Valeri Metelev

STAT3 inhibitors for cancer therapy: Have all roads been explored?

The signal transducer and activator of transcription STAT3 is a transcription factor which plays a key role in normal cell growth and is constitutively activated in about 70% of solid and hematological cancers. Activated STAT3 is phosphorylated on tyrosine and forms a dimer through phosphotyrosine/src homology 2 (SH2) domain interaction. The dimer enters the nucleus via interaction with importins and binds target genes. Inhibition of STAT3 results in the death of tumor cells, this indicates that it is a valuable target for anticancer strategies; a view that is corroborated by recent findings of activating mutations within the gene. Yet, there is still only a small number of STAT3 direct inhibitors; in addition, the high similarity of STAT3 with STAT1, another STAT family member mostly oriented toward apoptosis, cell death and defense against pathogens, requires that STAT3-inhibitors have no effect on STAT1. Specific STAT3 direct inhibitors consist of SH2 ligands, including G quartet oligodeoxynucleotides (ODN) and small molecules, they induce cell death in tumor cells in which STAT3 is activated. STAT3 can also be inhibited by decoy ODNs (dODN), which bind STAT3 and induce cell death. A specific STAT3 dODN which does not interfere with STAT1-mediated interferon-induced cell death has been designed pointing to the STAT3 DBD as a target for specific inhibition. Comprehensive analysis of this region is in progress in the laboratory to design DBD-targeting STAT3 inhibitors with STAT3/STAT1 discriminating ability.

Addressed fragmentation of RNA molecules

Many problems of molecular biology require specific fragmentation of nucleic acids. For DNAs this can be done with the help of specific endonucleases (restriction enzymes). However, no reliable and efflcient methods are known for strictly specific cleavage of RNA molecules. This paper describes an experimental approach to addressed fragmentation of polyribonucleotides. It is known that RNase H splits RNA in RNA-DNA heteroduplexes [l] or in the heteroduplexes made of monotonous synthetic polyribonucleotides and

Cell death in NF-κB-dependent tumour cell lines as a result of NF-κB trapping by linker-modified hairpin decoy oligonucleotide

The transcription factor NF‐κB is frequently activated in cancer, and is therefore a valuable target for cancer therapy. Decoy oligodeoxynucleotides (ODNs) inhibit NF‐κB by preventing its binding to the promoter region of target genes. Few studies have used NF‐κB‐targeting with ODNs in cancer. Using a hairpin NF‐κB‐decoy ODN we found that it induced growth inhibition and cell death in NF‐κB‐dependent tumour cell lines. The ODN colocalized with the p50 subunit of NF‐κB in cells and directly interacted with it in nuclear extracts. In TNFα‐treated cells the ODN and the p50 subunit were found in the cytoplasm suggesting that the complex did not translocate to the nucleus. Transcriptional activity of NF‐κB was efficiently inhibited by the ODN, whereas a scrambled ODN was without effect on transcription. Thus, ODN‐mediated inhibition of NF‐κB can efficiently promote cell death in cancer cells providing a potentially powerful approach to tumour growth inhibition.

Specific conjugation of DNA binding proteins to DNA templates through thiol–disulfide exchange

The double‐stranded oligodeoxyribonucleotides with single internucleotide disulfide linkages were successfully used for covalent trapping of cysteine containing protein. In particular, an efficient conjugation of DNA methyltransferase SsoII to sequence‐specific decoys was demonstrated. The obtained results assume that synthetic oligodeoxyribonucleotides bearing a new trapping site can be used as new tools to study and manipulate biological systems.

