Alternative Lengthening of Telomeres: Recurrent Cytogenetic Aberrations and Chromosome Stability under Extreme Telomere Dysfunction1,2
Despoina Sakellariou, Maria Chiourea, Christina Raftopoulou and Sarantis Gagos Laboratory of Genetics and Gene Therapy, Center of Basic Research II, Biomedical Research Foundation of the Academy of Athens, Athens, Greece
Abstract Human tumors using the alternative lengthening of telomeres (ALT) exert high rates of telomere dysfunction. Numerical chromosomal aberrations are very frequent, and structural rearrangements are widely scattered among the genome. This challenging context allows the study of telomere dysfunction–driven chromosomal instability in neoplasia (CIN) in a massive scale. We used molecular cytogenetics to achieve detailed karyotyping in 10 human ALT neoplastic cell lines. We identified 518 clonal recombinant chromosomes affected by 649 structural rearrangements. While all human chromosomes were involved in random or clonal, terminal, or pericentromeric rearrangements and were capable to undergo telomere healing at broken ends, a differential recombinatorial propensity of specific genomic regions was noted. We show that ALT cells undergo epigenetic modifications rendering polycentric chromosomes functionally monocentric, and because of increased terminal recombinogenicity, they generate clonal recombinant chromosomes with interstitial telomeric repeats. Losses of chromosomes 13, X, and 22, gains of 2, 3, 5, and 20, and translocation/deletion events involving several common chromosomal fragile sites (CFSs) were recurrent. Long-term reconstitution of telomerase activity in ALT cells reduced significantly the rates of random ongoing telomeric and pericentromeric CIN. However, the contribution of CFS in overall CIN remained unaffected, suggesting that in ALT cells whole-genome replication stress is not suppressed by telomerase activation. Our results provide novel insights into ALT-driven CIN, unveiling in parallel specific genomic sites that may harbor genes critical for ALT cancerous cell growth. Neoplasia (2013) 15, 1301–1313
Introduction Mitotic chromosome integrity in humans relies on efficient DNA damage responses (DDR), unfailing cell cycle checkpoints, as well as functional telomeres and centromeres [1–4]. Centrosomes, kinetochores, chromatid cohesion, and nuclear and microtubule architecture also play important roles in preserving faithful mitotic chromosome segregation [5,6]. Chromosomal instability in neoplasia (CIN) is an extremely aggravated form of ongoing mitotic infidelity that is observed in most cancer cell populations . Randomly dispersed CIN generates clonal tumorigenic chromosome aberrations, contributes dramatically to intratumor genomic heterogeneity, and is mainly responsible for cancer genome evolution that shapes the multistep process of neoplasia [3,7]. Even more, CIN is related to advanced, incurable malignancy and is thought to complicate all current and future oncotherapeutic strategies . Understanding the patterns and driving mechanisms of
replication stress, and telomere deprotection [9,10]. Replication stress due to chemical agents, activated oncogenes, or genetic interventions has been shown to cause random illegitimate recombinogenicity of cancer chromosomes that occurs frequently at common chromosomal fragile sites (CFSs) and can create novel clonal rearrangements [9–12]. CFSs are AT-rich chromosomal regions that preferentially form cytologically visible gaps or breaks on metaphase chromosomes under replication stress . The DNA polymerase inhibitor aphidicolin introduces replication stress and induces 77 of 88 known human CFSs . Fragile sites are conserved among mammals and are also found in lower eukaryotes including yeast and flies . CFSs are hotspots for gene amplification and viral integration, and they have been also implicated in sister chromatid exchanges and in the generation of constitutional or acquired deletions and translocations . Telomeres protect the ends of eukaryotic chromosomes . In most human somatic tissues, these specialized nucleoprotein complexes are challenged after each round of DNA replication. From yeast to humans, replicative loss of telomeric DNA is replenished by the action of the RNP enzyme telomerase or by the telomerase-independent alternative lengthening of telomeres (ALT) . Most normal human tissues do not possess a constitutive means to fully maintain their telomeres; thus, actively dividing cells demonstrate progressive telomeric loss and deprotection . Critical impairment of telomere protection activates DDR, and the cell cycle becomes arrested . In normal cells, senescence and apoptosis are biologic barriers that prevent neoplastic transformation . To overcome these barriers, human malignancies sustain continuous cellular growth by activating telomerase [14,17] or by using the alternative pathway of telomere lengthening (ALT) . The ALT pathway for telomere elongation was originally described in yeast and in mammalian immortalized and cancer cells lacking telomerase [15,18]. Although relatively rare in human neoplasia, the ALT pathway has been frequently observed in various types of aggressive human tumors such as osteosarcomas, undifferentiated pleomorphic sarcomas, leiomyosarcomas, astrocytic tumors (grades 2 and 3), and pancreatic neuroendocrine tumors . In addition, the engagement of the ALT pathway may confer acquired resistance to cancer therapy in telomerase-positive cancer cells treated with telomerase inhibitors and has been considered a major burden for current and future telomerebased antitumor therapeutics . Although not well understood, the mechanisms of ALT are thought to engage non-homologous end joining (NHEJ) to seed “neo-telomeres” at broken chromosome ends . However, the extent of this process still remains unknown . ALT also implies the assembly and activation of the complex homologous recombination-mediated DNA replication machinery that elongates telomeres in the absence of telomerase . Cells using the ALT pathway display extensive telomeric length heterogeneity, ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs), extrachromosomal telomeric C-circles, massive telomere dysfunction, large-scale epigenetic modifications at chromosome termini, as well as high rates of structural and numerical chromosome instability . Telomere dysfunction–driven genome damage is operated by frequent chromosomal break-fusion-bridge (B/F/B) cycles [24,25]. The B/F/B cycles can generate various types of oncogenic structural chromosome rearrangements and may lead to extensive loss of genomic material through anaphase lagging of whole chromosomes or chromosomal segments [3,26]. In highly proliferating ALT cells, a continuous process of frequent B/F/B cycles generates very complex karyotypes that up to now have not been analyzed in detail . Numerical chromosomal
