Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Interaction of Triplex Forming Oligonucleotides (TFOs) and Various Anti-cancer Drugs with a Promoter Regions of c-met and c-myc
Triplex forming oligonucleotides (TFO) targeted at cancer-promoting genes to achieve transcriptional gene silencing is one of the promising strategies in cancer therapy. A major advantage of TFOs resides in the possibility of a persistent down-regulation of transcription preventing the re-synthesis of RNA and protein. Besides the antigene approach, minor / major groove binding ligands, intercalating agents as competitors for DNA-binding proteins, also selectively cause inhibition at the promoter level. In general, proto-oncogenes share a common feature of having a promoter with high GC content which lacks TATA and CAAT boxes. This property of them is being e utilized by DNA binding antitumor drugs as well as TFOs. Various antitumor drugs have been shown to bind preferentially to GC rich sequences of DNA and inhibit their transcription.
Keeping this in mind, we have selected two very important members of cancer progression, c-myc and c-met which are implicated in various physiological processes-cell growth, proliferation, loss of differentiation, and apoptosis and overexpression has been implicated in the pathogenesis of most types of human cancer. Met is a growth factor receptor with tyrosine kinase activity which gained a lot of attention very recently because of its role in cell signaling.
We have selected two short GC rich DNA sequences from c-met and c-myc. The sequences were selected on the basis of their importance in the transcription process. Designing of TFOs were done against these sequences. Detailed sequence analysis of c-met promoter revealed that the major positive regulatory region is located at -233 to -68 within the promoter. We have selected a short guanine rich sequence (5?-GGGGCAGAGGCGGGAGGAAACGCG-3?) which is a part of this strong positive regulatory region at locations -142 to -119. A 15mer TFO was designed against this sequence (5?-AGGAGGGGGAGAGG-3?). Similarly a short defined 21bp long oligonucleotide (5?-TAAAGGGCCGGTGGGCGGAGA-3?) upstream to P1 and 178-bp upstream to P2 of c-Myc was selected. The TFO selected against this sequence is 5?-AGGAGGGGGGAGAGG-3?).
Interaction of DNA was also studied using the conventional anti-cancer drugs, Cisplatin, a DNA cross linking agent and Adriamycin and Actinomycin D, which are groove cum intercalators. The interaction of drugs/TFO with dDNAs has been undertaken in this study by using UV-Vis absorption, UV melting, fluorescence, circular dichroism spectroscopy and molecular modeling. The biophysical results are further corroborated with the cell cytotoxic data in HepG2 and HeLa cell lines.
Moganty R. Rajeswari
Department of Biochemistry,