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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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Myotubes reactivated by CKI KD undergo apoptosis, which can be delayed by forced Bcl-2 expression.(A) MSC-derived myotubes were transfected with the indicated siRNAs and harvested at the time points shown. The indicated proteins were analyzed by western blotting. GAPDH was used as a loading control. (B) MSC-derived myotubes were transfected with siRNAs to p21 and p27 and mock-treated (mock) or infected with an empty adenoviral vector or a Bcl-2-carrying virus at the indicated multiplicities of infection. The myotubes were continuously incubated with BrdU until fixation, 48 hours later. BrdU incorporation was detected by immunofluorescence and nuclei were counterstained with Hoechs 33258. The percentages of BrdU-positive (BrdU+) and mitotic or micronucleated myotubes (mitoses) are shown. Casp  =  caspase; cl.  =  cleaved; moi  =  multiplicity of infection.
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pone-0011559-g005: Myotubes reactivated by CKI KD undergo apoptosis, which can be delayed by forced Bcl-2 expression.(A) MSC-derived myotubes were transfected with the indicated siRNAs and harvested at the time points shown. The indicated proteins were analyzed by western blotting. GAPDH was used as a loading control. (B) MSC-derived myotubes were transfected with siRNAs to p21 and p27 and mock-treated (mock) or infected with an empty adenoviral vector or a Bcl-2-carrying virus at the indicated multiplicities of infection. The myotubes were continuously incubated with BrdU until fixation, 48 hours later. BrdU incorporation was detected by immunofluorescence and nuclei were counterstained with Hoechs 33258. The percentages of BrdU-positive (BrdU+) and mitotic or micronucleated myotubes (mitoses) are shown. Casp  =  caspase; cl.  =  cleaved; moi  =  multiplicity of infection.

Mentions: As already described (Fig. 1), myotubes quickly die following cell cycle reactivation. To determine whether they die by apoptosis, we assessed cleavage-mediated activation of several molecules that regulate and effect apoptosis. As shown in Fig. 5A, caspase 3, caspase 9, and PARP-1 are all cleaved in a time-dependent fashion in reactivated myotubes, but not in control-transfected cells.


DNA replication is intrinsically hindered in terminally differentiated myotubes.

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

Myotubes reactivated by CKI KD undergo apoptosis, which can be delayed by forced Bcl-2 expression.(A) MSC-derived myotubes were transfected with the indicated siRNAs and harvested at the time points shown. The indicated proteins were analyzed by western blotting. GAPDH was used as a loading control. (B) MSC-derived myotubes were transfected with siRNAs to p21 and p27 and mock-treated (mock) or infected with an empty adenoviral vector or a Bcl-2-carrying virus at the indicated multiplicities of infection. The myotubes were continuously incubated with BrdU until fixation, 48 hours later. BrdU incorporation was detected by immunofluorescence and nuclei were counterstained with Hoechs 33258. The percentages of BrdU-positive (BrdU+) and mitotic or micronucleated myotubes (mitoses) are shown. Casp  =  caspase; cl.  =  cleaved; moi  =  multiplicity of infection.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011559-g005: Myotubes reactivated by CKI KD undergo apoptosis, which can be delayed by forced Bcl-2 expression.(A) MSC-derived myotubes were transfected with the indicated siRNAs and harvested at the time points shown. The indicated proteins were analyzed by western blotting. GAPDH was used as a loading control. (B) MSC-derived myotubes were transfected with siRNAs to p21 and p27 and mock-treated (mock) or infected with an empty adenoviral vector or a Bcl-2-carrying virus at the indicated multiplicities of infection. The myotubes were continuously incubated with BrdU until fixation, 48 hours later. BrdU incorporation was detected by immunofluorescence and nuclei were counterstained with Hoechs 33258. The percentages of BrdU-positive (BrdU+) and mitotic or micronucleated myotubes (mitoses) are shown. Casp  =  caspase; cl.  =  cleaved; moi  =  multiplicity of infection.
Mentions: As already described (Fig. 1), myotubes quickly die following cell cycle reactivation. To determine whether they die by apoptosis, we assessed cleavage-mediated activation of several molecules that regulate and effect apoptosis. As shown in Fig. 5A, caspase 3, caspase 9, and PARP-1 are all cleaved in a time-dependent fashion in reactivated myotubes, but not in control-transfected cells.

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