Topoisomerase IIIα is required for normal proliferation and telomere stability in alternative lengthening of telomeres
2008; Springer Nature; Linguagem: Inglês
10.1038/emboj.2008.74
ISSN1460-2075
AutoresNassima Temime-Smaali, Lionel Guittat, Thomas Wenner, Émilie Bayart, Céline Douarre, Dennis Gómez, Marie‐Josèphe Giraud‐Panis, Arturo Londoño‐Vallejo, Éric Gilson, Mounira Amor-Guéret, Jean‐François Riou,
Tópico(s)Telomeres, Telomerase, and Senescence
ResumoArticle17 April 2008free access Topoisomerase IIIα is required for normal proliferation and telomere stability in alternative lengthening of telomeres Nassima Temime-Smaali Nassima Temime-Smaali Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France Search for more papers by this author Lionel Guittat Lionel Guittat Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France Laboratoire de Régulation et dynamique des génomes, INSERM U565, CNRS UMR5153, Muséum National d'Histoire Naturelle USM503, Paris, France Search for more papers by this author Thomas Wenner Thomas Wenner Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France Search for more papers by this author Emilie Bayart Emilie Bayart Institut Curie, Section de Recherche, CNRS UMR 2027, Centre Universitaire, Orsay, France Search for more papers by this author Céline Douarre Céline Douarre Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France Search for more papers by this author Dennis Gomez Dennis Gomez Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, Toulouse, France Search for more papers by this author Marie-Josèphe Giraud-Panis Marie-Josèphe Giraud-Panis Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5239, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Arturo Londono-Vallejo Arturo Londono-Vallejo Laboratoire Télomères et Cancer, CNRS UMR 7147, Institut Curie, Paris, France Search for more papers by this author Eric Gilson Eric Gilson Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5239, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Mounira Amor-Guéret Mounira Amor-Guéret Institut Curie, Section de Recherche, CNRS UMR 2027, Centre Universitaire, Orsay, France Search for more papers by this author Jean-François Riou Corresponding Author Jean-François Riou Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France Laboratoire de Régulation et dynamique des génomes, INSERM U565, CNRS UMR5153, Muséum National d'Histoire Naturelle USM503, Paris, France Search for more papers by this author Nassima Temime-Smaali Nassima Temime-Smaali Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France Search for more papers by this author Lionel Guittat Lionel Guittat Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France Laboratoire de Régulation et dynamique des génomes, INSERM U565, CNRS UMR5153, Muséum National d'Histoire Naturelle USM503, Paris, France Search for more papers by this author Thomas Wenner Thomas Wenner Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France Search for more papers by this author Emilie Bayart Emilie Bayart Institut Curie, Section de Recherche, CNRS UMR 2027, Centre Universitaire, Orsay, France Search for more papers by this author Céline Douarre Céline Douarre Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France Search for more papers by this author Dennis Gomez Dennis Gomez Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, Toulouse, France Search for more papers by this author Marie-Josèphe Giraud-Panis Marie-Josèphe Giraud-Panis Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5239, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Arturo Londono-Vallejo Arturo Londono-Vallejo Laboratoire Télomères et Cancer, CNRS UMR 7147, Institut Curie, Paris, France Search for more papers by this author Eric Gilson Eric Gilson Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5239, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Mounira Amor-Guéret Mounira Amor-Guéret Institut Curie, Section de Recherche, CNRS UMR 2027, Centre Universitaire, Orsay, France Search for more papers by this author