Sequence‐specific targeting of IGF‐I and IGF‐IR genes by camptothecins

Abstract

We and others have clearly demonstrated that a topoisomerase I (Top1) inhibitor, such as camptothecin (CPT), coupled to a triplex-forming oligonucleotide (TFO) through a suitable linker can be used to cause site-specific cleavage of the targeted DNA sequence in in vitro models. Here we evaluated whether these molecular tools induce sequence-specific DNA damage in a genome context. We targeted the insulin-like growth factor (IGF)-I axis and in particular promoter 1 of IGF-I and intron 2 of type 1 insulin-like growth factor receptor (IGF-IR) in cancer cells. The IGF axis molecules represent important targets for anticancer strategies, because of their central role in oncogenic maintenance and metastasis processes. We chemically attached 2 CPT derivatives to 2 TFOs. Both conjugates efficiently blocked gene expression in cells, reducing the quantity of mRNA transcribed by 70-80%, as measured by quantitative RT-PCR. We confirmed that the inhibitory mechanism of these TFO conjugates was mediated by Top1-induced cleavage through the use of RNA interference experiments and a camptothecin-resistant cell line. In addition, induction of phospho-H2AX foci supports the DNA-damaging activity of TFO-CPT conjugates at specific sites. The evaluated conjugates induce a specific DNA damage at the target gene mediated by Top1.

Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

“…Previously, camptothecin-TFOs have been shown to trap topoisomerase I proximal to the triplex complex in cell nuclei 39 and induce site specific, genomic cleavage. 40 The present results for the first time demonstrate than camptothecin-PNA complexes may significantly increase gene correction rates. We suggest that the observed effect from camptothecin-PNA in the nuclear extract is connected to camptothecin interaction with topoisomerase I, which can lead to single strand breaks.…”

Sequence selectivity of the cleavage sites induced by topoisomerase I inhibitors: a molecular dynamics study

“…The CPT class of anticancer drugs targets only Top1 and the poisoning of Top1-DNA cleavage complexes occurs only at specific cleavage sites along the DNA chain. This sequence selectivity is a key step in preferentially directing the action of drugs onto specific genomic sequences of therapeutic interest ( 5–8 ). The sequence selectivity exhibited by Top1 inhibitors is ‘intrinsically’ dependent on the base sequences immediately preceding (−1) and following (+1) the cleavage site.…”

Replication-Fork Stalling and Processing at a Single Psoralen Interstrand Crosslink in Xenopus Egg Extracts

“…Triplex-forming oligonucleotides (TFO) conjugates are widely used to introduce DNA lesions at specific sites in plasmids or in genomic DNA [23] , [24] . Since triplex DNA alone has been reported to interfere with DNA repair [25] , [26] , we devised a method to eliminate the TFO moiety after introducing a psoralen crosslink at a specific site in the pTUC plasmid.…”