Abstract

Epigenetic drugs are in use in clinical trials of various human cancers and are potent at reactivating genes silenced by DNA methylation and chromatin modifications. We report here the analysis of a set of normal fibroblast and cancer cell lines after combination treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and the histone deacetylase inhibitor 4-phenylbutyric acid (PBA). Low doses of the drug combination caused cell cycle arrest, whereas high doses induced apoptosis in T24 bladder carcinoma cells. Both p16 (CDKN2A/INK4) and p21 (CIP1/SDI1/WAF1) expression were induced to similar levels in normal and cancer cells in a dose-dependent fashion after combination treatments. We detected a distinct increase of histone H3 acetylation at lysine 9/14 near the transcription start sites, in both LD419 normal fibroblasts and T24 bladder carcinoma cells, whereas the acetylation changes in the p21 locus were less apparent. Interestingly, the levels of trimethylation of histone H3 on lysine 9, which usually marks inactive chromatin regions and was associated with the p16 promoter in silenced T24 cells, did not change after drug treatments. Furthermore, we provide evidence that the remethylation of the p16 promoter CpG island in T24 cells after 5-aza-CdR treatment cannot be halted by subsequent continuous PBA treatment. The p16 gene is resilenced with kinetics similar to 5-aza-CdR only-treated cells, which is also marked by a localized loss of histone acetylation at the transcription start site. Altogether, our data provide new insights into the mechanism of epigenetic drugs and have important implications for epigenetic therapy.

Highlights

  • DNA methylation occurs at the C-5 position of cytosine residues in CpG dinucleotides, which are found at a lower than expected frequency in mammalian DNA but are clustered in CpG-rich areas called ‘‘CpG islands’’ [1, 2]

  • CdR were determined by monitoring the effects of increasing doses of 5-aza-CdR on DNA demethylation and phenylbutyric acid (PBA) on histone hyperacetylation in T24 bladder cancer cells (Fig. 1). ‘‘Low dose’’ was defined as the dose of either drug leading to detectable changes and ‘‘high dose’’ as the dose leading to a plateau in change in DNA demethylation or histone acetylation

  • We observed a major decrease in cell growth in T24 cells in the high-dose group (3 Amol/L 5-aza-CdR for 24 h and 3 mmol/L PBA continuous), which was clearly due to the significant apoptosis caused by the drug combination

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Summary

Introduction

DNA methylation occurs at the C-5 position of cytosine residues in CpG dinucleotides, which are found at a lower than expected frequency in mammalian DNA but are clustered in CpG-rich areas called ‘‘CpG islands’’ [1, 2]. These islands are often found close to promoters and are usually unmethylated in normal somatic tissues, whether the gene is transcriptionally active. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for A.M. Aparicio: Department of Genitourinary Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Unit 1374, 1515 Holcombe Boulevard, Houston, TX 77030

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