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

Hande MP, Samper E, Lansdorp P, Blasco MA - J. Cell Biol. (1999)

Bottom Line: Interestingly, the most frequent fusions found in mTER-/- cells were homologous fusions involving chromosome 2.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.

View Article: PubMed Central - PubMed

Affiliation: Terry Fox Laboratory, British Columbia Cancer Research Center, Vancouver, British Columbia V5Z 1L3, Canada.

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.

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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.
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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.

Mentions: Fig. 1 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. 2 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. 1 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. 1 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. 1 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. 1, 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. 1, 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. 1 B). In contrast, there was a 6-kb increase in the telomere length at the 6th generation (Fig. 1, 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.


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

Hande MP, Samper E, Lansdorp P, Blasco MA - J. Cell Biol. (1999)

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.
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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.
Mentions: Fig. 1 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. 2 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. 1 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. 1 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. 1 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. 1, 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. 1, 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. 1 B). In contrast, there was a 6-kb increase in the telomere length at the 6th generation (Fig. 1, 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.

Bottom Line: Interestingly, the most frequent fusions found in mTER-/- cells were homologous fusions involving chromosome 2.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.

View Article: PubMed Central - PubMed

Affiliation: Terry Fox Laboratory, British Columbia Cancer Research Center, Vancouver, British Columbia V5Z 1L3, Canada.

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.

Show MeSH
Related in: MedlinePlus