Targeting the human mdr1 gene by 125I-labeled triplex-forming oligonucleotides.
Antigene radiotherapy is our approach to targeting specific sites in the genome 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). As a model, we used the multidrug resistance gene (mdr1) overexpressed and amplified nearly 100 times in the human KB-V1 carcinoma cell line. Phosphodiester pyrrazolopyrimidine dG (PPG)-modified TFO complementary to the polypurine-polypyrimidine region of the mdr1 gene were synthesized and labeled with 125I-dCTP at the C5 position of two cytosines by the primer extension method. 125I-TFO were delivered into KB-V1 cells with several delivery systems. DNA from the 125I-TFO-treated cells was recovered and analyzed for sequence-specific cleavage in the mdr1 target by Southern hybridization. Experiments with plasmid DNA containing the mdr1 polypurine-polypyrimidine region and with purified genomic DNA confirmed the ability of the designed 125I-TFO to bind to and introduce double-strand breaks into the target sequence. We showed that 125I-TFO in nanomolar concentrations can recognize and cleave a target sequence in the mdr1 gene in situ, that is, within isolated nuclei and intact digitonin-permeabilized cells. Our results demonstrate the ability of 125I-TFO to target specific sequences in their natural environment, that is, within the eukaryotic nucleus. The nearly 100-fold amplification of the mdr1 gene in KB-V1 cells affords a very useful cell culture model for evaluation of methods to produce sequence-specific DNA double-strand breaks for gene-specific radiotherapy.
Sedelnikova
O A
OA
Department of Nuclear Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892-1180, USA.
Panyutin
I G
IG
Luu
A N
AN
Reed
M W
MW
Licht
T
T
Gottesman
M M
MM
Neumann
R D
RD
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000235:genetics; D002467:Cell Nucleus / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000008:administration & dosage / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D016503:Drug Delivery Systems; D018390:Gene Targeting; D005822:Genetic Vectors; D006801:Humans; D007457:Iodine Radioisotopes; D007624:KB Cells; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D007457:Iodine Radioisotopes; D016376:Oligonucleotides, Antisense; C051802:triplex DNA; D004247:DNA