Differentiation-Induced Radioresistance in Muscle Cells

Abstract

DNA damage induces cell cycle arrest and DNA repair or apoptosis in proliferating cells. Terminally differentiated cells are permanently withdrawn from the cell cycle and partly resistant to apoptosis. To investigate the effects of genotoxic agents in postmitotic cells, we compared DNA damage-activated responses in mouse and human proliferating myoblasts and their differentiated counterparts, the myotubes. DNA double-strand breaks caused by ionizing radiation (IR) induced rapid activating autophosphorylation of ataxia-teleangiectasia-mutated (ATM), phosphorylation of histone H2AX, recruitment of repair-associated proteins MRE11 and Nbs1, and activation of Chk2 in both myoblasts and myotubes. However, IR-activated, ATM-mediated phosphorylation of p53 at serine 15 (human) or 18 (mouse) [Ser15(h)/18(m)], and apoptosis occurred in myoblasts but was impaired in myotubes. This phosphorylation could be enforced in myotubes by the anthracycline derivative doxorubicin, leading to selective activation of proapoptotic genes. Unexpectedly, the abundance of autophosphorylated ATM was indistinguishable after exposure of myotubes to IR (10 Gy) or doxorubicin (1 μM/24 h) despite efficient phosphorylation of p53 Ser15(h)/18(m), and apoptosis occurred only in response to doxorubicin. These results suggest that radioresistance in myotubes might reflect a differentiation-associated, pathway-selective blockade of DNA damage signaling downstream of ATM. This mechanism appears to preserve IR-induced activation of the ATM-H2AX-MRE11/Rad50/Nbs1 lesion processing and repair pathway yet restrain ATM-p53-mediated apoptosis, thereby contributing to life-long maintenance of differentiated muscle tissues.

We thank K. H. Vousden, J. Petrini, S. Elledge, C Poizat, M. Crescenzi, and S. Soddu for providing critical reagents (antibodies, plasmids, adenoviruses, and probes) used in this work; Y. Xu kindly provided MEFs from p53wt, p53 null, and p53 Ser18ala knock-in mice; and M. Rudnicki kindly provided primary satellite cells from wild-type and p53 null mice. We are also grateful to J. Falck for stimulating discussion and M. Crescenzi, C. Poizat, and E. Borges for critically reading the manuscript.

This work was supported by the Danish Cancer Society, European Union, and Marie Curie Individual Fellowship QLGA-CT-2000-52052 (L.L.). C.S. is supported by a Telethon fellowship and the Sbarro Health Research Organization. P.L.P. was partially supported by an American Heart Association grant and a Compagnia San Paolo di Torino grant to Dulbecco Telethon Institute.