Abstract
Terminally differentiated cells are defined by their inability to proliferate. When forced to re-enter the cell cycle, they generally cannot undergo long-term replication. Our previous work with myotubes has shown that these cells fail to proliferate because of their intrinsic inability to complete DNA replication. Moreover, we have reported pronounced modifications of deoxynucleotide metabolism during myogenesis. Here we investigate the causes of incomplete DNA duplication in cell cycle-reactivated myotubes (rMt). We find that rMt possess extremely low levels of thymidine triphosphate (dTTP), resulting in very slow replication fork rates. Exogenous administration of thymidine or forced expression of thymidine kinase increases deoxynucleotide availability, allowing extended and faster DNA replication. Inadequate dTTP levels are caused by selective, differentiation-dependent, cell cycle-resistant suppression of genes encoding critical synthetic enzymes, chief among which is thymidine kinase 1. We conclude that lack of dTTP is at least partially responsible for the inability of myotubes to proliferate and speculate that it constitutes an emergency barrier against unwarranted DNA replication in terminally differentiated cells.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cell Cycle / drug effects*
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Cell Cycle / genetics
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Cell Differentiation / genetics
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Cyclin-Dependent Kinase Inhibitor p21 / genetics
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Cyclin-Dependent Kinase Inhibitor p21 / metabolism
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Cyclin-Dependent Kinase Inhibitor p27 / genetics
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Cyclin-Dependent Kinase Inhibitor p27 / metabolism
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DNA Replication / drug effects*
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Deoxycytosine Nucleotides / metabolism
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Gene Expression Regulation
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Histones / genetics
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Histones / metabolism
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Mice
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Muscle Development / genetics
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Muscle Fibers, Skeletal / cytology
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Muscle Fibers, Skeletal / drug effects*
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Muscle Fibers, Skeletal / metabolism
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Primary Cell Culture
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Satellite Cells, Skeletal Muscle / cytology
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Satellite Cells, Skeletal Muscle / drug effects*
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Satellite Cells, Skeletal Muscle / metabolism
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Thymidine / pharmacology*
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Thymidine Kinase / genetics*
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Thymidine Kinase / metabolism
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Thymidine Monophosphate / metabolism
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Thymine Nucleotides / deficiency*
Substances
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Cyclin-Dependent Kinase Inhibitor p21
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Deoxycytosine Nucleotides
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Histones
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Thymine Nucleotides
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gamma-H2AX protein, mouse
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Cyclin-Dependent Kinase Inhibitor p27
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2'-deoxycytidine 5'-triphosphate
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Thymidine Monophosphate
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thymidine kinase 2
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Thymidine Kinase
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thymidine kinase 1
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thymidine 5'-triphosphate
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Thymidine