Inhibition of repair of radiation-induced DNA damage enhances gene expression from replication-defective adenoviral vectors.
Radiation has been shown to up-regulate gene expression from adenoviral vectors in previous studies. In the current study, we show that radiation-induced dsDNA breaks and subsequent signaling through the mitogen-activated protein kinase (MAPK) pathway are responsible, at least in part, for this enhancement of transgene expression both in vitro and in vivo. Inhibitors of ataxia-telangiectasia-mutated, poly(ADP-ribose) polymerase-mutated, and DNA-dependent protein kinase (DNA-PK)-mediated DNA repair were shown to maintain dsDNA breaks (gammaH2AX foci) by fluorescence-activated cell sorting and microscopy. Inhibition of DNA repair was associated with increased green fluorescent protein (GFP) expression from a replication-defective adenoviral vector (Ad-CMV-GFP). Radiation-induced up-regulation of gene expression was abrogated by inhibitors of MAPK (PD980059 and U0126) and phosphatidylinositol 3-kinase (LY294002) but not by p38 MAPK inhibition. A reporter plasmid assay in which GFP was under the transcriptional control of artificial Egr-1 or cytomegalovirus promoters showed that the DNA repair inhibitors increased GFP expression only in the context of the Egr-1 promoter. In vivo administration of a water-soluble DNA-PK inhibitor (KU0060648) was shown to maintain luciferase expression in HCT116 xenografts after intratumoral delivery of Ad-RSV-Luc. These data have important implications for therapeutic strategies involving multimodality use of radiation, targeted drugs, and adenoviral gene delivery and provide a framework for evaluating potential advantageous combinatorial effects.
Hingorani
Mohan
M
Targeted Therapy Team, The Institute of Cancer Research, Queen Mary's School of Medicine and Dentistry, Royal Marsden Hospital, London, United Kingdom.
White
Christine L
CL
Merron
Andrew
A
Peerlinck
Inge
I
Gore
Martin E
ME
Slade
Andrew
A
Scott
Simon D
SD
Nutting
Christopher M
CM
Pandha
Hardev S
HS
Melcher
Alan A
AA
Vile
Richard G
RG
Vassaux
Georges
G
Harrington
Kevin J
KJ
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D000818:Animals; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D004273:DNA, Neoplasm / Q000528:radiation effects; D051766:Early Growth Response Protein 1 / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000528:radiation effects; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D045325:HCT116 Cells; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000235:genetics*; D015398:Signal Transduction / Q000528:radiation effects; D015854:Up-Regulation / Q000528:radiation effects; D014779:Virus Replication
D004273:DNA, Neoplasm; D051766:Early Growth Response Protein 1; D049452:Green Fluorescent Proteins; D008156:Luciferases