Ribonuclease H attack of leukaemic fused transcripts AML1-MTG8 (ETO) by DNA/RNA chimeric hammerhead ribozymes.

Abstract

Catalytic anti-sense oligonucleotides might be useful tools for controlling specific gene expression. However, to obtain effective oligonucleotides of the desired function in vivo is still a difficult task. To evaluate the usefulness of synthesized DNA/RNA hammerhead ribozymes targeting AML1-MTG8 (ETO) leukaemic fusion transcripts in vivo, we analysed their effects on cell growth and the mechanism of action using isolated cell nuclei. These ribozymes inhibited the growth of leukaemic cell lines expressing the AML1 -MTG8 and degraded AML1-MTG8 mRNA in isolated nuclei of these cells. However, the reactions gave rise to additional cleavage products. Systematic cleavage analyses using an anti-sense oligonucleotide array revealed that the cleavage was induced by endogenous RNase H at specific sites, in accordance with their calculated melting temperature (Tm) values. With suppression of RNase H by sulfhydryl agents, the DNA/RNA ribozyme had a ribozyme catalytic activity. In addition, the ribozymes and anti-sense oligonucleotides suppressed the AML1-MTG8 protein in the leukaemic cells. The DNA/RNA ribozymes inhibited cell growth primarily via anti-sense effects, the main role of which was the activation of RNase H-digestion by their DNA arms. In addition, the isolated nuclei provided a useful assay system for modelling in vivo conditions for the quantitative evaluation of anti-sense/ribozyme activity.