Neoplasia Vol. 15, No. 11, 2013 aberrations are very frequent, whereas structural rearrangements affect almost every single chromosome . This extremely challenging context provides excellent grounds for comparative analysis of clonal and random chromosome anomalies and allows the study of processes related to recombinant chromosome functionality in a large scale. In several histopathologic types of malignancies, specific chromosomal aberrations have been associated with biologic mechanisms, along with the diagnosis or prognosis of the disease . Little is known about recurrent chromosome rearrangements that might characterize the ALT pathway and may reflect known or unravel unknown genes or biologic pathways operating in this type of malignancy [27,29]. Furthermore, our knowledge on the distribution of CIN between different chromosomes of the human genome and the processes related to clonal perpetuation of recombinant chromosomes in neoplasia still remains poor. We combined multicolor fluorescence in situ hybridization (M-FISH), inverted 4,6-diamidino-2-phenylindole (DAPI) banding, telomere- or centromere-specific FISH, strand-specific chromatid orientation FISH (CO-FISH), and immunocytochemistry to achieve detailed molecular karyotyping in 10 human ALT cell lines. In a cohort of 649 unique clonal structural aberrations, we present the frequencies and chromosomal locations of different types of structural rearrangements and describe cytogenetic markers of ALT continuous growth. Our data demonstrate that ALT-rearranged chromosomes are products of frequent ongoing telomeric, pericentromeric, and CFS recombinogenicity, and their clonality is preserved through extensive telomere healing and by widespread centromere epigenetic modifications that allow polycentric chromosomes to become functionally monocentric and perpetuate in culture. However, beyond extreme complexity compelled by frequent B/F/B cycles and despite the highly increased rates of pericentromeric and CFS instability, human ALT-neoplastic karyotypes revealed recurrent numerical and structural chromosome anomalies that unravel biologic pathways related to cancerous cellular growth. Materials and Methods
Cell Lines and Culture Conditions The osteosarcoma cell line Saos-2 was obtained from the American Type Culture Collection (Wesel, Germany), and the osteosarcoma cell line U2-OS was a gift from E. Gonos (National Hellenic Research Foundation, Athens, Greece). The SV-40 large T-antigen transformed ALT cell lines GM-847, VA-13, and IMR-90 were donated by A. Londoño-Vallejo (Institute Curie, Paris, France) . In addition, we used a VA-13 derivative cell line that stably expresses human telomerase RNA component (hTERC) and human telomerase reverse transcriptase (hTERT) and has reconstituted telomerase activity (VA-13TA) as described by Ford et al. . The VA-13+hTERC+hTERT (VA13TA) was a gift from J. W. Shay. The ALT liposarcoma Lisa-2 and Ls-2 and the SV-40–transformed human ovarian surface epithelium cell lines HIO107 and HIO118 were kindly provided by D. Broccoli (Fox Chase Cancer Center, Philadelphia, PA) . The T-24 bladder cancer cells were provided by T. Vlahou (Biomedical Research Foundation of the Academy of Athens, Athens, Greece). HeLa and breast cancer MCF-7 cells were gifts from I. Irminger-Finger (Geneva Medical School, Geneva, Switzerland). The colon cancer SW-480 cell line was obtained from the American Type Culture Collection. The osteosarcoma cell line KH-OS was provided by E. Gonos (Greek National Institute for Research, Athens, Greece). The HT-29 colon cancer and NCI-H-460 lung cancer cell lines, as well as the glioblastoma cell line SF-268, were provided by C. Dimas (Biomedical Research Foundation of the
Neoplasia Vol. 15, No. 11, 2013 Academy of Athens). All cell cultures were grown at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) supplemented with 10% FBS (Gibco), 25 units/ml penicillin (Sigma, St Louis, MO), and 25 pg/ml streptomycin (Invitrogen, Grand Island, NY).
Construction of Representative Karyotypes Logarithmically growing cell cultures were exposed to colcemid (0.1μg/ml) (Gibco) for 1 to 3 hours, at 37°C in 5% CO2. Cells were harvested by trypsinization (Gibco), suspended in medium, and spun down (10 minutes at 1000 rpm). Supernatant was removed completely, and 5 ml of 0.075 M KCl (Sigma) at room temperature was added drop by drop. The cells were incubated for 20 minutes at room temperature, and then 1 ml of fixative [3× methanol (Applichem GmbH, Darmstadt, Germany)/1× CH3COOH (Merck, Darmstadt, Germany)] was added. Cells were spun down (10 minutes at 1000 rpm), supernatant was removed, fixative was added, and the cells were recentrifuged for 10 minutes at 1000 rpm. Finally, cells were dropped onto wet microscope slides and left to air-dry. For karyotypic analysis, we combined inverted DAPI staining, G-banding, and molecular karyotyping by M-FISH (MetaSystems GmbH, Altlussheim, Germany). G-banding was performed after treatment with 0.25% trypsin (Gibco) and Giemsa (Carl Roth GmbH, Karlsruhe, Germany) staining. M-FISH was performed according to the manufacturer’s protocols (MetaSystems). For inverted DAPI banding, slides were counterstained and mounted with 0.1μg/ml DAPI in VECTASHIELD antifade medium (Vector Laboratories, Burlingame, CA). Cytogenetic analyses were performed using a ×63 magnification lens on a fluorescent Axio-Imager Z1, Zeiss microscope, equipped with a MetaSystems charge-coupled device camera and the MetaSystems Ikaros or Isis software. As representatives, we considered the karyotypes of the major clone per cell harvest (at least 25 metaphases stained with M-FISH and inverted DAPI banding from one harvest were completely analyzed). Because of extreme complexity of chromosomal rearrangements, karyotypes were written in the extended form according to International System for Human Cytogenetic Nomenclature (ISCN) 2009 [33,34].
Fluorescence In Situ Hybridization For FISH, we used centromere-specific satellite DNA probes targeting either all human centromeres or specific for chromosomes 1 [yellow, red + green signals; green, fluorescein isothiocyanate (FITC); red, rhodamine], 9 (green, fluorescein), and 18 (green, FITC or purple/ blue, spectrum aqua). Probes were purchased from Vysis (Abbott Park, IL) and Cytocell (Cambridge, United Kingdom). In brief, our protocol was based on pepsin (Invitrogen) pretreatment, formamide (Applichem) or NaOH (Sigma) target denaturation, overnight hybridization, and high stringency post-hybridization washes. Telomeric peptide nucleic acid (PNA) FISH was performed using a Cy3-(CCCTAA)3 PNA Probe according to the manufacturer’s instructions (Dako Cytomation, Glostrup, Denmark). Briefly slides were incubated at 3.7% formaldehyde (Carlo Erba Reagenti SpA, Milano, Italy), washed with 1× TBS (Dako Cytomation), immersed in pretreatment solution (Dako Cytomation), and dehydrated with cold ethanol series (VWR, Radnor, PA). Probe and target DNA were denatured at 80°C for 5 minutes, and then slides were incubated for 1 hour at room temperature in the dark. For M-FISH, we used the 24XCyte Kit from MetaSystems GmbH. Staining was performed according to the manufacturer’s instructions. All FISH preparations were mounted and counterstained with VECTASHIELD antifade medium containing 0.1μg/ml DAPI
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(Vector Laboratories). Digital images were captured and enhanced in a MetaSystems workstation as described above.
Immunocytochemistry Metaphase preparations were obtained through a modification of the standard cytogenetic harvest technique that excluded acetic acid fixation. Freshly fed and subconfluent cell cultures were incubated with colcemid (0.01-0.04 mg/ml) for 0.5 to 16 hours at 37°C. Cells were dislodged with trypsin-EDTA solution and centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. Hypotonic solution (0.075 M KCl, prewarmed to 37°C) was added to the cell pellet, gently mixed by pipetting, and incubated for 20 to 30 minutes at 37°C. The cells were washed with methanol. Preparations were cytospined for 10 minutes at 2000 rpm, and slides were left to air-dry and immediately placed to 4°C. Fluorescence immunocytochemistry was performed after phosphate-buffered saline (PBS) washes, permeabilization with Triton X100 (Applichem), and normal serum blocking. The CENP-A (Stressgen Biotechnologies Corporation San Diego, CA) and CENP-C (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were applied at dilutions of 1:50 to 1:500 for 2 hours at room temperature. For antibody detection, we used a suitable Alexa Fluor–conjugated secondary antibody (1:500-1:1000 in PBS; Molecular Probes, Grand Island, NY). Slides were incubated for 1 hour and washed with PBS, and then FISH was applied as described above. For antibody detection, we used Alexa Fluor 568 (Molecular Probes)– and IgG-FITC (Santa Cruz Biotechnology)–conjugated antibodies, respectively (1:500 in 1% BSA).
