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Phosphorothioate-capped antisense oligonucleotides to Ras GAP inhibit cell proliferation and trigger apoptosis but
J R White1, E C Gordon-Smith, T R Rutherford
1Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London, United Kingdom.
Abstract:
We have studied the effects of an antisense oligonucleotide to Ras GAP in leukaemia cell lines. When terminal phosphorothioate linkages were introduced into this oligonucleotide, it caused major growth inhibition and apoptosis in the chronic myeloid leukaemia (CML) cell line K562, but had little effect on the promyelocytic leukaemia cell line HL60. Neither the expression of Ras GAP mRNA nor p120 GAP protein was downregulated by the antisense oligonucleotide, suggesting a non-antisense mechanism for growth inhibition. The antisense oligonucleotide contained GGC triplets which have previously been reported to inhibit the activity of p210bcr-abl both in vitro and in vivo. However, cellular phosphotyrosine levels were found to be unaffected, suggesting that the activity of p210bcr-abl was normal and that the antisense oligonucleotide may be interacting aptamerically with a different cellular protein. Since K562 is very resistant to apoptotic cell death, the identity of the putative target molecule would be of considerable interest.
Insights
An antisense oligonucleotide induced apoptosis in chronic myeloid leukaemia (CML) cells, but not other leukaemia cells. This effect was not due to targeting Ras GAP, suggesting a novel mechanism for leukaemia treatment.
Area of Science:
- Molecular Biology
- Cancer Research
- Leukemia Therapeutics
Background:
- Antisense oligonucleotides are investigated for targeted cancer therapy.
- Ras GTPase-activating protein (Ras GAP) plays a role in cell signaling pathways relevant to cancer.
- Chronic myeloid leukemia (CML) is a type of blood cancer characterized by specific genetic mutations.
Purpose of the Study:
- To investigate the effects of a modified antisense oligonucleotide targeting Ras GAP in leukemia cell lines.
- To elucidate the mechanism of action of the oligonucleotide, particularly its impact on cell growth and apoptosis.
Main Methods:
- Treatment of K562 (CML) and HL60 (promyelocytic leukemia) cell lines with a phosphorothioate-linked antisense oligonucleotide.
- Analysis of Ras GAP mRNA and p120 GAP protein expression levels.
- Assessment of cellular phosphotyrosine levels to evaluate p210bcr-abl activity.
- Evaluation of growth inhibition and apoptosis induction.
Main Results:
- The modified antisense oligonucleotide significantly inhibited growth and induced apoptosis in K562 cells, but not HL60 cells.
- No downregulation of Ras GAP mRNA or p120 GAP protein was observed.
- The oligonucleotide did not affect cellular phosphotyrosine levels, indicating normal p210bcr-abl activity.
- The findings suggest a non-antisense mechanism and potential aptameric interaction with an unknown cellular target.
Conclusions:
- The studied antisense oligonucleotide exhibits potent anti-leukemic activity in K562 cells via a non-canonical mechanism.
- The GGC triplet motif may interact with a target other than Ras GAP or p210bcr-abl.
- Identifying the specific target is crucial, given K562's resistance to apoptosis, for developing novel CML therapies.