Overexpression of Ribonucleotide Redactase in Transfected Human KB Cells Increases Their Resistance to Hydroxyurea: M2 but not Ml Is Sufficient to Increase Resistance to Hydroxyurea in Transfected Cells

Ribonucleotide reducÃ-ase (RR) is a rate-limiting enzyme in DNA syn thesis. The enzyme consists of two subunits, Ml and M2. Hydroxyurea (III ) is an M2-specific inhibitor. It has been shown that a I II'-résistant clone derived from stepwise exposure to HU overexpresses the M2 mRNA and the RR protein (Y. Yen et al, Cancer Res., 54: 3868-3691, 1994). In this study, we established stable clones by transfecting human KB cells with the cDNA of human wild-type RR in which each subunit was overexpressed by a SV40 promoter. The mammalian cell expression vector l>II/i APr-1 was used for constructing Ml, M2, and M1/M2 subunit cDNA. The transfected cells were selected with G418. The clones designated M2-D, Ml-D, X-D, and KIÃŽ-Vrepresent transfectant clones which contain M2 cDNA, Ml cDNA, M1/M2 cDNA, and vector alone, respectively. The parental KB cells and clones containing vector plasmid KB-V express equally low amounts of M2 and Ml mRNA from the endogenous genes. The expression of M2 mRNA and Ml mRNA is elevated 2-3-fold in the X-D transfectants. M2-D clone demonstrated a 6-fold higher M2 mRNA level although the Ml mRNA expression remains the same as parental cells. Ml-D transfectants have a 3-fold increase in Ml mRNA expression relative to parental cells, but reveal no alteration of M2 mRNA. Southern analysis of genomic DNA suggested the incorporation of the plasmid into the genome. The X-D clone revealed both integration of the A/2 and Ml gene while the M2-D clone only showed M2 gene integration. The Ml-D clone revealed Ml gene integration relative to the parental cells. The Western blot of M2 protein showed a 3-fold increase in the X-D and M2-D clones whereas the M2 protein level in Ml-D was the same as it was in parental cells. The Ml protein was increased 3-fold in X-D and 1.5-fold in Ml-D over that of parental cells. However, lower Ml protein levels were identified in the M2-D clone. The specific activity of the RR enzyme from each transfectant showed a 3-fold increase in both the X-D and M2-D clones and slightly increased in Ml-D clone over that of parental cells. However, X-D and M2-D both demonstrated a 3-fold increase in resist ance to HU as compared to Ml-D which showed the same sensitivity as the parental enzyme. From these results, we propose that the "enzymatic" activity and the resistance of RR to HU are greatly affected by the amount of the M2 subunit in the cell. These transfectants will be utilized for obtaining detailed information regarding the RR enzyme.