Chromatid Orientation FISH Strand-specific telomeric CO-FISH was performed according to Bailey et al. , with minor modifications. In brief, subconfluent cell monolayers were cultured for 24 hours into medium containing 3 × 10−3 mg/ml 5′-bromo-2’-deoxyuridine (Sigma). Colcemid (0.1μg/ml; Gibco) was added for an hour before cell harvest. Metaphase spreads were prepared by conventional cytogenetic methods. Chromosome preparations were treated with 0.5 mg/ml RNase-A (Roche, Athens, Greece) for 10 minutes at 37°C, stained with Hoechst 33258 (0.5μg/ ml; Sigma), incubated in 2× SSC (Invitrogen) for 15 minutes at room temperature, and exposed to 365-nm UV light (Stratalinker 1800 UV irradiator) for 30 minutes. The 5′-bromo-2’-deoxyuridine–substituted DNA was digested with Exonuclease III (Promega, Madison, WI) in a buffer supplied by the manufacturer (5 mM DTT, 5 mM MgCl2, and 50 mM Tris-HCl, pH 8.0) for 10 minutes at room temperature. Digested strand denaturation was performed at 70°C, with 70% formamide (Applichem) in 2× SSC for 1 minute. The slides were then dehydrated through a cold ethanol series (70%, 85%, and 100%) and airdried. PNA-FISH was performed as above.
Statistical Analysis One-way analysis of variance (ANOVA) or paired t tests were performed using the MINITAB software. Results
ALT Karyotypes Derive from Polyploidization Followed by Frequent Chromosome Gains and Losses The representative chromosome counts in metaphase spreads of the 10 cell lines of our panel varied from hypo-triploidy to near-heptaploidy (according to ISCN 2009) . High chromosome numbers, and frequent karyotypic presence of two or multiple copies of specific clonal
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recombinant chromosomes, indicated that one, two, or more rounds of genome reduplication have taken place and were selected to prevail in the cell lines of this panel (Figures W1 and W2 and Supplementary Data Set 1). In fact, all 10 karyotypes exhibited deviant modal chromosome numbers from those expected after complete dosage duplication or multiplication of the whole normal human genome content. Consistent with the hypothesis of Duesberg et al. [36,37], this discrepancy can be explained by the increased susceptibility of polyploid cancer cells to undergo chromosome losses. However, in the highly aberrant ALT background, clonal chromosomal gains into the representative ploidy index were equally frequent to clonal losses (79 vs 83, respectively). In accordance to our recent report , all 10 cell lines of our panel exerted a continuous propensity to generate subclones with reduplicated or multiplied genomic material compared to their major clones. Typically, the majority of observed polyploid metaphases were derivatives of whole-genome duplication of a preexisting subclone that had the size of the representative chromosome number (Figure W3). Hyperpolyploid cells (i.e., mitotic nuclei that had undergone three or more rounds of genome reduplication) were present but rare (<1%). These results indicate that the ALT karyotypes examined in this study are clonal evolution products of an ongoing pressure for polyploidization that is accompanied by equally frequent whole chromosome gains and losses.
Large Deletions, Terminal Structural Rearrangements, and Whole-Genome Pericentromeric Recombinogenicity Compared to telomerase-positive examples [39–42], the incidence of clonal structural chromosome anomalies in the karyotypes of the
Neoplasia Vol. 15, No. 11, 2013 ALT cell lines of this panel was elevated by 3.7-fold (Figure 1A). Although monoclonal in origin, all ALT cell lines displayed variable percentages of co-existing cytogenetically distinct subpopulations, characterized by unique clonal rearrangements (Figure W3). According to ISCN 2009 , clonality of structural chromosome aberrations in a cancer cell population is defined by the presence of an identical marker chromosome, in at least two co-dividing cells. As representative, we considered a particular karyotype of a series of at least 25 fully analyzed mitotic cells from the same harvest, containing most, if not all, of the identical chromosome markers shared by the majority of the cells. In the representative karyotypes of the 9 unique ALT cell lines of our panel, consisting of a total of 867 clonal autonomous chromosomes, 518 were identified as structurally aberrant (59.75%). Several rearranged chromosomes were found in multiple copies or exerted more than one structural anomaly (Figures W1–W3 and Supplementary Data Set 1). In a total of 649 identified unique clonal structural aberrations, deletions of large genomic segments mostly spanning up to chromosomal termini were the most frequent (41.4%), indicating a robust pressure toward ALT chromosome trimming. More than half of total deletions (65.7%) encompassed pericentromeric breakpoints. Translocations between chromosomal segments composed the 30.1% of total aberrations. They were mostly unbalanced and often complex (involving more than two chromosomes). Consistent to our previous work , a high proportion of the translocation breakpoints was pericentromeric (52.8%). Clonal pseudo-dicentric or pseudo-polycentric chromosomes were frequent (13.3%). Inverted duplications were encountered in 4.8% of the total clonal rearrangements, whereas
Figure 1. Clonal structural chromosome rearrangements in human neoplastic cell lines: (A) Significant differences in the frequencies of clonal structural aberrations in the karyotypes of eight ALT and eight telomerase-positive human cancer or transformed continuous cell lines based on the results presented in this study and in [39–42] (ANOVA). (B) Frequencies of different types of aberrations in 649 unique clonal chromosome rearrangements, identified by M-FISH and inverted DAPI banding in nine human ALT cell lines.
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Figure 2. Terminal fusions, pericentromeric rearrangements, and telomere healing: Virtually all chromosome arms of the human karyotype are involved in ALT clonal end-to-end fusions, and all centromeres are capable of taking part in clonal translocations and of undergoing telomere capture (p-arms of acrocentric chromosomes were not examined). (A) Chromosome 9 and the small chromosomes (18-22) seemed more prone to take part in terminal or pericentric rearrangements (P < .01 by ANOVA). (B) A karyogram of the ALT U2-OS cell line, stained by inverted DAPI banding and a telomere-specific PNA FISH probe, labeled with Cy3 (red), indicates chromosomal sites of telomere healing (arrows). (C) Telomere healing in the ALT pathway occurs in a massive scale because 45.1% of the recombinant chromosome arms maintain clonality through capture of telomeric repeats. (D) Sites of frequent telomere capture.
isochromosome formations, inversions, or duplications were more rarely detected (0.15%-1%; Figures 1B, W1, and W2). Hence, the increased rates of telomere dysfunction operating in the ALT pathway lead predominantly to large-scale deletions and unbalanced translocations that frequently occur at the pericentromeric regions.
