The depletion of DNA methyltransferase-1 and the epigenetic effects of 5-aza-2'deoxycytidine (decitabine) are differentially regulated by cell cycle progression - PubMed (original) (raw)

The depletion of DNA methyltransferase-1 and the epigenetic effects of 5-aza-2'deoxycytidine (decitabine) are differentially regulated by cell cycle progression

Mazin Al-Salihi et al. Epigenetics. 2011 Aug.

Abstract

5-Aza-2'-deoxycytidine (decitabine) is a drug targeting the epigenetic abnormalities of tumors. The basis for its limited efficacy in solid tumors is unresolved, but may relate to their indolent growth, their p53 genotype or both. We report that the primary molecular mechanism of decitabine-depletion of DNA methyltransferase-1 following its "suicide" inactivation-is not absolutely associated with cell cycle progression in HCT 116 colon cancer cells, but is associated with their p53 genotype. Control experiments affirmed that the secondary molecular effects of decitabine on global and promoter-specific CpG methylation and MAGE-A1 mRNA expression were S-phase dependent, as expected. Secondary changes in CpG methylation occurred only in growing cells ~24-48 h after decitabine treatment; these epigenetic changes coincided with p53 accumulation, an index of DNA damage. Conversely, primary depletion of DNA methyltransferase-1 began immediately after a single exposure to 300 nM decitabine and it progressed to completion within ~8 h, even in confluent cells arrested in G 1 and G 2/M. Our results suggest that DNA repair and remodeling activity in arrested, confluent cells may be sufficient to support the primary molecular action of decitabine, while its secondary, epigenetic effects require cell cycle progression through S-phase.

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Figures

Figure 1

Cell cycle progression is unnecessary for depletion of DNMT1 enzyme in HCT 116 cells exposed to decitabine. (A) Western immunoblots of DNMT1 enzyme and β-tubulin. Serum-starved, growing or confluent HCT116 p53+/+ cells were treated once with 300 nM decitabine or 1 µM 6-azacytidine. DNMT1 was detected in the cell lysates by western immunoblotting. β-tubulin served as a loading control. The blots are representative of five experiments for the growing and confluent cells and two experiments for the starved cells. (B) Time course of DNMT1 depletion by decitabine. Plots of the relative depletion of DNMT1 versus time were based on the densitometry of DNMT1 immunoblots normalized to β-tubulin. *Denotes statistical significance (p < 0.05), error bars are 1 SD (n = 5 confluent and growing, n = 2 starved). The Table shows the t½ ± SD of DNMT1 depletion for each experimental condition and the corresponding percentage of the cell population in s-phase.

Figure 2

The p53 tumor suppressor modulates the rate of depletion of DNMT1 in HCT 116 cells exposed to decitabine. (A) Immunoblots of DNMT1 enzyme and tubulin Confluent HCT 116 cells that were wild type p53+/+ or null p53−/− were treated once with 300 nM decitabine. DNMT1 enzyme levels and β-tubulin loading control were detected by western immunoblotting. The blots are representative of five experiments for the wild type p53+/+ and two experiments for null p53−/− HCT 116 cells. (B) Time course of DNMT1 depletion by decitabine. Plots of the relative depletion of DNMT1 versus time were based on the densitometry of the DNMT1 immunoblots normalized to β-tubulin. *Denotes statistical significance, error bars are 1 SD. The table shows the t½ ± SD of DNMT1 depletion for each experimental condition and the corresponding percentage of the cell population in s-phase.

Figure 3

Epigenetic effects of decitabine in HCT 116 cells are cell cycle dependent: MAGE-A1 re-expression is s-phase dependent. Reverse transcription PCR and ethidium bromide detection of MAGE-A1 mRNA expression at 24 and 48 h after a single exposure to 300 nM decitabine in growing, confluent or confluent HCT 116 cells released from arrest. GAPDH and actin were used as procedural controls. Agarose gels shown are representative of three experiments.

Figure 4

A p53 response to DNA damage coincides with the epigenetic effects of decitabine. (A) Immunoblot of p53 tumor suppressor protein and tubulin in growing cells. Growing wild-type p53+/+ HCT116 cells were treated once with 300 nM decitabine. p53 Protein and α-tubulin loading control in cell lysates were measured at 12 h intervals by western immunoblotting. (B) Immunoblot of p53 tumor suppressor protein and tubulin in confluent cells. Confluent wild-type p53+/+ HCT116 cells were treated once with 300 nM 5decitabine. p53 Protein and β-tubulin loading control in cell lysates were measured at 12 h intervals by western immunoblotting.

References

1. Esteller M. Epigenetics in cancer. N Engl J Med. 2008;358:1148–1159. - PubMed
1. Issa JP, Kantarjian HM. Targeting DNA Methylation. Clin Cancer Res. 2009;15:3938–3946. - PMC - PubMed
1. Wijermans P, Lubbert M, Verhoef G, Bosly A, Ravoet C, Andre M, et al. Low-dose 5-aza-2′-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: a multicenter phase II study in elderly patients. J Clin Oncol. 2000;18:956–962. - PubMed
1. Issa JP, Garcia-Manero G, Giles FJ, Mannari R, Thomas D, Faderl S, et al. Phase I study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2′-deoxycytidine (Decitabine) in hematopoietic malignancies. Blood. 2004;103:1635–1640. - PubMed
1. Samlowski WE, Leachman SA, Wade M, Cassidy P, Porter-Gill P, Busby L, et al. Evaluation of a 7-day continuous intravenous infusion of decitabine: inhibition of promoter-specific and global genomic DNA methylation. J Clin Oncol. 2006;23:3897–3905. - PubMed

The depletion of DNA methyltransferase-1 and the epigenetic effects of 5-aza-2'deoxycytidine (decitabine) are differentially regulated by cell cycle progression - PubMed (original) (raw)