Antigene radiotherapy: targeted radiodamage with 125i-labeled triplex-forming oligonucleotides.
Antigene radiotherapy is based upon damaging selected genes by a high dose of radiation from radionuclides delivered to this gene by a sequence-specific DNA-binding molecule. Here we describe our recent trials of antigene radiotherapy using the human mdr1 gene over-expressed in KB-V1 cells as a model. As a delivery molecule, we used a triplex-forming oligonucleotide (TFO) with a binding site in intron 14 of mdr1. This TFO was labeled with an Auger-electron-emitting radionuclide 125I. Decay of 125I releases a shower of low energy electrons that produce DNA strand breaks mostly within 10 bp from the decay site. Targeting in situ was assessed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. Double-strand breaks in the target sequence were detected in purified nuclei and digitonin-permeabilized cells, but not in the intact cells when TFO were delivered with liposomes. On the basis of these observations we hypothesized that there are cytoplasmic factors that bind such TFO and deliver them into the nucleus, but do not release them inside the nucleus, thus preventing TFO from binding their genomic targets. To test this hypothesis we (i) delivered TFO along with an excess of unlabeled oligonucleotide with an arbitrary sequence ("ballast") and (ii) conjugated TFO with a nuclear localization sequence peptide (NLS). We have found that TFO/NLS conjugates cleaved the target in a concentration-dependent manner regardless of the presence of the "ballast" oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the "ballast." These results may provide a new insight into the mechanism of intracellular transport of oligonucleotides.
Panyutin
I G
IG
Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA. ogorp@helix.nih.gov
Sedelnikova
O A
OA
Karamychev
V N
VN
Neumann
R D
RD
D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects / Q000528:radiation effects; D004247:DNA / Q000187:drug effects* / Q000528:radiation effects*; D019450:Genes, MDR / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D007457:Iodine Radioisotopes / Q000494:pharmacology*; D009369:Neoplasms / Q000188:drug therapy* / Q000532:radiotherapy*; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*
D007457:Iodine Radioisotopes; D009838:Oligodeoxyribonucleotides; D004247:DNA