Distribution of Telomere Dysfunction–Driven Clonal Rearrangements along Chromosomal Termini and Telomere Healing of Recombinant Chromosomes From 1036 chromosome arms involved in ALT clonal structural aberrations, 235 (22.7%) took part in terminal fusions. This percentage is raised up to 39%, if we take into account the incidence of clonal pseudo-polycentric chromosomes and inverted terminal duplications. Interestingly, with the exception of chromosome 9, which showed remarkably increased rates of telomeric recombinogenicity in both arms, smaller chromosomes (13-22) showed a significantly higher propensity to undergo clonal terminal p- or q-fusions (P = .031 by one-way ANOVA; Figure 2A). In a highly proliferating cell population, clonal maintenance of structurally rearranged chromosomes requires the presence and functionality of the two main chromosomal organelles: the centromere and the telomere [3,43]. ALT recombinant chromosomes may obtain telomeres by using a preexisting telomere of another chromosome (canonical capping) or by introducing a tract of telomeric repeats through homologous recombination or NHEJ at a “blunt” chromosome end to heal it (telomere capture or healing; Figure 2B) [21,44]. From 1036 arms of the rearranged chromosomes described in this study, 54.9% were found canonically capped. A substantial proportion (31.7%) of the recombinant arms that were terminally protected by telomere healing acquired telomeres at pericentromeric regions (Figure 2C ). Expanding our earlier
observations , every human ALT centromere involved in pericentromeric whole-arm deletions was found capable to capture telomeres and to become stabilized as a neo-acrocentric chromosome. Centromeres of chromosomes 18 to 20 were proved highly recombinogenic in both telomeric and centromeric regions (Figure 2A). From 219 noncentromeric breakpoints healed by telomere capture, 89 (40.6%) coincided with common CFSs, whereas 21.9% were found at genomic regions bearing conserved interstitial telomeric repeats (ITRs; Figure 2D) . These results indicate that in the ALT context, every chromosome of the human karyotype can be involved in telomeric or pericentromeric recombinogenicity and can acquire telomeric repeats at broken ends. However, specific chromosomes display elevated propensity for telomeric or centromeric instability. In addition, in a significant proportion of structural rearrangements, clonality is entailed through the acquisition of “neo-telomeres” near CFSs or conserved ITRs.
Clonal Pseudo-Polycentric Chromosomes and Recombinant Chromosomes with Large ITRs Compared to telomerase-positive examples, the ALT cell lines of this panel demonstrated significantly higher frequencies of pseudo-dicentric or pseudo-polycentric recombinant chromosomes as well as rearranged chromosomes with cytologically visible blocks of telomeric repeats at the recombination breakpoints (Figure 3, A and C). In humans, clonality of polycentric recombinant chromosomes implies activity of one centromere and inactivation of all other extra centromeres . A proportion of the rearranged chromosomes of our cell line panel was interpreted as pseudo-dicentric by M-FISH and inverted DAPI banding (Figures 3A, W1, and W2 and Supplementary Data Set 1). We further investigated these peculiar entities of ALT genomic instability
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by centromere- and telomere-specific FISH and immunocytochemistry using antibodies specific for CENP-A and CENP-C proteins. Fluorescent microscopy revealed that the excessive centromeres of clonal ALT dicentric or polycentric chromosomes are indeed inactivated  (Figure 3B). In addition, telomere-specific FISH showed that several ALT clonal aberrant chromosomes deriving from end-to-end fusions maintained cytologically visible blocks of telomeric repeats at recombination sites (Figure 3D). Examination of the orientation of recombinant interstitial telomeric sequences, by strand-specific CO-FISH, indicated that they represented NHEJ processes between telomeres at an antiparallel orientation or between telomeres and non-telomeric genomic sites. The above results demonstrate that in the ALT pathway the inert tendency for aberrant large-scale epigenetic modifications is not confined only at the telomeres [23,48] but may affect several centromeric regions, facilitating inactivation of centromeres in excess, in polycentric chromosomes produced by B/F/B cycles. In addition, increased terminal recombinogenicity generates recombinant chromosomes with large ITRs.
Recurrent Chromosomal Rearrangements in the ALT Pathway We then attempted to identify recurrent chromosome aberrations that might be related to the genetic pathways driving ALT continuous
Neoplasia Vol. 15, No. 11, 2013 growth or may reflect specific patterns of ALT chromosome evolution under increased telomeric instability. As recurrent, we considered all unique chromosome anomalies, affecting the same chromosome, in at least three of nine ALT cell lines. Losses of genomic material from whole chromosome 13 were recurrently observed in seven of nine karyotypes (77.7%). Other chromosomes found to be frequently lost were 22 (77.7%), X (55.5%), 8, 14, and 18 (44.4%), as well as 1, 7, 9, 10, 11, 15, 16, and 21 (33.3%; Table W1). The same chromosomes suffered also from large terminal or whole-arm deletions. Recurrent chromosome gains beyond ploidy index involving chromosomes 2, 3, 5, and 20 were observed in 50% of the cases (Table W1). Recurrent pericentromeric rearrangements are described in Table W2. Deletions or unbalanced translocations of both arms of chromosome X were observed in all the cell lines of this panel. The breakpoint that was more frequently affected by terminal deletions was between Xq21.1 and Xq22.3 (in six of nine lines). A recombination/deletion hotspot was localized at the short arm of chromosome 1 (between 1p32 and 1p36; observed in six of nine cell lines); in addition, chromosome 1 was also found to be affected by terminal deletions originating at 1q21 (four cases) with the other cases displaying either larger 1q deletions or losses of the whole chromosome 1. Chromosome 2 exerted a recombination hotspot at 2q31-2q36 (five of nine lines), whereas seven of
Figure 3. Distinctive cytogenetic findings of the ALT pathway: (A) Incidence of clonal pseudo-polycentric chromosomes in eight telomerase-positive and eight ALT cell lines (P = .019 by ANOVA). Examples of two “cryptic,” clonal pseudo-dicentric recombinant chromosomes of the ALT U2-OS karyotype: At the upper row, a neo-acrocentric derivative of chromosome 1, lacking the whole 1p arm, displays alphoid centromeric repeats specific for human centromeres 1 and 18, located in close proximity to the telomere (M-FISH: yellow, specific for chromosome 1; purple, specific for chromosome 18; inverted DAPI: gray/black, ×630). At the lower row, a complex rearrangement between chromosomes 1 and 9 maintains alphoid repeats from both centromeres 1 and 9. (B) Lack of chromatid cohesion and negative staining for antibodies specific for CENP-A or CENP-C indicate that the terminally positioned centromere 1 is inactivated, whereas centromere 9 remains active. (C) Frequencies of clonal rearranged chromosomes bearing cytologically visible ITRs in a panel of eight telomerase-positive and eight ALT cell lines (P = .014 by ANOVA). (D) Strand-specific CO-FISH reveals two types of clonal ITR: those representing NHEJ-mediated telomeric fusions between telomeres at antiparallel orientation (yellow arrows) and fusions of telomeric repeats with non-telomeric genomic regions (white arrows; ×630).
