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The Pennsylvania State University The Pennsylvania State University The Graduate School Department of Molecular Toxicology FUNCTIONAL SIGNIFICANCE OF THE GAG TRINUCLEOTIDE-REPEAT POLYMORPHISM IN THE CATALYTIC SUBUNIT OF THE GLUTATHIONE BIOSYNTHETIC ENZYME, -GLUTAMYLCYSTEINE LIGASE A Dissertation in Integrative Biosciences by Sailendra N Nichenametla 2010 Sailendra N Nichenametla Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2010 The dissertation of Sailendra Nichenametla was reviewed and approved* by the following: John P Richie Jr. Professor of Pharmacology & Health Evaluation Sciences Dissertation Advisor and Chair of Committee Philip Lazarus Professor of Pharmacology & Health Evaluation Sciences Joshua Muscat Professor of Public Health Sciences Curtis J Omiecinski Professor of Veterinary Science Scot Kimball Professor of Cellular and Molecular Physiology Peter Hudson Professor of Biology Head of the Department of Integrative Biosciences *Signatures are on file in the Graduate School iii Abstract Glutathione (GSH), the most abundant intracellular antioxidant, is proposed to prevent cancer through multiple biochemical pathways including carcinogen detoxification, scavenging and neutralizing free radicals, modulating gene expression and cell signaling. Low glutathione levels have been associated with higher risk for diseases such as chronic obstructive pulmonary disease, heart disease, arthritis and diabetes. -glutamylcysteine ligase (GCL), the rate limiting enzyme for glutathione biosynthesis is made up of a catalytic subunit (GCLC) and a modifier subunit (GCLM). A guanine-adenine-guanine (GAG) trinucleotide repeat polymorphism in the 5’ UTR of GCLC, the gene that codes for catalytic subunit, has been identified with five different alleles consisting of 4, 7, 8, 9 or 10 GAG repeats. The GAG polymorphism is associated with glutathione levels in certain cell lines and also with risk for diseases such as chronic beryllium diseases, chronic obstructive pulmonary disease and schizophrenia. In the present work, in vitro and in vivo studies were conducted to investigate if and how genetic variation (the GAG repeat polymorphism) in the GCL, the rate limiting enzyme for glutathione biosynthesis, affects glutathione levels in healthy adults and whether its effect on glutathione levels alters risk for cancer. In the first study presented here, it was hypothesized that the GAG repeat polymorphism in GCLC is associated with altered GCL activity and blood GSH levels in vivo. Healthy adult individuals were genotyped for the GAG iv polymorphism in the GCLC gene and related phenotypes, blood GCLC protein levels, blood GCL activity and GSH levels were also determined. Among the three most prevalent genotypes i.e., GAG-7/7, GAG-7/9 and GAG-9/9, individuals with genotype GAG-9/9 had lower GSH levels compared to those with genotype GAG-7/9, lower GCL activity compared to those with genotype GAG-7/7 and lower GCLC protein levels compared to those with either genotypes GAG-7/7 and GAG-7/9. These findings suggest that the GAG repeat polymorphism contributes to lower blood GSH levels in GAG-9/9 individuals than GAG-7/7 individuals apparently due to a decrease in GCLC protein levels and hence GCL activity. The specific impact of GAG repeat number on GCLC expression was investigated by in vitro transcription and in vitro translation assays. Through luciferase reporter assays of cell cultures and transcription and translation assays of cell free lysates, it was found that the GAG polymorphism in the GCLC 5’-UTR affects the translation of the luciferase mRNA but not its transcription. With an increasing number of GAG repeats in the constructs, luciferase activity and protein levels reached maximal levels in GAG-8 or GAG-9 and then decreased with GAG-10. The finding that luciferase expression was greater in GAG-9 than in GAG-7 was in contrast to in vivo findings where higher blood GCLC protein levels and blood GCL activity were observed for genotype 7/7 compared to genotype 9/9. These observations are likely a result of differential regulation of glutathione levels in red blood cells, in which all of the phenotypes were measured in our in vivo study. v Based on the functional consequences of the GAG repeat polymorphism in GCLC, we hypothesized that the GAG polymorphism is associated with risk for certain cancers. To test this hypothesis, individuals with cancers whose etiology is thought to be linked with oxidative stress i.e., lung, oral, colorectal and breast cancers and matched controls were genotyped for GAG repeats in GCLC. Statistical analyses showed that individuals with GAG-7/7 were at approximately 2-fold higher risk compared to those with GAG-9/9 for lung cancer (p=0.05) and oral cancer (p=0.03) but not for colorectal and