Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice

Abstract

To study the effect of continued telomere shortening on chromosome stability, we have analyzed the telomere length of two individual chromosomes (chromosomes 2 and 11) in fibroblasts derived from wild-type mice and from mice lacking the mouse telomerase RNA (mTER) gene using quantitative fluorescence in situ hybridization. Telomere length at both chromosomes decreased with increasing generations of mTER-/- mice. At the 6th mouse generation, this telomere shortening resulted in significantly shorter chromosome 2 telomeres than the average telomere length of all chromosomes. Interestingly, the most frequent fusions found in mTER-/- cells were homologous fusions involving chromosome 2. Immortal cultures derived from the primary mTER-/- cells showed a dramatic accumulation of fusions and translocations, revealing that continued growth in the absence of telomerase is a potent inducer of chromosomal instability. Chromosomes 2 and 11 were frequently involved in these abnormalities suggesting that, in the absence of telomerase, chromosomal instability is determined in part by chromosome-specific telomere length. At various points during the growth of the immortal mTER-/- cells, telomere length was stabilized in a chromosome-specific man-ner. This telomere-maintenance in the absence of telomerase could provide the basis for the ability of mTER-/- cells to grow indefinitely and form tumors.

Figure 1

Telomere dynamics in wild type and mTER^−/− primary cells from different mouse generations. (A) Telomere fluorescence of all chromosomes (top), chromosome 2 (center), and chromosome 11 (bottom) from primary MEFs derived from embryos of the indicated genotype and generation. Fluorescence is expressed in TFU, where 1 TFU corresponds to 1 kb of TTAGGG repeats in plasmid DNA (Martens et al., 1998). Each value represents the mean of 15 or more metaphases. Primary cells from wt and mTER^−/− embryos from 2nd (KO2-G2), 4th (KO7-G4), and 6th (KO9-G6, KO11-G6, KO1-G6, KO2-G6, KO3-G6, KO4-G6, KO5-G6) generation were used in the study. Black squares, average of q- and p-telomeres; white diamonds, q-telomeres; and open circles, p-telomeres. all chr., All chromosomes; chr.2, chromosome 2; chr.11, chromosome 11. The standard error is indicated with a bar. Despite the wide heterogeneity in individual telomere fluorescence intensity values (see for example Fig. 2 D), the standard errors of the mean were usually very small due to the large number of data points (See Materials and Methods for details). As a result, the error bars are not always visible in the graphs (i.e., A). (B) The average telomere length of q- and p-telomeres together, and of q-telomeres and p-telomeres, separately, in the different embryos studied from each generation was plotted and the data was analysed by least square methods to calculate the average telomere shortening per generation. In the case of chromosome 11 telomeres, we obtained a low r² value (0.67) when we included all the data up to the G6 generation. The low r² value indicates that it is not correct to include G6 chromosome 11 telomere values to calculation of rate of shortening, further suggesting that chromosome 11 did not suffer the predicted telomere shortening from G4 to G6. To calculate the telomere shortening per generation in chromosome 11, we excluded G6 telomeres (B) obtaining a linear equation with r² close to 1. Primary cells from wt and mTER^−/− embryos from 2nd (G2), 4th (G4), and 6th (G6) generation were used in the study. Black squares, average of q- and p-telomeres; white diamonds, q-telomeres; open circles, p-telomeres.

Figure 2

Telomere dynamics in wt and mTER^−/− cell lines. (A–C) The telomere fluorescence, measured as TFU, of all telomeres (A), chromosome 2 telomeres (B), and chromosome 11 telomeres (C) in wt (Wt14) and mTER^−/− cell lines at different PDs is shown. Black bars, fluorescence of q-telomeres; gray bars, fluorescence of p-telomeres. Despite the wide heterogeneity in individual telomere fluorescence intensity values, and due to the large number of data points used in the analysis, the error bars are not always visible in the graphs (i.e., A). (D) The distribution of telomere fluorescence intensity values of 2p-, 2q-, 11p-, and 11q-telomeres (black bars) in wt cell line (Wt14) and the indicated mTER^−/− cell lines at increasing PDs.

Figure 2

Telomere dynamics in wt and mTER^−/− cell lines. (A–C) The telomere fluorescence, measured as TFU, of all telomeres (A), chromosome 2 telomeres (B), and chromosome 11 telomeres (C) in wt (Wt14) and mTER^−/− cell lines at different PDs is shown. Black bars, fluorescence of q-telomeres; gray bars, fluorescence of p-telomeres. Despite the wide heterogeneity in individual telomere fluorescence intensity values, and due to the large number of data points used in the analysis, the error bars are not always visible in the graphs (i.e., A). (D) The distribution of telomere fluorescence intensity values of 2p-, 2q-, 11p-, and 11q-telomeres (black bars) in wt cell line (Wt14) and the indicated mTER^−/− cell lines at increasing PDs.