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Figure 4. Structural chromosome instability before and after long-term reconstitution of telomerase activity in ALT cells: Reconstitution of telomerase activity in ALT VA-13 cells significantly reduced overall genomic structural CIN in two independent harvests (a and b) of the double-transfected isogenic VA-13TA cell line, grown in the presence of telomerase activity for more than 250 PDs. We analyzed 1095 VA-13 and 3416 VA-13TA chromosomes (1657 from harvest a and 1759 from b) using M-FISH/inverted DAPI. (A and B) Distribution of random (non-clonal) structural rearrangements per chromosome, in the VA-13 cells, reveals that, in the ALT pathway, any chromosome can be affected by increased recombinogenicity that predominantly affects terminal and pericentromeric regions and can be substantially suppressed on long-term activation of telomerase. Random chromosome rearrangements were classified as telomeric, pericentromeric, genomic (non-telomeric or centromeric), and unidentified. (C) Long-term expression of telomerase activity led to significant suppression of telomeric and centromeric vulnerability (P < .0001 for VA-13TAa and for b, by paired t test). (D) The contribution of CFS in the proportion of identified random chromosome breakpoints was not decreased, indicating that telomerase activation cannot repress ALT whole-genome replication stress.
nine ALT lines exerted increased rates of recombinogenicity for the regions between 3q21 and 3q26. Chromosome 4 was found highly recombinogenic at bands 4q21 to 4q23 (six of nine lines). Deletions of the short arm of chromosome 5 were observed in six lines (66.6%). A recombination hot region on this chromosome was identified between 5q31 and 5q35 (in six lines). Six of nine lines displayed rearrangements into the regions 6q22.1 to 6q24.3, whereas four of them and two other cell lines displayed a hot region between 6p21.3 and 6p23. Deletions of the short arm of chromosome 8 were present in six of nine lines, whereas five lines showed breakage at 8q21-8q24. The terminal regions of chromosomes 7, 9, 11, and 18 to 21 showed a highly increased tendency to undergo end-to-end fusions or terminal translocations (88.8%). Pericentromeric rearrangements of chromosome 10 were
found in 88.8% of the ALT lines. A clustering of breakpoints was also noted between bands 10q21.2 and 10q22.1. Six cell lines displayed terminal deletions of 14q21-22. The pericentromeric region of chromosome 16 exerted high rates of breakage and recombination, mainly affecting p11.2 (in seven cell lines). Chromosome 18 showed also remarkable pericentromeric recombinogenicity. Seven of nine cell lines displayed large deletions of the short arm of chromosome 18, whereas the other two lines showed full losses of the same chromosome. Similar was the case with 18q. In addition to increased fragility that gave rise to terminal deletions of either the short or the long arms, pericentromeric regions of chromosome 18 were often taking part in translocations with pericentric sites of other chromosomes such as 1, 7, 9, 10, 15, 16, 17, and 20. Losses of 19p were observed in six lines of our panel.
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Chromosome 20 showed increased propensity for pericentromeric rearrangements and terminal fusions (88.8%). The long arm of chromosome 21 showed recurrent propensity to undergo deletions or translocations. A recombinatorial hotspot was found at 21q22.1. A significant proportion (37.3%) of the above recurrent rearrangements was localizing at common CFS (Tables W2 and W3), suggesting that virtually all ALT chromosomes suffer from generalized replication stress. Therefore, beyond extreme complexity, ALT karyotypes are characterized by numerous recurrent chromosomal imbalances that may uncover genes and biologic pathways related to ALT.
Reconstitution of Telomerase Activity in ALT Cells Suppresses Terminal and Pericentromeric CIN Reconstitution of telomerase activity in human ALT cells has been related to decreased frequencies of telomere dysfunction and reduced CIN . The VA-13+hTERC+hTERT (VA-13TA) cells are stably transfected to express telomerase activity for more than 250 population doublings (PDs) and display high prevalence of near-pentaploidy [31,38]. To address, in a cell-by-cell basis and in a chromosome bandby-band basis, the extent of suppression of ALT-driven CIN between co-dividing cells, we examined, before and after long-term restoration of telomerase activity, the frequencies of non-clonal (unsystematic) structural rearrangements in 15 randomly selected metaphase spreads obtained from the same harvest of the ALT VA-13 and 30 mitoses from two independent harvests of the telomerase-positive VA-13TA stained by M-FISH and inverted DAPI (Figures W3–W5). The VA-13TA karyograms maintained, in duplicate, most of signature anomalies observed in parental VA-13 cells (Figures W4 and W5). As expected by the high rates of identified ALT clonal rearrangements, 238 of 1095 VA-13 chromosomes (21.7%) demonstrated random telomeric, pericentromeric, or genomic recombinogenicity (Figure 4A). Reconstitution of telomerase activity resulted in a 2.3- to 3.6-fold decrease of this phenotype (Figure 4B). Similar to the distribution of clonal anomalies, random terminal or pericentromeric CIN affected all of the chromosomes of VA-13 and VA-13TA karyotypes (Figure 4A). Telomerase activity significantly suppressed telomere fusions (4.6- up to 7.4-fold decrease). Although frequent in both cell lines, unsystematic pericentromeric instability was also significantly repressed after restoration of telomere dysfunction by ectopic telomerase expression (1.9- to 3.6-fold; Figure 4C). However, in both VA-13 and VA-13TA cell lines, a significant proportion of identified random genomic rearrangements coincided with common CFSs (16.2% in VA-13 and 20.5% to 28.6% for VA-13TAa and b, respectively; Figure 4D). These results suggest that the breakpoint patterns of random ongoing chromosomal instability in ALT cells are analogous to those of clonal rearrangements. In addition, a proportion of the extremely high pericentromeric, but not of CFS, recombinogenicity in the ALT karyotypes may be attributed to mechanical forces generated during telomere dysfunction–driven B/F/B cycles, because they are repressed by the introduction of telomerase . Discussion Tumor genome evolution in humans is considered a rather slow process that may take several years to produce a highly malignant genome [7,8,49,50]. Cellular immortalization and continuous culture growth through the ALT pathway are accompanied by extremely high rates of CIN that generate a plethora of random and clonal structural chromosome anomalies . Complicated more by high frequencies of telomere dysfunction–driven polyploidization and extensive chromosome gains or losses [38,52], the ALT karyotype provides an ideal
