Telomere loss provokes multiple pathways to apoptosis and produces genomic instability in Drosophila melanogaster

Abstract

Telomere loss was produced during development of Drosophila melanogaster by breakage of an induced dicentric chromosome. The most prominent outcome of this event is cell death through Chk2 and Chk1 controlled p53-dependent apoptotic pathways. A third p53-independent apoptotic pathway is additionally utilized when telomere loss is accompanied by the generation of significant aneuploidy. In spite of these three lines of defense against the proliferation of cells with damaged genomes a small fraction of cells that have lost a telomere escape apoptosis and divide repeatedly. Evasion of apoptosis is accompanied by the accumulation of karyotypic abnormalites that often typify cancer cells, including end-to-end chromosome fusions, anaphase bridges, aneuploidy, and polyploidy. There was clear evidence of bridge-breakage-fusion cycles, and surprisingly, chromosome segments without centromeres could persist and accumulate to high-copy number. Cells manifesting these signs of genomic instability were much more frequent when the apoptotic mechanisms were crippled. We conclude that loss of a single telomere is sufficient to generate at least two phenotypes of early cancer cells: genomic instability that involves multiple chromosomes and aneuploidy. This aneuploidy may facilitate the continued escape of such cells from the normal checkpoint mechanisms.

Figure 1.—

Dicentric/acentric chromosome production and segregation. FLP-induced recombination between oppositely oriented FRTs on sister chromatids produces a dicentric chromosome and an acentric chromosome. At anaphase the dicentric chromosome is stretched between the poles and usually breaks. Centromeres are indicated as filled circles, telomeres as filled squares, FRTs as arrows.

Figure 2.—

Dicentric-inducible chromosomes used in this work. (a–d) The DcY chromosomes: H1 has inverted FRTs inserted near the tip of the long arm; H2 and H3 have inverted FRTs inserted near the tip of the short arm; K2 has inverted FRTs inserted within a duplication of virtually the entire chromosome 4 attached to the long arm of the Y. Chromosomes a and b are based on the standard B^SYy⁺ chromosome (Ashburner et al. 2005). FrTr4B1A is inserted on the short arm of a Y chromosome that also carries y⁺, although the location of y⁺ is uncertain. (e–h) X and autosomes with inverted FRT insertions. The location of each insertion is given, along with a schematic indication of the approximate cytological location. The location of Dc3(FrTr1D) is known only approximately by metaphase cytology.

Figure 3.—

Dicentric bridges and breakage, in vivo. (A) Mitotic divisions in a fixed preparation of the eye imaginal disc [genotype: y w P{70FLP}3F/DcY(K2)]. Chromatin is visualized with anti-phospho-histone H3 antibody shortly after FLP-induced formation of dicentric chromosomes. Anaphase bridges (i, ii, and iv) are easily seen, and one bridge (iii) appears to have broken. (B) Still images from a time-lapse movie of a mitotic division from a cellularized Drosophila embryo that shows breakage of the dicentric bridge. Chromosomes fluoresce because of a His2AvDGFP transgene (Clarkson and Saint 1999). These embryos also carried a dicentric-inducible chromosome and the FLP-expressing transgene [genotype: y w/DcY(K2); P{70FLP}10/+; His2AvDGFP/+].

Figure 4.—

Apoptosis in imaginal discs. Represented here are different severities of apoptosis after dicentric induction. Apoptosis was visualized by indirect immunofluorescence using an antibody against cleaved caspase-3. Dark spots indicate cells undergoing apoptosis in these negative images.

Figure 5.—

Frequency of the wild-type karyotype in larval brains after dicentric induction. The y-axis shows the percentage of cells with a wild-type karyotype among all metaphase spreads in the entire larval brain at different time points after dicentric induction as denoted on the x-axis. Usually 3–4 brains were counted with an average of 64 metaphases per brain. Genotypes and complete data are given in Table 3.

Figure 6.—

Larval neuroblasts with altered karyotypes after dicentric formation. The genotype of each cell is indicated, along with the time after 70FLP induction. Acentric chromatids of expected size are indicated with arrowheads. (A–D) Karyotypes of cells with Dc2(FrTr1B) [genotype: y w P{70FLP}3F/y w; Dc2(FrTr1B)/+]. (A) A cell at the first metaphase showing dicentric (Dc2) and acentric of chromosome 2 (Ac). (B) A cell showing two acentric chromatids and a short centric fragment (Fr2) with apparently unfused ends. (C) A cell with 14 acentric fragments, including some that differ in size (yellow arrow). (D) A cell showing four circular acentric chromatids. (E and F) Karyotypes of mei-41 chk2 cells with Dc3(FrTr1D) [genotype: mei-41^29D/Y; lok^p6; P{70FLP}4A/Dc3(FrTr1D)]. (E) An octaploid cell for all chromosomes except the fourth, carrying a stable Y;3 translocation that was successfully replicated three times (yellow arrows). (F) A tetraploid cell with an end–end fusion between two chromosome 3′s that was successfully replicated (yellow arrow). (G–I) Karyotypes isolated from cells with DcX(105). (G and H) Telophase nuclei exhibiting lagging acentric chromatids [genotype: y w P{70FLP}3F/DcX(105); p53^5A-1-4]. (I) A nucleus with 54 acentric chromatids of three different sizes (white, yellow, and red arrows) [genotype: y w P{70FLP}3F/DcX(105)].

Figure 7.—

A mechanism for circular acentric chromosome formation. Symbols are as in Figure 1.