MODULATION OF DNA METHYLATION BY CATECHOL-O-METHYLTRANSFERASE AND CATECHOL-CONTAINIG DIETARY POLYPHENOLS

국문 초록 (Abstract)

본연구에서는 catechol-O-methyltransferase (COMT)와 S-adenosyl-L-homocysteine (SAH)가 DNA 메틸화의 조절기전에 미치는 영향을 연구하였다. 생리학적으로 알맞은 양의 COMT가 in vitro DNA 메틸화를 증가시켰으며, COMT siRNA의 transfection을 암세포에 실시한 결과 COMT 유전자발현이 약 50%정도 줄어드는 결과를 얻었다. 이러한 COMT의 감소는 MCF-7 세포내의 RAR-beta유전에서의 DNA 메틸화를 현저히 감소시켰다. 효소역학실험결과는 SAH가 매우 강력한 그리고 비경쟁적 (noncompetitive)인 DNA 메틸화 억제물이며, 이러한 억제는 DNA methyltransferase (DNMT)와의 결합을 SAH와 S-adenosyl-L-methionine (SAM) 경쟁으로 인한 결과이다. 본 연구에서는 커피 폴리페놀 (polyphenols), 차, 그리고 bioflavonoids가 DNA 메틸화에 미치는 영향과 암억제 유전자의 promoter지역 DNA 메틸화에 미치는 영향을 연구하였다. 두가지 커피 폴리페놀 (caffeic acid and chlorogenic acid), 차 catechins (catechin, epicatechin, and EGCG), 그리고 bioflavonoids (quercetin, fisetin, and myricetin)이 DNMTs에 의한 DNA 메틸화를 농도의존적형태로 억제하는 결과를 보였다. 효소역학결과는 폴리페놀이 DNA 메틸화 억제시키는 주요원인은 식이성 폴리페놀의 메틸화의 부산물인 SAH의 농도증가에 의한 것임을 증명하였다. 반면, 매우 강한 DNA 메틸화 억제물인 EGCG는 그 자체의 메틸화와는 상관없이 DNMT에 직접 결합함으로서 DNA 메틸화를 억제시키는 것으로 사료되었다. 그리고 이러한 EGCG와 DNMT와의 강한 결합에서 마그네슘이온이 결정적인 중요한 역할을 하는 것으로 나타났다.

다국어 초록 (Multilingual Abstract)

The DNA methyltransferase (DNMT)-mediated methylation of DNA at the C-5 position of cytosine within the CpG dinucleotides represents a basic mechanism for epigenetic control of gene expression and maintenance of genome integrity. In the present study, we examined the modulating effects of catechol-O-methyltransferase (COMT) and S-adenosyl-L-homocysteine (SAH) on DNA methylation catalyzed by prokaryotic M.SssI DNMT and human DNMT1. We found that the presence of COMT (at physiologically-relevant concentrations) enhanced the rate of DNA methylation in vitro catalyzed by M.Sssl DNMT and human DNMT1. Transfection of the COMT siRNAs (at 100 nM) into these cells caused ~50% reduction of COMT expression, and this reduction of the COMT levels resulted in a decrease of the methylation status of the RARβ gene (a representative gene) in MCF-7 cells. Kinetic studies showed that SAH strongly and noncompetitively inhibited .the methylation of DNA by competing S-adenosyl-L-methionine (SAM) off the DNMT, thus shifting more enzyme molecules to a form that is bound with SAH. Our data showed that the enhancement of DNA methylation by COMT likely is due to the sequestration of SAH by COMT, which reduces the availability of the free SAH for DNMT inhibition.
We also studied the modulating effects of coffee polyphenols, tea catechins and bioflavonoids on the in vitro methylation of synthetic DNA substrates and also on the methylation status of the promoter regions of two representative tumor suppressor genes in cultured cells. Each of the coffee polyphenols, tea catechins (catechin, epicatechin, and [-]-epigallocatechin-3-O-gallate [EGCG]) and bioflavonoids (quercetin, fisetin, and myricetin) inhibited M.SssI DNMT- and DNMT1-mediated DNA methylation in a concentration-dependent manner. Kinetic analysis showed that the mechanism by which this catechol-containing dietary polyphenols inhibited DNA methylation predominantly through a noncompetitive mechanism, and this inhibition was largely due to increased formation of SAH (a potent feedback inhibitor of DNA methylation), resulting from the COMT-mediated 0-methylation of these dietary catechols. In comparison, the strong inhibitory effect of EGCG on the DNMT-mediated DNA methylation was independent of its own methylation, and it is largely due to its direct inhibition of the DNMTs. This inhibition is strongly enhanced by Mg^(2+). Computational modeling studies showed that the gallic acid moiety of EGCG plays a crucial role in its direct inhibitory interaction with the catalytic site of the human DNMT1, and its binding with the enzyme is stabilized by Mg^(2+), suggesting that the inhibition potency of EGCG for human DNMT1 in the presence of Mg^(2+) would be markedly enhanced, which is in perfect agreement with our experimental finding.