Neoplasia Vol. 15, No. 11, 2013 context to study massive tumor genome alterations occurring in a “fast-forward” mode. In this first comparative detailed molecular cytogenetic analysis of human ALT cancer and immortalized cell lines, we identified a relatively large cohort of recombinant chromosomes suitable for statistical analyses concerning distribution of imbalances, breakpoints, and clonality potential of cancerous rearrangements under extreme telomere dysfunction. Telomere dysfunction has been related to polyploidization through genome endoreduplication as well as to chromosome losses through B/F/B cycles and anaphase lagging [53,54]. Our results suggest that extreme telomere deprotection might also be capable of triggering single chromosomal non-disjunctions, or ALT immortalized cells may gain chromosomes either by whole chromosome reduplication or widespread hyperpolyploidy reduction . Despite the increase of genomic content because of polyploidization, the great extent of large-scale deletions and unbalanced translocations identified in this study confirmed that the ALT genome suffers from extensive dosage depletion of large segments of genomic material, as described in a recent SNP array report from 22 human ALT cell lines (including four of the lines of our panel: U-2OS, GM847, SaOS-2, and VA13) . The rarity of the ALT pathway in neoplasia and the tightly regulated stochastic activation of ALT-like mechanisms in embryonic stem (ES) and induced pluripotent stem (iPS) cells [56,57] imply that a number of critical suppressor pathways are abrogated during massive engagement of ALT in cancer. Hence, several ALT suppressors may exist and might be recurrently depleted in the ALT cell lines of our panel. We have shown in the past that, in addition to the expected frequent telomeric vulnerability, ALT cells present also high rates of pericentromeric instability . Centromere mitotic recombination is increased in cells lacking the DNA (cytosine-5) methyltransferase 3a (DNMT3a) and DNMT3b , suggesting an altered state of ALT centromeric heterochromatin that facilitates local recombination activity. Our current data expand these previous observations, proposing that, in the ALT context, every human telomere, or centromere, can be involved in clonal rearrangements. Clustering of breakpoints at specific genomic regions might correspond to peculiarities of local chromosome architecture or represent selection-adaptation processes favoring ALT critical genome alterations. Although the presence of one or a few dysfunctional telomeres has been associated with initiation of CIN , little is known about the degree of involvement of every specific telomere of the human genome in dysfunction-driven chromosome rearrangements. Our results reveal that virtually all telomeres of the ALT human karyotype can be affected by random or clonal recombinatorial events. However, specific chromosome termini were found more fusion-prone, and some were resistant. This may suggest that specific human chromosome termini are more vulnerable to generalized telomere protection failure than others. However, selection and adaptation of marker chromosomes that confer oncogenic advantages to the ALT cells should not be disregarded. For example, our results indicate high telomeric vulnerability of 9qter, but this region contains important cancer-related genes such as Notch1, TRAF2, and COBRA1 [59–61], depletion, duplication, or alteration of which might be important for ALT. To become clonal and to contribute into critical genome imbalances that are responsible for cancerous growth, CIN-generated aberrant chromosomes with blunt chromosome ends get stabilized through extensive telomere healing [21,62]. In the absence of telomerase activ-
Neoplasia Vol. 15, No. 11, 2013 ity, clonal perpetuation of terminally deleted chromosomes implies seeding of telomeres at the terminal breakpoint. Telomere healing can be achieved through homologous recombination, NHEJ, or by replication fork stalling and template switching . We identified 219 genomic regions at which microscopically detectable terminal deletions were mitotically stabilized by telomere healing so to become clonally maintained. Virtually all pericentromeric regions were capable to acquire telomeric repeats and to ensure mitotic perpetuation of structurally altered chromosomes. CFSs and ITRs were also frequent sites of telomere healing. Hence, our results demonstrate that, in the ALT pathway, telomere healing occurs in a massive scale and may involve both NHEJ and homologous recombination (HR). Centromeric inactivation renders human meiotic or mitotic, recombinant, poly-centric chromosomes to become functionally monocentric by a largely unknown process of epigenetic modifications that ameliorate binding of centromere-specific CENP proteins and restrict chromatid cohesion that shapes primary constriction of metaphase chromosomes despite the presence of alphoid DNA repeats . The high frequencies of clonal pseudo-polycentric chromosomes in the ALT pathway are described for the first time. They reflect the well-established elevated rates of B/F/B cycles, owing to inert continuous stochastic telomere failure [22,53], but may also represent a peculiar ALT heterochromatin epigenetic status that is highly permissive for the inactivation of excessive centromeres. Loss of function of DNMT3a and DNMT3b in mice was associated with defects in telomeric and subtelomeric heterochromatin structure, altered telomere length homeostasis, and activation of ALT . Along the clonal pseudo-polycentrics, recombinant chromosomes with large ITRs, due to increased telomere recombinogenicity, may also represent typical cytogenetic markers of the ALT karyotype. Similar to variable in size, species-specific microscopically visible ITRs, observed in hamsters, pigs, and muntjacs [64–66], ALT ITRs remained relatively stable for several PDs and did not impair clonal perpetuation of ITR-carrier chromosomes. Oncogene-induced replication stress affects DNA integrity at numerous CFSs within the cancer cell genome [11,67]. The great extent of recombinogenicity at CFS, observed in the cell lines of our panel, indicates that in ALT cells extreme replication stress affects the whole genome. Another issue might be the implication of CFS genes in ALT critical genomic rearrangements. Several CFS regions harbor genes that may be involved in ALT, such as the CENPA-CAD, localized in close proximity with FRAXB at Xq22.1 . CENPA-CAD encodes a complex recruited to centromeres to facilitate incorporation of newly synthesized CENP-A that entails centromeric functionality . Deficiency of CENPA-CAD may explain the increased propensity for the formation of ALT clonal pseudo-polycentric chromosomes demonstrated in this study. Other important genes may also be affected by recurrent losses of chromosome X, whole-arm deletions, or translocations. For example, mutations in ATRX, sited at Xq21.1 (recurrently affected in our study) have been considered capable to activate ALT [29,68,70]. Interestingly, ATRX deficiency also disturbs sister chromatid cohesion and causes mitotic aberrations . Partial or full monosomies of the short arm of chromosome 1 have been frequently reported in human malignant tumors that, in great majority, express telomerase activity [72,73]. The ALT pathway seems to take advantage of negatively unbalanced genome material from both arms of this chromosome that is also proved highly recombinogenic [ and this study]. However, chromosome 1qter might harbor important genes for ALT, because its tip showed a remarkable re-
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sistance to clonal telomere dysfunction. Indeed, EXO1 is located at 1q42-q43 . EXO1 is important for generating both types of telomerase knockdown survivors (I and II) in budding yeast . Terminal deletions, affecting a large portion of 3q material including the hTERC gene at 3q26.2, were identified in 55.5% of our ALT cell lines including VA-13 and U2-OS. In study of Christodoulidou et al., we verified the lack of hTERC expression in both VA-13 and U2-OS . Four of nine ALT cell lines showed large terminal deletions involving 4q31. At least three genes that might associate with ALT continuous growth are located here: the transcriptional modulator of cell growth and apoptosis SMAD1 [68,75]; SMARCA5, an SWItch/ sucrose non fermentable (SWI/SNF)-related matrix-associated actindependent regulator of chromatin [68,76]; and the anaphase promoting complex subunit 10 . The TERT gene is located at the tip of the short arm of chromosome 5 (p15.33) [68,77]. Interestingly, six of nine cell lines showed variable size terminal deletions of 5p. The TERT gene is silenced in all ALT cell lines of our panel [3,38,78–80]. The gene for the CENP-C binding protein, DAXX, is located at 6p21.3 . DAXX has been shown to be a negative regulator of ALT [29,81]. Consistent with Lovejoy et al., deletions or unbalanced translocations of 6p21-22 were