Characterization of BIX-01294, and its analogs E67 and E11 on growth and reactivation of epigenetically silenced tumor suppressor genes in human breast cancer cells. Open Access

Pattabiraman, Vaishnavi (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/gh93h026k?locale=en
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Abstract

Epigenetic silencing of tumor suppressor genes in cancers are characterized by DNA hypermethylation and local alterations in histone modifications, including increased repressive histone modifications such as histone H3K9me2/3, H3K27me3 and a decrease in activating histone modifications such as histone H3K4me2/3. G9a and G9a-like protein (GLP) are euchromatin associated histone methyltransferases that repress transcription by methylating histone H3 at K9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen for specific inhibitors against histone methyltransferases (HMTases); E67 and E11 are lysine mimics and analogs of BIX-01294 that have been reported to have better inhibitory activity towards GLP in comparison to BIX-01294 in in vitro enzyme assays. In this study, we have characterized the impact of three small molecule inhibitors- BIX-01294, E67 and E11 in breast cancer cells. We show that all three agents effectively inhibit the growth of breast cancer cells with IC 50 values between 3.0 μM and 10.0 μM. BIX-01294 and E11 are the most potent; E67 is the least potent. As single agents, these inhibitors have minimal effects on the expression of epigenetically silenced tumor suppressor genes in breast cancer cells. We also show BIX-01294 and E67 are antagonistic to 5-Aza-2'-Deoxycytidine (5-azaCdR) induced demethylation at the TMS1 locus. Our findings indicate that these inhibitors exert their effects in vivo without altering the protein levels of G9a and DNMT1. Finally, we show a significant decrease in global levels of histone H3K9me2 in cancer cells treated with these agents. Our results propose that these agents harbor different growth inhibitory activity, differential effects on DNA methylation of a tumor suppressor gene and similar effects on down-regulation of H3K9me2 modification, in breast cancer cells. Studying the mechanism of action of BIX-01294, E67 and E11 by themselves or in combination with 5'azaCdR will allow for the further development of epigenetic therapy of human breast cancer.

Characterization of BIX-01294, its analogs E67 and E11 on growth, and reactivation of epigenetically silenced tumor suppressor genes in human breast cancer cells.
By
Vaishnavi Pattabiraman
M.Sc , Bharathiar University, 2002
Advisor: Paula M. Vertino, Ph.D.
A thesis submitted to the Faculty of the
James T. Laney School of Graduate Studies of Emory University
in partial fulfillment of the requirements for the degree of
Master of Science
in the Graduate Division of Biological and Biomedical Sciences
Genetics and Molecular Biology
2011

Table of Contents

Table of Contents
Chapter 1: Introduction...1

Figures...26

Chapter 2: Material and Methods...35

Figures...35

Chapter 3: Results...42

Figures...54

Chapter 4: Discussion...81

Figures...91

References...93


List of Figures and Tables
Chapter 1: Introduction

Figure 1: DNA methyltransferase reaction
Figure 2: The pictorial representation of various post translational histone modifications and their biological roles.
Figure 3: Domain organization of the core G9a components: G9a, GLP and Wiz
Figure 4: Schematic representation of the mouse G9a locus
Figure 5: DNA methylation and histone modification patterns are altered in cancers
Figure 6: Epigenetic drugs for cancer therapy
Figure 7: Chemical structure of the compound BIX-01294
Figure 8: Chemical structures of E67 and E11(BIX-01294 derivatives)
Figure 9: Diagram of the TMS1 gene

Chapter 2: Materials and Methods

Figure 10: Schematic of the experimental design

Chapter 3: Results

Figure 11: Effect of BIX-01294 and its analogs on the growth of breast cancer cells
Figure 12: Effect of BIX-01294 and its analogs on the growth of normal immortalized breast epithelial cells
Figure 13-16: Effect of BIX-01294, its analogs and/or 5-azaCdR on gene expression in MDA-MB231 cells
Figure 17: Effect of BIX-01294 and/or 5-azaCdR on DNA methylation at the TMS1 locus
Figure 18: Effect of E67 and/or 5-azaCdR on DNA methylation at the TMS1 locus
Figure 19: Effect of E11 and/or 5-azaCdR on DNA methylation at the TMS1 locus
Figure 20: Effect of BIX-01294, its analogs and/or 5-azaCdR on G9a and DNMT1 protein levels in breast cancer cells
Figure 21: Effect of BIX-01294, its analogs and/or 5-azaCdR on total levels of histone H3K9me2 modification in breast cancer cells
Figure 22: Effect of BIX-01294, its analogs and/or 5-azaCdR on total levels of histone H3K9me3 modification in breast cancer cells
Figure 23: Effect of BIX-01294, its analogs and/or 5-azaCdR on total levels of histone H3K9me1 modification in breast cancer cells
Figure 24: Effect of BIX-01294, its analogs and/or 5-azaCdR on total levels of histone H3K27me2 modification in breast cancer cells
Figure 25: Effect of BIX-01294, its analogs and/or 5-azaCdR on total levels of histone H3K4me2 modification in breast cancer cells

Chapter 4: Discussion

Figure 26: Model for epigenetic regulation of BIX-01294, its analogs and 5'azaCdR at the promoters of epigenetically silenced tumor suppressor genes in MDA-MB231 cells

Tables: Results

Table 1: Approximate IC 50 values of BIX-01294, E67 and E11
Table 2: Percent CpG demethylation at the TMS1 locus in untreated and treated MDA-MB231 cells

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