Effects Of Mammalian Ribonucleotide Reductase Deregulation On Redox Homeostasis And Genomic Integrity
Ribonucleotide reductase catalyzes the rate-limiting step in de novo deoxyribonucleoside triphosphate (dNTPs) biosynthesis and is essential for providing balanced dNTP pools for nuclear and mitochondrial genome maintenance. RNR contains two components R1 and R2. R2 generates free radicals that are transferred to R1 and used for catalysis. RNR is tightly regulated through several mechanisms, including the control of R2 expression levels. Broad overexpression of R2 in transgenic mice causes lung neoplasms through a mutagenic mechanism. Because R2 produces free radicals, I hypothesized that R2 deregulation results in mutagenic perturbations of cellular redox status which could contribute to R2-induced tumorigenesis. This dissertation aims to (A) elucidate the effect of RNR deregulation on redox homeostasis and dissect the molecular mechanism of RNR-induced mutagenesis and tumorigenesis, and (B) use RNR mice as a lung tumor model in imaging studies to assess lung tumor growth patterns. For the first aim, we generated cells that overexpress R2 and showed that this overexpression leads to increased reactive oxygen species (ROS) production. By generating a series of R2 mutants, we subsequently identified the source of R2-induced ROS production. In addition, some R2 mutants showed dominant negative effects by interfering with endogenous RNR, leading to mitochondrial DNA depletion and mitochondrial redox imbalance. These findings indicate the importance of RNR regulation in maintaining cellular ROS levels and suggest the possibility that R2-induced ROS may play a role in mutagenesis. For the second aim, we adapted an automated algorithm for the measurement of pulmonary nodules on human chest CT scans and used it to measure mouse lung tumors. Euthanized mice were first imaged to optimize scan parameters and refine computational algorithms for tumor volume measurement. Lung tumor-bearing mice were then scanned sequentially for tumor growth rate determination. Findings from this study establish new automated algorithms to measure lung tumor volume in mice and confirm an exponential growth model for murine lung neoplasms. Together, these studies demonstrate the importance of RNR regulation in maintaining cellular redox homeostasis and genome integrity, and that RNR mice serve as an authentic model of human lung cancer in translational studies.
Ribonucleotide Reductase; Redox Homeostasis; Genomic Integrity
Weiss, Robert S.
Schimenti, John C.; Soloway, Paul
Molecular & Cell Biology
Ph.D. of Molecular & Cell Biology
Doctor of Philosophy
dissertation or thesis