Alternative lengthening of telomeres: recurrent cytogenetic aberrations and chromosome stability under extreme telomere dysfunction

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.

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.

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.

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, x630). 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; x630).

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.