found in 55% of the ALT cell lines of our panel (five of nine) . In proximity to DAXX, at 6p22 , the DNA replication inhibitor Geminin (GMNN) resides, depletion of which might be associated to increased rates of polyploidization through whole-genome endoreduplication observed in ALT [[38,52] and this study]. The same region (6p21.2) harbors RNF8 , an E3 ubiquitin–protein ligase gene, involved in DDR. RNF8 mediates the ubiquitination of histones H2A and H2AX, promoting the formation of TP53BP1 and BRCA1 ionizing radiation–induced foci . The long arm of chromosome 6 displayed a recurrent breakpoint hotspot at q21-q23.3. This region harbors ASF1, an H3/H4 chaperone that is required for the formation of senescence associated heterochromatin foci, as well as the DNA replication licensing factor MCM9 . Recurrent deletions/translocations affecting 7q22.17q22.3 were found in four of nine cell lines. This is the site for MLL5 gene, responsible for a histone methyltransferase that specifically monomethylates and dimethylates “Lys-4” of histone H3 (H3K4me1 and H3K4me2) [68,84]. Losses of whole chromosome 8 and/or 8p arm deletions were observed in seven of nine ALT cell lines. The WRN helicase gene is located at 8p12 . Despite its involvement in APBs, WRN is dispensable for ALT . Another gene that might be related to the formation of ALT pseudo-polycentrics is the ESCO2, located at 8p21.1, that is important for sister chromatid cohesion [68,85]. PIN2/TERF1 interacting, telomerase inhibitor 1 (PINX1) at 8p23.1 is a microtubule-binding protein essential for faithful chromosome segregation [68,86]. PINX1 mediates telomeric repeat binding factor 1 (TRF1) and TERT accumulation in nucleoli and enhances TRF1 binding to telomeres . Tankyrase, a telomere interacting factor related to mitotic fidelity and telomere integrity, is also located at 8p23.1 [68,88]. The telomeric region of 9p was involved in the structural rearrangements in four of nine cell lines. The SMARCA gene that encodes a protein participating in the large ATP-dependent chromatin remodeling complex SNF/SWI is located at 9p24.3 [68,89]. Deletions and/or translocations affecting the short arm of chromosome 9 clustered between bands p21.2 and p21.3. This is the genomic site for the tumor suppressor cyclin-dependent kinase inhibitor 2A (p16) [68,90]. The cyclin-dependent kinase inhibitor 2A locus regulates the two tumor-suppressive pathways (Rb and p53) that are
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most commonly disrupted in a wide range of human malignancies [90,91]. The product of p16 induces cell cycle arrest by preventing phosphorylation of pRb. Loss of function of p16 is reported to be involved in immortalization as an alternative to inactivation of pRb [91–93]. The tip of the short arm of chromosome 11 was involved in clonal translocations or fusions in 66% of the nine lines. The HRAS oncogene is located in close proximity to 11pter . This region harbors also insulin-like growth factor 2 growth. It is an imprinted gene, expressed only from the paternal allele, and epigenetic changes at this locus are associated with rhabdomyosarcomas [90,94,95]. The long arm of chromosome 11 showed recurrent involvement in deletions or translocations at bands q23.1 to q23.3; the MLL1 gene is located here . MLL1 is another histone methyltransferase implicated in telomere metabolism . MLL1 depletion in human diploid fibroblasts resulted in reduced levels of telomere H3/K4 methylation, the induction of a DDR at telomeres, a p53-dependent growth arrest, and cellular senescence . Almost 50% of retinoblastomas use the ALT pathway to maintain their telomeres . The Rb1 gene localizes at 13q14.2 . Recurrent losses of whole chromosome 13 and/or deletions/translocations involving 13q14.2 were evident in all cell lines of our panel. Fibroblast-deriving human ALT cell lines immortalized either spontaneously or by physical or chemical carcinogens, lack expression of p16INK4a, and display hyperphosphorylation of pRb . The region 15q13.1 was recurrently involved in translocations affecting different chromosomes, such as 12, 18, and 20, and terminal deletions. The NDNL2 gene that resides at 15q13.1 is a part of the SMC5-SMC6 complex, involved in homologous recombination DNA repair [68,93]. The complex is required for telomere maintenance through recombination in ALT cell lines. The same region hosts the gene HERC2, responsible for an E3 ubiquitin–protein ligase that regulates ubiquitin-dependent retention of DDR proteins on damaged chromosomes . The great majority of known ALT cell lines is impaired in the p53 pathway, either due to the expression of viral oncoproteins or through a p53 mutation . Immortalized cell lines, derived from Li-Fraumeni breast fibroblasts carrying a germline mutation in p53, use ALT for telomere maintenance [29,98]. Deletions and/or negatively unbalanced translocations affecting the region of the TP53 gene at 17p13.1 were observed in five of nine cell lines of our panel. In contrast, 17q might harbor important genes for ALT because 17q was found to be unaffected in eight of nine cell lines. The APB component BRCA1 is located at 17q21.31, RECQL5 at q25, MAP3K3 at q23.3, MAPKK6 at q24.3, as well as the topoisomerase TOP2A located at q21.2 [68,99–103]. Gains of genomic material from 18p and pericentromeric rearrangements of both the p- and q-arms of this chromosome were frequent in the ALT cell lines of our study and might implicate genes such as transforming growth factor β–induced factor homeobox 1 and methyltransferaselike 4 at 18p11.3, whereas the 18q11.2 region harbors the BRCA1 modulator retinoblastoma binding protein 8 (RBBP-8) [68,104]. Several genes located at 18p such as CENTRIN1, Yamaguchi sarcoma viral oncogene homolog 1 (YES1), methyltransferase-like 4, transforming growth factor β–induced factor homeobox 1, establishment of sister chromatid cohesion N-acetyltransferase 1 (ESCO1), GATA-binding factor 6 (GATA6 ), or cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1) may confer advantages to the ALT phenotype [68,103]. Losses of 19p and gains of 19q were observed in all lines of our panel. The DNMT1 located at 19p13.2 may be an ALT repressor
Neoplasia Vol. 15, No. 11, 2013 . DNA methylation is enriched at pericentromeric, centromeric, and subtelomeric regions and is regarded to undergo late replication and to prevent frequent recombination events . The product of the ZSCAN4 gene at the highly recombinogenic tip of 19q is responsible for the stochastic engagement of an ALT-like phenotype in ES and iPS cells [56,64]. Amplification of the long arm of chromosome 20 was observed in 44.4% of our cell line panel. The gene responsible for the immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) syndrome, the DNMT3B, localizes at 20q11.21. Hypomethylation of subtelomeric and epigenetic modifications at telomeric regions was shown to affect the formation of telomere sister chromatid exchange . TOP1 (20q12) and AURKA (20q13.2) may also be candidates for the list with important genes for ALT continuous growth [68,107,108]. The 21q22.1-3 was involved in structural aberrations in 50% of our cell line panel. Specific genes, like RUNX1, ERG, TMPRSS2, and TFF, located in this region have been involved in tumorigenesis . Moreover, Pericentrin, a cell cycle regulation protein, necessary for centrosomal function, is located at the tip of 21q (q22.3) [68,109]. Extensive loss of genomic material from chromosome 22 was verified in six of nine lines. Chromosome 22 hosts, among many others, CHK2 (q12.1), Merlin (q12.2), and EP300 (q13.2) . Mutations of the CHK2 gene involved in DDR and the control of the cell cycle are found in osteosarcomas . Deletions in Merlin are responsible for neurofibromatosis 2, associated with predisposition to the development of the nerve sheath tumors schwannomas . Interestingly, telomerase activity could not be detected in all schwannomas examined, implying