Efficient killing of SW480 colon carcinoma cells by a signal transducer and activator of transcription (STAT) 3 hairpin decoy oligodeoxynucleotide – interference with interferon‐γ‐STAT1‐mediated killing

The signal transducers and activators of transcription (STATs) convey signals from the membrane to the nucleus in response to cytokines or growth factors. STAT3 is activated in response to cytokines involved mostly in cell proliferation; STAT1 is activated by cytokines, including interferon‐γ, involved in defence against pathogens and the inhibition of cell proliferation. STAT3, which is frequently activated in tumour cells, is a valuable target with respect to achieving inhibition of tumour cell proliferation. Indeed, its inhibition results in cell death. We previously observed that inhibition of the transcription factor nuclear factor‐κB, a key regulator of cell proliferation, with decoy oligodeoxynucleotides results in cell death. We used a similar approach for STAT3. A hairpin STAT3 oligodeoxynucleotide was added to a colon carcinoma cell line in which it induced cell death as efficiently as the STAT3 inhibitor stattic. The hairpin STAT3 oligodeoxynucleotide co‐localized with STAT3 within the cytoplasm, prevented STAT3 localization to the nucleus, blocked a cyclin D1 reporter promoter and associated with STAT3 in pull‐down assays. However, the same cells were efficiently killed by interferon‐γ. This effect was counteracted by the STAT3 oligodeoxynucleotide, which was found to efficiently inhibit STAT1. Thus, although it can inhibit STAT3, the hairpin STAT3 oligodeoxynucleotide appears also to inhibit STAT1‐mediated interferon‐γ cell killing, highlighting the need to optimize STAT3‐targeting oligodeoxynucleotides.

Influence of probe structure on unique (regiospecific) cleavage of RNA by RNase H.

Chimeric oligo(ribo‐deoxyribo)nucleotides with an internucleotide pyrophosphate bond are novel probes for regiospecific hydrolysis of RNA by RNase H. It has been shown that the use of d(TGTGTAT)ppGCCAU leads to unique hydrolysis of the TMV RNA fragment pAAUGGCAUACAC between C10 and A11.

Hairpin-shaped DNA duplexes with disulfide bonds in sugar-phosphate backbone as potential DNA reagents for crosslinking with proteins

Convenient approaches were described to incorporate ‐OP(=O)O−‐SS‐O−(O=)PO‐ bridges in hairpin‐shaped DNA duplexes instead of regular phosphodiester linkages: (i) H2O2‐ or 2,2′‐dipyridyldisulfide‐mediated coupling of 3′‐ and 5′‐thiophosphorylated oligonucleotides on complementary template and (ii) more selective template‐guided autoligation of a preactivated oligonucleotide derivative with an oligomer carrying a terminal thiophosphoryl group. Dithiothreitol was found to cleave completely modified internucleotide linkage releasing starting oligonucleotides. The presence of complementary template as an intrinsic element of the molecule protects the hairpin DNA analog from spontaneous exchange of disulfide‐linked oligomer fragments and makes it a good candidate for auto‐crosslinking with cysteine‐containing proteins.

Site-specific enzymatic cleavage of TMV RNA directed by deoxyribo- and chimeric (deoxyribo-ribo)oligonucleotides

The TMV RNA molecule can be cleaved at a single site by RNase H directed by chimeric oligo(deoxyribo‐ribo)nucleotide with an internucleotide pyrophosphate bond

Specific covalent binding of a NF-κB decoy hairpin oligonucleotide targeted to the p50 subunit and induction of apoptosis

The NF‐κB transcriptional factor regulates various functions such as immune responses, cellular growth and development, and is frequently activated in tumor cells. Thus, inhibition of NF‐κB could suppress tumor cell growth. Using a decoy synthetic hairpin‐shaped oligodeoxyribonucleotide (ODN) containing the κB site with an integrated single diphosphoryldisulfide linkage, we demonstrate its covalent binding to the p50 subunit of NF‐κB. Furthermore, this decoy ODN induces apoptosis in a lymphoblastoma cell line. Thus, such chemically modified decoys could be valuable tools for blocking nuclear factors and tumor cell growth.

Regulation of activity of transcription factor NF-κB by synthetic oligonucleotides

Eukaryotic dimeric nuclear factor-κB (NF-κB) is one of the main transcription factors that activate expression of genes, products of which play the key role in development of cardiovascular pathologies, carcinogenesis, and inflammatory and viral diseases. In this review, the main attention is given to modulation of the transcription factor NF-κB activity by antisense oligonucleotides and oligonucleotide decoys. Also, current concepts about interactions between NF-κB dimers and DNA and general problems that arise in experimental use of synthetic oligonucleotides in vivo are discussed.