Jean-François Riou Corresponding Author Jean-François Riou Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France Laboratoire de Régulation et dynamique des génomes, INSERM U565, CNRS UMR5153, Muséum National d'Histoire Naturelle USM503, Paris, France Search for more papers by this author Author Information Nassima Temime-Smaali1,‡, Lionel Guittat1,2,‡, Thomas Wenner1,‡, Emilie Bayart3, Céline Douarre1, Dennis Gomez4, Marie-Josèphe Giraud-Panis5, Arturo Londono-Vallejo6, Eric Gilson5, Mounira Amor-Guéret3 and Jean-François Riou 1,2 1Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France 2Laboratoire de Régulation et dynamique des génomes, INSERM U565, CNRS UMR5153, Muséum National d'Histoire Naturelle USM503, Paris, France 3Institut Curie, Section de Recherche, CNRS UMR 2027, Centre Universitaire, Orsay, France 4Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, Toulouse, France 5Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5239, Ecole Normale Supérieure de Lyon, Lyon, France 6Laboratoire Télomères et Cancer, CNRS UMR 7147, Institut Curie, Paris, France ‡These authors contributed equally to this work *Corresponding author. Laboratoire de Régulation et dynamique des génomes, INSERM U565, CNRS UMR5153, Muséum National d'Histoire Naturelle USM503, 43 rue Cuvier, CP26, 75231 Paris Cedex 5, France. Tel.: +33 1 40 79 36 98; Fax: +33 1 40 79 37 05; E-mail: [email protected] The EMBO Journal (2008)27:1513-1524https://doi.org/10.1038/emboj.2008.74 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Topoisomerase (Topo) IIIα associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIα colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIα immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIα depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIα expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIα is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function. Introduction DNA topoisomerases (Topo) are ubiquitous enzymes that are required for nearly all aspects of DNA metabolism (Wang, 1996; Nitiss, 1998; Champoux, 2001). DNA topoisomerases induce transient breaks in DNA that are associated with a covalent topoisomerase/DNA complex to allow strand passage or enzyme swivelling, which results in the topological state modification of DNA. DNA topoisomerases are divided into four groups, type IA, IB, IIA, and IIB (Wang, 2002). Type IA DNA topoisomerases are conserved in all organisms and function to remove highly negative supercoils; they are assumed to have a fundamental role in the regulation of the topology of replication and recombination intermediates (Wu and Hickson, 2001; Wang, 2002). The budding yeast Saccharomyces cerevisiae expresses a single type IA topoisomerase encoded by the TOP3 gene, initially identified in the top3Δ strain by a slow growth phenotype and hyper-recombination between repetitive DNA sequences, such as rDNA (Wallis et al, 1989). The deletion of SGS1, whose product belongs to the RecQ family of DNA helicases involved in the maintenance of genome stability, suppresses the TOP3 loss-of-function phenotype. It was subsequently shown that Sgs1p forms a complex with Top3p (Gangloff et al, 1994; Hickson, 2003). Humans have two members of the IA subfamily, Topo IIIα and Topo IIIβ (Wang, 2002). Topo IIIα exists in a complex with BLM, a RecQ helicase whose mutation is responsible for Bloom's syndrome (BS) (Johnson et al, 2000; Wu et al, 2000). In biochemical assays, Topo IIIα has a weak DNA relaxation activity on double-stranded DNA but is able to bind, cleave, and religate single-stranded DNA (Goulaouic et al, 1999). The interaction of BLM with Topo IIIα stimulates the strand passage activity of Topo IIIα (Wu and Hickson, 2002). Furthermore, Topo IIIα and BLM cooperate to convert double Holliday junctions (DHJ) to decatenated products in vitro, suggesting a putative role of the complex in the resolution of recombination intermediates (Wu and Hickson, 2003). Recently, BLAP75/RMI1 was identified as a third component of the BLM/Topo IIIα complex; this