INFIDELITY OF CYTOSINE METHYLATION AND HUMAN CANCER
Principal Investigator: Lawrence C Sowers
Abstract: DESCRIPTION (provided by applicant): The development of cancer in man requires a series of genetic changes including both mutations and epigenetic alterations. Promoter hypermethylation (epigenetic) leads to the silencing of multiple tumor suppressor genes. Very little is known about mechanisms by which methylation patterns are altered. In the previous period of funding, we focused on inflammation-mediated DNA damage due to the long-standing association between inflammation and the development of cancer. Through a series of studies using in vitro and model systems, we established that reactive molecules generated by activated neutrophils and eosinophils can generated an array of DNA adducts. Among these products are 5-chlorocytosine (5ClC) and 5-bromocytosine (5BrC). We established that proteins containing methyl-binding domains, as well as the human maintenance methyltransferase DNMT1, do not distinguish these adducts from 5-methylcytosine. Therefore, these inflammation- generated DNA damage products could act as fraudulent epigenetic signals resulting in local hypermethylation. Several other adducts were shown to interfere with DNA-protein interactions, potentially leading to loss of methylation. Although multiple studies have measured chlorinated and brominated amino acids associated with human disease, the literature is essentially silent on the presence of 5ClC and 5BrC in human tissues. Several methodological issues are discussed here that have hampered these measurements. As described in Aim 1, we have the reagents and expertise needed to develop the required analytical methods. In human tissues, reactive molecules generated by immune cells must cross the cell membrane and enter the nucleus in order to react with the DNA. Preliminary data support the hypothesis that the formation of chloramines or bromamines might facilitate the delivery of reactive molecules to the nucleus, and that some tertiary amines, including nicotine, might catalyze halogen transfer from the haloamines to cytosine. This hypothesis will be tested in Aim 2 using methods developed in Aim 1. The presence of the 5-halocytosines within promoter regions could serve to both silence transcription and "seed" further methylation. In order to begin testing this hypothesis, we present new methods in Aim 3 that both selectively generate 5-halocytosine in the DNA of human cells in culture and allow its detection at the DNA sequence level. In Aim 4, we propose to use methods developed in the previous aims to directly measure the presence of 5- halocytosines in the DNA of normal and tumor tissues. We present for the first time, the measurement of 5ClC and 5BrC in human surgical tissues. Completion of the aims proposed here will allow an in- depth examination of the connection between inflammation, DNA damage and cancer etiology.
Funding Period: 2000-05-01 - 2017-03-31
more information: NIH RePORT
- Endogenous cytosine damage products alter the site selectivity of human DNA maintenance methyltransferase DNMT1Victoria Valinluck
Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA
Cancer Res 67:946-50. 2007..These data may provide a mechanistic link for the associations documented between inflammation and cancer...
- Inflammation-mediated cytosine damage: a mechanistic link between inflammation and the epigenetic alterations in human cancersVictoria Valinluck
Department of Biochemistry and Microbiology, School of Medicine, Loma Linda University, Loma Linda, California 92354, USA
Cancer Res 67:5583-6. 2007..These inflammation-mediated cytosine damage products may provide, in some cases, a mechanistic link between inflammation and cancer...
- Polymerase incorporation and miscoding properties of 5-chlorouracilCherine H Kim
Department of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, California 92350, USA
Chem Res Toxicol 23:740-8. 2010..When mispaired with G, ClU is targeted for removal by human glycosylases. The formation, incorporation, and repair of ClU could promote transition mutations and other forms of heritable DNA damage...
- Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specificationShinsuke Ito
Howard Hughes Medical Institute
Nature 466:1129-33. 2010..Thus, our studies not only uncover the enzymatic activity of the Tet proteins, but also demonstrate a role for Tet1 in ES cell maintenance and inner cell mass cell specification...
- Impact of base analogues within a CpG dinucleotide on the binding of DNA by the methyl-binding domain of MeCP2 and methylation by DNMT1Victoria Valinluck Lao
Department of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, California 92350, United States
Biochemistry 49:10228-36. 2010..The enhanced binding of the MBD to oligonucleotides containing several cytosine analogues observed here is better explained by a DNA-protein interface mediated by structured water as opposed to hydrophobic interactions...
- Mass spectrometric studies on epigenetic interaction networks in cell differentiationLei Xiong
Department of Chemistry, University of California, Riverside, California 92521, USA
J Biol Chem 286:13657-68. 2011..Collectively, our approach has provided an unprecedentedly systemic set of insights into the role of epigenetic regulation in leukemia cell differentiation...
- Enthalpy-entropy compensation in biomolecular halogen bonds measured in DNA junctionsMegan Carter
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, United States
Biochemistry 52:4891-903. 2013..Thus, the overall free energy of an X-bonding interaction balances the stabilizing electrostatic effects of the σ-hole against the competing effects on the local structural dynamics of the system. ..