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.
Our laboratory has been studying how inflammation can result in DNA damage because of the well known correlations between inflammation and cancer. We have studied the types of DNA damage induced by reactive molecules generated during inflammation, and we have identified specific forms of DNA damage including 5-bromocytosine (5BrC) and 5-chlorocytosine (5ClC) that we propose could contribute to aberrant methylation in tumor cells leading to malignant transformation. Results of these studies could lead to rational approaches to reduce cancer incidence.
|Theruvathu, Jacob A; Yin, Y Whitney; Pettitt, B Montgomery et al. (2013) Comparison of the structural and dynamic effects of 5-methylcytosine and 5-chlorocytosine in a CpG dinucleotide sequence. Biochemistry 52:8590-8|
|Carter, Megan; Voth, Andrea Regier; Scholfield, Matthew R et al. (2013) Enthalpy-entropy compensation in biomolecular halogen bonds measured in DNA junctions. Biochemistry 52:4891-903|
|Seiberling, Kristin A; Church, Christopher A; Herring, Jason L et al. (2012) Epigenetics of chronic rhinosinusitis and the role of the eosinophil. Int Forum Allergy Rhinol 2:80-4|
|Xiong, Lei; Darwanto, Agus; Sharma, Seema et al. (2011) Mass spectrometric studies on epigenetic interaction networks in cell differentiation. J Biol Chem 286:13657-68|
|Ito, Shinsuke; D'Alessio, Ana C; Taranova, Olena V et al. (2010) Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466:1129-33|
|Lao, Victoria Valinluck; Darwanto, Agus; Sowers, Lawrence C (2010) Impact of base analogues within a CpG dinucleotide on the binding of DNA by the methyl-binding domain of MeCP2 and methylation by DNMT1. Biochemistry 49:10228-36|
|Kim, Cherine H; Darwanto, Agus; Theruvathu, Jacob A et al. (2010) Polymerase incorporation and miscoding properties of 5-chlorouracil. Chem Res Toxicol 23:740-8|
|Theruvathu, Jacob A; Kim, Cherine H; Rogstad, Daniel K et al. (2009) Base pairing configuration and stability of an oligonucleotide duplex containing a 5-chlorouracil-adenine base pair. Biochemistry 48:7539-46|
|Herring, Jason L; Rogstad, Daniel K; Sowers, Lawrence C (2009) Enzymatic methylation of DNA in cultured human cells studied by stable isotope incorporation and mass spectrometry. Chem Res Toxicol 22:1060-8|
|Theruvathu, Jacob A; Kim, Cherine H; Darwanto, Agus et al. (2009) pH-Dependent configurations of a 5-chlorouracil-guanine base pair. Biochemistry 48:11312-8|
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