protein is also highly conserved among eukaryotes (Chang et al, 2005; Yin et al, 2005). BLAP75/RMI1 recruits Topo IIIα to DHJ and its depletion by RNA interference increases the frequency of sister chromatid exchanges (Yin et al, 2005; Wu et al, 2006). Under normal cell growth conditions, BLM and Topo IIIα colocalized in promyelocytic (PML) nuclear bodies together with many other proteins, including Rad50, Mre11, NBS1, p53, and Sp100 (Johnson et al, 2000; Yankiwski et al, 2000), and this localization is disrupted in BS cells (Johnson et al, 2000). The Topo IIIα/BLM complex was also proposed to function as a repair complex in response to replication defects and may restart stalled replication forks (Ababou et al, 2000; Wang et al, 2000; Amor-Gueret, 2006). Following camptothecin treatment, a phosphorylated form of BLM dissociates from the Topo IIIα/BLM complex at its PML storage sites and accumulates with γ-H2AX at replication damage sites (Rao et al, 2005). Telomeres consist of repetitive G-rich DNA sequences that protect the chromosome ends from fusion and illegitimate recombination. In human somatic cells, telomere length decreases at each round of division and cellular mechanisms that counteract this degradation are able to confer indefinite proliferation potential. Two classes of mechanisms have been described in human tumor cells that allow the maintenance of telomere length. The first requires a specialized enzyme, called telomerase, which is able to copy, as a reverse transcriptase, the short TTAGGG motif at the 3′ end of telomeres. Telomerase is composed of a catalytic subunit, hTERT, associated with an RNA containing the template of the telomere repeat unit, hTR. Telomerase is overexpressed in a large number of tumours (about 85%) and is involved in the capping of telomere ends (McEachern et al, 2000). The second mechanism is observed in tumours (about 15%) as well as in immortalized cell lines lacking telomerase activity and involves recombination between telomeres, a mechanism known as alternative lengthening of telomeres (ALT) (Dunham et al, 2000; Londono-Vallejo et al, 2004). ALT cells display a heterogeneous telomere length and particular nuclear foci termed ALT-associated PML bodies (APB) that contain, in addition to PML, telomeric DNA, telomere binding proteins (TRF1 and TRF2), and several proteins involved in DNA synthesis, repair, and recombination. The latter include the MRE11/RAD50/NBS1 complex and the RecQ helicases WRN and BLM (Kim et al, 1995; Yeager et al, 1999; Grobelny et al, 2000; Wu et al, 2000; Huang et al, 2001; Lillard-Wetherell et al, 2004; Tsai et al, 2006). In yeast, telomerase-negative survivors with heterogeneous telomere sequences (type II) also involve a telomere–telomere recombination mechanism that requires Rad50p, Rad52p, and Sgs1p (Huang et al, 2001) as well as Top3 (Kim et al, 1995; Tsai et al, 2006). Telomeres end in a 3′ single-stranded overhang that may be involved in different DNA conformations such as t-loop and/or G-quadruplexes (Mergny et al, 2002; de Lange, 2005). T-loops are created through strand invasion of the 3′ telomeric overhang into the duplex part of the telomere and are thought to represent a strategy to protect chromosome ends. They might also correspond to recombination intermediates. TRF2 is required for the establishment of t-loops and these structures are presumably involved in protecting the DNA at the telomeric single/double-strand junction (de Lange, 2005). RecQ helicases associated with the telomeric complex, such as WRN or BLM (Lillard-Wetherell et al, 2004), cooperate with POT1 (protection of telomere 1), a protein that binds specifically to the single-strand telomeric sequence, to unwind telomeric forked duplexes and D-loop structures (Opresko et al, 2005). Therefore, Topo IIIα might participate in solving topological issues arising from the action of a RecQ helicase during telomere recombination/replication in the ALT pathway. We have examined whether Topo IIIα is required for the correct maintenance of