title: Imaging DNA damage in vivo using gammaH2AX-targeted immunoconjugates.
abstract: DNA damage responses (DDR) occur during oncogenesis and therapeutic responses to DNA damaging cytotoxic drugs. Thus, a real-time method to image DNA damage in vivo would be useful to diagnose cancer and monitor its treatment. Toward this end, we have developed fluorophore- and radioisotope-labeled immunoconjugates to target a DDR signaling protein, phosphorylated histone H2A variant H2AX (γH2AX), which forms foci at sites of DNA double-strand breaks. Anti-γH2AX antibodies were modified by the addition of diethylenetriaminepentaacetic acid (DTPA) to allow (111)In labeling or the fluorophore Cy3. The cell-penetrating peptide Tat (GRKKRRQRRRPPQGYG) was also added to the immunoconjugate to aid nuclear translocation. In irradiated breast cancer cells, confocal microscopy confirmed the expected colocalization of anti-γH2AX-Tat with γH2AX foci. In comparison with nonspecific antibody conjugates, (111)In-anti-γH2AX-Tat was retained longer in cells. Anti-γH2AX-Tat probes were also used to track in vivo DNA damage, using a mouse xenograft model of human breast cancer. After local X-ray irradiation or bleomycin treatment, the anti-γH2AX-Tat probes produced fluorescent and single photon emission computed tomography signals in the tumors that were proportionate to the delivered radiation dose and the amount of γH2AX present. Taken together, our findings establish the use of radioimmunoconjugates that target γH2AX as a noninvasive imaging method to monitor DNA damage, with many potential applications in preclinical and clinical settings.
authors.affiliation: CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom.
authors.forename: Bart
authors.initials: B
authors.lastname: Cornelissen
authors.forename: Veerle
authors.initials: V
authors.lastname: Kersemans
authors.forename: Sonali
authors.initials: S
authors.lastname: Darbar
authors.forename: James
authors.initials: J
authors.lastname: Thompson
authors.forename: Ketan
authors.initials: K
authors.lastname: Shah
authors.forename: Kate
authors.initials: K
authors.lastname: Sleeth
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hill
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Vallis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000008:administration & dosage / Q000276:immunology; D001943:Breast Neoplasms / Q000235:genetics* / Q000628:therapy; D002232:Carbocyanines / Q000008:administration & dosage; D045744:Cell Line, Tumor; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000032:analysis*; D005260:Female; D015696:Gene Products, tat / Q000008:administration & dosage / Q000493:pharmacokinetics; D006657:Histones / Q000276:immunology*; D006801:Humans; D018796:Immunoconjugates / Q000276:immunology* / Q000493:pharmacokinetics; D007205:Indium Radioisotopes / Q000008:administration & dosage; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D004369:Pentetic Acid / Q000008:administration & dosage / Q000493:pharmacokinetics; D014183:Transplantation, Heterologous
chemical_list: D000906:Antibodies; D002232:Carbocyanines; D004273:DNA, Neoplasm; D015696:Gene Products, tat; C468783:H2AX protein, human; D006657:Histones; D018796:Immunoconjugates; D007205:Indium Radioisotopes; C089187:cyanine dye 3; D004369:Pentetic Acid