplemented with 10% fetal bovine serum (FBS), 0.2 units/ml penicillin G, and 100 lg/ ml streptomycin (Wisent Bioproducts, Becton Dickinson, Franklin Lakes, NJ). They were maintained on culture until 20 population doublings, which we established by counting cells on a hemacymeter with trypan blue. Human normal primary skin fibroblasts and SW480 cell line were grown in DMEM and McCoy’s 5A medium, respectively, supplemented with 10% (FBS), 0.2 units/ml penicillin G, and 100 mg/ml streptomycin (Wisent Bioproducts, Becton Dickinson, Franklin Lakes, NJ). Telomere Restriction Fragment Technique

We used the telomere restriction fragment (TRF) technique to assess average telomere length, and from this average length, we estimated the length of individual telomeres in kb as described in Samassekou et al. (2009). DNA was extracted from all four LoVo cell lines using the standard phenol-chloroform method. The TRF Genes, Chromosomes & Cancer DOI 10.1002/gcc

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technique was performed using the TeloTAGGG Telomere Length Assay kit (Roche Applied Science, Laval, Quebec, Canada). Harvesting, Metaphase, and Interphase Nucleus Preparation

For metaphase preparation, LoVo cells were harvested according to the protocol described in Rochette et al. (2005). LoVo cells were treated with 0.25 mg/ml of Colcemid (Gibco, Burlington, Ontario, Canada) for 30 min and with 0.75% (w/v) sodium citrate for 30 min. Then, they were fixed four times in a mixture of methanol and acetic acid (3:1, v/v). Ten microliters of fixed nucleus suspension was spread onto cleaned slides in a modified environmental control unit Thermotron (CDS-5, Thermotron, Amsterdam, the Netherlands; Spurbeck et al., 1996), where temperature and relative humidity remained constant at 28 C and 55%, respectively. Prepared slides were left at 37 C for 24 hr for telomere fluorescence in situ hybridization (FISH) on metaphases. For three-dimensional (3D) interphase nuclei, LoVo cells were harvested as described previously (Guffei et al., 2007). Cells were treated with 5 ml of 0.075 M KCl for 10 min at room temperature (RT). Next, they were prefixed with 1 ml of freshly prepared fixative (methanol:acetic acid, 3:1, v/v). Thereafter, pellets were fixed twice with 5 ml of fixative for 10 min. Finally, 10 ml of fixed cells were gently dropped on slides at RT and telomere FISH for 3D was immediately performed. Telomere FISH

For metaphases, to determine length of individual telomeres, we performed FISH using a peptide nuclei acid (PNA) probe labeled with Cy3, specific to telomeres (Dako, Glostrup, Denmark). Hybridizations were performed following the protocol provided by the manufacturer, with modifications based on protocols described in our previous reports (Yan et al., 2004; Samassekou et al., 2009). Prepared slides for metaphases were fixed in 3.7% formaldehyde in 13 phosphate-buffered saline (PBS) pH 7.4 followed by a proteinase K treatment. Next, 7 ml of PNA telomere probe was applied on each slide after which they were incubated at 80 C for 3 min followed by hybridization at 30 C for 2 hr using a Hybrite (Vysis; Abbott Diagnostics, Des Plains, IL). A posthybridization wash was done using 0.43 saline–sodium citrate buffer (SSC) containing 0.3% NP-40 substitute at Genes, Chromosomes & Cancer DOI 10.1002/gcc

65 C for 2 min and 23 SSC containing 0.1% NP-40 substitute for 2 min. The slides were counterstained using 125 ng/ml 40 ,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, St Louis, MO). The slides were then air-dried and cover slipped with Vestashield (Vector Laboratories, Burlington, Ontario, Canada). For 3D interphase nuclei, the procedure was performed as described previously (Gadji et al., 2010). Briefly, slides were incubated in 3.7% formaldehyde in 13 PBS (pH 7.4) for 20 min and washed three times in 13 PBS. Then, slides were treated with 0.5% Triton X-100 in 13 PBS for 10 min followed by an incubation in 20% glycerol for 1 hr. Thereafter, slides were immersed in liquid nitrogen four times (Solovei et al., 2002) and washed twice in 13 PBS for 5 min each followed by a 5-min incubation in 0.1N HCl. After washing twice in 13 PBS for 5 min each, slides were incubated in 70% formamide in 23 SSC (pH 7.0) for 1 hr. Next, 8 ml of PNA Cy3-labeled telomeric probe (Dako) was applied on each slide. The slides were incubated at 80 C for 3 min followed by hybridization at 30 C for 2 hr using a Hybrite (Vysis; Abbott Diagnostics, Des Plains, IL). A posthybridization wash was done with 70% formamide in 10 mM Tris (pH 7.4) at RT for 15 min, followed by two washes in 0.13 SSC at 55 C for 5 min each, and two washes in 23 SSC/0.05% Tween 20 twice for 5 min each at RT. Cells were counterstained with 1 mg/ml DAPI (Sigma Aldrich). The slides were then air-dried and cover slipped with Vestashield (Vector Laboratories, Burlington, Ontario, Canada) for analysis. Multicolor Fluorescence In Situ Hybridization (M-FISH) Karyotype

