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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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Phosphorylation of histone H2AX in myotubes but not fibroblasts reactivated by different strategies.C2C12 myotubes and quiescent C3H-10T1/2 fibroblasts were infected with cell cycle-reactivating viruses expressing either E1A (dl520) or cyclin D1 and cdk4 (D1/K4). The cells were also infected with an empty control virus (Ctr-Ad) or mock-infected. In addition, fibroblasts were reactivated with 10% FBS or treated with Phleomycin to provide a positive control for DNA damage. γ-H2AX was analyzed by western blotting. Multiplicities of infection as follows. Myotubes: Ctr-Ad 400, dl520 200, cyclin D1 60, cdk4 760. C3H-10T1/2: Ctr-Ad 300, cyclin D1 30, cdk4 300, dl520 200. l.c.  =  loading control.
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pone-0011559-g007: Phosphorylation of histone H2AX in myotubes but not fibroblasts reactivated by different strategies.C2C12 myotubes and quiescent C3H-10T1/2 fibroblasts were infected with cell cycle-reactivating viruses expressing either E1A (dl520) or cyclin D1 and cdk4 (D1/K4). The cells were also infected with an empty control virus (Ctr-Ad) or mock-infected. In addition, fibroblasts were reactivated with 10% FBS or treated with Phleomycin to provide a positive control for DNA damage. γ-H2AX was analyzed by western blotting. Multiplicities of infection as follows. Myotubes: Ctr-Ad 400, dl520 200, cyclin D1 60, cdk4 760. C3H-10T1/2: Ctr-Ad 300, cyclin D1 30, cdk4 300, dl520 200. l.c.  =  loading control.

Mentions: Conceivably, the DNA damage observed in reactivated myotubes might be linked to the specific means used to trigger cell cycle reentry. To exclude this possibility, we reactivated both myotubes and quiescent fibroblasts by infection with the mutant adenovirus, dl520 [17], or expression of cyclin D1 and cdk4 [9]. As shown in Fig. 7, myotubes reactivated in either way displayed strong increases in γ-H2AX levels, while fibroblasts brought into the cell cycle by the same means did not.


DNA replication is intrinsically hindered in terminally differentiated myotubes.

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

Phosphorylation of histone H2AX in myotubes but not fibroblasts reactivated by different strategies.C2C12 myotubes and quiescent C3H-10T1/2 fibroblasts were infected with cell cycle-reactivating viruses expressing either E1A (dl520) or cyclin D1 and cdk4 (D1/K4). The cells were also infected with an empty control virus (Ctr-Ad) or mock-infected. In addition, fibroblasts were reactivated with 10% FBS or treated with Phleomycin to provide a positive control for DNA damage. γ-H2AX was analyzed by western blotting. Multiplicities of infection as follows. Myotubes: Ctr-Ad 400, dl520 200, cyclin D1 60, cdk4 760. C3H-10T1/2: Ctr-Ad 300, cyclin D1 30, cdk4 300, dl520 200. l.c.  =  loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011559-g007: Phosphorylation of histone H2AX in myotubes but not fibroblasts reactivated by different strategies.C2C12 myotubes and quiescent C3H-10T1/2 fibroblasts were infected with cell cycle-reactivating viruses expressing either E1A (dl520) or cyclin D1 and cdk4 (D1/K4). The cells were also infected with an empty control virus (Ctr-Ad) or mock-infected. In addition, fibroblasts were reactivated with 10% FBS or treated with Phleomycin to provide a positive control for DNA damage. γ-H2AX was analyzed by western blotting. Multiplicities of infection as follows. Myotubes: Ctr-Ad 400, dl520 200, cyclin D1 60, cdk4 760. C3H-10T1/2: Ctr-Ad 300, cyclin D1 30, cdk4 300, dl520 200. l.c.  =  loading control.
Mentions: Conceivably, the DNA damage observed in reactivated myotubes might be linked to the specific means used to trigger cell cycle reentry. To exclude this possibility, we reactivated both myotubes and quiescent fibroblasts by infection with the mutant adenovirus, dl520 [17], or expression of cyclin D1 and cdk4 [9]. As shown in Fig. 7, myotubes reactivated in either way displayed strong increases in γ-H2AX levels, while fibroblasts brought into the cell cycle by the same means did not.

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