Development of DNA-based radiopharmaceuticals carrying Auger-electron emitters for antigene radiotherapy

Sedelnikova, Olga A.; Luu, Andrew; Karamychev, Valeri N.; Panyutin, Igor G.; Neumann, Ronald D.

doi:10.1016/s0360-3016(00)01486-3

Cited by 25 publications

(12 citation statements)

References 15 publications

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“…The site-directed and sequence-specific induction of DSB in a DNA target sequence, with a breakage percentage of 40% after 143 days (∼ 1.5 × 10 9 decays/ng target DNA) as shown in Figure 4 is in good agreement with the results of Sedelnikova et al (2001), who described a percentage of 50% TFO-induced DSB in plasmid and genomic DNA after 136 days of decay accumulation. Additionally, we showed that no DSB occur at similar accumulated decays when the target DNA is exposed to non-specific I-125 labeled TFO (Figure 4; lane 2), demonstrating that the DNA damage caused by non-DNA bound I-125 can be neglected.…”

Section: Discussionsupporting

confidence: 88%

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Cytotoxic effects and specific gene expression alterations induced by I-125-labeled triplex-forming oligonucleotides

Dahmen

Kriehuber

2012

International Journal of Radiation Biology

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Purpose:Triplex-forming oligonucleotides (TFO) bind to the DNA double helix in a sequence-specific manner. Therefore, TFO seem to be a suitable carrier for Auger electron emitters to damage exclusively targeted DNA sequences, e.g., in tumor cells. We studied the influence of I-125 labeled TFO with regard to cell survival and induction of DNA double-strand breaks (DSB) using TFO with different genomic targets and target numbers. Furthermore, the ability of TFO to alter the gene expression of targeted genes was examined.Materials and methods:TFO were labeled with I-125 using the primer extension method. DNA triplex formation and sequence-specific DSB were demonstrated in vitro. Cell survival was analyzed by colony-forming assay and DNA damage was assessed by microscopic quantification of protein 53 binding protein 1 (53BP1) foci in the human squamous carcinoma cell line II (SCL-II). Quantitative real-time polymerase-chain-reaction (qRT-PCR) was performed to analyze gene expression alterations.Results:The sequence-specific induction of a single DSB in a 1695 bp long DNA double stranded fragment was demonstrated in vitro. I-125-labeled TFO binding to single and multiple targets were shown to induce a pronounced decrease in cell survival and an increase of DSB. TFO targeting multiple sites differing in the total target number showed a significant different cell killing per decay that is also in good accordance with the observed induction of DSB. Single gene targeting I-125-labeled TFO significantly decreased cell survival and altered gene expression in the targeted gene.Conclusions:I-125-labeled TFO enable specific targeting of DNA in vitro as well as in a cellular environment and thus induce sequence-specific complex DNA lesions. Therefore I-125-labeled TFO might be a very useful tool for basic DNA repair research.

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Section: Discussionsupporting

confidence: 88%

“…The breakage efficiency in the target fragment was calculated as the ratio of the sum of the EtBr band intensities of the two breakage fragments to the sum of the intensities of all three fragments (Sedelnikova et al 2001) leading to a percentage of breaks of ∼40% after 143 days of decay accumulation (∼ 1.5 × 10 9 decays/ng target DNA).…”

Section: Resultsmentioning

confidence: 99%

Cytotoxic effects and specific gene expression alterations induced by I-125-labeled triplex-forming oligonucleotides

Dahmen

Kriehuber

2012

International Journal of Radiation Biology

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“…Yet oligonucleotide-based inhibitors offer high binding affinities and specificity, which, if combined with the radiotoxicity of short-range emitters such as 111 In, have the potential to generate gene-targeted radiotherapeutics. This concept was proposed by Neumann and colleagues, who examined sequence-specific triplex-forming oligonucleotides to carry 125 I for this purpose (45,46). The use of modified nucleic acids confers improved characteristics on therapeutic oligonucleotides.…”