After imaging the slides used for TelomereFISH on metaphases, the same slides were used for M-FISH. The M-FISH procedure was performed according to the protocol of the manufacturer (MetaSystems, Altlussheim, Germany). Image Acquisition of Telomere-FISH and M-FISH on Metaphase

After telomere-FISH on metaphases, the slides were examined under an epifluorescence microscope (Olympus BX61, Olympus America Inc., Center Valley, PA). Metaphases were captured with a Compulog IMAC-CCD S30 camera (MetaSystems Group Inc., Waltham, MA), and the corresponding coordinates were recorded. For each cell line, 30 metaphases were captured. The same

TP53 MUTATIONS, CHROMOSOME INSTABILITY, AND TELOMERES

metaphase images, captured for quantitative FISH (Q-FISH), were recaptured for M-FISH. The image processing for both Q-FISH and M-FISH was described in Rochette et al. (2005) and Samassekou et al. (2011). Analysis of Chromosomal Abnormalities and Individual Telomere Lengths

To analyze chromosome abnormalities, we referred to the criteria in the International System for Human Cytogenetic Nomenclature (Shaffer et al., 2009). A chromosomal abnormality was defined as major when at least three mitoses presented the same chromosomal abnormality. For a missing chromosome, the same change had to be present in at least six mitoses to be considered as a chromosomal abnormality. To measure individual telomere lengths, three methods were used: (1) according to TRF measurement, we converted fluorescence intensity of individual telomeres in kb as previously described (Perner et al., 2003; Samassekou et al., 2009); (2) the relative lengths of individual telomeres were assessed according to a previous report (LondonoVallejo et al., 2001); and (3) we used the z-value to determine the presence of relatively long telomeres as previously described (Samassekou et al., 2011). 3D Image Acquisition and Analysis

Following telomere-FISH for 3D, slides were examined under an AxioImager Z1 microscope (Carl Zeiss Canada Ltd., Toronto, Canada) and an AxioCam HRm charge-coupled device (Carl Zeiss Canada Ltd.). A 1003 oil objective lens (Carl Zeiss Canada Ltd.) was used for the acquisition of 30 nuclei per sample. The image processing was described in Gadji et al. (2010). mRNAs Quantitative Reverse Transcription-PCR

To determine the expression profile of telomere shelterin, telomerase, and DNA damage response genes, we used mRNAs quantitative reverse transcription-PCR (polymerase chain reaction) (primers are described in Supporting Information Table S1). For all cell lines, total RNA was extracted using Trizol, as described by the manufacturer (Invitrogen, Ontario, Canada). Reverse transcription of 1 mg RNA was done in triplicate R Reverse Transcription using the TaqManV Reagents (Applied Biosystems, Ontario, Canada). The triplicates were then pooled and quantitative real-time-PCR of 1.25 ng/ml cDNA was performed

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in triplicate with the Rotor-Gene 3000 (Corbett Life Science). The SYBR Green Master Mix (Applied Biosystems) was used for the PCR amplification applying the following temperatures and times: 95 C for 10 min, followed by 40 cycles of 95 C for 40 sec, 56 C for 40 sec, and 72 C for 40 sec. We did an internal normalization for each gene using GAPDH gene as the internal gene of control. We used the DDCt method to determine mRNA induction ratios. Statistical Analysis

Experimental results from 3D-Q-FISH and QFISH on metaphases were analyzed with Mann– Whitney U or the Kolmogorov–Smirnov test to determine significant differences between various findings among the data sets. A P value of less than 0.05 was considered statistically significant. RESULTS Cellular Growth

The mutant LoVo cells had a tendency to grow more in clusters than those of the wild type LoVo. However, their population doubling was different. The mutant TP53-R273H was the fastest growing whereas the mutant TP53-R175H mutation was the slowest (Fig. 1). Chromosome Instability

To determine the extent of CIN, we compared the M-FISH karyotypes of 30 metaphases for each LoVo cell line. The major chromosomal abnormalities are represented in Table 1. The karyotype analysis of the wild-type TP53 showed a modal number of 49 chromosomes (ranging from 44 to 49), and the major chromosomal abnormalities were t(2;12)(q13;p11.2), 15, 17, 112, and i(15) (q10) (Fig. 2). These chromosomal abnormalities are the same as those found by others in the original LoVo cell line; for the reciprocal translocation t(2;12), we used the breakpoints reported in previous reports after G-bands by trypsin using Giemsa karyotyping and comparative genomic hybridization (Masramon et al., 2000; Melcher et al., 2002). Therefore, the LoVo with wild-type TP53 is chromosomally stable despite transfection and a long period of culture. The karyotype analysis of mutant TP53-R273H showed the presence of a near diploid chromosome constitution in almost all of the cells (29/30). In contrast, the mutant TP53V143A line was composed of two groups: one was near diploid (15/30) and the other was near Genes, Chromosomes & Cancer DOI 10.1002/gcc

