title: Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.
abstract: Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.
authors: Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, United States of America. mhirsch@email.unc.edu
authors: Matthew L
authors: ML
authors: Hirsch
authors: B Matthew
authors: BM
authors: Fagan
authors: Raluca
authors: R
authors: Dumitru
authors: Jacquelyn J
authors: JJ
authors: Bower
authors: Swati
authors: S
authors: Yadav
authors: Matthew H
authors: MH
authors: Porteus
authors: Larysa H
authors: LH
authors: Pevny
authors: R Jude
authors: RJ
authors: Samulski
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D002478:Cells, Cultured; D004249:DNA Damage / Q000235:genetics; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000378:metabolism* / Q000473:pathology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; C468783:H2AX protein, human; D006657:Histones; D012333:RNA, Messenger; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53