that they might use ALT to sustain continuous growth . The most consistent cytogenetic change reported in benign meningiomas is partial del(22)(q12) or total deletion of chromosome 22 . Many meningiomas were found to use ALT . Moreover, cells without the p300 protein cannot effectively restrain growth and division, allowing cancerous tumors to develop and grow . In the highly aberrant ALT karyotypes, the identification of structural chromosomal rearrangements that might be harboring important genes for the ALT pathway is hampered by stochastic events driven by telomeric, pericentromeric, or CFS vulnerability. Notably, several of the above recurrent breakpoints or imbalances correspond very well to recently published SNP array data from 22 ALT cell lines (four of those are included in our panel) . Hence, despite the relatively lower throughput and resolution, compared to whole-genome comparative genomic hybridization (CGH) approaches, spectral karyotyping (SKY)/M-FISH/DAPI banding analysis is capable of identifying with great accuracy major ALT imbalances and recurrent genomic recombinatorial hotspots. Exogenous reconstitution of telomerase activity reduced significantly the rates of B/F/B cycles and suppressed overall CIN in the ALT VA-13 cells [this study and [31,38]]. Telomeres, centromeres, and subtelomeric regions are considered to be highly sensitive to B/F/B-induced double-strand breaks . Our results show that a proportion of ALT pericentromeric instability may be driven by mechanical forces operating during B/F/B cycles. However, replication stress persists even after stable restoration of telomerase activity, because the proportion of random rearrangements affecting CFS in VA-13TA cells remained similar or even increased compared to the isogenic ALT VA-13. In contrast to many hematologic malignancies [114,115], and to a small number of solid tumors [114,116], our results did not reveal clear evidence for recurrent translocation-driven gene fusions in the ALT
Neoplasia Vol. 15, No. 11, 2013 pathway. However, a thorough examination of specific breakpoints such as those at 15q and 17q12 that were found consistently involved in recurrent but variable translocations may—in the near future— reveal if such entities exist. Our data are consistent to previous studies [29,117] suggesting that the major oncosuppressor pathway abrogated in ALT cells is p53/Rb/ INK4. In addition, the ALT cells take advantage of high rates of endogenous CIN and undergo selection adaptation processes that shape their genome toward the depletion of several regulators of the epigenome such as ATRX and DAXX [29,117]. Hence, the ALT karyotype is shaped by massive stochastic chromosome mutation events that are selected to cluster in particular genomic sites. In this context, clonality of cancerous recombinant chromosomes is preserved through high rates of telomere seeding and frequent deactivation of extra centromeres of fused chromosomes. Our results shed light into the dynamics of clonal perpetuation of cancerous recombinant chromosomes under extreme telomere dysfunction, unveiling, in parallel, specific genomic sites that may harbor genes critical for ALT cancerous cell growth. Acknowledgments We thank J. Shay for providing the VA-13+hTERC+hTERT cells. We also thank all cell line donators, as indicated in the Materials and Methods section, as well as G. Grigoriadis for continuous support and valuable discussions. References  Halazonetis TD, Gorgoulis VG, and Bartek J (2008). An oncogene-induced DNA damage model for cancer development. Science 319, 1352–1355.  Kazda A, Zellinger B, Rössler M, Derboven E, Kusenda B, and Riha K (2012). Chromosome end protection by blunt-ended telomeres. Genes Dev 26, 1703–1713.  Gagos S and Irminger-Finger I (2005). Chromosome instability in neoplasia: chaotic roots to continuous growth. Int J Biochem Cell Biol 37, 1014–1033.  Roschke AV and Kirsch IR (2010). Targeting karyotypic complexity and chromosomal instability of cancer cells. Curr Drug Targets 11, 1341–1350.  Eckert CA, Gravdahl DJ, and Megee PC (2007). The enhancement of pericentromeric cohesin association by conserved kinetochore components promotes highfidelity chromosome segregation and is sensitive to microtubule-based tension. Genes Dev 21, 278–291.  Silva P, Barbosa J, Nascimento AV, Faria J, Reis R, and Bousbaa H (2011). Monitoring the fidelity of mitotic chromosome segregation by the spindle assembly checkpoint. Cell Prolif 44, 391–400.  Greaves M and Maley CC (2012). Clonal evolution in cancer. Nature 481, 306–313.  Stratton MR (2011). Exploring the genomes of cancer cells: progress and promise. Science 331, 1553–1558.  Durkin SG and Glover TW (2007). Chromosome fragile sites. Annu Rev Genet 41, 169–192.  Dillon LW, Burrow AA, and Wang YH (2010). DNA instability at chromosomal fragile sites in cancer. Curr Genomics 11, 326–337.  Sideridou M, Zakopoulou R, Evangelou K, Liontos M, Kotsinas A, Rampakakis E, Gagos S, Kahata K, Grabusic K, Gkouskou K, et al. (2011). Cdc6 expression represses E-cadherin transcription and activates adjacent replication origins. J Cell Biol 195, 1123–1140.  Debatisse M, Le Tallec B, Letessier A, Dutrillaux B, and Brison O (2012). Common fragile sites: mechanisms of instability revisited. Trends Genet 28, 22–32.  Ma K, Qiu L, Mrasek K, Zhang J, Liehr T, Quintana LG, and Li Z (2012). Common fragile sites: genomic hotspots of DNA damage and carcinogenesis. Int J Mol Sci 13, 11974–11999.  Blasco MA (2005). Telomeres and human disease: ageing, cancer and beyond. Nat Rev Genet 6, 611–622.
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Chromosome ends are protected by telomeres that prevent DNA damage response and degradation. Telomerase expression extends telomeres and inhibits DNA damage response. Telomeres are also maintained by the recombination-based alternative lengthening pa
Telomerase activation and alternative lengthening of telomeres are two major mechanisms of telomere length maintenance. Soft tissue sarcomas appear to use the alternative lengthening of telomeres more frequently. Loss of α-thalassemia/mental retardat
Alpha thalassemia/mental retardation syndrome X-linked (ATRX) is mutated in nearly a third of pediatric glioblastoma (GBM) patients. We developed an animal model of ATRX-deficient GBM. Using this model combined with analysis of multiple human glioma
Alternative lengthening of telomeres (ALT) involves homology-directed telomere synthesis. This multistep process is facilitated by loss of the ATRX or DAXX chromatin-remodeling factors and by abnormalities of the telomere nucleoprotein architecture,
The mechanism of activation of the alternative lengthening of telomeres (ALT) pathway of mammalian chromosome-end maintenance has been unclear. We have now discovered that co-depletion of the histone chaperones ASF1a and ASF1b in human cells induced
Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere length maintenance mechanism that enables the unlimited proliferation of a subset of cancer cells. Some neuroblastoma (NB) tumors appear to maintain telomere length by ac
Telomeres are responsible for protecting chromosome ends in order to prevent the loss of coding DNA. Their maintenance is required for achieving immortality by neoplastic cells and can occur by upregulation of the telomerase enzyme or through a homol
Mutations in isocitrate dehydrogenase 1 (IDH1) have been found in the vast majority of low grade and progressive infiltrating gliomas and are characterized by the production of 2-hydroxyglutarate from α-ketoglutarate. Recent investigations of maligna
The alternative lengthening of telomeres (ALT) mechanism allows cancer cells to escape senescence and apoptosis in the absence of active telomerase. A characteristic feature of this pathway is the assembly of ALT-associated promyelocytic leukemia (PM
Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might favor onset of Alternative Lengthening of Telomeres