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Figure 1. Images of bright field microscope of the wild-type and three mutant TP53 cell lines. Images were captured at 3100 and 3400 magnifications. The LoVo cells were fusiform and grew in clusters. The doubling population time was 29.40; 26.90; 38.11; and 21.80 hr for wild type TP53, TP53-V143A, TP53-R175H, and TP53-R273H, respectively. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

tetraploid (15/30). The same results were obtained for the mutant TP53-R175H but with a lower proportion of near tetraploid cells (11/30). In addition, the mutant TP53 lines were distinguishable from the wild-type line by the presence of a der(11)t(11;14)(p?;q?), der(18)t(7;18)(q?;q?), the absence of 112, and i(15)(q10) (Table 1 and Fig. 2). Moreover, each mutant showed some specific features. While tetraploidy was characteristic for the mutant TP53-V143A (Fig. 3), loss of the Y chromosome, trisomy of chromosome 15, and the presence of a secondary group of cells with del(4)(p10) and der(4;15)(p10;q10) (Figs. 2 and 3) were typical for the mutant TP53-R175H. The

mutant TP53-R273H was characterized by the presence of del(5)(q10) (Fig. 2). Furthermore, 10 more structural abnormalities, considered minors, were observed in the mutant TP53-R175H, whereas none or less than three were found in the wild type and the other mutants, respectively (Table 2). In conclusion, the cell line bearing the TP53-R175H mutation was polyclonal and the most chromosomally instable. The 3D Signatures of Telomeres

Ongoing chromosomal rearrangements have been associated with the remodeling of the

TABLE 1. Major Chromosomal Abnormalities in LoVo Cell Lines (wild-type TP53, the mutant TP53-V143A, TP53-R175H, and TP53-R273) Number of metaphases presenting chromosomal abnormalities Chromosomal abnormalities

wt

V143A

R175H

R273H

2Y t(2;12)(q13;p11.2) del(4)(p10) der(4;15)(p10;q10) 15 del(5)(q10) 17 der(11)t(11;14)(p?;q?) 112 115 i(15)(q10) del(15)(q?) der(18)t(7;18)(q?;q?) Modal number of chromosomes

NS(3) 30 – – 30 – 29 – 30 – 29 – – 49 (44249)a

NS(2) 30 – – 6 – 30 30 – – – 3 29 47 and 94 (45296)a

30 29 12 12 27 – 29 29 – 30 – – 29 48 (42297)a

NS(3) 30 – – NS(1) 28 29 30 – – – – 30 48 (39294)a

The number of analyzed metaphases was 30 for each sample. wt: wild type, NS: nonsignificant. a The number in parenthesis indicated the range of chromosome number in the 30 cells analyzed for each cell line.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

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Figure 2. Multicolor fluorescent in situ hybridization (M-FSH) on metaphases of LoVo cells. M-FISH images of metaphases from of the LoVo wild-type TP53, the mutant TP53-V143A, TP53-R175H, and TP53-R273H. (A) is the pseudo color image of a metaphase, (B) is the

inverted DAPI of the metaphase, and (C) is the karyotype table of the same metaphase as in (A), which shows the classification of chromosomes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

nuclear architecture of telomeres (Louis et al., 2005). To understand the impact of TP53 mutations and their resulting CIN on the nuclear architecture of telomeres, we performed 3D imaging and quantitative 3D analysis of telomeres in interphase nuclei of LoVo cell lines (Fig. 4). First, the analysis of telomere lengths showed that the mutant TP53-R175H presented longer telomeres than the mutant TP53-V143A (P < 0.0001) whose

telomeres were longer than those of the two other cell lines (P < 0.001). We did not find significant differences between telomere lengths of wild-type TP53 and the mutant TP53-R273H (P 5 0.814) (Fig. 5A). Further, the number of telomeres in the mutant TP53-V143A and TP53-R175H were higher than those in wild-type TP53 and the mutant TP53-R273H (Fig. 5B). This high number of telomeres in these mutants was mostly due to Genes, Chromosomes & Cancer DOI 10.1002/gcc

