Ribonucleotide reductase R2 component is a novel malignancy determinant that cooperates with activated oncogenes to determine transformation and malignant potential

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Abstract
Ribonucleotide reductase is a highly regulated cell cycle-controlled activity that is essential for DNA synthesis and repair. A retroviral vector for the R2 component of mammalian ribonucleotide reductase, the rate-limiting protein for enzyme activity and DNA synthesis in proliferating cells, was constructed and introduced into mammalian cells. Expression of Myc epitope-tagged R2 protein in benign BALB/c 3T3 and NIH 3T3 cells leads to a greatly increased frequency of focus formation in cooperation with H-ras transformation. Four lines of H-ras-transformed mouse 10T½ fibroblasts showed increased growth efficiency in soft agar after infection with the recombinant R2 expression virus vector. Furthermore, cells with altered R2 expression also exhibited significantly reduced subcutaneous tumor latency and increased tumor growth rates in syngeneic mice, and showed markedly elevated metastatic potential in lung metastasis assays. The results indicate that altered R2 gene expression cooperates with ras in mechanisms of malignant progression. A major Ras pathway involves the Raf-1 protein, which is recruited to the plasma membrane for activation. We show that recombinant R2 expression leads to significant increases in membrane-associated Raf-1 protein and mitogen-activating protein kinase-2 activity suggesting a mechanism for the observed Ras/R2 synergism. In support of this finding, we observed that activated Rac-1, which operates parallel to Raf-1 and cooperates with Raf-1 in Ras activated pathways, also cooperates with R2 in cellular transformation. These studies demonstrate that the R2 protein can participate in other critical cellular functions in addition to ribonucleotide reduction, and that deregulated R2 is a novel tumor progressor determinant that cooperates in oncogene-mediated mechanisms, which control malignant potential.