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Mechanistic Studies on the NAT2 Genetic Polymorphism: A Potential Factor that Modifies Individual Breast Cancer Risk
The breast cancer incidence rate among US women continues to increase. My research emphasizes breast cancer prevention, especially study of individual breast cancer susceptibility. Epidemiologic and animal research have found that environmental or dietary exposure to chemical carcinogens contributes to the etiology of breast cancer. Aromatic amines in cigarette smoke and heterocyclic amines in foods are mammary carcinogens. Human arylamine-N-acetyltransferase 2 (NAT2) metabolizes these carcinogens in a complex manner. NAT2 detoxifies aromatic amines by its N-acetylation activity, whereas it bioactivates N-hydroxyl heterocyclic amines by its O-acetylation activity into electrophilic products that may form DNA adducts. NAT2 is highly polymorphic within the human population. Most polymorphisms are single nucleotide substitutions in the coding region of NAT2 gene, some of which cause functional variations. Considering the role of NAT2 in the metabolism of mammary carcinogens, NAT2 polymorphisms may modify the association of carcinogen exposure with breast cancer by influencing the individual response to these compounds. However, molecular epidemiologic studies on breast cancer concerning both NAT2 status and lifestyle carcinogen exposures are limited and the results are inconsistent. Due to the poor understanding of NAT2 polymorphism at present, it is possible that the accuracy and validity of these results are compromised. The objective of this proposal is to improve the knowledge of NAT2 polymorphism by accomplishment of the following three research aims. Aim 1: Establish a comprehensive NAT2 phenotype-genotype association. Aim 2: Elucidate the possible mechanisms for how nucleotide changes in NAT2 coding region cause functional changes. 3. Build a molecular structure model for human NAT2. In specific aim 1, I will use a mammalian cell expression system to functionally characterize different NAT2 SNPs and alleles. The coding region of wildtype NAT2 allele will be inserted into a mammalian expression vector and the variant NAT2 alleles will be constructed by site-directed mutagenesis. The NAT2 alleles will be transiently expressed in COS-1 cells and the NAT2-catalyzed N-acetylation and O-acetylation of mammary carcinogens will then be measured in harvested cells so that the phenotypes are identified. In specific aim 2, I will focus on the allozymes with lower activities. We anticipate to find heterogeneous mechanisms for different NAT2 allozymes. First, kinetic parameters Vmax and Km will be determined to examine whether the substrate binding region or catalytic center of the enzyme has been changed. NAT2 mRNA level will be determined by real-time RT-PCR to find out whether the transcription is impaired. Moreover, NAT2-specific immunoblotting will also be performed to quantify expressed protein. If less immunoreactive protein has been detected, protein thermostability and degradation assays will be performed. In specific aim 3, I will build a homology based structure model for human NAT2. The experimental data in aim 2 will be fitted into the model. Site-directed mutagenesis and docking experiments will be performed to test and optimize the model. This is the first comprehensive study that functionally characterizes different NAT2 alleles in mammalian culture. The results of this research will ultimately contribute to the understanding of gene-environmental interactions in breast cancer. First, better study design and proper interpretations can be made by avoiding misclassification of study subjects. Therefore, the role of NAT2 and some potential carcinogens in breast cancer risk can be clearer. Second, the structure model helps predict the fate of chemical carcinogens and provide valuable information on cancer prevention. In conclusion, understanding the mechanisms for activity changes in different NAT2 allozymes is essential for identifying high-risk persons, assessing individual cancer risk, and developing individual breast cancer prevention strategies.
The number of women diagnosed with breast cancer keeps on growing. In fact, it is estimated by American Cancer Association that breast cancer will rank first in the 2003 US cancer cases among women. Thus, efforts in breast cancer prevention are very important. In order to develop effective prevention schemes, risk factors for breast cancer need to be discovered and identified. Arylamine-N-acetyltransferase 2 (NAT2) is a potential factor that influences breast cancer risk. This enzyme plays a significant role in the clearance and biotransformation of some mammary carcinogens. There are about twenty-five forms of NAT2 gene, named alleles, in populations across the world. People with different NAT2 allele combinations (genotypes) are found to display different levels of NAT2 activities (phenotypes) in their body. Therefore, they should respond differently to the same carcinogen and have different chances of getting breast cancer. Previous studies have shown that NAT2 phenotype modifies individual breast cancer risk from cigarette smoking or consumption of well-done red meat. However, additional studies have reported inconsistent results. Due to the technical impracticality of measuring NAT2 phenotype from samples, the phenotypes were usually predicted by the subject’s genotype in these studies. Since the relationship between NAT2 genotype and phenotype hasn’t been fully studied in mammalian cells, some subjects may be classified into wrong categories and lead to biased results. We believe that the lack of knowledge about NAT2 polymorphism can compromise accuracy of etiological studies. Also, since the mechanism of NAT2 action is not very clear and how different alleles result in different NAT2 activities is still unknown, it is impossible to assess individual cancer risk based on their NAT2 status and carcinogen exposure. The goal of my dissertation research is to improve our understanding of NAT2 polymorphisms. Three specific aims will be accomplished in my studies. In the first specific aim, I plan to establish a complete NAT2 genotype-phenotype relationship in mammalian cells by functionally characterizing most NAT2 alleles, including some newly discovered ones. I will first make different NAT2 alleles by artificially mutating an existing wild type NAT2 gene, and then express them into culture cells separately. The cells will produce different NAT2 proteins based on the type of allele they received. After I get different NAT2 proteins produced by the cells, I will measure the enzyme activities of these various NAT2 proteins to establish an allele-function relationship. With the knowledge of this relationship, more scientific studies can be designed in order to explore the role of NAT2 in breast cancer risk. In specific aim 2, I will focus on alleles that encode NAT2 proteins with lower activity, and try to figure out how these variant NAT2 proteins lose their normal function. Our preliminary studies indicate that there are multiple mechanisms involved. The variant proteins may lose the catalytic capability or lose the stability. Both of them can be caused by changes in protein structure. I will perform experiments to measure mRNA and protein levels, kinetic parameters, protein stability and degradation for each of those alleles. Our expectation is to see different mechanisms for different alleles. In aim 3, I will build a theoretical three-dimensional structure model of NAT2 on a computer. This model will help us understand the mechanism how NAT2 works. It will also help to explain the results in aim 2. This information is highly valuable because only if we understand the mechanism why people with different form of NAT2 gene have different NAT2 activity can we predict the individual cancer risk and set up individualized breast cancer prevention strategies.