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Figure 3. Multicolor FISH on metaphases of polyploid cells of mutant TP53-V143A and TP53-R175H. Representation of metaphases of polyploid cells from the mutant TP53-V143A and TP53-R175H. The metaphase of R175H is from groups of cells with t(4;15). (A) is the

pseudo color image of a metaphase, (B) is the inverted DAPI of the metaphase, and (C) is the karyotype table of the metaphase as in (A), which shows the classification of chromosomes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

the near tetraploidy status in half of the mutant TP53-V143A and TP53-R175H cells. Next, we verified the presence of telomere aggregates (TAs). TAs are fused or close telomeres that cannot be further resolved at a resolution of 200 nm, the physical resolution limit for conventional microscopes (Vermolen et al., 2005). They are characteristic of tumor cells and a higher number of TAs reflects increased genomic instability (Louis et al., 2005). We found that both the wildtype TP53 and the mutant TP53-R273H cells presented some TAs, and no statistical difference was noted between their TA frequencies (P 5 0.438). In contrast, the number of TAs in the mutant TP53-V143A and TP53-R175H were significantly higher than those of the wild type (P < 0.0001; Fig. 5C). Next, we determined the nuclear distribution of all telomeres, which is reflected by the parameter of the a/c ratio (Vermolen et al., 2005). The nuclear space occupied by telomeres is measured by two main axes, a and b, that are equal in length, and a third axis, c, that has a different length. A low a/c ratio represents an oblate spheroid shape while a high a/c ratio corresponds to disk-like shape (Vermolen et al., 2005). We found that telomeres in the mutant cells usually presented an oblate spheroid shape while those in the wild type

formed a disk-like shape. In addition, cells of the mutant TP53-V143A were uniform with respect to their nuclear telomere shape (Fig. 5D). Finally, we measured the distance of each telomere from the nucleus center. With respect to their nuclear volume (Fig. 5E), telomeres of the mutant TP53 were more peripherally located within

Genes, Chromosomes & Cancer DOI 10.1002/gcc

TABLE 2. Minor Structural Chromosomal Abnormalities in Wild-Type and Mutant TP53 Cells Chromosomal abnormalities

wt

V143A

R175H

R273H

dic(1;3)(q?;?) der((2;13)(p10;q10) der(4;15)(q10;q10) t(8;22)(q?;q10) der(11;21)(p10;q10) del(2)(p10) der(12;15)(p10;q10) t(8; 13)(q?; q10) der(14;15)(q10;q10) t(14;16)(q?;q10) der(15;22)(q10;q10)

– – – – – – – – – – –

– – – – – – – 1 – 2 –

1 1 1 1 1 1 2 – 1 – 1

1 – – – – – – – – – –

These chromosomal abnormalities were not major according to our criteria because they failed to appear in at least three metaphases. Therefore, we considered them as minor chromosomal abnormalities. The TP53-R175H had the particularities to present 10 of these abnormalities in eight different metaphases (30 metaphases were scored). The wild-type TP53 presented none of these abnormalities.

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Figure 4. Representative 2D and 3D images of LoVo cells. 2D and 3D images of LoVo nuclei (blue) and their telomeres (red) imaged and visualized with AxioVision 4.6. The wild-type TP53 and the mutant TP53-R273H showed less telomere signals and aggregates, whereas the mutant TP53-V143A, and TP53-R175H presented more telomere signals and aggregates. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

nuclei than those of the wild-type TP53 (Fig. 5F). Also, we found only in the wild-type TP53 that TAs were more nuclear centered than the rest of telomeres (P 5 0.028). In conclusion, the mutant TP53 presented different nuclear architecture of telomeres than the wild type, and the remodeling of the nuclear architecture of telomeres was more pronounced in the mutant TP53-V143A and TP53R175H. Telomere Lengths

To understand further the impact of TP53 mutations and the associated CIN on telomeres, we studied the lengths of individual telomeres in LoVo cells (Fig. 6). The length profiles of individual telomeres were dissimilar either in kb or in relative length (Supporting Information Figs. S1 and S2). Among the mutant TP53, the ones that presented more similar profile as that of the wild-type TP53 were the mutant TP53-R273H for absolute lengths in kb (Spearson’s correlation coefficient, q 5 0.514) and the mutant TP53-V143A for the relative lengths (Spearson’s correlation coefficient, q 5 0.502). Interestingly, when we analyzed the five shortest and longest individual telomeres, we found recurrence of individual telomeres being

among the longest or the shortest telomeres in the wild type and the mutant cells. For example, the telomeres on 1p and 3q were recurrent of being the longest while telomeres on 21p and Yq were being among the shortest (Table 3). In summary, mutant TP53 influenced the profile of the individual telomeres even though some few telomeres kept the same profile regardless the TP53 mutation. To decipher the relation between chromosomal abnormalities and lengths of individual telomeres, we measured telomere lengths on the ends of abnormal chromosomes. First, we studied the difference of telomere length between near tetraploid and near diploid cells. We found that near tetraploid cells presented longer telomeres than near diploid cells in the mutant TP53-R175H (P < 0.0001), whereas no significant difference was observed in the mutant TP53-V143A (P 5 0.495; Fig. 7A). Next, the analysis of telomere length on structural abnormal chromosomes revealed that: (1) in the mutant TP53-R175H, the cells bearing the translocation der(4;15)(p10;q10) presented longer telomere than those negative for this translocation (P < 0.0001; Fig. 7B); (2) telomeres on the p-arms of the reciprocal t(2;12)(q13;p11.2) in TP53-mutants were similar, and they were longer than those of the wild type (P < 0.0001; Fig. 7C); (3) the presence of telomeres on the centromeric region Genes, Chromosomes & Cancer DOI 10.1002/gcc

