Telomere Dynamics of Individual Chromosomes in mTER−/− Telomerase-deficient Embryos from Different Generations
Primary cells (passage 1 mouse embryonic fibroblasts, MEFs) derived from wt embryos and from mTER−/−
embryos from the 1st (G1) to the 6th (G6) generation were obtained following the scheme previously described (Blasco et al., 1997
). The telomere length of individual chromosomes (chromosomes 2 and 11) was measured using Q-FISH and chromosome painting. Chromosome 2 was chosen because it consistently has relatively short telomeres in several mouse strains (Hande, M.P., and P. Lansdorp, unpublished results; this paper). Chromosome 11 is the mouse homologue of human chromosome 17, which was found to have relatively short telomeres in all individuals analyzed to date (Martens et al., 1998
Fig. shows the mean and standard error of telomere fluorescence intensity of all telomeres together (average of q- and p-arms), and also of q- and p-arms separately from primary MEFs of both wt and mTER−/− embryos of the 2nd (KO2-G2), 4th (KO7-G4), and 6th generation (littermate embryos KO9-G6 and KO11-G6; littermate embryos KO1-G6 to KO4-G6 and embryo KO5-G6). Despite considerable variation between individual telomere fluorescence values (see for example Fig. D), the large number of data points (>1,000) resulted in insignificant standard error values in the telomere values of all chromosomes. The standard error was also small for individual telomeres on chromosomes 2 and 11, with smaller number of data points (50–100; see Figs. A and 2, B and C). The standard error rather than the standard deviation is shown for clarity and presentation purposes only. The average telomere fluorescence of all chromosomes decreased linearly during successive generations of mTER−/− mice. The average telomere shortening was 3.9 kb per generation (calculated as described in Fig. B). This shortening affected both q-telomeres (telomeres of the q-arms) that showed a shortening of 4.17 kb per generation, and p-telomeres (telomeres of the p-arms) that showed a shortening of 3.7 kb per generation. As a result, the difference in telomere length between p- and q-arm telomeres was maintained throughout the six mouse generations (Fig. B). The loss of telomere repeats in mTER−/− mice resulted in an average length of 14.5 and 22.4 kb for p- and q-telomeres, respectively, in cells derived from the 6th generation. When we measured the mean telomere fluorescence of chromosome 2, the estimated rate of telomere shortening per generation was 3.4 kb for both 2q- and 2p-telomeres (Fig. , A and B). This telomere shortening resulted in 6th generation 2p- and 2q-telomeres of an average length of 7.6 kb and 16.2 kb, respectively (embryo KO9-G6 had an estimated 2p-telomere length of only 0.15 ± 0.1 kb), shorter than the average of all chromosomes. In the case of chromosome 11, the average telomere fluorescence of 11q and 11p-telomeres decreased at an average rate of 5.2 and 5.6 kb per generation, respectively, up to the 4th generation (Fig. , A and B). Interestingly, from the 4th (embryo KO-G4) to the 6th generation (the average of seven different embryos) we did not detect the expected telomere shortening in any of chromosome 11 telomeres (Fig. B). In contrast, there was a 6-kb increase in the telomere length at the 6th generation (Fig. , A and B). Altogether, these results suggest that in the absence of telomerase activity, telomere shortening occurs at a similar rate in all chromosome ends. However, it appears that mechanisms that prevent telomere shortening in the absence of telomerase act differentially on different telomeres. In our study, chromosome 11 telomeres did not show the predicted shortening with increasing generations in seven different embryos, while chromosome 2 telomeres continued to shorten throughout the six generations of mTER−/− mice (see Discussion). In this study, we cannot rule out that telomerase independent mechanisms of telomere maintenance are also operating in early generation mTER−/− or wt mice.
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 (more ...)
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 (more ...)