telomeres of ALT and telomerase-positive cells. We show that Topo IIIα interacts with TRF2 independently of its interaction with BLM and forms a complex with TRF2 and BLM at APB in ALT cells. Topo IIIα interacts with telomeric DNA as shown by chromatin immunoprecipitation (ChIP) assays in ALT cells and its depletion by small interfering RNA (siRNA) leads to the accumulation of anaphase bridges and to telomere uncapping and is associated with cell growth arrest. As Topo IIIα interacts in vitro with single-stranded telomeric sequences, we suggest that the BLM/Topo IIIα/TRF2 complex at APB is involved in the maintenance of telomeres in ALT cells, possibly through the resolution of intermediate DNA structures that arise during telomere recombination. Results Colocalization of Topo IIIα at APB with PML and shelterin subunits in ALT cells Indirect immunofluorescence staining with an anti-Topo IIIα antibody detected Topo IIIα protein organized into multiple nuclear foci in the ALT cell lines WI38-VA13, MRC5-V1, and U2OS (Figure 1A and Supplementary Figure S1A). Antibodies that recognize the human TRF2 and PML proteins were used to identify APB in these cells. Topo IIIα foci were large and bright and an almost complete colocalization (>90%) of Topo IIIα, TRF2, and PML was observed (Figure 1A and Supplementary Figure S1A). To confirm that Topo IIIα is a component of APB, MRC5-V1 cells were stably transfected with YFP–Topo IIIα and stained with TRF2 or PML antibodies (Figure 1B and D) or cotransfected with other CFP-tagged shelterin components (CFP–TRF2, CFP–POT1, or CFP–TIN2) (Figure 1C and D). The results showed a strong colocalization of YFP–Topo IIIα, POT1, TIN2, TRF2, and PML in agreement with the indirect immunofluorescence results. Similar experiments were performed in two telomerase-positive cell lines (EcR293 and HT1080) and indicated that YFP–Topo IIIα perfectly colocalizes with PML (Supplementary Figure S1B). In contrast to ALT cells, only rare colocalizations were observed between TRF2 foci and YFP–Topo IIIα in interphase nuclei from telomerase-positive cells (Figure 1C, arrows, and Supplementary Figure S2). During mitosis, the Topo IIIα signal is absent from condensed chromosomes in both ALT and telomerase-positive cells (Supplementary Figure S2). These results indicate that Topo IIIα is localized at APB in ALT cells. In contrast, Topo IIIα foci mostly correspond to PML foci in telomerase-positive cells in agreement with previous studies (Johnson et al, 2000; Wu et al, 2000). Figure 1.Topo IIIα colocalizes with PML and shelterin components at APB in MRC5-V1 ALT cells. The stable expression of a YFP-tagged Topo IIIα protein was also used to determine its localization. (A) Colocalization of Topo IIIα (red, detected by immunofluorescence) with TRF2 or PML (green, detected by immunofluorescence. (B) Representative images of colocalization in MRC5-V1 of Topo IIIα tagged with YFP (Topo IIIα∷YFP, green), with TRF2 or PML (red, detected by immunofluorescence). (C) Representative images of colocalization in MRC5-V1 of Topo IIIα tagged with YFP (Topo IIIα∷YFP, green) with TIN2 tagged with CFP (blue, TIN2∷CFP), POT1 tagged with CFP (blue, POT1∷CFP), or TRF2 tagged with CFP (blue, TRF2∷CFP). (D) Representative images of colocalization in MRC5-V1 of Topo IIIα tagged with YFP (Topo IIIα∷YFP, green), TRF2 tagged with CFP (TRF2∷CFP, blue), and PML (red detected by immunofluorescence). Download figure Download PowerPoint Topo IIIα interacts with TRF2 To test for a physical interaction between telomeric proteins and Topo IIIα, we performed co-immunoprecipitation experiments in U20S, MRC5-V1 ALT, HT1080, and 293T telomerase-positive cell extracts. In the complex precipitated by the Topo IIIα D6 antibody, we detected TRF2 by immunoblotting (Figure 2). A similar result was obtained for EcR293 telomerase-positive cells and WI38-VA13 ALT cells (data not shown). Immunoblotting with an anti-BLM antibody also revealed the presence of BLM in the Topo IIIα complex (data not shown). To investigate this further, the