Targeting the human mdr1 gene by I-125-labeled triplex-forming oligonucleotides

Antigene radiotherapy is our approach to targeting specific sites in the ge nome by combining the highly localized DNA damage produced by the decay of Auger electron emitters, such as I-125, With the sequence-specific action o f tripler-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 pyrrazolopyrinidine dG (BP G)-modified TFO complementary to the polypurine-polypyrimidine region of th e mdr1 gene were synthesized and labeled with I-125-dCTP at the C5 position of two cytosines by the primer extension method,I-125-TFO were delivered i nto KB-V1 cells with several delivery systems. DNA from the I-125-TFO-treat ed cells was recovered and analyzed for sequence-specific cleavage in the m dr1 target by Southern hybridization, Experiments with plasmid DNA containi ng the mdr1 polypurine-polypyrimidine region and with purified genomic DNA confirmed the ability of the designed I-125-TFO to bind to and introduce do uble-strand breaks into the target sequence. We showed that I-125-TFO in na nomolar concentrations can recognize and cleave a target sequence in the md r1 gene in situ, that is, within isolated nuclei and intact digitonin-perme abilized cells. Our results demonstrate the ability of I-125-TFO to target specific sequences in their natural environment, that is, within the eukary otic nucleus, The nearly 100-fold amplification of the mdr1 gene in KB-V1 c ells affords a very useful cell culture model for evaluation of methods to produce sequence-specific DNA double-strand breaks for gene-specific radiot herapy.