The relationship between spontaneous telomere loss and chromosome instability in a human tumor cell line

Abstract

Chromosome instability plays an important role in cancer by promoting the alterations in the genome required for tumor cell progression. The loss of telomeres that protect the ends of chromosomes and prevent chromosome fusion has been proposed as one mechanism for chromosome instability in cancer cells, however, there is little direct evidence to support this hypothesis. To investigate the relationship between spontaneous telomere loss and chromosome instability in human cancer cells, clones of the EJ-30 tumor cell line were isolated in which a herpes simplex virus thymidine kinase (HSV-tk) gene was integrated immediately adjacent to a telomere. Selection for HSV-tk-deficient cells with ganciclovir demonstrated a high rate of loss of the end these "marked" chromosomes (10-4 events/cell per generation). DNA sequence and cytogenetic analysis suggests that the loss of function of the HSV-tk gene most often involves telomere loss, sister chromatid fusion, and prolonged periods of chromosome instability. In some HSV-tk-deficient cells, telomeric repeat sequences were added on to the end of the truncated HSV-tk gene at a new location, whereas in others, no telomere was detected on the end of the marked chromosome. These results suggest that spontaneous telomere loss is a mechanism for chromosome instability in human cancer cells.

Figure 1

The characterization of pNCT-tel plasmid sequences integrated in the EJ-30 clones A3 and B3. (A) The structure of a telomere created by the integration of a single copy of the linearized pNCT-tel plasmid on the end of a chromosome. The location of the restriction sites BamHI (Bm), BgIII (Bg), ClaI (CI), NruI (N) PvuI (Pv), SacI (Sa), SspI (Sp), XbaI (Xb), and XhoI (Xh) in the plasmid are shown. (B) Southern blot analysis of genomic DNA from clones A3 and B3 digested with PvuI, BamHI, or XbaI, which cut once within the plasmid, or BgIII, which cuts at either end of the HSV-tk gene. Hybridization was performed with the pNCT-Δ plasmid probe that does not contain telomeric repeat sequences. (C) Digestion of genomic DNA from A3 and B3 with BAL31 exonuclease to identify terminal restriction fragments. After digestion of genomic DNA with BAL31, the DNA was digested with BamHI and Southern blot analysis was performed with the pNCT-Δ plasmid as a probe.

Figure 2

Southern blot analysis of the integrated plasmid sequences in HSV-tk^- subclones of A3 and B3 that retained some portion of the plasmid sequences. (A) Genomic DNA from A3 and 11 of its HSV-tk^- subclones (G1–G39), and (B) genomic DNA from B3 and 12 of its HSV-tk^- subclones (G41–G74) was digested with BamHI, which cuts once in the plasmid, and hybridization was performed with the pNCT-Δ probe. The absence of the diffuse band indicates the loss of the telomere, whereas the appearance of a new discrete band indicates the presence of nontelomeric DNA fused on to the end of the chromosome.

Figure 3

Further analysis of HSV-tk^- subclones that showed no detectable change in the plasmid sequences by Southern blot analysis of genomic DNA digested with BamHI. Genomic DNAs from A3 and four of its HSV-tk^- subclones, G1, G6, G8, and G38, that showed no change with BamHI, were digested with (A) BamHI and ClaI or (B) BgIII, and hybridization was performed with the pNCT-Δ probe. The presence of diffuse bands indicates that although the plasmid is still telomeric, the BgIII and ClaI sites between the HSV-tk gene and the telomeric repeat sequences are no longer present.

Figure 4

Nucleotide sequence analysis of the sites of addition of telomeric repeat sequences following the loss of the end of the HSV-tk gene. (A) The nucleotide sequences at the sites of addition of the telomeric repeat sequences on to the end of the HSV-tk gene in subclones G6, G8, and T4 are compared with the sequence of the HSV-tk gene (TK) and telomeric repeat sequence (Tel). Regions of homology (asterisks) and short regions of complementarity between the plasmid and telomeric repeat sequences at the site of addition (bold) are indicated. The sequences of the telomere-specific primers used for PCR are also shown (underlined). (B) The structure of the integrated plasmid sequences in the parental clone B3 compared with the structure of the plasmid sequences in the G6, G8 and T4 subclones with terminal deletions involving the HSV-tk gene. The location of restriction sites for BamHI (Bm), BgIII (Bg) and ClaI (Cl) are shown.

Figure 5

Analysis of the DNA fused on to the end of the marked chromosome in the HSV-tk^- subclones G55 and G71. The plasmid sequences rescued from subclone G55 after a partial Sspl digestion, or subclone G71 after partial BamHI digestion (bold lines), indicated the presence of an inverted repeat of the plasmid sequences in both subclones. The location of the restriction sites BamHI (Bm), BgIII (Bg), ClaI (Cl), NruI (N) PvuI (Pv), SacI (Sa), SspI (Sp), XbaI (Xb), and XhoI (Xh) are shown.

Figure 6

Analysis of the site of integration of the pNCT-tel plasmid in clone B3 using fluorescence in situ hybridization. (A) A single integration site of the pNCT-tel plasmid is observed in clone B3 at the end of the short arm of a chromosome. Hybridization was performed with the pNCT-Δ plasmid and chromosomes were counterstained with propidium iodide. The chromosome was identified by chromosome-specific probes to be chromosome 16 (data not shown). (B) Hybridization with both a telomere-specific PNA probe (red) and a chromosome-16-specific probe (green) demonstrated that telomeric repeat sequences are located on the ends of both homologs of chromosome 16 in clone B3. Chromosomes were counterstained with DAPI. (C) The hybridization signal observed with the telomere-specific probe alone showing detectable telomeres on both ends of all the chromosomes.

Figure 7

Fluorescence in situ hybridization demonstrating the instability of the marked chromosome in subclones G60 and G71 that have nontelomeric DNA joined on to the end of the marked chromosome. Metaphase chromosomes from subclones G60 (A, B, E, F) and G71 (C, D) were hybridized with either the pNCT-Δ plasmid probe (A, C, E) or a chromosome-16-specific probe (B, D, F) and the chromosomes were counter stained with propidium iodide. Extensive heterogeneity was observed in the structure of the marked chromosome, including the presence of chromosome 16 fragments of different lengths joined on to the end (A, B), dicentric chromosomes involving the marked chromosome and other chromosomes (C, D), and amplification of the marked chromosome with chromosome-16-specific fragments interspersed with fragments of other chromosomes (E, F).

Figure 8

Analysis of telomeres on the end of the unstable marked chromosome in subclone G71. Hybridization was performed with both a telomere-specific PNA probe (red) and a chromosome 16-specific probe (green). Chromosomes were counter stained with DAPI. Hybridization signals are shown for both probes together (A, C, and E) and with the telomere-specific PNA probe alone (B, D, and F). The results are shown for a cell (A, B) in which the DNA joined on to the end of the marked chromosome originated from chromosome 16 and had no detectable telomere on the end (arrow); a cell (C, D) in which the DNA joined on to the end of the marked chromosome originated from chromosome 16 and had a telomere on the end (arrow); and a cell (E, F) in which the DNA joined on to the end of the marked chromosome originated from both chromosome 16 and another chromosome and had a telomere on the end. The location of the interstitial plasmid sequences (as determined by hybridization with the pNCT-Δ probe, data not shown) at the junction between the marked chromosome and a fragment of chromosome 16 is shown (arrow, E).