reverse experiments using an anti-TRF2 antibody was performed in telomerase-positive and ALT cells (Figure 2). For unknown reason that could include the presence of the epitope close to the protein interaction region or the relative abundance of these proteins in nuclei, we were unable to reproducibly co-immunoprecipitate Topo IIIα using TRF2 antibody (Figure 2 and data not shown). Despite modifications of the immunoprecipitation protocol using milder detergent conditions, the use of nuclear extract, and the use of other TRF2 antibodies, we were unable to recover significant amounts of Topo IIIα in TRF2 immunoprecipitates (Supplementary Figure S3). Figure 2.Topo IIIα/TRF2 complex is detected in telomerase-positive and ALT cells as revealed by immunoprecipitation with D6 Topo IIIα antibody. The antibodies (TRF2, Topo III, or control IgG) used for immunoprecipitation (IP) are listed at the top of each panel and the antibodies (BLM, Topo III, and TRF2) used for western blot analysis (WB) are listed at the left of each panel. Topo IIIα co-immunoprecipitates TRF2 in HT1080 or 293T (telomerase-positive) and in U2OS or MRC5-V1 (ALT) cell lines. Reciprocal immunoprecipitation of Topo IIIα by TRF2 antibody (4A794, mouse) is not detected in 293T, U2OS, or MRC5-V1 and is unreproducible in other cells. Download figure Download PowerPoint Next, we investigated whether the BLM/Topo IIIα/TRF2 complex depended on the presence of DNA by evaluating its resistance to DNase treatment. The MRC5-V1 protein extract was treated with DNase I (data not shown). This treatment did not impair the recovery of TRF2 and BLM from Topo IIIα immunoprecipitates, suggesting the existence of DNA-independent interactions among BLM, Topo IIIα, and TRF2 in ALT cells. We further determined whether the TRF2/Topo IIIα complex was stable in the absence of the BLM protein by using the telomerase-positive and BLM-deficient GM08505 cell line (data not shown). Immunoprecipitation experiments indicated that TRF2 and Topo IIIα interacted, suggesting that these two proteins form a BLM-independent complex. Thus, we concluded that the presence of BLM is not required for Topo IIIα/TRF2 interaction. These data suggest that the Topo IIIα/TRF2 complex might exist as part of a multiprotein complex in telomerase-positive and ALT cells. Topo IIIα associates with telomeric DNA in ALT cells Next, we studied the in vitro interaction of purified recombinant Topo IIIα (Goulaouic et al, 1999) with telomeric DNA by electrophoretic mobility shift assay (EMSA) using oligonucleotides corresponding to double-stranded telomeric repeats with a 3′ G-rich single-stranded extension (Tel-1) or without the single-stranded region (Tel-2) (Figure 3). Gel shift assays with increasing concentrations of Topo IIIα were performed in the presence of 5 nM of radiolabelled oligonucleotide. Topo IIIα bound to the telomeric substrate with a 3′ G-overhang (Tel-1) with a band shift detectable at a concentration as low as 50 nM (Figure 3, left panel). Under the same experimental conditions, a Topo IIIα band shift was detected on the DNA substrate without the G-overhang extension (Tel-2) at a concentration equal to 150 nM (Figure 3, right panel), suggesting that Topo IIIα bound to the G-overhang sequence, in agreement with the known preference of Topo IIIα for single-stranded DNA (Goulaouic et al, 1999; Chen and Brill, 2007). To determine whether TRF2 and Topo IIIα form a complex at single-stranded telomeric DNA, retardation assays were also performed using the 21G oligonucleotide (Supplementary Figure S4). Owing to its short size (21 nucleotides), the binding of Topo III or TRF2 requires higher protein concentrations (>350 nM). The addition of Topo IIIα and TRF2 induces the formation of a higher molecular weight complex (see arrow), suggesting the formation of a ternary Topo IIIα/TRF2/DNA complex. A similar result was obtained with the 21Gmu3 oligonucleotide in which three guanines of the telomeric sequence have been replaced by cytosines (Supplementary Figure S4), indicating