Interestingly, in the different mTER−/−
cells derived from 6th generation embryos, we observed a marked heterogeneity in the mean telomere fluorescence. This heterogeneity affected both chromosome 2 and 11 telomeres and was also observed in cells derived from littermate embryos. This variation in telomere length could be the basis for the variable penetrance of the phenotypes described in 6th generation mTER−/−
mice (Lee et al., 1998
; Herrera et al., 1999
Telomere Dynamics of Individual Chromosomes in Spontaneously Immortalized mTER−/− Cell Lines
Serial passage of mouse embryonic fibroblasts allows the selection of oligoclonal populations with the capacity to stably proliferate in culture. We had previously described that serial passage of mTER−/−
MEFs according to a 3T3 protocol resulted in the selection of immortal cell lines in a manner similar to that of mTER+/+
MEFs, indicating that telomerase activity is not essential for the immortalization of mouse cells (Blasco et al., 1997
). Although some differences were observed in the growth rate between wt and mTER−/−
cell lines, all cultures have shown a continued growth that exceeded 500 PDs for G1 MEFs and 250 PDs for G6 MEFs (not shown). To understand the basis for the continuous growth of these telomerase negative cells we have analyzed their telomere dynamics. Fig. A shows the mean telomere fluorescence of q- (black bars) and p-telomeres (gray bars), separately, during increasing PDs of wt and mTER−/−
cells. We calculated that telomeres of wt cells, Wt14, underwent a modest shortening at an estimated rate of 24.8 bp per PD (Fig. A). The occurrence of telomere shortening in wt MEFs could indicate that the level of telomerase activity present in these cells is not sufficient to prevent telomere erosion as it has been proposed previously for other cell types (Counter et al., 1994
; Chiu et al., 1996
) or, alternatively, that telomere length in these cultured cells is not tightly regulated around a fixed length.
In contrast to wt cells, mTER−/− cell lines derived from 1st (KO16-G1 and KO19-G1), 2nd (KO2-G2), and 4th generation (KO7-G4) embryos, showed a marked decrease in the telomere fluorescence of both p- and q-telomeres (Fig. A). The estimated average telomere loss in the different cell lines ranged between 65 and 108 bp per PD, similar to the shortening rate described for human cells that do not express telomerase. This indicates that in these mTER−/− cells that had escaped senescence and are immortal, the mean telomere length continues to shorten with increasing passage number. Interestingly, the rate of telomere shortening at both p- and q-telomeres decreased at later passages (PDs 215 and 322) of the KO16-G1 cell line, suggesting the activation of telomere maintenance mechanisms when telomeres shorten to a critical length (Fig. A). In the case of two different mTER−/− cell lines, KO9-G6 and KO11-G6, derived from 6th generation embryos, the telomere fluorescence at both p- and q-telomeres was maintained or increased during the different PDs analyzed (Fig. A). In KO9-G6 cells, p- and q-telomeres were maintained at an average length of 10.8 and 24.8 kb, respectively, and in KO11-G6 cells at an average length of 16.3 and 25.7 kb. These observations point to telomerase-independent mechanisms for the maintenance of telomeres in the immortal cells derived from the 6th generation mTER−/− MEFs.
Representative FISH images of metaphase spreads from wt and mTER−/−
cell lines at early and late passages are shown in Fig. . As previously described for immortal MEF cultures (Zindy et al., 1997
), most of the cell lines studied here were aneuploid at late passages (Fig. , A, C, and D). Fig. A shows two metaphases of Wt14 cells before, PD 2, and after immortalization, PD 243. At PD 243, all chromosome ends had TTAGGG repeats and the cells did not show an increase of end-to-end fusions except for a very long chromosome that was clonal (indicated by an arrowhead in Fig. A). Metaphases of KO16-G1 and KO7-G4 mTER−/−
cell lines (Fig. , B and C, respectively) show a decrease in telomere fluorescence when early and late PDs are compared. In contrast, KO9-G6 cells showed a similar telomere fluorescence signal at both early, PD 2, and late, PD 88, PDs, in agreement with the observation that the mean telomere length is maintained in these cells (Fig. D, see above). Finally, all mTER−/−
cell lines contain many chromosomes lacking detectable telomere signal at late PDs, as well as a significant increase of end-to-end fusions (Fig. arrows; see below).
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 (more ...)
