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Disease-associated CAG·CTG triplet repeats expand rapidly in non-dividing mouse cells, but cell cycle arrest is insufficient to drive expansion.

Gomes-Pereira M, Hilley JD, Morales F, Adam B, James HE, Monckton DG - Nucleic Acids Res. (2014)

Bottom Line: Importantly, the rates of expansion in non-dividing cells were at least as high as those of proliferating cells.Although expansions can accrue in non-dividing cells, we also show that cell cycle arrest is not sufficient to drive instability, implicating other factors as the key regulators of tissue-specific instability.Our data reveal that de novo expansion events are not limited to S-phase and further support a cell division-independent mutational pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK Inserm UMR 1163, Laboratory of CTG Repeat Instability and Myotonic Dystrophy Type 1, 75015 Paris, France Paris Descartes-Sorbonne Paris Cité University, Imagine Institute, 75015 Paris, France mario.pereira@inserm.fr.

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BrdU incorporation patterns. To measure levels of DNA synthesis we performed a BrdU incorporation assay. Representative low magnification images (left) reveal the relative proportion of BrdU immunostaining (green) nuclei counter-stained with DAPI (blue). Representative high-magnification images (right) illustrate BrdU immunostaining patterns within individual cells and reveal differences between chemical treatments.
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Figure 2: BrdU incorporation patterns. To measure levels of DNA synthesis we performed a BrdU incorporation assay. Representative low magnification images (left) reveal the relative proportion of BrdU immunostaining (green) nuclei counter-stained with DAPI (blue). Representative high-magnification images (right) illustrate BrdU immunostaining patterns within individual cells and reveal differences between chemical treatments.

Mentions: To develop a non-proliferative cell model of repeat size instability and to determine directly if DNA replication and cell division are required to generate trinucleotide repeat expansions, we used chemical inhibitors to arrest a clonal transgenic mouse cell line at various phases of the cell cycle. The D2763Kc2 cell line was selected to perform this study because it carries an unstable CAG·CTG repeat that expands rapidly with time, recreating the step-wise, expansion-biased somatic instability of trinucleotide repeat expansions (31). These cells provide a suitable model system to investigate the core mechanisms of trinucleotide repeat dynamics and identify factors that modify repeat expansion rates. Cells were arrested in S and G2/M by exposure to MMC (35), in S with hydroxyurea (HU) (36), in G1 with roscovitine (37), and in G1 and G2 with apicidin and trichostatin A (TSA) (38,39). For each chemical treatment, six replicate cultures of arrested cells were maintained for up to 121 days in parallel with six replicate control cell cultures (Supplementary Figure S1). To monitor the effect on cell cycle progression, we carefully examined the number of viable cells by trypan blue exclusion assays, the levels of DNA synthesis using BrdU incorporation and protein levels of PCNA (Table 1, Figure 1, Supplementary Figures S2 and S3). The rapidly proliferating control cells (population doubling time, PDT ∼25 h) expressed high levels of PCNA in the nucleus and nearly all cells incorporated high levels of BrdU (94%). The number of viable cells decreased significantly following the initial period of exposure to the chemical, as a result of high mortality, and it remained low throughout the treatment. No additional signs of cell death were detected (e.g. overt cell detachment, cell membrane breakdown, cell/organelle swelling) as the treatment progressed, hence having minimal impact on the PDT measured. Viable cells did not accumulate in any treated cultures and only in HU-treated cultures was there an increase in viable cell numbers at the end of the experimental period (Figure 1A). All the other chemical treatments resulted in highly statistically significant reductions in the rate of incorporation of BrdU (Figure 1B, Supplementary Figure S2) and dramatic decreases in PCNA protein levels (Figure 1C, Supplementary Figure S3). In particular, homogenous nuclear incorporation of BrdU was not detected in TSA-treated cells and was only observed at very low levels (<2%) in apicidin-treated cells, possibly as a result of a small fraction of cells that escaped cell cycle arrest. None of the MMC-treated cells displayed the homogenous nuclear incorporation of BrdU typical of the dividing cells, but a low proportion (∼6%) did present with discrete nuclear BrdU-positive intranuclear foci (Figure 2). Such foci have been observed previously in MMC-treated cells and shown to be sites of active DNA repair (40). The punctuated cytoplasmic staining in apicidin- and TSA-treated cells likely results from BrdU incorporation into mitochondrial DNA (41). Consistent with an S-phase arrest, HU-treated cells showed only a slight drop in PCNA expression and BrdU incorporation (91%) following exposure to the chemical for one week. However, HU-treated cultures displayed a dramatic reduction in cell number after 25 days that only slowly recovered with time and only exceeded starting cell numbers at the end of the treatment period (121 days) (Figure 1A). HU-treated cells thus continued to divide, but with a dramatically increased PDT of ∼120 days. Thus, all of the chemical treatments resulted in either complete cell cycle arrest, or at least a 100-fold increase in the PDT.


