bridge cycles initiated by spontaneous telomere loss in a human cancer cell line - PubMed (original) (raw)

DNA amplification by breakage/fusion/bridge cycles initiated by spontaneous telomere loss in a human cancer cell line

Anthony W I Lo et al. Neoplasia. 2002 Nov-Dec.

Abstract

The development of genomic instability is an important step in generating the multiple genetic changes required for cancer. One consequence of genomic instability is the overexpression of oncogenes due to gene amplification. One mechanism for gene amplification is the breakage/fusion/bridge (B/F/B) cycle that involves the repeated fusion and breakage of chromosomes following the loss of a telomere. B/F/B cycles have been associated with low-copy gene amplification in human cancer cells, and have been proposed to be an initiating event in high-copy gene amplification. We have found that spontaneous telomere loss on a marker chromosome 16 in a human tumor cell line results in sister chromatid fusion and prolonged periods of chromosome instability. The high rate of anaphase bridges involving chromosome 16 demonstrates that this instability results from B/F/B cycles. The amplification of subtelomeric DNA on the marker chromosome provides conclusive evidence that B/F/B cycles initiated by spontaneous telomere loss are a mechanism for gene amplification in human cancer cells.

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Figures

Figure 1

Structure of the integrated plasmid sequences and length of seeded telomeres in EJ-30 clones A3 and B3. (A) Clones A3 and B3 contain a single copy of the pNCT-tel plasmid integrated on the end of a chromosome. The locations of the cellular DNA, plasmid vector (amp/ori), neo gene, HSV-tk gene, and telomeric repeat sequences are shown. The location of BamHI restriction sites (Bm) used for Southern blot analysis and the size of the BamHI restriction fragments are shown. (B) Southern blot analysis of BamHI-digested genomic DNA from clones A3 and B3, and subclones 1 through 5 selected at random from each clone. Hybridization was performed with the pNCT-Δ probe, which is identical to pNCT-tel except that it does not contain telomeric repeat sequences. (C) Southern blot analysis of BamHI-digested genomic DNA from clone B3 and its subclones B3-1 and B3-2 after different numbers of cell doublings using the pNCT-Δ plasmid as a probe. The internal BamHI fragments in clones A3 and B3 are both approximately 4.5 kb in length. The terminal fragments containing 4.4 kb of plasmid sequences are heterogeneous in length due to variability in the length of the telomeres in different cells in the population. Molecular size markers consisting of Lambda bacteriophage HindIII restriction fragments are shown.

Figure 2

The mechanism of gene amplification involving B/F/B cycles. B/F/B cycles are initiated when sister chromatids fuse following the loss of a telomere. The resulting dicentric chromosome forms a bridge during anaphase and breaks again, continuing the cycle until the chromosome obtains a new telomere. Breakage at locations other than the site of fusion results in amplification of sequences in one daughter cell and deletions in the other daughter cell. The distance between the amplified arrays is dependent upon the distance of the break from the site of fusion. The telomeres (gray squares), centromeres (circles), and orientation of the subtelomeric sequences (arrow) are shown.

Figure 3

Anaphase bridges specific to chromosome 16 in subclone G71. The staining of total chromatin with DAPI (left panel, pseudo-colored orange) is used to identify anaphase cells without (A) and with (B,C) chromosome bridges (arrows). Hybridization with a chromosome 16-specific painting probe (center panel, green) demonstrates that most anaphase bridges in subclone G71 involve chromosome 16. The merged image showing both DAPI staining and hybridization with the chromosome 16-specific probe is also shown (right panel).

Figure 4

Gene amplification resulting from the spontaneous telomere loss. (A) Metaphase chromosomes from subclone G71 hybridized with (i) a subtelomeric cosmid probe RT99 and counterstained with propidium iodide; (ii) a telomere-specific PNA probe and counterstained with DAPI; (iii) a chromosome 16-specific painting probe and counterstained with propidium iodide; and (iv) both a chromosome 16q-specific BAC clone GS-240-G10 (red) and a chromosome 16p-specific BAC clone GS-121-I4 (green). The marker chromosome is identified by the absence of hybridization with the GS-121-I4 BAC clone due to the terminal deletion associated with integration of the pNCT-tel plasmid. (B) Two additional metaphase chromosomes of subclone G71 demonstrating increased hybridization with the subtelomeric cosmid probe RT99 and variability in the size of the fragment joined onto the end of the marker chromosome. (C) Three metaphase spreads containing stretched chromosomes were hybridized with fluorescein-labeled cosmid RT99 (green) located immediately adjacent to the telomere and rhodamine-labeled cosmid 317H7 (red) located 1 Mb from the telomere. Overlapping RT99 and 317H7 hybridization signals appear yellow. Chromosomes were counterstained with DAPI (blue). The inserts are magnified views of the amplified regions.

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DNA amplification by breakage/fusion/bridge cycles initiated by spontaneous telomere loss in a human cancer cell line - PubMed (original) (raw)