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DNA replication is intrinsically hindered in terminally differentiated myotubes.

Pajalunga D, Puggioni EM, Mazzola A, Leva V, Montecucco A, Crescenzi M - PLoS ONE (2010)

Bottom Line: Similar results are obtained when myotubes and fibroblasts are reactivated by forced expression of E1A or cyclin D1 and cdk4.We conclude that the inability of myotubes to complete DNA replication must be ascribed to peculiar features inherent in their TD state, rather than to the reactivation method.These results define an unexpected basis for the well known incompetence of mammalian postmitotic cells to proliferate.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neurosciences, National Institute of Health, Rome, Italy.

ABSTRACT

Background: Terminally differentiated (TD) cells permanently exit the mitotic cycle while acquiring specialized characteristics. Although TD cells can be forced to reenter the cell cycle by different means, they cannot be made to stably proliferate, as attempts to induce their replication constantly result in cell death or indefinite growth arrest. There is currently no biological explanation for this failure.

Principal findings: Here we show that TD mouse myotubes, reactivated by depletion of the p21 and p27 cell cycle inhibitors, are unable to complete DNA replication and sustain heavy DNA damage, which triggers apoptosis or results in mitotic catastrophe. In striking contrast, quiescent, non-TD fibroblasts and myoblasts, reactivated in the same way, fully replicate their DNA, do not suffer DNA damage, and proliferate even in the absence of growth factors. Similar results are obtained when myotubes and fibroblasts are reactivated by forced expression of E1A or cyclin D1 and cdk4.

Conclusions: We conclude that the inability of myotubes to complete DNA replication must be ascribed to peculiar features inherent in their TD state, rather than to the reactivation method. On reviewing the literature concerning reactivation of other TD cell types, we propose that similar mechanisms underlie the general inability of all kinds of TD cells to proliferate in response to otherwise mitogenic stimuli. These results define an unexpected basis for the well known incompetence of mammalian postmitotic cells to proliferate. Furthermore, this trait might contribute to explain the inability of these cells to play a role in tissue repair, unlike their counterparts in extensively regenerating species.

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BrdU incorporation and death in cells reactivated by CKI KD.(A) quiescent C3H-10T1/2 and myotubes were transfected with the indicated siRNAs. The indicated proteins were analyzed by western blotting 48 or 20 hours later, respectively. β-tubulin is a loading control. (B) Serum-starved, quiescent C3H-10T1/2 fibroblasts and MSC-derived myotubes were treated as indicated and incubated with BrdU for the next 44 hours. BrdU incorporation was detected by indirect immunofluorescence. (C) Myotubes were transfected with a control siRNA (to GFP) or siRNAs to p21 and p27. The number of BrdU-positive myotubes (BrdU, myosin double-positive cells) was counted in 10 random, low-power microscopic fields at the indicated times post-transfection. Ctr  =  control.
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pone-0011559-g001: BrdU incorporation and death in cells reactivated by CKI KD.(A) quiescent C3H-10T1/2 and myotubes were transfected with the indicated siRNAs. The indicated proteins were analyzed by western blotting 48 or 20 hours later, respectively. β-tubulin is a loading control. (B) Serum-starved, quiescent C3H-10T1/2 fibroblasts and MSC-derived myotubes were treated as indicated and incubated with BrdU for the next 44 hours. BrdU incorporation was detected by indirect immunofluorescence. (C) Myotubes were transfected with a control siRNA (to GFP) or siRNAs to p21 and p27. The number of BrdU-positive myotubes (BrdU, myosin double-positive cells) was counted in 10 random, low-power microscopic fields at the indicated times post-transfection. Ctr  =  control.

Mentions: To study in depth the consequences of cell cycle reactivation in TD vs. non-TD cells, we subjected TD myotubes derived from mouse satellite cells (MSC) and serum-deprived, quiescent C3H-10T1/2 mouse fibroblasts to RNA interference (RNAi) for p21 and p27 (Fig. 1A). As previously reported, interference with relevant CKIs induces reentry into the cell cycle of quiescent, senescent, and TD cells alike [10]. Fig. 1B shows that 37% of the fibroblasts and 62% of the myotubes incorporated 5-bromo-2′-deoxyuridine (BrdU) in these conditions. However, the eventual fates of the two cell types diverge strikingly. While the fibroblasts, as already shown, proliferate in the following days in the continuing absence of serum [10], the reactivated myotubes die within 3 days (Fig. 1C).