We have also studied the telomere fluorescence of chromosomes 2 and 11 as a function of the accumulated number of cell doublings (Fig. , B and C). When telomere fluorescence of both chromosomes 2 and 11 was measured in the wt cell line Wt14, we observed a slight decrease with increasing PDs, (Fig. , B and C). The calculated average rate of telomere shortening for chromosomes 2 and 11 in the Wt14 cells was 10 and 11 bp per PD, respectively. Interestingly, when we studied telomere dynamics of chromosome 2 and 11 in the different generation of mTER−/− cell lines, we could not detect the predicted pattern of telomere shortening with increasing PDs (Fig. , B and C). The length of p- and q-telomeres at chromosomes 2 and 11 with increasing PDs suggests the activation of telomere maintenance mechanisms at different points during the growth of the cell lines. In this regard, it is interesting to note that in KO16-G1 cells, 11q-telomeres did not shorten from PD 19 to PD 81 or from PD 215 to PD 322. However, 11p-telomeres continued to shorten to an average length of only 5.6 kb at PD 215 and then were stabilized. The involvement of 11p telomere in the chromosomal instability of this cell line will be discussed later in this paper. The maintenance of telomere length was particularly clear in cumulative PDs of the two cell lines derived from the 6th generation mTER−/− embryos (Fig. , B and C). Interestingly, in these cells telomeres from different chromosomes were maintained at different length and, in general, chromosome 2 telomeres were stabilized at shorter lengths than chromosome 11 telomeres (Fig. , B and C).
Fig. D shows the distribution of fluorescence intensity values for 2q, 2p, 11q, and 11p telomeres with increasing PDs in wt (Wt14) and in mTER−/− cell lines from the first (KO16-G1) and from the 6th (KO9-G6 and KO11-G6) generation. The telomere fluorescence in wt cells at 2p, 2q, 11p, and 11q telomeres with increasing PDs remained similar, in agreement with the fact that, overall, telomeres were maintained in this cell line. In contrast, the number of telomeres with low fluorescence values (0–10 TFU) increased with passage number in the mTER−/− cell lines. Interestingly, in the mTER−/− cell lines the heterogeneity in fluorescence intensity values increased with increasing PDs for some telomeres (i.e., 11q telomeres in KO9-G6 cell line), again suggesting the existence of alternative telomere maintenance mechanisms in these cells.
Analysis of End-to-End Fusions
To analyze the nature of the chromosomal fusions promoted by the absence of telomerase, we performed FISH on wt and mTER−/− metaphases using telomeric and centromeric probes, as well as chromosomes 2 and 11 painting probes (Materials and Methods). Fig. shows the diagrams of the different fusions characterized in this study together with representative images. End-to-end fusions were classified into different types according to their structure as shown in Fig. . Types I, II, and III involve p-to-p arms fusions. Type I fusions contain telomeric repeats at the fusion point (Fig. a) and two copies of minor satellite centromere repeat sequences (b). Type II fusions do not contain detectable telomeric sequences at the fusion point (Fig. a) and yield two centromere signals (b). Type III fusions lack telomeric signals at the fusion point (Fig. a) and only have one centromere signal (b). Type IV and V fusions involve q-to-q arm fusion, and have or lack detectable telomeric signals at the fusion point, respectively. Finally, type VI involves p-to-q arm fusion. In some cases, we performed chromosome painting to determine whether the fusions were homologous (for example, chromosome 2-to-chromosome 2 in panel c of type II fusions) or nonhomologous (for example, chromosome 11 to an undetermined chromosome in panel c of type VI fusions).
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 (more ...)
Chromosomal Instability in Primary mTER−/− Cells
No fusions were detected in metaphases analyzed from early passage primary wt cells (Table ). In the case of mTER−/−
primary cells, the frequency of fusions increased significantly from 0.07 fusions per metaphase in mTER−/−
cells from the 1st generation (KO19-G1) to an average of 1.04 fusions (range from 0.5 to 1.72) per metaphase in seven independently derived mTER−/−
cells from 6th generation embryos (KO9-G6, KO11-G6, and KO1-G6 to KO5-G6; Table ). Interestingly, cytogenetic analysis of the cells derived from seven independent 6th generation embryos revealed that an average of 41% of the fusions was type II fusions. Chromosome painting showed that 70% of these type II fusions were homologous fusions involving 2p (2p-to-2p fusions; see Fig. for example) and only 2% involved chromosome 11. The dramatic increase in chromosome 2 but not chromosome 11p-arm fusions in 6th generation mTER−/−
cells, is probably the consequence of 2p-telomeres shortening from 26.0 ± 2.8 kb in the wt cells, to an average of 7 kb, shorter than the average of all telomeres in cells from the 6th generation. In this regard, in 6th generation cells derived from embryo KO9-G6, 2p-telomeres were only an estimated 0.15 kb long and 100% of type II fusions were 2p-to-2p fusions. The homologous nature of these fusions indicates that they are likely to be the result of a failure to separate sister chromatids during mitosis (see model in Fig. ). Interestingly, fusions involving chromosome 2 were stably maintained in two different G6 cell lines studied, KO9-G6 and KO11-G6, at least for >80 PDs (Table , see Discussion). Other fusions found in primary KO9-G6 cells included type I fusions (35%), and less frequently, type III (8%) and type V (18.4%) fusions (see Fig. for examples and Table for data). Taken together, chromosome 2 seems to be more frequently involved in chromosome fusions than other chromosomes in the mTER−/−
MEFs, although we can not rule out that other chromosomes might occasionally be involved in fusions in mTER−/−
cells (Lee et al., 1998
Different Types of End-to-End Fusions Detected in mTER−/− Primary MEFs
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 (more ...)
