Modified Oligonucleotides as Bona Fide Antagonists of Proteins Interacting with DNA

Abstract

The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report describes the design of potent DNA based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cellular transformation, human lung non-small carcinoma cells were treated with the DNA MeTase antagonists. DNA-binding proteins that regulate gene expression play an important biological role and are potentially attractive therapeutic targets. However, the study of their role in different physiological and pathological processes has been hindered by the lack of specific inhibitors. These proteins are especially appealing as drug targets because their ligand is a DNA sequence that can be identified by standard molecular biology techniques and can be synthesized and modified by well established chemistries (1). In addition, recent observations suggest that double-stranded DNA-based oligonucleotides bearing transcription factor recognition sites can be delivered into cells in culture and in vivo and exhibit pharmacological effects (2, 3). A major limitation of this approach is that oligonucleotide antagonists that are identical to the transcription factors cognate site act as stochiometric competitors. Therefore, very high intracellular concentrations are required to effectively engage all the transcription factor available in the cell at all times. An ideal DNA-binding protein antagonist should exhibit higher affinity to the protein than the cognate sequence and bear a slow off rate. In this report we have tested the hypothesis that DNA-based inhibitors of DNA-binding proteins that address these requirements could be developed, using the DNA methyltransferase enzyme (DNA MeTase) 1 as a model DNA-binding protein.Basic oncogenic pathways such as the Ras-Jun signaling pathway have been shown to up-regulate DNA MeTase mRNA (4 -6) and the hyperactivation of DNA MeTase observed in many cancer cells (7,8) occurs in parallel with the development of the aberrant patterns of DNA methylation that these cells exhibit (9, 10). A number of studies suggest that the hyperactivation of DNA MeTase plays a causal role in oncogenesis. For example, the intraperitoneal injection of antisense oligonucleotide to DNA MeTase mRNA into LAF/1 mice bearing tumors derived from the syngeneic tumor cell line Y1 (11) inhibits tumor growth; and in vivo reduction of DNA MeTase levels by either 5-azaCdR treatment or by bearing one mutated allele of DNA MeTase reduces the frequency of appearance of intestinal adenomas in the Min mouse bearing a mutation in the adenomatosis polyposis coli gene (12).Uncovering the biological role of DNA MeTase in vivo requires the a...

Epigenomic Stress Response

“…The intra-S-phase arrest of replication following MG88 treatment possibly protects A549 cells from genome-wide loss of methylation. We have previously observed that hemimethylated inhibitors of DNMT1 that inhibit DNA replication also cause only limited demethylation of DNA (33).…”

“…Inhibition of DNMT1 mRNA by MG88 was also confirmed in a gene array expression analysis presented in Table II. (33). One possible explanation for the reduced cell growth is that knockdown of DNMT1 results in inhibition of firing of DNA replication origins (17).…”

“…Agents such as MG88, that reduce the availability of DNMT1 at the replication fork, are strong inhibitors of cell growth and should be effective in inhibiting tumor growth but will not cause extensive demethylation (33). There might be an advantage for therapeutic agents that do not cause extensive demethylation, since extensive hypomethylation has been previously associated with metastasis (54) and possibly induction of silenced repetitive elements (55,56).…”

Reactivating the expression of methylation silenced genes in human cancer

“…More recently, two alternative inhibitors of DNMT1 have been described (Bigey et al, 1999;Fournel et al, 1999). These inhibitors are of interest because they circumvent a number of the potential drawbacks associated with 5-aza-CdR treatment, including the requirement for DNA incorporation, the formation of toxic protein-DNA adducts, and the lack of selectivity for the different DNA methyltransferase enzymes.…”

“…A second novel method for inhibiting DNMT1 consists of hairpin-structured oligonucleotide substrate mimics. These inhibitors act competitively and inhibit purified DNMT1 at nanomolar concentrations in vitro (Bigey et al, 1999). However, for reasons that are unclear, the hairpin inhibitors of DNMT1 are unable to induce DNA methylation changes or activate methylation-silenced genes in treated cells (Bigey et al, 1999).…”