Disease-associated CAG·CTG triplet repeats expand rapidly in non-dividing mouse cells, but cell cycle arrest is insufficient to drive expansion.

Gomes-Pereira M, Hilley JD, Morales F, Adam B, James HE, Monckton DG - Nucleic Acids Res. (2014)

BrdU incorporation patterns. To measure levels of DNA synthesis we performed a BrdU incorporation assay. Representative low magnification images (left) reveal the relative proportion of BrdU immunostaining (green) nuclei counter-stained with DAPI (blue). Representative high-magnification images (right) illustrate BrdU immunostaining patterns within individual cells and reveal differences between chemical treatments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4066746&req=5

Figure 2: BrdU incorporation patterns. To measure levels of DNA synthesis we performed a BrdU incorporation assay. Representative low magnification images (left) reveal the relative proportion of BrdU immunostaining (green) nuclei counter-stained with DAPI (blue). Representative high-magnification images (right) illustrate BrdU immunostaining patterns within individual cells and reveal differences between chemical treatments.
Mentions: To develop a non-proliferative cell model of repeat size instability and to determine directly if DNA replication and cell division are required to generate trinucleotide repeat expansions, we used chemical inhibitors to arrest a clonal transgenic mouse cell line at various phases of the cell cycle. The D2763Kc2 cell line was selected to perform this study because it carries an unstable CAG·CTG repeat that expands rapidly with time, recreating the step-wise, expansion-biased somatic instability of trinucleotide repeat expansions (31). These cells provide a suitable model system to investigate the core mechanisms of trinucleotide repeat dynamics and identify factors that modify repeat expansion rates. Cells were arrested in S and G2/M by exposure to MMC (35), in S with hydroxyurea (HU) (36), in G1 with roscovitine (37), and in G1 and G2 with apicidin and trichostatin A (TSA) (38,39). For each chemical treatment, six replicate cultures of arrested cells were maintained for up to 121 days in parallel with six replicate control cell cultures (Supplementary Figure S1). To monitor the effect on cell cycle progression, we carefully examined the number of viable cells by trypan blue exclusion assays, the levels of DNA synthesis using BrdU incorporation and protein levels of PCNA (Table 1, Figure 1, Supplementary Figures S2 and S3). The rapidly proliferating control cells (population doubling time, PDT ∼25 h) expressed high levels of PCNA in the nucleus and nearly all cells incorporated high levels of BrdU (94%). The number of viable cells decreased significantly following the initial period of exposure to the chemical, as a result of high mortality, and it remained low throughout the treatment. No additional signs of cell death were detected (e.g. overt cell detachment, cell membrane breakdown, cell/organelle swelling) as the treatment progressed, hence having minimal impact on the PDT measured. Viable cells did not accumulate in any treated cultures and only in HU-treated cultures was there an increase in viable cell numbers at the end of the experimental period (Figure 1A). All the other chemical treatments resulted in highly statistically significant reductions in the rate of incorporation of BrdU (Figure 1B, Supplementary Figure S2) and dramatic decreases in PCNA protein levels (Figure 1C, Supplementary Figure S3). In particular, homogenous nuclear incorporation of BrdU was not detected in TSA-treated cells and was only observed at very low levels (<2%) in apicidin-treated cells, possibly as a result of a small fraction of cells that escaped cell cycle arrest. None of the MMC-treated cells displayed the homogenous nuclear incorporation of BrdU typical of the dividing cells, but a low proportion (∼6%) did present with discrete nuclear BrdU-positive intranuclear foci (Figure 2). Such foci have been observed previously in MMC-treated cells and shown to be sites of active DNA repair (40). The punctuated cytoplasmic staining in apicidin- and TSA-treated cells likely results from BrdU incorporation into mitochondrial DNA (41). Consistent with an S-phase arrest, HU-treated cells showed only a slight drop in PCNA expression and BrdU incorporation (91%) following exposure to the chemical for one week. However, HU-treated cultures displayed a dramatic reduction in cell number after 25 days that only slowly recovered with time and only exceeded starting cell numbers at the end of the treatment period (121 days) (Figure 1A). HU-treated cells thus continued to divide, but with a dramatically increased PDT of ∼120 days. Thus, all of the chemical treatments resulted in either complete cell cycle arrest, or at least a 100-fold increase in the PDT.

Bottom Line: Importantly, the rates of expansion in non-dividing cells were at least as high as those of proliferating cells.Although expansions can accrue in non-dividing cells, we also show that cell cycle arrest is not sufficient to drive instability, implicating other factors as the key regulators of tissue-specific instability.Our data reveal that de novo expansion events are not limited to S-phase and further support a cell division-independent mutational pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK Inserm UMR 1163, Laboratory of CTG Repeat Instability and Myotonic Dystrophy Type 1, 75015 Paris, France Paris Descartes-Sorbonne Paris Cité University, Imagine Institute, 75015 Paris, France mario.pereira@inserm.fr.

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Related in: MedlinePlus