The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments

Abstract

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.

Figure 1

Analysis of Myc-tagged R1 expression from pSHD/mR1 transiently transfected BHK cells by the indirect immunofluorescence assay (A), and from the stable retroviral packaging cell line PA/mR1 by radioimmunoprecipitation (B). Anti-Myc epitope antibody 9E10 was used for both assays.

Figure 2

Reduced growth efficiency in soft agar with recombinant R1 expressing cells. Cell lines stably infected with the R1 viral vector were compared with appropriate empty vector-infected control cell lines (see Table 1 and below). Data presented were obtained from at least three independent experiments, each consisting of triplicate plates per cell line. Inoculum sizes (cells/plate) were as follows: 5 × 10⁵ for C1/SHD and C1/mR1 cells, 1 × 10⁵ for C1/mR2 and C1/mR2/mR1 cells, 1 × 10⁴ for C1/mR2a/SHD and C1/mR2a/mR1 cells, ras-3/SHD and ras-3/mR1 cells, and 1 × 10³ for Colo/SHD and Colo/mR1 cells. In all cases, the difference in numbers of colonies formed between recombinant R1 expressing cells and the control cells were statistically significant (P < 0.001).

Figure 3

Increased colony-forming efficiency in soft agar with N/ras & ASR1 cells (b) as compared with control N/ras cells (a). Each plate was inoculated with 1 × 10⁴ cells. N/ras & ASR1 cells formed at least four times more colonies, which were generally larger than those formed by N/ras cells. The colonies shown in a developed after 3 weeks and those shown in b developed after 2 weeks of incubation. When data from six experiments each consisting of four plates per cell line were analyzed, the difference in colony-forming efficiencies exhibited by the two cell lines were found to be highly significant (P < 0.0001).

Figure 4

(A) Western blot analysis showing reduced R1 protein in N/ras & ASR1 (b) as compared with control N/ras cells (a). (B) India ink staining of the nitrocellulose membrane shown in A, demonstrating approximately equal loading of cell extracts. Densitometric analysis indicated a decrease of 3.2-fold in R1 protein level in N/ras & ASR1 cells when compared with N/ras cells.