Our recent studies have shown that deregulated expression of R2, the rate-limiting component of ribonucleotide reductase, enhances transformation and malignant potential by cooperating with activated oncogenes. We now demonstrate that the R1 component of ribonucleotide reductase has tumor-suppressing activity. Stable expression of a biologically active ectopic R1 in ras-transformed mouse fibroblast 10T 1 ⁄2 cell lines, with or without R2 overexpression, led to significantly reduced colony-forming efficiency in soft agar. The decreased anchorage independence was accompanied by markedly suppressed malignant potential in vivo. In three ras-transformed cell lines, R1 overexpression resulted in abrogation or marked suppression of tumorigenicity. In addition, the ability to form lung metastases by cells overexpressing R1 was reduced by >85%. Metastasis suppressing activity also was observed in the highly malignant mouse 10T 1 ⁄2 derived RMP-6 cell line, which was transformed by a combination of oncogenic ras, myc, and mutant p53. Furthermore, in support of the above observations with the R1 overexpressing cells, NIH 3T3 cells cotransfected with an R1 antisense sequence and oncogenic ras showed significantly increased anchorage independence as compared with control ras-transfected cells. Finally, characteristics of reduced malignant potential also were demonstrated with R1 overexpressing human colon carcinoma cells. Taken together, these results indicate that the two components of ribonucleotide reductase both are unique malignancy determinants playing opposing roles in its regulation, that there is a novel control point important in mechanisms of malignancy, which involves a balance in the levels of R1 and R2 expression, and that alterations in this balance can significantly modify transformation, tumorigenicity, and metastatic potential.Ribonucleotide reductase is the only enzyme in the cell that is responsible for converting ribonucleotides into deoxyribonucleotides, the eventual substrates for DNA polymerase (1-3). In mammalian cells, this enzyme contains two dissimilar protein components, called R1 and R2, which are encoded by two different genes located on different chromosomes (4). Protein R1 is a homodimeric structure, with a molecular mass of 168 kDa, and has substrate sites and allosteric effector sites that control enzyme activity and substrate specificity (3, 5). Protein R2 is a homodimer, with a molecular mass of 88 kDa, and forms two equivalent dinuclear iron centers that stabilize a tyrosyl free radical required for the initiation of electron transformation during catalysis (5, 6). R1 and R2 proteins interact at their C-terminal ends to form an active holoenzyme (5, 7).Expression of ribonucleotide reductase is highly regulated (3,5,8). Nonproliferating cells do not contain ribonucleotide reductase activity. In proliferating cells the R2 protein is found primarily in S phase of the cell cycle (9). Although the R1 protein can be detected throughout the cell cycle, synthesis of R1 mRNA, like R2 mRNA...

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 10T 1 ⁄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 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 mitogenactivating 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.The first unique step leading to DNA synthesis is the conversion of ribonucleotides to their corresponding deoxyribonucleotides, a reaction that is catalyzed in a cell cycle-specific manner by ribonucleotide reductase (1-3). The enzyme is composed of two dissimilar components often called R1 and R2, which are differentially regulated during the cell cycle. Although the levels of the R1 protein do not appear to change substantially during the cell cycle, there is an S-phase correlated increase in the R2 protein resulting from its de novo synthesis (1, 4). Interestingly, the activity of ribonucleotide reductase, and therefore DNA synthesis and cell proliferation, is controlled during the cell cycle by the synthesis and degradation of the R2 component (5). The rate-limiting R2 component is a phosphoprotein capable of being phosphorylated by the CDC2 and CDK2 protein kinase mediators of cell cycle progression (6), and contains non-heme iron that stabilizes a unique tyrosyl-free radical required for enzyme activity (1, 2, 7). Chemotherapeutic compounds like hydroxyurea inhibit ribonucleotide reductase activity by destabilizing the iron center of the R2 protein causing the destruction o...

We have constructed a retroviral expression vector for the mammalian ribonucleotide reductase R2 component. Stable infectants, which express a myc epitopo tagged R2 protein from the vector eDNA were obtained and described for the first time. Cells containing the recombinant protein exhibited increased ribonucleotide reductase activity, and were resistant to the antitumour agent hydroxyurea, which targets the R2 component of ribonucleotide reductase. Furthermore, a direct link between ferritin gene expression and R2 protein was observed, since cells containing vector expressed recombinant R2 protein exhibited Increased H-chain and L.chain ferritin gone expression.

A human Myc epitope is frequently used to tag proteins for expression experiments in nonhuman cells. We used the monoclonal 9E10 antibody specific for this epitope to analyse the expression of four proteins carrying the Myc tag in cells transfected with expression vectors. While all four proteins can be detected by immunofluorescence and immunoprecipitation assays, surprisingly, only two proteins could be detected in Western blot analysis, indicating that epitope recognition by the monoclonal antibody can be blocked in some membrane-retained ectopic proteins. Other techniques such as immunofluorescence and immunoprecipitation assays can be successfully used with the 9E10 antibody to determine potential expression of Myc-tagged proteins.

A human Myc epitope is frequently used to tag proteins for expression experiments in nonhuman cells. We used the monoclonal 9E10 antibody specific for this epitope to analyse the expression of four proteins carrying the Myc tag in cells transfected with expression vectors. While all four proteins can be detected by immunofluorescence and immunoprecipitation assays, surprisingly, only two proteins could be detected in Western blot analysis, indicating that epitope recognition by the monoclonal antibody can be blocked in some membrane-retained ectopic proteins. Other techniques such as immunofluorescence and immunoprecipitation assays can be successfully used with the 9E10 antibody to determine potential expression of Myc-tagged proteins.