RIBONUCLEOTIDE REDUCTASE R2 COMPONENT IS A NOVEL MALIGNANCY DETERMINANT THAT COOPERATES WITH ACTIVATED ONCOGENES TO DETERMINE TRANSFORMATION AND MALIGNANT POTENTIAL

Ribonucleotide reductase is a highly regulated cell cycle-controlled a ctivity that is essential for DNA synthesis and repair, A retroviral v ector for the R2 component of mammalian ribonucleotide reductase, the rate-limiting protein for enzyme activity and DNA synthesis in prolife rating cells, was constructed and introduced into mammalian cells, Exp ression 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-transforme d mouse 10T1/2 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 significa ntly reduced subcutaneous tumor latency and increased tumor growth rat es in syngeneic mice, and showed markedly elevated metastatic potentia l 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 t o the plasma membrane for activation, We show that recombinant R2 expr ession leads to significant increases in membrane-associated Raf-1 pro tein and mitogen-activating protein kinase-2. activity suggesting a me chanism for the observed Ras/R2 synergism. In support of this finding, we observed that activated Rac-1, which operates parallel to Raf-1 an d cooperates with Raf-1 in Ras activated pathways, also cooperates wit h R2 in cellular transformation, These studies demonstrate that the R2 protein can participate in other critical cellular functions in addit ion to ribonucleotide reduction, and that deregulated R2 is a novel tu mor progressor determinant that cooperates in oncogene-mediated mechan isms, which control malignant potential.