Investigation of Potential Targets for Gene Therapy in Acute Myeloid Leukaemia: Targeting PML/RARα Transcript by DNAzymes

• Main Author: Kabuli, Majid
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2004
• Subjects: Biochemistry; Medicine; Molecular biology
• ISBN: 9780438202238; 0438202236

The effects of ribozymes have been investigated in a number of leukaemias. More recently, increased interest was shown in the activity of new molecules called DNAzymes. Acute promyelocytic leukaemia (APL) is characterized by the reciprocal translocation t(15;17) which produces the fusion gene PML/RARα. To investigate the activity and specificity of nucleic acid enzymes in cleaving the PML/RARα fusion transcript, and to compare the specificity of ribozymes and DNAzymes, the activity of a hammerhead ribozyme (RZl) which cleaves the PML/RARα transcript at the AUU triplet located four nucleotides downstream of the fusion site in the RARα portion was compared with four DNAzymes (DZl, DZ2, DZ3, and MDZl) which were designed to cleave the PML/RARα transcript at the GC nucleotides at the fusion site of the mRNA (for DZl and MDZl), at the AU site, placed in the RARα portion, three nucleotides downstream of the fusion site in the RARα portion (for DZ2), and at the GC site, three nucleotides upstream of that, in the PML portion (for DZ3). Disabled ribozyme and disabled DNAzymes were synthesized as controls. To investigate enzymatic activity and specificity of RZl and DZs for the fusion RNA, cleavage reactions were performed firstly in a cell-free system on total RNA from NB4 cell line and PML/RARα and RARα RNA fragments. Post cleavage examination showed that DZl, DZ3, and MDZl cleave PML/RARα efficiently and specifically. Furthermore the effects of DZl, DZS, and MDZl were investigated on APL cells by transfecting an ATRA-sensitive clone of NB4 cells (NB4-S), and an ATRA- resistant clone (NB4-R) in the presence and absence of all-trans retinoic acid (ATRA), using cationic liposome reagent (DOTAP). The inhibitory effects of the DNAzymes on NB4 cells were evaluated by XTT assay, trypan blue exclusion, western blotting, and FACScan analysis. DZl and DZ3 inhibited the proliferation rate of NB4 cells and reduced the cell viability rate. They also reduced the level of the target protein and increased the rate of apoptosis significantly. The disabled DNAzymes had no effect on NB4 cells. K562 cells transfected with the DNAzymes and disabled controls did not show any detectable effect. DNAzymes are less expensive to synthesize than ribozymes and are more resistant to serum. These data show that targeting the PML/RARα fusion transcript with DNAzymes can induce apoptosis in APL cells and may have a role in the treatment of APL. They also show that DNAzymes are promising tools for targeting specific genes in leukaemia.