RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult.
Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (gammaH2AX) foci, which indicate DNA double-strand breaks. The formation of gammaH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak gammaH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these gammaH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce gammaH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.
Bosco
Emily E
EE
Department of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
Mayhew
Christopher N
CN
Hennigan
Robert F
RF
Sage
Julien
J
Jacks
Tyler
T
Knudsen
Erik S
ES
D000818:Animals; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects; D015536:Down-Regulation; D005347:Fibroblasts; D051379:Mice; D009153:Mutagens / Q000494:pharmacology*; D016160:Retinoblastoma Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D009153:Mutagens; D016160:Retinoblastoma Protein; D002945:Cisplatin; D002166:Camptothecin