목차 (Table of Contents)

• Title Page = ⅰ
• Copyright Page = ⅱ
• DEDICATION = ⅲ
• ACKNOWLEDGEMENTS = ⅳ
• ABSTRACT = ⅵ
• TABLE OF CONTENTS = ⅷ
• LIST OF TABLES = ⅹ
• LIST OF FIGURES = xi
• LIST OF ABBREVIATIONS = xiii
• CHAPTER 1 Introduction = 1
• Overview = 2
• DNA methylation = 3
• Global hypomethylation and gene-specific hypermethylation in cancer = 6
• DNA methyltransferases (DNMTs) = 10
• Diet and Cancer = 12
• Catechol-O-methyltransferases (COMT) = 14
• Purpose of the study and specific aims = 16
• Significance = 17
• CHAPTER 2 Modulation of the Rate of Enzymatic DNA Methylation by Catechol-O-Methyltransferase = 24
• ABSTRACT = 25
• INTRODUCTION = 27
• MATERIALS AND METHODS = 29
• Chemicals = 29
• Assay of enzymatic DNA methylation in vitro = 30
• Determination of the methylation status of RARβ gene in MCF-7 cells = 32
• RESULTS = 36
• Modulation of M.SssI DNMT-mediated DNA methylation by COMT = 36
• Modulation of human DNMT1-mediated DNA methylation by COMT = 39
• Effect of COMT siRNAs on the DNA methylation status in cultured MCF-7 cells = 40
• DISCUSSION = 41
• CHAPTER 3 Inhibition of DNA Methylation by Caffeic Acid and Chlorogenic Acid, Two Common Catechol-Containing Coffee Polyphenols = 55
• ABSTRACT = 56
• INTRODUCTION = 58
• MATERIALS AND METHODS = 61
• Chemicals = 61
• Assay of enzymatic DNA methylation in vitro = 61
• Determination of the methylation status of p16 and RARβ genes in cultured cancer cells = 62
• RESULTS = 64
• Optimization of the conditions for enzymatic DNA methylation in vitro = 64
• Modulation of DNMT-mediated DNA methylation by caffeic acid and chlorogenic acid = 65
• Kinetic mechanisms for the inhibition of DNA methylation by caffeic acid and chlorogenic acid = 66
• Inhibition of the promoter region DNA methylation in cultured cells by caffeic acid and chlorogenic acid = 67
• Discussion = 70
• CHAPTER 4 Mechanisms for the Inhibition of DNA Methyltransferases by Tea Catechins and Bioflavonoids = 87
• ABSTRACT = 88
• INTRODUCTION = 89
• MATERIALS AND METHODS = 91
• Chemicals = 91
• Assay of enzymatic DNA methylation in vitro = 91
• Determination of the methylation status of p16 and RARβ genes in cultured cancer cells = 92
• Computational modeling of the binding of EGCG to human DNMT1 = 93
• RESULTS = 96
• Inhibition of DNMT-mediated DNA methylation by tea polyphenols and bioflavonoids = 96
• Inhibition of the promoter region DNA methylation in cultured cells by EGCG and catechin = 99
• Kinetic mechanisms for the inhibition of DNA methylation by tea polyphenols = 101
• Computational analysis of the interaction of EGCG with human DNMT1 = 103
• DISCUSSION = 106
• FUTURE STUDIES = 135
• REFERENCES = 136