Development of DNA-based radiopharmaceuticals carrying Auger-electron emitters for anti-gene radiotherapy.
Targeting of radiation damage to specific DNA sequences is the essence of antigene radiotherapy. This technique also provides a tool to study molecular mechanisms of DNA repair on a defined, single radiodamaged site. We achieved such sequence-specific radiodamage by combining the highly localized DNA damage produced by the decay of Auger-electron-emitters such as 125I with the sequence-specific action of triplex-forming oligonucleotides (TFO). TFO complementary to polypurine-polypyrimidine regions of human genes were synthesized and labeled with 125I-dCTP by the primer extension method. 125I-TFO were delivered into cells with several delivery systems. In addition, human enzymes capable of supporting DNA single-strand-break repair were isolated and assessed for their role in the repair of this lesion. Also, the mutagenicity and repairability of 125I-TFO-induced double strand breaks (DSB) were assessed by repair of a plasmid possessing a site-specific DSB lesion. Using plasmids containing target polypurine-polypyrimidine tracts, we obtained the fine structure of sequence-specific DNA breaks produced by decay of 125I with single-nucleotide resolution. We showed that the designed 125I-TFO in nanomolar concentrations could bind to and introduce double-strand breaks into the target sequences in situ, i.e., within isolated nuclei and intact digitonin-permeabilized cells. We also showed 125I-TFO-induced DSB to be highly mutagenic lesions resulting in a mutation frequency of nearly 80%, with deletions comprising the majority of mutations. The results obtained demonstrate the ability of 125I-TFO to target specific sequences in their natural environment--within eucaryotic nucleus. Repair of 125I-TFO-induced DNA damage should typically result in mutagenic gene inactivation.
Panyutin
I G
IG
Department of Nuclear Medicine, National Institutes of Health, Bethesda, MD 20892-1180, USA.
Winters
T A
TA
Feinendegen
L E
LE
Neumann
R D
RD
D000818:Animals; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair; D006801:Humans; D007457:Iodine Radioisotopes / Q000494:pharmacology / Q000627:therapeutic use; D009369:Neoplasms / Q000000981:diagnostic imaging / Q000235:genetics / Q000532:radiotherapy; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000494:pharmacology* / Q000627:therapeutic use; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000494:pharmacology* / Q000627:therapeutic use
D007457:Iodine Radioisotopes; D009841:Oligonucleotides; D019275:Radiopharmaceuticals; D004247:DNA