Molecular Mechanisms Of Mutagenesis And Lung Tumorigenesis Caused By Ribonucleotide Reductase Deregulation
Ribonucleotide reductase catalyzes the rate-liming step in deoxyribonucleotide triphosphate biosynthesis and is a major determinant of genomic integrity. Unbalanced dNTP pools can cause genetic abnormality and cell death. Although a number of elaborate regulatory mechanisms govern RNR activity, the physiological impact of RNR deregulation had not previously been examined in an animal model. The aim of this dissertation is to elucidate the physiological effect of RNR deregulation using transgenic mouse models and to further dissect the molecular mechanisms of RNRinduced mutagenesis and lung tumorigenesis. We generated transgenic mice that broadly overexpress individual RNR genes, and found that overexpression of the small RNR subunit potently and selectively induces lung neoplasms. RNR deregulation was found to promote lung carcinogenesis through a mutagenic mechanism, as evidenced by increased mutation rates in RNR overexpressing 3T3 cells and enhanced mutagenesis and carcinogenesis when combining RNR deregulation with defects in DNA mismatch repair. Moreover, the proto-oncogene K-ras was identified as a frequent mutational target in RNR-induced lung neoplasms. Importantly, RNR-induced lung neoplasms histopathologically resemble human papillary adenocarcinoma, making RNR transgenic mice a particularly authentic model for lung cancer. We initially hypothesized that RNR-induced mutagenesis and carcinogenesis was due to disturbed dNTP pools. However, we observed no alteration of dNTP levels or ratios in RNR overexpressing cells, suggesting that RNR-induced mutagenesis might be independent of RNR enzyme activity. Moreover, RNR overexpression was not associated with acute transforming activity. Alternatively, excess free radical production by the small RNR subunit may account for lung specific carcinogenesis in RNR transgenic mice. To further assess the requirements for free radical production and RNR enzyme activity in RNR-induced mutagenesis, we generated Rrm2 mutants and found that Rrm2 overexpressing cells exhibited significantly higher intracellular reactive oxygen species levels and Rrm2 mutants that are defective for RNR enzyme activity still promote mutagenesis in cultured 3T3 cells and exhibited elevated reactive oxygen species levels. Our data suggest that increased ROS production, rather than increased RNR enzyme activity, is the major driving force of RNR-induced mutagenesis, and potentially lung tumorigenesis. These studies establish a new oncogenic activity for the small subunit of RNR. RNR-induced lung tumors arose with moderate latency in a stochastic process associated with an elevated mutation rate and increased ROS production. This novel mouse lung cancer model holds great promise for providing insights into basic mechanisms in human lung cancer and developing effective strategy for prevention and therapy of lung cancer.
dissertation or thesis