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Epigenetic changes by zebularine leading to enhanced differentiation of human promyelocytic leukemia NB4 and KG1 cells

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Abstract

Aberrant DNA methylation is a critical epigenetic process involved in gene expression of tumor cells. Diverse DNA methyltransferase inhibitors are being studied as potential anticancer drugs, and there is interest in developing novel and more effective DNMTIs. We evaluated zebularine, a stable and low-toxic cytidine analog, effects on human promyelocytic leukemia cell lines, NB4 and KG1. Zebularine caused a dose- and time-dependent NB4 and KG1 cell growth inhibition, did not induce myeloid differentiation but triggered concentration-dependent apoptosis as manifested by procaspase-3 and PAR-1 cleavage and the occurrence of early apoptosis detected by Annexin-V-propidium iodide. Zebularine co-treatment with all-trans retinoic acid (RA) at pharmacological dose (1 μM for NB4 cells) and higher (3 μM for KG1 cells) increased granulocytic differentiation in both cell lines. Pretreatment for 24 or 48 h with zebularine before the treatment with different doses of RA alone or RA with histone deacetylase inhibitors, phenyl butyrate, and BML-210, resulted in significant acceleration and enhancement of differentiation and cell cycle arrest at G0/1. Zebularine alone or in sequential combination with RA decreased expression of DNMT1, caused fast and time-dependent expression of pan-cadherin and partial demethylation of E-cadherin but not tumor suppressor p15. When used in combination with RA, zebularine increased expression of both genes transcript and protein. Zebularine induced regional chromatin remodeling by local histone H4 acetylation and histone H3-K4 methylation in promoter sites of methylated E-cadherin and also in the promoter of unmethylated p21 as evidenced by chromatin immunoprecipitation assay. Our results extend the spectrum of zebularine effects and the evaluation its utility in acute myeloid leukemia therapy based on epigenetics.

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Abbreviations

APL:

Acute promyelocytic leukemia

RA:

All-trans retinoic acid

ChIP:

Chromatin immunoprecipitation

DMSO:

Dimethyl sulphoxide

GAPDH:

Glyceraldehide-3-phosphate dehydrogenase

HDACI:

Histone deacetylase inhibitor

NBT:

Nitro blue tetrazolium

PBS:

Phosphate-buffered saline

PMA:

Phorbol myristate acetate

PARP:

Poly(ADP-ribose) polymerase

PB:

Sodium phenyl butyrate

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Acknowledgments

This research was financed by the Research Council of Lithuania (project No. LIG-20/2010).

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Authors and Affiliations

  1. Department of Molecular Cell Biology, Institute of Biochemistry, Vilnius University, Mokslininkų St. 12, 08662, Vilnius, Lithuania

    Jurate Savickiene, Grazina Treigyte, Renata Bruzaite, Veronika-Viktorija Borutinskaite & Ruta Navakauskiene

  2. Department of Biochemistry and Biophysics, Vilnius University, Ciurlionio St. 21, 03101, Vilnius, Lithuania

    Violeta Jonusiene

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  1. Jurate Savickiene
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  2. Grazina Treigyte
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  6. Ruta Navakauskiene
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Correspondence to Ruta Navakauskiene.

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Savickiene, J., Treigyte, G., Jonusiene, V. et al. Epigenetic changes by zebularine leading to enhanced differentiation of human promyelocytic leukemia NB4 and KG1 cells. Mol Cell Biochem 359, 245–261 (2012). https://doi.org/10.1007/s11010-011-1019-7

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  • DOI: https://doi.org/10.1007/s11010-011-1019-7

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