Chromosome End-to-End Fusions in Wild-type and mTER−/− Cell Lines as a Function of the Population Doubling
Chromosomal Instability in mTER−/− Cell Lines
To study the consequences of continuous proliferation in the absence of telomerase activity on chromosomal stability, we also analyzed chromosomal aberrations in wt and mTER−/− cell lines as a function of the accumulated number of PDs (Table ). The wt cell line, Wt14, did not show any end-to-end chromosome fusions in the first 20 PDs, except for a very long chromosome that was clonal. We determined that this long chromosome was the result of a terminal translocation between chromosome 11 and another chromosome (see arrowhead in Fig. A), and was stably transmitted throughout all the PDs analyzed. In later passages of the Wt14 cell line (PD 350), a low percentage of p-arm fusions, 0.2 per metaphase, was also detected. These fusions could be a consequence of the moderate telomere shortening detected in these cells (see above).
A dramatic increase in end-to-end fusions was observed with increasing PDs in all the mTER−/− cell lines studied (see also Fig. , B–D for examples of metaphases). This chromosomal instability furthermore increased with the generation number (Table ). The high frequency of p-arm fusions (89%) versus q-arm fusions (11%) in the cell lines could be due to the fact that mouse p-telomeres are shorter than q-telomeres. Whereas type I and type II fusions were the most common fusions in primary cells (Table ), type III fusions (with only one pair of centromere signals) were the most abundant in the cell lines (65%; see also Table ). Interestingly, 80% of the type II fusions detected in the two different G6 cell lines, KO9-G6 and KO11-G6 involved chromosome 2, in agreement with the observation that chromosome 2 telomeres were shorter than the average of all telomeres in the 6th generation MEFs and/or that there is an increased stability of fusions involving chromosome 2 relative to fusions involving other chromosomes. By chromosome painting, we determined that type III fusions present in mTER−/− cell lines, usually involve nonhomologous chromosomes. Interestingly, 20% of these fusions involved chromosome 11p fused to other chromosomes. In the KO16-G1 PD 81 cell line, >75% of all type III fusions involved chromosome 11, in agreement with the fact that 11p-telomeres were specially short in this particular cell line (for example, Figs. A and 4). Type VI fusions, visualized as chromosome rings, were also present in mTER−/− cell lines (for examples see Fig. , B1 and B2). Other chromosomal rearrangements appear at late passage in mTER−/− cells. For example, in the case of KO16-G1 (PD 215) cells, these rearrangements included reciprocal and terminal translocations involving chromosomes 2 or 11. The frequency of such chromosome exchanges was 0.06 and 0.1 per metaphase, respectively (not shown).
Fusion between nonhomologous chromosomes could originate by the simultaneous existence of two different chromosomes with critically short telomeres. To estimate the minimal telomere length that triggers chromosome fusions, we have calculated the mean telomere length of intrachromosomal telomere repeats in all fusions involving the p-arm of one chromosome and q-arm of a different chromosome (Type VI fusions; see Fig. ) and in terminal translocations detected at PD 215 and at PD 322 of the KO16-G1 cell line (Type I fusions were excluded from the analysis). The average length of intrachromosomal telomere repeats (not considering the type I fusions) was 2.3 kb (ranging between 0.1 and 5.7 kb), indicating that this length is not sufficient to prevent chromosome fusions in mouse cells. Altogether, these results suggest that end-to-end fusions and other chromosomal aberrations detected in the mTER−/− cell lines are the result of telomere shortening to a critical length, and that chromosomes 2 and 11 are commonly involved in these fusions.