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Figure 5. Parameters defining nuclear architecture of telomeres in LoVo cell lines. Boxplot representations of signal intensities of individual telomeres within interphase nuclei (A), the number of telomere aggregates (C), the a/c ratio (D), and the nuclear volume (E). (B) represents the

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number of telomeres in function of their intensities; x-axis is in logarithm scale base 2. (F) is the cumulative distribution functions of the relative distance of telomeres from nuclear center (distance of each telomere from nuclear center divided by the nuclear volume of a given cell).

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Figure 6. Telomere-specific FISH and corresponding M-FISH. (a) Telomere specific FISH. (b) Corresponding multicolor FISH of (a). (A) A metaphase of a near tetraploid cell of the mutant R175H. (B) Partial metaphases of (A) showing structurally abnormal chromosomes,

pointed out by white arrows. (C) A partial metaphases of the mutant R273H, showing telomere signal on the centromeric region of the del(5)(q10), indicated by white arrows. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

of del(4)(p10) and del(5)(q10) (Figs. 6B, 6C, and 7C) could be due to either an involvement of telomeric regions or telomere healing; (4) the mutant TP53R175H was the only cell line that showed length difference on individual telomeres that are involved in abnormal chromosomes (Supporting Information Table S2). In conclusion, individual telomere length is associated with chromosomal rearrangements and this association tends to be more present in the mutant TP53-R175H.

upregulated, the other shelterin genes were downregulated or had a normal expression. TERF1, POT1, and TERF2IP were repressed in all cell lines, except for the mutant TP53-R273H, and this repression was more pronounced in the wild type. TERF2 was repressed in the mutant TP53-V143A and TP53-R273H, whereas it had normal induction in the wild-type TP53 and the mutant TP53R175H. The reverse expression profile for TERF2 was observed for TIN2 (Fig. 8A). In summary, the wild-type TP53 and the mutant TP53-R175H presented similar expression profiles while those of the mutant TP53-V143A and TP53-R273H were alike. Next, we found that TERT was highly expressed in all LoVo cell lines and the wild type presented significantly higher expression than the mutants. In contrast, TERC was repressed in all cell lines (Fig. 8B). Moreover, we found that

Gene Expression Profile

To investigate the consequence of TP53 mutations and a rearranged genome on the telomere shelterin genes and those genes involved in DNA damage response, we examined their expression profiles (Fig. 8). Except for TTP1, which was

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TABLE 3. Median of the Five Longest and Shortest Telomeres in Wild-Type and Mutant TP53 Cells Long telomeres

wt

V143A

R175H

R273H

Short telomeres

Tel

Length (median)

Tel

Length (median)

9p 3q 16p 1p 18p 1p 2p 3q 5p 8p 3q 1p 4q 12p 18p 3q 1q 5p 19q 1p

5.01 4.98 4.97 4.53 4.51 5.67 5.60 4.94 4.94 4.75 5.51 5.20 5.08 5.01 4.96 5.40 5.24 4.78 4.64 4.62

22p 15p 21p 13p Yq 4q 13p 21p 15p Yq 17q 21p 20p 22q 21q 10q 21p 11q 22q Yq

3.22 3.07 3.01 2.66 1.97 3.57 3.45 3.44 3.40 2.49 3.92 3.83 3.61 3.28 3.01 3.30 3.23 3.21 3.01 2.32

Telomere lengths are expressed in kb. The bold text indicates recurrent individual telomeres being among either the longest or the shortest telomeres in the wild type and the mutant cells.

telomerase activity positively correlated with the expression of TERT of all LoVo lines (data not shown). Although some TP53 mutations such as R273H have been associated with alternative lengthening of telomere (ALT) mechanism (Gocha et al., 2013), in our study, LoVo cell lines did not display features of ALT phenotype (data not shown). Moreover, the upregulation of TERT and the high activity of telomerase render the possibility of ALT mechanism less likely. Finally, we studied the expression of TP53 and some upstream and downstream gene targets of TP53. TP53 was upregulated in the mutant TP53-R175H and CDKN1A was upregulated in all LoVo cell lines and more importantly in the mutant TP53R175H. All the studied sensor genes of TP53 were downregulated (Fig. 8C). Importantly, we found that the ATM/ATR genes were less expressed in the most unstable genomic lines (TP53-R175H and TP53-V143A) and MRE11 were more downregulated in TP53-R175H than the other lines (Fig. 8C). DISCUSSION

