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Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

Published on: October 15, 2018

Exposure to hypomethylating 5-aza-2'-deoxycytidine (decitabine) causes rapid, severe DNA damage, telomere elongation

Caroline Bull¹, Graham Mayrhofer², Michael Fenech³

¹CSIRO Health & Biosecurity, Gate 13 Kintore Avenue, Adelaide, South Australia, Australia; School of Molecular and Biomedical Sciences, University of Adelaide, North Terrace, Adelaide, South Australia, Australia.

Mutation Research. Genetic Toxicology and Environmental Mutagenesis

|August 29, 2021

Abstract

Background:

5-aza-2'-deoxycytidine (5azadC, decitabine) is a DNA hypomethylating agent used in the treatment of myelodysplastic syndromes. Due to cytotoxic side effects dose optimization is essential. The aim of this study was to define and quantify the effects of 5azadC on biomarkers of chromosomal stability, and telomere length, in human lymphoblastoid cell line, WIL2-NS, at clinically relevant dosages.

Methods:

Human WIL2-NS cells were maintained in complete medium containing 0, 0.2 or 1.0 μM 5azadC for four days, and analysed daily for telomere length (flow cytometry), chromosomal stability (cytokinesis-block micronucleus cytome (CBMN-cyt) assay), and global methylation (%5me-C).

Results:

DNA methylation decreased significantly in 1.0 μM 5azadC, relative to control (p < 0.0001). Exposure to 1.0 μM 5azadC resulted in 1.7-fold increase in telomere length (p < 0.0001), in parallel with rapid increase in biomarkers of DNA damage; (micronuclei (MN, 6-fold increase), nucleoplasmic bridges (NPB, a 12-fold increase), and nuclear buds (NBud, a 13-fold increase) (all p < 0.0001). Fused nuclei (FUS), indicative of mitotic dysfunction, showed a 5- and 13-fold increase in the 0.2 μM and 1.0 μM conditions, respectively (p = 0.001) after 4 days.

Conclusions:

These data show that (i) clinically relevant concentrations of 5azadC are highly genotoxic; (ii) hypomethylation was associated with increased TL and DNA damage; and (iii) longer TL was associated with chromosomal instability. These findings suggest that lower doses of 5azdC may be effective as a hypomethylating agent, while potentially reducing DNA damage and risk for secondary disease.

Keywords:

5-aza-2′-deoxycytidine (5azadC)CBMN-cytome assay DNA methylation Decitabine Fused nuclei Micronuclei Myelodysplastic syndromes

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