Employment of PNA molecule for gene suppression in mammalian cells

Abstract

Background and Objective: The terminal deoxynucleotidyl transferase (TdT) is over-expressed in almost all cases of acute lymphoblastic leukemia (ALL). This enzyme catalyzes incorporation of deoxyribonucleotides at the 3´-end in the absence of a DNA template, may have a role in DNA repair in neoplastic and apoptotic cells. In addition, it has been reported that high level of TdT correlates with poor clinical prognosis due to enhanced malignancy. Aim: Employment of PNA molecule for gene suppression (TdT) in Molt-4 cells Methods: We examined PNAs targeting 5' and 3' junctions of intron 7 and addressed their mRNA splicing modulation effects using RT-PCR analysis. Moreover, we investigated alteration of transcription and translation for PNAs targeted to 5´UTR and AUG region using flow cyrometry and real-time PCR. The rate of cell apoptosis and cell survival was analyzed. Results: The results of splicing part showed that full match PNAs could alterate the splicing process, thereby produce a longer mRNA still including intron 7, or mRNA without exon 7. The results of PNAs directed to5´UTR and AUG region revealed TdT downregulation. Of note, efficient translation inhibition was observed when cells treated simultaneously with both antisense PNAs. In addition, reduced level of TdT protein in Molt-4 cells was accompanied by an increased rate of apoptosis and decreased the level of cell survival. Conclusion: Overall, downregulation of TdT gene expression with antisense PNAs through modulation of splicing events and translation process and also at transciptional level with antigene PNA may be a promising route for the discovery of novel anticancer agents.