The study of the effect of specific mutations of TP53 occurring during the neoplastic process remains difficult because of the difference in Genes, Chromosomes & Cancer DOI 10.1002/gcc

genomic alterations between different cancer cells or cell models. Mouse models used to study specific TP53 mutations present germline mutations, which are more suitable to study the Li–Fraumeni syndrome (Borresen et al., 1995; Olive et al., 2004). Therefore, the use of an isogenic model of cancer cells is essential in understanding the impact of specific mutations of TP53 to genomic instability during the neoplasic process. In this study, we took advantage of a unique isogenic model of the LoVo colon cancer cell lines (Pocard et al., 1996), which consist of wild-type TP53 and mutant TP53 (V143A, R175H and R273H) to investigate the role of TP53 mutants in CIN, remodeling of nuclear architecture of telomeres, and dynamic changes in length of individual telomeres. This model has the advantage that LoVo cells are chromosomally stable (Masramon et al., 2000; Melcher et al., 2002) and their transfection per se does not lead to chromosomal rearrangements. Therefore, the model represents a unique tool to study TP53 mutant and wildtype functions on parameters such as genomic instability and nuclear architecture of telomeres. In this study, we showed that specific TP53 mutations in LoVo cells of the colon cancer can induce additional genomic instability characterized by chromosomal rearrangements (tetraploidy and structural chromosome abnormalities), remodeling of telomeric nuclear architecture (TAs and telomere nuclear peripheral location), dynamic alteration of individual telomere lengths (changes of telomere length profile and clonal lengthening); the results are summarized in Table 4. In considering all these genomic changes, the mutant TP53-R175H was the most susceptible to induce genomic instability in LoVo cells. Chromosomal Instability

The numerical CIN was mainly characterized by tetraploidy and its occurrence in mutant TP53 can be explained by the specificity of the mutations. The tetraploid G1 checkpoint is abolished in the mutant TP53-V143A and TP53-R175H, whereas it is still functional in the wild type and the mutant TP53-R273H when these different LoVo cells were exposed to mitotic spindle inhibitor (Dridi et al., 2003). The mechanism behind endoreplication leading to tetraploidization might be slippage, cytokinesis failure, or loss of telomere protection (Ganem et al., 2007; Davoli et al., 2010). We can speculate that loss of telomere protection through deregulation of telomere shelterin proteins can be one of the mechanisms responsible

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Figure 7. Telomere lengths on abnormal chromosomes. (A) Comparison between telomere lengths in diploid and polyploidy cells in TP53-V143A and TP53-R175H. The black and gray columns represent near diploid and near tetraploid cells, respectively. (B) Comparison between clones with and without t(4;15) in the mutant R175H. The black and gray columns represent clones with and without t(4;15), respectively. (C) Individual telomere lengths of major structural chromosomal abnormalities represented in Table 1. Telomere lengths on the short arms (p) and the long arm (q) of each major chromosomal abnormality were measured. The number of cells analyzed to determine the median of telomere length on these short and long arms was the same as the number of cells bearing these major chromosomal abnormalities (Table 1). The median length of telomeres is expressed in kb. Upper panel represents telomere length on the p-arms and lower panel represents telomere length on the q-arms. Black, gray, white, and hatch, columns represent the wild-type TP53, the mutant TP53-V143A, TP53-R175H, and TP53-R273H, respectively. The

t(2;12)(q13;p11.2)* and t(2;12)(q13;p11.2)** indicate the derivative 2 and 12 with this translocation, respectively. Error bars are at 95% confidence interval. We compared the length of individual telomeres (comparison between telomere on the same arm) for the same chromosomal abnormality between different cell lines (wild type and mutants). We observed the following statistical differences: (1) For the telomeres on the p-arms of the t(2;12)(q13;p11.2)*, each of the mutant TP53 presented longer telomeres than the wild type; (2) For the telomeres on the p-arms of the der(11)(11;14)(p?;q?), TP53-V143A and TP53-R273H presented longer telomeres than TP53-R273H; (3) For the telomeres on the q-arms of the t(2;12) (q13;p11.2)**, TP53-V143A and TP53-R273H presented longer telomeres than the wild type; (4) For telomeres on the p-arms of the del(14)(q?), TP53-R175H presented longer telomeres than TP53-R273H; (5) For telomeres on the q-arms of the der(18)(7;18)(p?;q?), TP53-R175 and TP53-R273H presented longer telomeres than the TP53-V143A.

for tetraploidization in the mutant TP53-V143A and TP53-R175H cells. Recently, it was shown that loss of telomere protection due to POT1 deregulation in the context of TP53 deficiency can induce tetraploidization. POT1 deregulation leads to prolonged DNA damage signal, which was caused by persistent telomere dysfunction, mitosis failure, and endoreplication. Then, cells re-

entered in S phase because of TP53 deficiency (Davoli et al., 2010; Davoli and de Lange, 2012). In our study, POT1 was downregulated in wildtype TP53, and the mutant TP53-V143A and TP53-R175H (Fig. 8A). The presence of functional TP53-dependent tetraploid G1 checkpoint in wild-type cells (Dridi et al., 2003) might prevent them from being tetraploid, whereas its Genes, Chromosomes & Cancer DOI 10.1002/gcc

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Figure 8. Gene expression profile. Mean of relative mRNA induction of three experiments. The mRNA levels of relevant genes (except for TERT) in LoVo cell lines were normalized to those of fibroblasts. For TERT mRNA, the expression level of that gene in SW480 was used to normalize those of LoVo cell lines. Error bars are standard deviation. (A) Telosome genes, (B) telomerase component genes, and (C) TP53 upstream and downstream target genes.