breast cancers. Stratified analysis of lung cancer patients indicated that current smokers with GAG-7/7 were at significantly higher risk (p=0.01) but not ex-smokers or never smokers. These results are consistent with in vitro studies linking the genotype GAG-7/7 is associated with lower glutathione levels and higher risk for cancer. In addition these results highlight the importance of the GSH biosynthetic pathway in determining the risk for tobacco related cancers such as lung and oral cancers. Overall, findings from these studies suggest that genetic variation in the GSH biosynthetic pathway alters risk for cancer by contributing to different levels of GSH among individuals. Since GSH is involved in numerous biochemical functions of the cell, this genetic variation might also play a role in other diseases whose etiology is linked to GSH or oxidative stress. Furthermore, the GAG genotype information along with other polymorphisms and risk factors can be used as a genetic biomarker to identify individuals predisposed to diseases and toxicities that involve GSH metabolism or oxidative stress. vi TABLE OF CONTENTS LIST OF FIGURES .............................................................................................. ix LIST OF TABLES ............................................................................................... xi ACKNOWLEDGEMENTS .................................................................................. xii Chapter 1 - Introduction ..................................................................................... 1 1. 1. OXIDATIVE STRESS AND CANCER ..................................................................... 2 1.1.A. Sources of Oxidative Stress ................................................................................. 2 1.1.B. Consequences of Oxidative Stress ...................................................................... 8 1.1.C. Role of Oxidative Stress in Cancer .................................................................... 11 1.1.D. Antioxidant Systems .......................................................................................... 12 1.2. GLUTATHIONE AND CANCER .............................................................................20 1.2.A. Introduction ......................................................................................................... 20 1.2.B. Protective Role of Glutathione in Cancer ........................................................... 22 1.2.C. GSH Metabolism ................................................................................................ 28 1.3. FACTORS REGULATING GLUTATHIONE LEVELS ..................................................30 1.3.A. Dietary Factors Affecting Glutathione Levels ..................................................... 30 1.3.B. Genetic Factors Affecting Glutathione Levels .................................................... 33 1.3.C. Miscellaneous Factors Affecting Glutathione Levels ......................................... 34 1.4. TRINUCLEOTIDE REPEAT POLYMORPHISMS .......................................................35 1.4.A. Occurrence and Distribution of Microsatellites ................................................... 35 1.4.B. Functional Significance ...................................................................................... 37 1.4.C. Microsatellites and Cancer ................................................................................. 42 1.5. HYPOTHESES ..................................................................................................45 vii Chapter 2 - Effect of GAG repeat polymorphism in GCLC on GCLC protein levels, GCL activity and GSH levels in vivo ................................................... 48 2.1 INTRODUCTION .................................................................................................49 2.2 METHODS ........................................................................................................50 2.2.A. Recruitment of Study Subjects ........................................................................... 50 2.2.B. Biospecimen Collection ...................................................................................... 52 2.2.C. Determining GCLC Genotype, DNA Amplification ............................................. 52 2.2.D. Determining GCLC Genotype, Polyacrylamide Gel Electrophoresis ................. 54 2.2.E. Determination of Blood GSH .............................................................................. 54 2.2.F. Determination of GCL Activity ............................................................................ 56 2.2.G. Determination of GCLC Protein Levels ............................................................. 57 2.2.H. Statistical
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