Limits...
Azacytidine and decitabine induce gene-specific and non-random DNA demethylation in human cancer cell lines.

Hagemann S, Heil O, Lyko F, Brueckner B - PLoS ONE (2011)

Bottom Line: Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context.These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes.Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.

View Article: PubMed Central - PubMed

Affiliation: Division of Epigenetics, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

ABSTRACT
The DNA methyltransferase inhibitors azacytidine and decitabine represent archetypal drugs for epigenetic cancer therapy. To characterize the demethylating activity of azacytidine and decitabine we treated colon cancer and leukemic cells with both drugs and used array-based DNA methylation analysis of more than 14,000 gene promoters. Additionally, drug-induced demethylation was compared to methylation patterns of isogenic colon cancer cells lacking both DNA methyltransferase 1 (DNMT1) and DNMT3B. We show that drug-induced demethylation patterns are highly specific, non-random and reproducible, indicating targeted remethylation of specific loci after replication. Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context. We also identified a subset of genes that were never demethylated by drug treatment, either in colon cancer or in leukemic cell lines. These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes. Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.

Show MeSH

Related in: MedlinePlus

Genome-wide methylation analysis of HCT116 cells.A, Global methylation analysis (CE) of HCT116 cells treated with the indicated concentrations of AZA and DAC for 24 h. B, Time-course CE measurement of drug-induced demethylation using 1 µM AZA or DAC; Co, untreated HCT116 cells. C, Kernel density estimates of Infinium methylation data for untreated (Co) and drug-treated cells. D, Validation of Infinium methylation data using 454 bisulfite sequencing; methylation data of 4 CG loci, measured either by Infinium analysis or by 454 sequencing, correlate strongly (Pearson's correlation coefficient r  = 0.84); treatment with AZA and DAC is indicated by A and D, respectively; different loci are indicated by colors: PIK3CG (blue), NTRK3 (green), Ells1 (red), and AFF2 (black).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3049766&req=5

pone-0017388-g001: Genome-wide methylation analysis of HCT116 cells.A, Global methylation analysis (CE) of HCT116 cells treated with the indicated concentrations of AZA and DAC for 24 h. B, Time-course CE measurement of drug-induced demethylation using 1 µM AZA or DAC; Co, untreated HCT116 cells. C, Kernel density estimates of Infinium methylation data for untreated (Co) and drug-treated cells. D, Validation of Infinium methylation data using 454 bisulfite sequencing; methylation data of 4 CG loci, measured either by Infinium analysis or by 454 sequencing, correlate strongly (Pearson's correlation coefficient r  = 0.84); treatment with AZA and DAC is indicated by A and D, respectively; different loci are indicated by colors: PIK3CG (blue), NTRK3 (green), Ells1 (red), and AFF2 (black).

Mentions: As a first step towards a systematic characterization of demethylation responses induced by azacytidine (AZA) and decitabine (DAC), we aimed to maximize demethylation efficiency, to minimize drug toxicity [31], [32] and to prevent remethylation as observed during long-term treatment [33]. To this end, we treated HCT116 cells with increasing drug concentrations (Figure 1A) and over various periods of time (Figure 1B) to determine global genomic methylation levels by capillary electrophoresis. The results clearly showed for both drugs that maximum demethylation was reached with concentrations of 1 µM after 24–36 h with DAC showing a 60% reduction and AZA showing a 50% reduction of global DNA methylation.


Azacytidine and decitabine induce gene-specific and non-random DNA demethylation in human cancer cell lines.

Hagemann S, Heil O, Lyko F, Brueckner B - PLoS ONE (2011)

Genome-wide methylation analysis of HCT116 cells.A, Global methylation analysis (CE) of HCT116 cells treated with the indicated concentrations of AZA and DAC for 24 h. B, Time-course CE measurement of drug-induced demethylation using 1 µM AZA or DAC; Co, untreated HCT116 cells. C, Kernel density estimates of Infinium methylation data for untreated (Co) and drug-treated cells. D, Validation of Infinium methylation data using 454 bisulfite sequencing; methylation data of 4 CG loci, measured either by Infinium analysis or by 454 sequencing, correlate strongly (Pearson's correlation coefficient r  = 0.84); treatment with AZA and DAC is indicated by A and D, respectively; different loci are indicated by colors: PIK3CG (blue), NTRK3 (green), Ells1 (red), and AFF2 (black).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3049766&req=5

pone-0017388-g001: Genome-wide methylation analysis of HCT116 cells.A, Global methylation analysis (CE) of HCT116 cells treated with the indicated concentrations of AZA and DAC for 24 h. B, Time-course CE measurement of drug-induced demethylation using 1 µM AZA or DAC; Co, untreated HCT116 cells. C, Kernel density estimates of Infinium methylation data for untreated (Co) and drug-treated cells. D, Validation of Infinium methylation data using 454 bisulfite sequencing; methylation data of 4 CG loci, measured either by Infinium analysis or by 454 sequencing, correlate strongly (Pearson's correlation coefficient r  = 0.84); treatment with AZA and DAC is indicated by A and D, respectively; different loci are indicated by colors: PIK3CG (blue), NTRK3 (green), Ells1 (red), and AFF2 (black).
Mentions: As a first step towards a systematic characterization of demethylation responses induced by azacytidine (AZA) and decitabine (DAC), we aimed to maximize demethylation efficiency, to minimize drug toxicity [31], [32] and to prevent remethylation as observed during long-term treatment [33]. To this end, we treated HCT116 cells with increasing drug concentrations (Figure 1A) and over various periods of time (Figure 1B) to determine global genomic methylation levels by capillary electrophoresis. The results clearly showed for both drugs that maximum demethylation was reached with concentrations of 1 µM after 24–36 h with DAC showing a 60% reduction and AZA showing a 50% reduction of global DNA methylation.

Bottom Line: Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context.These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes.Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.

View Article: PubMed Central - PubMed

Affiliation: Division of Epigenetics, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

ABSTRACT
The DNA methyltransferase inhibitors azacytidine and decitabine represent archetypal drugs for epigenetic cancer therapy. To characterize the demethylating activity of azacytidine and decitabine we treated colon cancer and leukemic cells with both drugs and used array-based DNA methylation analysis of more than 14,000 gene promoters. Additionally, drug-induced demethylation was compared to methylation patterns of isogenic colon cancer cells lacking both DNA methyltransferase 1 (DNMT1) and DNMT3B. We show that drug-induced demethylation patterns are highly specific, non-random and reproducible, indicating targeted remethylation of specific loci after replication. Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context. We also identified a subset of genes that were never demethylated by drug treatment, either in colon cancer or in leukemic cell lines. These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes. Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation.

Show MeSH
Related in: MedlinePlus