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TABLE 4. Comparative Summary of Major Findings from Chromosomal Abnormalities, Nuclear Architecture of Telomeres, and Telomere Length Chromosomal abnormalities

Cell lines

Tetraploidy

Wt V143A R175H R273H

– 11 11 –

Structural abnormalities 2 4 5 4

absence in the mutant TP53-V143A and TP53R175H might favor tetraploidy. Regardless of the proposed mechanisms, the occurrence of tetraploidy in LoVo cells was mutant-TP53 dependent. The two nonreciprocal translocations leading to two derivative chromosomes, der(18)t(7;18)(q?;q?) and der(11)t(11;14)(p?;q?), were among the structural chromosomal abnormalities featuring CIN (Table 1). These two translocations were present in all the mutant-TP53 cell lines and this might denote a common mechanism behind these structural chromosome abnormalities regardless of the mutation type. Contrary to the tetraploidy and the other chromosomal abnormalities, which were mutant-TP53 dependent, these two translocations might be caused by a loss of function of wild-type TP53. The implication of TP53 in suppressing structural chromosome abnormalities has been highlighted in HCT116 cells from human colon carcinoma cells (Dalton et al., 2010). In these cells, more structural chromosome alterations were present in null TP53 cells than in wild-type TP53 cells after a prolonged mitotic arrest. The same study found that polyploid cells were more prone to elicit structural chromosome changes than diploid cells (Dalton et al., 2010). Contrary to the latter finding, we did not find differences in structural chromosomal abnormalities between diploid and polyploid cells. This discrepancy can be explained by (1) the occurrence of structural chromosomal abnormalities prior to tetraploidization in our study, (2) the abilities of TP53 mutations to promote more structural chromosome abnormalities than null TP53 mutations (Song et al., 2007), or (3) the propensity of the genetic background of the mutant-TP53 cells to enhance genomic instability (Artandi et al., 2000; Ward et al., 2005). Another mechanism for the occurrence of these nonreciprocal translocations might be the impairment of ATM/ATR/MRE11-dependent DNA damage responses. Recent findings in mouse model showed an increase in chromosomal translocations

Nuclear architecture of telomeres Nuclear location of telomere

Telomere aggregates

Clonal lengthening of telomeres

Central Peripheral Peripheral Peripheral

– 11 11 –

– 1 11 –

when the TP53-R175H and TP53-R273H mutants inactivated MRE11/ATM-dependent DNA damage responses (Liu et al., 2010). The downregulated ATM/ATR/MRE11 genes and the impairment of TP53 in the mutant cells (Fig. 8C) might support this mechanism in our study. Remodeling of the Nuclear Architecture of Telomeres

Remodeling of nuclear architecture of telomeres is another indicator of an ongoing genomic instability in LoVo mutant cells. A shift in the location of telomeres toward the nuclear periphery was characteristic of the mutant TP53 cells (Fig. 5F). This peripheral location of telomeres has been associated with nuclear scaffold dysfunction (Gonzalez-Suarez et al., 2009) and found in some cancers such as chronic myeloid leukemia (Samassekou et al., 2013). Furthermore, the telomere displacement from central (wild type) to peripheral (mutants) nuclear location might be going along with nuclear remodeling of chromosomes that is associated with structural chromosome abnormalities (Louis et al., 2005). Neighboring organization of telomeres favors nuclear organization of chromosomes and their recombination (De Vos et al., 2009). Some evidence suggests that telomere positioning is one of the determinant factors governing chromosome territory (Louis et al., 2005; Pandita et al., 2007; Pampalona et al., 2010) whose disruption can affect gene expression (Marella et al., 2009), generate chromosomal abnormalities (Louis et al., 2005), and disrupt cell function (Solovei et al., 2009). From our data, we suggest that the peripheral location of telomeres is TP53-mutation dependant and is an enhanced factor of genomic instability. The mutant TP53-V143A and TP53-R175H presented higher number of TAs (Figs. 4 and 5B) as well as high CIN (Tables 1 and 2). In mouse Genes, Chromosomes & Cancer DOI 10.1002/gcc

