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Antisense oligodeoxyribonucleotides suppress hematologic cell growth through stepwise release of deoxyribonucleotides
J L Vaerman1, P Moureau, F Deldime
1Laboratoire de Biologie Moleculaire Hématologique, Cliniques St Luc, Université Catholique de Louvain, Clos Chapelle-aux-champs, Brussels, Belgium.
Abstract:
Antisense oligodeoxyribonucleotides (ODNs) are now being extensively investigated in an attempt to achieve cell growth suppression through specific targeting of genes related to cell proliferation, despite increasing evidence of non-antisense cytotoxic effects. In the context of anti-BCR/ABL antisense strategies in chronic myeloid leukemia, we have reexamined the antiproliferative effect of phosphodiester and phosphorothioate ODNs on the leukemic cell line BV173 and on CD34+ bone marrow cells in liquid culture. The 3' sequences of the ODNs determine their effect. At concentrations of 10 micromol/L (for phosphorothioate ODNs) or 25 micromol/L (for phosphodiester ODNs), all the tested ODNs exert an antiproliferative activity, except those that contain a cytosine residue at either their two most terminal 3' positions. We show that this antiproliferative effect is due to the toxicity of the d-NMPs (5' monophosphate deoxyribonucleosides), the enzymatic hydrolysis products of the ODNs in culture medium. The toxicity of the d-NMPs on hematologic cells depends on their nature (d-CMP [2'deoxycytidine 5'-monophosphate] is not cytotoxic), on their concentration (d-GMP [2'-deoxyguanosine 5'-monophosphate], TMP [thymidine 5'-monophosphate], and d-AMP [2'-deoxyadenosine 5'-monophosphate] are cytotoxic at concentrations between 5 and 10 micromol/L), and on the coincident presence of other d-NMPs in the culture medium (d-CMP neutralizes the toxicity of d-AMP, d-GMP, or TMP). The antiproliferative activity of ODNs is thus restricted to conditions where the 3' hydrolysis process by exonucleases generates significant amounts of d-NMPs with a low proportion of d-CMP. Our results reveal a novel example of a nonantisense effect of ODNs, which should be taken into account when performing any experiment using assumed antisense ODNs.
Insights
Antisense oligodeoxyribonucleotides (ODNs) show antiproliferative effects on leukemia cells due to toxic hydrolysis products, not antisense activity. The 3' sequence and d-NMP composition determine toxicity, revealing a non-antisense mechanism.
Area of Science:
- Molecular Biology
- Pharmacology
- Hematology
Background:
- Antisense oligodeoxyribonucleotides (ODNs) are investigated for cancer therapy by targeting genes involved in cell proliferation.
- Non-antisense cytotoxic effects of ODNs are increasingly recognized, necessitating re-evaluation of their mechanisms of action.
- Chronic myeloid leukemia (CML) research utilizes anti-BCR/ABL antisense strategies, making ODN effects on leukemic cells critical.
Purpose of the Study:
- To re-examine the antiproliferative effects of phosphodiester and phosphorothioate ODNs on leukemic and bone marrow cells.
- To elucidate the mechanism behind the observed antiproliferative activity, distinguishing between antisense and non-antisense effects.
- To identify factors influencing ODN-induced cytotoxicity, specifically the role of 3' sequences and hydrolysis products.
Main Methods:
- Tested phosphodiester and phosphorothioate ODNs on BV173 leukemic cell line and CD34+ bone marrow cells in liquid culture.
- Varied ODN concentrations and sequences, focusing on the 3' terminal positions.
- Analyzed the hydrolysis products of ODNs (5' monophosphate deoxyribonucleosides - d-NMPs) and their cytotoxic effects.
Main Results:
- All tested ODNs showed antiproliferative activity at specific concentrations, except those with cytosine at the two 3' terminal positions.
- The antiproliferative effect was attributed to the toxicity of d-NMPs, enzymatic hydrolysis products of ODNs.
- d-NMP toxicity depended on their type (d-CMP non-cytotoxic), concentration, and the presence of other d-NMPs (d-CMP neutralized toxicity).
Conclusions:
- The antiproliferative activity of ODNs is primarily a non-antisense effect mediated by toxic d-NMPs generated during hydrolysis.
- ODN efficacy is restricted to conditions favoring d-NMP generation with low d-CMP levels.
- These findings highlight the importance of considering non-antisense mechanisms, particularly d-NMP toxicity, in ODN-based research and therapy.