that the complex formation is not sequence dependent. A ternary complex was also obtained using the double-stranded DS26 oligonucleotide (Supplementary Figure S4, arrow). In these conditions, the binding of TRF2 on this nontelomeric sequence is nearly undetectable, indicating that Topo IIIα recruits TRF2 onto DNA by direct interaction. Figure 3.Topo IIIα interacts with telomeric sequences in vitro. EMSA were performed using 5 nM of telomeric template with a 3′ single-stranded extension (Tel-1, left panel) or without the single-stranded region (Tel-2, right panel) (see Materials and methods for details) in the presence of increasing concentrations (50, 150, 250, and 350 nM) of purified recombinant Topo IIIα (Goulaouic et al, 1999). For Tel-1, a Topo IIIα/DNA bandshift was detected at 50 nM, whereas the Tel-2/Topo IIIα bandshift was detected at 150 nM, suggesting a preferential binding to the 3′ single-stranded extension. Download figure Download PowerPoint To evaluate whether Topo IIIα is associated with telomeric DNA, ChIP experiments were performed in ALT and telomerase-positive cells (293T, HeLa, HT1080, MRC5-V1, U2OS, and WI38-VA13). In a first set of experiments, telomeric DNA immunoprecipitated by Topo IIIα was analysed by PCR amplification (Cawthon, 2002) and a smear indicative of telomeric DNA was detected in all cell lines tested (result not shown). Determination of the relative Topo IIIα amount at the telomere, as compared to other repeated sequences in the genome, was performed using a dot blot hybridization assay using telomeric and Alu repeat probes in 293T, MRC5V1, and U2OS cells (Figure 4). TRF2 was used as a positive control. The results indicated that the amount of telomeric DNA recovered by TRF2 immunoprecipitation was nearly equivalent in the telomerase-positive and ALT cell lines (Figure 4B and data not shown). In contrast, the amount of telomeric DNA recovered by Topo IIIα immunoprecipitation was higher in ALT cells than in 293T cell line, and corresponded to a specific association with telomeric sequence when compared to Alu (Figure 4A and B). The relative increase of telomeric DNA immunoprecipitation by Topo IIIα in ALT cells, as compared to 293T (defined as 1), was equal to 3.2±0.7-fold for U2OS and 5.6±2.9-fold for MRC5-V1 (Figure 4B and data not shown). These data suggest that Topo IIIα has a marked preference for telomeric DNA in ALT cells. As a hallmark of ALT cells is their extreme heterogeneity in telomere length due to homologous recombination and the presence of extra chromosomal telomeric repeats (ECTR or t-circles) (Dunham et al, 2000; Cesare and Griffith, 2004; Londono-Vallejo et al, 2004; Wang et al, 2004), we cannot exclude the possibility that the increased ratio of Topo IIIα at telomeric DNA relative to Alu DNA in ALT cells was due to binding to ECTR rather than to telomeres. Figure 4.Topo IIIα interacts with telomeric sequences in vivo as shown by ChIP. Three different cell lines (telomerase-positive 293T and MRC5-V1 and U2OS ALT cell lines) were evaluated after ChIP with TRF2 or Topo IIIα antibodies. IgG antibodies were used as negative controls. Total input fractions (2.5 and 1%) and antibody-recovered fractions (10% of input) were subjected to Southern blot analysis using telomeric or ALU repeat-specific probes. (A) Representative ChIP experiment. (B) Quantification of radioactivity by Imagequant™ software of the experiment presented in (A). The percentage of precipitated DNA was calculated as a ratio of input (telomeric or Alu) signals and plotted. Download figure Download PowerPoint siRNA directed against Topo IIIα inhibits ALT cell growth To examine the importance of Topo IIIα for ALT cell growth, Topo IIIα gene expression was downregulated by RNA interference using specific siRNA oligonucleotide duplexes. Three siRNAs, Si-1, Si-2, and Si-3 Topo IIIα, were designed to target the Topo IIIα cDNA sequence (NM004618) and evaluated for effects on Topo IIIα expression levels in the MRC5-V1 cell line 72 h after siRNA transfection. Immunoblots