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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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Aberrant mitoses in CKI KD-reactivated myotubes but not in fibroblasts.(A) MSC-derived myotubes were transfected with siRNAs to p21 and p27. The cells were fixed and stained 46 hours later. Bar  = 25 µm. (B) Serum-starved, quiescent C3H-10T1/2 cells were transfected with siRNA to p21 and p27 and kept in medium containing 0.1% FBS. The cells were fixed and stained 44 hours later. Bar  = 12.5 µm.
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pone-0011559-g002: Aberrant mitoses in CKI KD-reactivated myotubes but not in fibroblasts.(A) MSC-derived myotubes were transfected with siRNAs to p21 and p27. The cells were fixed and stained 46 hours later. Bar  = 25 µm. (B) Serum-starved, quiescent C3H-10T1/2 cells were transfected with siRNA to p21 and p27 and kept in medium containing 0.1% FBS. The cells were fixed and stained 44 hours later. Bar  = 12.5 µm.

Mentions: Microscopic observation showed that the mitoses occurring in the reactivated myotubes are invariably extremely aberrant. While some mitoses display a large number of anomalies such as chromosomal breaks, DNA bridges, and chromosome missegregation, most others show widely scattered and/or pulverized chromosomes. In addition, myotubes surviving the mitotic phase show an often extreme degree of micronucleation, the result of nuclear membrane reassembly around isolated chromosome fragments (Fig. 2A). On the contrary, quiescent fibroblasts reactivated by the same means undergo morphologically normal mitoses (Fig. 2B).


DNA replication is intrinsically hindered in terminally differentiated myotubes.

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

Aberrant mitoses in CKI KD-reactivated myotubes but not in fibroblasts.(A) MSC-derived myotubes were transfected with siRNAs to p21 and p27. The cells were fixed and stained 46 hours later. Bar  = 25 µm. (B) Serum-starved, quiescent C3H-10T1/2 cells were transfected with siRNA to p21 and p27 and kept in medium containing 0.1% FBS. The cells were fixed and stained 44 hours later. Bar  = 12.5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011559-g002: Aberrant mitoses in CKI KD-reactivated myotubes but not in fibroblasts.(A) MSC-derived myotubes were transfected with siRNAs to p21 and p27. The cells were fixed and stained 46 hours later. Bar  = 25 µm. (B) Serum-starved, quiescent C3H-10T1/2 cells were transfected with siRNA to p21 and p27 and kept in medium containing 0.1% FBS. The cells were fixed and stained 44 hours later. Bar  = 12.5 µm.
Mentions: Microscopic observation showed that the mitoses occurring in the reactivated myotubes are invariably extremely aberrant. While some mitoses display a large number of anomalies such as chromosomal breaks, DNA bridges, and chromosome missegregation, most others show widely scattered and/or pulverized chromosomes. In addition, myotubes surviving the mitotic phase show an often extreme degree of micronucleation, the result of nuclear membrane reassembly around isolated chromosome fragments (Fig. 2A). On the contrary, quiescent fibroblasts reactivated by the same means undergo morphologically normal mitoses (Fig. 2B).

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