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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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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.
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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.

Mentions: Representative FISH images of metaphase spreads from wt and mTER−/− cell lines at early and late passages are shown in Fig. 3. As previously described for immortal MEF cultures (Zindy et al., 1997), most of the cell lines studied here were aneuploid at late passages (Fig. 3, A, C, and D). Fig. 3 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. 3 A). Metaphases of KO16-G1 and KO7-G4 mTER−/− cell lines (Fig. 3, 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. 3 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. 3 arrows; see below).


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

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132934&req=5

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.
Mentions: Representative FISH images of metaphase spreads from wt and mTER−/− cell lines at early and late passages are shown in Fig. 3. As previously described for immortal MEF cultures (Zindy et al., 1997), most of the cell lines studied here were aneuploid at late passages (Fig. 3, A, C, and D). Fig. 3 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. 3 A). Metaphases of KO16-G1 and KO7-G4 mTER−/− cell lines (Fig. 3, 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. 3 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. 3 arrows; see below).

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