Section: Discussionmentioning

confidence: 99%

Radiolabeled Oligonucleotides Targeting the RNA Subunit of Telomerase Inhibit Telomerase and Induce DNA Damage in Telomerase-Positive Cancer Cells

et al. 2019

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Telomerase is expressed in the majority (>85%) of tumors, but has restricted expression in normal tissues. Long-term telomerase inhibition in malignant cells results in progressive telomere shortening and reduction in cell proliferation. Here we report the synthesis and characterization of radiolabeled oligonucleotides that target the RNA subunit of telomerase, hTR, simultaneously inhibiting enzymatic activity and delivering radiation intracellularly. Oligonucleotides complementary (Match) and noncomplementary (Scramble or Mismatch) to hTR were conjugated to diethylenetriaminepentaacetic dianhydride (DTPA), allowing radiolabeling with the Auger electron-emitting radionuclide indium-111 (111 In). Match oligonucleotides inhibited telomerase activity with high potency, which was not observed with Scramble or Mismatch oligonucleotides. DTPA-conjugation and 111 In-labeling did not change telomerase inhibition. In telomerase-positive cancer cells, unlabeled Match oligonucleotides had no effect on survival, however, 111 In-labeled Match oligonucleotides significantly reduced clonogenic survival and upregulated the DNA damage marker gH2AX. Minimal radiotoxicity and DNA damage was observed in telomerase-negative cells exposed to 111 In-Match oligonucleotides. Match oligonucleotides localized in close proximity to nuclear Cajal bodies in telomerasepositive cells. In comparison with Match oligonucleotides, 111 In-Scramble or 111 In-Mismatch oligonucleotides demonstrated reduced retention and negligible impact on cell survival. This study indicates the therapeutic activity of radiolabeled oligonucleotides that specifically target hTR through potent telomerase inhibition and DNA damage induction in telomerase-expressing cancer cells and paves the way for the development of novel oligonucleotide radiotherapeutics targeting telomerase-positive cancers. Significance: These findings present a novel radiolabeled oligonucleotide for targeting telomerase-positive cancer cells that exhibits dual activity by simultaneously inhibiting telomerase and promoting radiation-induced genomic DNA damage.

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“…Incorporated into DNA, the decay of 125 I produces DSB localized mostly within one turn of the double-helix around the decay site (10 bp) with an efficiency of 0.8 DSB/ decay. This extremely short range of radiodamage produced by 125 I led to the idea of targeting this Auger electron emitter to specific genes within genomic or plasmid DNA (29).…”

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confidence: 99%

Repair of Sequence-specific 125I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts

Odersky

Panyutin²,

Panyutin³

et al. 2002

Journal of Biological Chemistry

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In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of doublestrand break (DSB) repair. Rejoining of DSB produced by decay of 125 I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the 125 I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin 125 I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the 125 I-TFO within its target site. We show that NHEJ of 125 I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after 125 I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.Mammalian genomes constantly suffer a variety of types of damage, of which double-strand breaks (DSB) 1 are considered the most dangerous. DSB may arise spontaneously in the cell or may be induced by exogenous agents, such as ionizing radiation. The estimation that mammalian cells suffer at least 10 spontaneous DSB/day suggests that efficient repair of DSB is critical for cell survival (1). Failure to do so can result in deleterious genomic rearrangements, cell cycle arrest, or cell death.Recent studies have revealed that DSB in the genomes of higher eukaryotes can be repaired by at least three different pathways (2): (i) Homologous recombination repair, the most accurate process, is able to restore the original sequence at the break. Because of its strict dependence on extensive sequence homology, this mechanism is suggested to be active mainly during the S and G 2 phases of the cell cycle (3, 4). (ii) Singlestranded annealing is another homology-dependent but less accurate process that can repair DSB between direct repeats and thereby produces mainly interstitial deletions (4). (iii) Nonhomologous DNA end joining (NHEJ) comprises at least two different processes (5). The major and best investigated NHEJ pathway depends on the Ku70/80 heterodimer, the catalytic subunit of the DNA-dependent protein kinase, DNA ligase IV, and its essential co-factor XRCC4 (6, 7). In contrast to homologous recombination repair and single-stranded annealing, NHEJ can operate in the absence of sequence homology (although short sequence homologies, so-called microhomologies, may facilitate the process) and is able to rejoin broken ends directly (2). This process is supposed to occur mainly in the G 0 and G 1 phases of th...

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Development of DNA-based radiopharmaceuticals carrying Auger-electron emitters for antigene radiotherapy

Cited by 25 publications

References 15 publications

Cytotoxic effects and specific gene expression alterations induced by I-125-labeled triplex-forming oligonucleotides

Cytotoxic effects and specific gene expression alterations induced by I-125-labeled triplex-forming oligonucleotides

Radiolabeled Oligonucleotides Targeting the RNA Subunit of Telomerase Inhibit Telomerase and Induce DNA Damage in Telomerase-Positive Cancer Cells

Repair of Sequence-specific 125I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts

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