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

Citation
T. Kozu et al., Ribonuclease H attack of leukaemic fused transcripts AML1-MTG8 (ETO) by DNA/RNA chimeric hammerhead ribozymes, GENES CELLS, 5(8), 2000, pp. 637-647
Citations number
42
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Molecular Biology & Genetics
Journal title
GENES TO CELLS
ISSN journal
1356-9597 → ACNP
Volume
5
Issue
8
Year of publication
2000
Pages
637 - 647
Database
ISI
SICI code
1356-9597(200008)5:8<637:RHAOLF>2.0.ZU;2-7
Abstract
Background: Catalytic anti-sense oligonucleotides might be useful tools for controlling specific gene expression. However, to obtain effective oligonu cleotides of the desired function in vivo is still a difficult task. Results: To evaluate time usefulness of synthesized DNA/RNA hammerhead ribo zymes targeting AML1-MTG8 (ETO) leukaemic fusion transcripts in vivo, we an alysed their effects on cell growth and the mechanism of action using isola ted cell nuclei. These ribozymes inhibited the growth of leukaemic cell lin es expressing the AML1-MTG8 and degraded AML1-MTG8 mRNA in isolated nuclei of these cells. However, the reactions gave rise to additional cleavage pro ducts. Systematic cleavage analyses using an anti-sense oligonucleotide arr ay revealed that the cleavage was induced by endogenous RNase H at specific sites, in accordance with their calculated melting temperature (T-m) value s. With suppression of RNase H by sulfhydryl agents, the DNA/RNA ribozyme h ad a ribozyme catalytic activity. In addition, the ribozymes and anti-sense oligonucleotides suppressed the AML1-MTG8 protein in the leukaemic cells. Conclusions: The DNA/RNA ribozymes inhibited cell growth primarily via anti -sense effects, the main role of which was the activation of RNase H-digest ion by their DNA arms. In addition, the isolated nuclei provided a useful a ssay system for modelling in vivo conditions for the quantitative evaluatio n of anti-sense/ribozyme activity.