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model, deregulation of the myc box II of MYC lead to the formation of TAs and their high number correlated with changes of chromosome relative positions and an increase of structural chromosomal abnormalities (Louis et al., 2005). Moreover, in human Hodgkin’s lymphoma, TAs favor genomic instability and contribute to the transition from mononuclear Hodgkin cells to multinucleated Reed–Sternberg cells (Knecht et al., 2009). The number of TAs is an indicator of genomic instability (Mai, 2010) and specific TP53 mutations might be contributing factors to their genesis. From our data, we can infer that specific TP53 mutations such as V143A and R175H, leading to conformation changes of TP53-mutant protein, are more potent to induce TA formation in tumor cells. Telomere Length Changes

Individual telomere lengths and their profiles were also remodeled in the TP53 mutants. Telomeres on the Yq were among the shortest telomeres in all lines (Table 3), except for the mutant TP53-R175H in which it was lost. The loss of the Y chromosome in this mutant might be telomere length dependent, which was evoked in chronic myeloid leukemia (Samassekou et al., 2009). It is generally acknowledged that short telomeres have been associated with genomic instability (Londono-Vallejo, 2008). This observation was at odd with our findings. The mutant TP53-R175H and TP53-V143A presented relative long telomeres, whereas the wild type, the most genomic stable line in our study, had relative short telomeres (Fig. 5A) and the highest TERT expression (Fig. 8B) and telomerase activity. The lengthening of telomeres might be due to TP53 mutation, which could increase the processing of telomerase by gain of function (Li et al., 1999). Another possibility might be the association between telomere lengthening and CIN. In the mutant TP53R175H, on the one hand, the polyploid cells had longer telomeres than diploid cells, and, on the other hand, cells having a der(4;15) also presented longer telomeres than the main line (Figs. 7A and 7B). In human tumors, poor prognosis has been associated with long telomeres in colon cancers (Garcia-Aranda et al., 2006), Barrett carcinoma (Gertler et al., 2008), and hepatocellular carcinoma (Oh et al., 2008). In this report, the hypothesis that long telomeres might be advantageous for tumor progression (Svenson and Roos, 2009) by sustaining cell proliferation and survival can be Genes, Chromosomes & Cancer DOI 10.1002/gcc

taken into consideration. The understanding of the mechanisms behind this observation can further shed light on the interplay between TP53 mutations, CIN, and telomeres. TP53 and Genomic Instability

Previous reports have shown the impact of TP53 mutations on genomic instability (Gualberto et al., 1998; Caulin et al., 2007). Moreover, data from human cancers and the TP53 mutant knock-in mice illustrate that different TP53 mutations have distinct phenotypes (Borresen et al., 1995; Olive et al., 2004). However, our findings pointed out that features defining genomic instability (chromosomal abnormalities, telomeric nuclear localization) were common in the TP53 mutants regardless of the type of mutation. In addition, the mutant-TP53 tumors were more aggressive than null-TP53 tumors, indicating an acquisition of new properties such as inducing genomic instability (Caulin et al., 2007), and its degree can depend on the mutation types. For instance, the most studied and frequent mutant TP53, R175H or its mouse equivalent (R172H), induces genomic instability (Caulin et al., 2007; Liu et al., 2010), whereas the impact of R273H on genomic instability is more cellular context dependent (Song et al., 2007). Although the TP53 mutations were germline in those studies, their findings were in agreement with ours where the mutant TP53-R175H and TP53-V143A were the most genomic instable lines, defined by the degrees of chromosomal abnormalities and remodeling of the nuclear architecture of telomeres. The disruption of the nuclear architecture of telomeres might be another novel pathway through which TP53 mutants induce genomic instability in cancer cells. The different effect on genomic instability in the same cellular context can be mostly due to the nature of the mutation. Both the R175H and V143A mutations are structural mutations that dramatically change TP53 conformation while the R273H mutation is a DNA-contact mutation, which directly disrupts the TP53 binding to its target genes. Also, the TP53-R273H mutation was shown to have native conformation with residual transcriptional activity (Joerger and Fersht, 2007). Compared to other TP53 mutants, the TP53 upregulation of the mutant TP53-R175H might also contribute to the genomic instability of this cell line. The genetic background difference of mutant-TP53 cells and how mutant TP53 interacts

TP53 MUTATIONS, CHROMOSOME INSTABILITY, AND TELOMERES

with other TP53 family proteins, such as TP63 and TP73, can also be factors explaining their difference of inducing genomic instability. In this study, we deciphered to some extent the impact of some of most frequent TP53 mutations on genomic instability in cancer cells. This study better simulates the occurrence of TP53 mutation over a course of a cancer. In many tumors, TP53 mutations are not an early event and they have a huge influence on the prognosis. Predicting genomic instability by knowing a mutant TP53 status in a specific tumor can help formulate a better therapeutic response. Similar studies in different cancers are needed to deepen our knowledge about mutant TP53 roles in tumor progression. ACKNOWLEDGMENTS

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