Figure 3

Metaphase spreads from wt and mTER^−/− cell lines at selected PDs. (A) Representative metaphase spreads from wt cells (Wt14) at the indicated PD. The arrowhead points to a long chromosome present in all the metaphases analyzed at PD 243. (B) Metaphase spreads from 1st generation mTER^−/− cells, KO16-G1, at the indicated PDs. Note the weaker telomere fluorescence at PD 215 compared with PD 19. A chromosome with intrachromosomal TTAGGG signal is indicated with a white arrowhead. (C) Metaphase spreads from 4th generation mTER^−/− cell line, KO7-G4, at the indicated PDs. Note that telomere fluorescence decreased at PD 159 compared with PD 2. (D) Metaphase spreads from 6th generation mTER^−/− cell line, KO9-G6. Note the strong heterogeneity in telomere fluorescence. Red arrows, chromosomal ends lacking detectable telomere fluorescence. White arrows, end-to-end fusions. These are representative images from individual metaphase spreads after FISH showing fluorescent spots on telomeres for illustration purpose only.

Figure 3

Metaphase spreads from wt and mTER^−/− cell lines at selected PDs. (A) Representative metaphase spreads from wt cells (Wt14) at the indicated PD. The arrowhead points to a long chromosome present in all the metaphases analyzed at PD 243. (B) Metaphase spreads from 1st generation mTER^−/− cells, KO16-G1, at the indicated PDs. Note the weaker telomere fluorescence at PD 215 compared with PD 19. A chromosome with intrachromosomal TTAGGG signal is indicated with a white arrowhead. (C) Metaphase spreads from 4th generation mTER^−/− cell line, KO7-G4, at the indicated PDs. Note that telomere fluorescence decreased at PD 159 compared with PD 2. (D) Metaphase spreads from 6th generation mTER^−/− cell line, KO9-G6. Note the strong heterogeneity in telomere fluorescence. Red arrows, chromosomal ends lacking detectable telomere fluorescence. White arrows, end-to-end fusions. These are representative images from individual metaphase spreads after FISH showing fluorescent spots on telomeres for illustration purpose only.

Figure 4

Examples of the different end-to-end fusions detected in mTER^−/− cells. The chromosomal arms involved in the fusion are inferred by the morphology of the fused chromosome. Chromosomes probed with telomeric PNA to characterize the fusion according to the presence or absence of detectable telomeric sequences at the fusion point are depicted in panels a. Chromosomes probed with minor satellite DNA to characterize the fusion according to the number and location of centromeres are depicted in panels b (in p-arm and p-arm/q-arm fusions). Panels b (in q-arm examples) depict DAPI staining of the fusion. Panels c depict chromosome painting with whole- chromosome DNA from chromosome 2 (example of type II fusion) and chromosome 11 (examples of type III fusion and p-arm/q-arm fusion). Chromosomes are counterstained with DAPI for telomere probe and propidium iodide for minor satellite and chromosome probes.

Figure 5

Models for the generation of chromosomal fusions by telomere loss. (Top) Replication and segregation of a mouse chromatid with normal telomeres. (Middle and bottom) Shortening of p- or q-arm telomeres to a critical length leads to fusion of sister chromatids after replication. The subsequent failure in the separation of these fusions might result in a daughter cell (1) harboring a Robertsonian-like configuration (middle) or a dicentric chromosome (bottom) and a second daughter cell (2) that has lost a chromosome. Fused chromosomes may undergo successive cycles of breakage-fusion-bridge and are inherently unstable (reviewed in de Lange, 1995).

Figure 6

Model of telomere dynamics and chromosomal instability during tumor progression. Telomere shortening could occur during the initial stages of tumor progression if cells divide in the absence of compensating telomere lengthening mechanisms. Telomere shortening to a critical length eventually triggers chromosomal instability as described in this paper. At this point, telomere maintenance mechanisms can be activated and selected to allow immortal growth. The preferred mechanism to maintain telomeres in tumor cells is the activation of the enzyme telomerase (reviewed in Shay and Bacchetti, 1996). However, the results shown in this paper indicate that in the absence of telomerase activity alternative telomere-maintaining mechanisms are activated as a consequence telomere shortening.