Tretyakova Research Group Research Areas

Mapping carcinogen-induced DNA damage within critical genes by mass spectrometry

The human genome is under a continuous attack by chemicals present in the environment, our diet, and also produced naturally as a result of normal metabolism. Many of the unwanted chemicals undergo metabolic processing to convert them to species more easily excreted from the body. Unfortunately, some of these metabolites are capable of chemically reacting with DNA, altering the DNA bases to form DNA adducts. Since DNA adducts have distorted biophysical properties, if not repaired, they can cause a sequence of DNA to be incorrectly copied during DNA replication, resulting in permanent mutations (Scheme 1). Mutations that take place in critical genes that control cell growth and differentiation can transform normal cell into a cancerous cell. For example, proto-oncogenes are present in an inactive form in all normal cells, but a single mutation in these genes can create a cancerous cell.

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Since carcinogens modify DNA in sequence-selective manner, it should be possible to link the genetic alterations observed in cancer to specific types of DNA damage. The most commonly mutated sites (mutational "hot spots") within the DNA sequence should co-incide with the sites targeted by chemical carcinogens (Scheme 1). However, it is a challenging task because humans are exposed to complex mixtures of chemicals on a daily basis. Furthermore, existing tools for locating chemical damage within gene sequences cannot distinguish DNA modifications caused by different carcinogens. This knowledge gap limits our understanding of cancer etiology and hinders the development of new strategies for decreasing cancer risk.

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We have developed a way to determine the chemical structure and sequence location of carcinogen-induced DNA damage. Our approach uses mass spectrometry to measure the masses of individual nucleobases within gene sequence. Until recently, mass spectral analysis of DNA was impossible due to the large chemical size and complexity of DNA biopolymers. However, recent advances in the technique have made it possible to analyze intact DNA and protein molecules by mass spectrometry. We have taken advantage of the recent breakthrough in biological mass spectrometry to develop novel methods that enable us to map the location of carcinogen-induced DNA damage within critical genes. In one approach, carcinogen-damaged DNA is incubated with an exonuclease enzyme that sequentially removes DNA bases from one end of a DNA chain. The resulting DNA "ladders" are analyzed by mass spectral analysis of (Scheme 2). Since mass spectrometry provides the molecular mass of each fragment, mass differences between each two adjacent peaks correspond to nucleobases within DNA sequence. Each normal DNA nucleobase has a unique mass, enabling us to directly "read" DNA sequence from the mass spectra (Scheme 2). Chemically modified nucleobases are detected and identified based on the characteristic changes in mass of the DNA bases involved in carcinogen binding (Scheme 2). The final result of such analysis is a complete DNA sequence, indicating the exact position of carcinogen-damaged DNA bases.

One major research area in our laboratory focuses on tobacco carcinogenesis. Cigarette smoking is responsible for at least 85% of total lung cancer cases in the US. Although over 60 carcinogens have been isolated from tobacco smoke, their relative importance for lung cancer initiation is unknown. NIH supported project "Sequence effects on the formation of tobacco carcinogen-DNA adducts" investigates the distribution of tobacco carcinogen-DNA adducts within the context of the K-ras proto-oncogene and the p53 tumor suppressor gene, two primary genetic targets in lung cancer.

The ultimate goal of this research is to elucidate the biological mechanisms of chemical carcinogens by establishing a link between specific types of DNA damage and the genetic changes observed in human cancers. By improving our understanding of the early critical steps in cancer initiation, we hope to offer new strategies for its prevention and treatment.

Molecular mechanisms of DNA-DNA and DNA-protein cross-linking agents

This major area of investigation in our laboratory concerns the molecular mechanisms of action of bifunctional antitumor drugs and carcinogens, e.g. nitrogen mustards and diepoxybutane. These molecules are capable of simultaneously reacting with two sites within the DNA duplex, cross-linking DNA. Depending on their structure, DNA cross-links can either cause cell death (this is the basis of action of bifunctional antitumor drugs) or give rise to heritable mutations as depicted in Scheme 1. It is important to identify the structural features of the cross-linking agents that render them biologically active. Our NIH supported project "DNA cross-linking by diepoxybutane" investigates chemical structures and biochemical characteristics of diepoxybutane induced DNA cross-links. This involves preparation of authentic nucleobase cross-links by an independent synthesis and their detection in DNA by isotope dilution HPLC-MS-MS.

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Cross-linking of DNA-binding proteins to their DNA targets

In addition to DNA-DNA cross-links, bifunctional lesions involving DNA and proteins are likely to play an important role in cytotoxicity of bifunctional electrophiles. DNA binding proteins such histones, High Mobility Group (HMG) proteins, transcription factors, and DNA repair proteins, are at risk of becoming covalently linked to their DNA targets in the presence of bifunctional alkylating agents. The resulting bulky adducts block DNA replication and transcription. Importantly, many DNA repair proteins are overproduced in tumor cells. We hypothesize that the cross-linking of DNA repair proteins to their DNA targets contributes to the mechanisms by which tumor cells are sensitized to anticancer DNA-damaging drugs.

In collaboration with Prof. Anthony Pegg at Penn State, we are investigating the cross-linking of human O⁶-alkylguanine DNA alkyltransferase (AGT) to DNA in the presence of 1,2,3,4-diepoxybutane (DEB). Initial DNA reactions with DEB generate N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)guanine monoadducts which retain one of the epoxy groups. We have shown that AGT binding to DEB-modified DNA leads to the formation of DNA-protein lesions that involve the active site cysteine of AGT (Cys 145). This was determined by HPLC-ESI-MS/MS analyses of tryptic digests of AGT and peptide sequencing. Studies are now in progress at our laboratory involving other DNA-binding proteins (e.g. histine H4) and bifunctional electrophiles (nitrogen mustards) to establish the role of DNA-protein cross-linking in their biological activity.

New biomarkers of oxidative stress

Another area of interest deals with DNA oxidation by reactive oxygen species produced in normal aerobic metabolism, immune responses, and inflammation. The oxidative degradation of DNA has been implicated in aging, cancer, and in some degenerative diseases. We are identifying new biomarkers of oxidative stress that can potentially serve as indicators of carcinogenic risk based on the highly sensitive and specific detection of oxidized DNA bases by mass spectrometry.

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