DNA replication is intrinsically hindered in terminally differentiated myotubes.

Pajalunga D, Puggioni EM, Mazzola A, Leva V, Montecucco A, Crescenzi M - PLoS ONE (2010)

BrdU incorporation and death in cells reactivated by CKI KD.(A) quiescent C3H-10T1/2 and myotubes were transfected with the indicated siRNAs. The indicated proteins were analyzed by western blotting 48 or 20 hours later, respectively. β-tubulin is a loading control. (B) Serum-starved, quiescent C3H-10T1/2 fibroblasts and MSC-derived myotubes were treated as indicated and incubated with BrdU for the next 44 hours. BrdU incorporation was detected by indirect immunofluorescence. (C) Myotubes were transfected with a control siRNA (to GFP) or siRNAs to p21 and p27. The number of BrdU-positive myotubes (BrdU, myosin double-positive cells) was counted in 10 random, low-power microscopic fields at the indicated times post-transfection. Ctr  =  control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903488&req=5

pone-0011559-g001: BrdU incorporation and death in cells reactivated by CKI KD.(A) quiescent C3H-10T1/2 and myotubes were transfected with the indicated siRNAs. The indicated proteins were analyzed by western blotting 48 or 20 hours later, respectively. β-tubulin is a loading control. (B) Serum-starved, quiescent C3H-10T1/2 fibroblasts and MSC-derived myotubes were treated as indicated and incubated with BrdU for the next 44 hours. BrdU incorporation was detected by indirect immunofluorescence. (C) Myotubes were transfected with a control siRNA (to GFP) or siRNAs to p21 and p27. The number of BrdU-positive myotubes (BrdU, myosin double-positive cells) was counted in 10 random, low-power microscopic fields at the indicated times post-transfection. Ctr  =  control.
Mentions: To study in depth the consequences of cell cycle reactivation in TD vs. non-TD cells, we subjected TD myotubes derived from mouse satellite cells (MSC) and serum-deprived, quiescent C3H-10T1/2 mouse fibroblasts to RNA interference (RNAi) for p21 and p27 (Fig. 1A). As previously reported, interference with relevant CKIs induces reentry into the cell cycle of quiescent, senescent, and TD cells alike [10]. Fig. 1B shows that 37% of the fibroblasts and 62% of the myotubes incorporated 5-bromo-2′-deoxyuridine (BrdU) in these conditions. However, the eventual fates of the two cell types diverge strikingly. While the fibroblasts, as already shown, proliferate in the following days in the continuing absence of serum [10], the reactivated myotubes die within 3 days (Fig. 1C).

Bottom Line: Similar results are obtained when myotubes and fibroblasts are reactivated by forced expression of E1A or cyclin D1 and cdk4.We conclude that the inability of myotubes to complete DNA replication must be ascribed to peculiar features inherent in their TD state, rather than to the reactivation method.These results define an unexpected basis for the well known incompetence of mammalian postmitotic cells to proliferate.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neurosciences, National Institute of Health, Rome, Italy.

ABSTRACT

Background: Terminally differentiated (TD) cells permanently exit the mitotic cycle while acquiring specialized characteristics. Although TD cells can be forced to reenter the cell cycle by different means, they cannot be made to stably proliferate, as attempts to induce their replication constantly result in cell death or indefinite growth arrest. There is currently no biological explanation for this failure.

Principal findings: Here we show that TD mouse myotubes, reactivated by depletion of the p21 and p27 cell cycle inhibitors, are unable to complete DNA replication and sustain heavy DNA damage, which triggers apoptosis or results in mitotic catastrophe. In striking contrast, quiescent, non-TD fibroblasts and myoblasts, reactivated in the same way, fully replicate their DNA, do not suffer DNA damage, and proliferate even in the absence of growth factors. Similar results are obtained when myotubes and fibroblasts are reactivated by forced expression of E1A or cyclin D1 and cdk4.

Conclusions: We conclude that the inability of myotubes to complete DNA replication must be ascribed to peculiar features inherent in their TD state, rather than to the reactivation method. On reviewing the literature concerning reactivation of other TD cell types, we propose that similar mechanisms underlie the general inability of all kinds of TD cells to proliferate in response to otherwise mitogenic stimuli. These results define an unexpected basis for the well known incompetence of mammalian postmitotic cells to proliferate. Furthermore, this trait might contribute to explain the inability of these cells to play a role in tissue repair, unlike their counterparts in extensively regenerating species.

Show MeSH
Related in: MedlinePlus