Study of DNA replication in mammalian terminally differentiated cells upon cell cycle reactivation

Terminally differentiated (TD) cells are characterized by the permanent inability to proliferate. In culture, skeletal muscle myotubes (Mt), a model system of TD, can be forced to reenter the cell cycle by several means, including expression of adenovirus E1A, overexpression of cyclin D1 and CDK4/6, or depletion of CDK inhibitors. Nonetheless, cell cycle reactivation in Mt never results in a long-term survival and effective proliferation. Reactivated Mt suffer heavy DNA damage, and die by apoptosis or mitotic catastrophe. Critically, they are not able to fully duplicate their DNA. The purpose of this thesis is to understand the molecular bases that prevent Mt to lead a regular and complete replication. We investigated through functional (i.e., biochemical) problems and structural obstacles. Functionally, we found that Mt attempt to duplicate their DNA with extremely low levels of deoxythimidine triphosphate (dTTP). This is explained by failure to upregulate thymidine kinase (TK) upon cell cycle reentry. Exogenous administration of deoxythymidine or expression of TK increased DNA synthesis, though it never attained completion. We then used Xenopus laevis egg extracts (XEE) to study DNA replication in nuclei from Mt and proliferating (P) or quiescent (Q) myoblasts. XEE are able to complement any functional defect, highlighting structural obstacles. Maximal DNA replication in Mt nuclei was strikingly lower than in those from Q and P myoblasts, revealing the presence of a barrier that prevent Mt to replicate the whole genome. To investigate at which level of nuclear complexity this obstacle lies, we studied replication kinetics in naked and nucleosome-assembled DNA from Mt, in comparison with similar samples from P myoblasts. Together, all results showed that both functional and structural obstacles prevent full DNA duplication in myotubes. Furthermore, they suggest that hitherto unexplored peculiarities of their chromatin are ultimately responsible for the inability of these cells to proliferate.