indicated that the Topo IIIα protein level decreased when cells were treated with Si-1 and Si-2 (60–70%) as compared to the control siRNA (Figure 5A). No significant decrease was observed when cells were treated with Si-3. An evaluation of the time course of the effect of Si-1 in MRC5-V1 cells indicated a marked decrease in Topo IIIα levels at 72–96 h after transfection (76–83%) (Figure 5B). Figure 5.Effect of Topo IIIα depletion by RNA interference on telomerase-positive and ALT cells growth. (A) Representative example of effect of three different siRNAs targeting Topo IIIα (Si-1, Si-2, and Si-3; see sequences in Materials and methods) and a control siRNA (C) on the amount of intracellular Topo IIIα protein at 72 h in the MRC5-V1 (ALT) cell line. The loading control was β-actin. The per cent decrease of Topo IIIα compared to treatment with the control is indicated at the bottom. (B) Data from a representative experiment in MRC5-V1 (ALT) cell line with Si-1. Negative control siRNA at 72 h (c72) and the amount of Topo IIIα before treatment (t0) are shown as controls. The per cent decrease of Topo IIIα relative to the control is indicated at the bottom. (C) Effect of Topo IIIα depletion (Si-1, 100 nM) on the growth of telomerase-positive (EcR293 and HT1080) and ALT (MRC5-V1, U2OS, and WI38-VA13) cell lines for up to 96 h after transfection. The results are expressed relative to control siRNA-treated cells, defined as 100%, and correspond to the mean value and standard deviation of three independent experiments. As a control for Topo IIIα depletion, a western blot experiment performed in parallel is presented at the bottom of each growth curve. Protein extracts from Si-1-treated cells at 72 h (Si-1/72 h) or from control siRNA-treated cells (C) were blotted using antibodies against Topo IIIα and β-actin. Download figure Download PowerPoint We next determined the importance of Topo IIIα for the growth of various ALT (MRC5-V1, U2OS, and WI38-VA13) and telomerase-positive (EcR293 and HT1080) cell lines 24–96 h after Si-1 transfection (Figure 5C). In parallel, the efficiency of the Topo IIIα depletion was evaluated by immunoblotting using the anti-Topo IIIα antibody 72 h after transfection (Figure 5C). For the five cell lines tested, the decrease in Topo IIIα protein level ranged from 60 to 80%. Treatment of the three ALT cell lines with Si-1 Topo IIIα induced a strong reduction in cell growth, as compared to control siRNA-treated cells. Growth inhibition began at 48 h after Si-1 transfection. Cell growth inhibition induced by Si-1 treatment was also associated with a reduction in cell viability determined by Trypan blue exclusion, which decreased to 32, 22, and 50% of controls for MRC5-V1, U2OS, and WI-38-VA13 cells, respectively, but without significant induction of apoptosis, measured by PARP1 and caspase-3 cleavage (Supplementary Figure S6). In contrast, depletion of Topo IIIα by RNA interference using Si-1 in telomerase-positive cell lines (EcR293 and HT1080) did not alter the proliferation of these cells compared to cells treated with control siRNA (Figure 5C). This difference between telomerase-positive and ALT cells was also observed when HT1080 and U2OS cells were treated with Si-2 (Supplementary Figure S5A). These results showed that Topo IIIα is essential for the proliferation of ALT cells but not for telomerase-positive cells over the time course of the assay. Topo IIIα depletion induces anaphase bridge formation, G-overhang signal decrease, and TRF2 depletion in ALT cells Telomere binding proteins such as TRF2 and its partners such as TIN2, RAP1, and Apollo have a pivotal role in the maintenance of the integrity of telomeres (de Lange, 2005; Lenain et al, 2006; van Overbeek and de Lange, 2006). As our results indicated that Topo IIIα forms a complex with TRF2 in ALT cells, we examined the importance of Topo IIIα for telomere integrity by analysing the formation of anaphase bridges, usually associated with a telomere dysfunction (Fig
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