DETERMINATION OF DEOXYRIBONUCLEOSIDE TRIPHOSPHATE POOL SIZES IN RIBONUCLEOTIDE REDUCTASE CDNA TRANSFECTED HUMAN KB CELLS

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthes is, which is responsible for controlling deoxyribonucleoside triphosph ate (dNTP) pool size. It has been shown that transfection of RR M2 cDN A in human KB cells (M2-D clone) results in overexpression for the M2 subunit and resistance to hydroxyurea (HU). In this study, dNTP pool a ssays were performed to measure the pool sizes in six cell lines: two controls, three transfectants, and drug-induced HU-resistant (HUR) cel ls. Total dNTP levels among the six tell lines rose in the following o rder: KB wild-type, KB vector-only transfectant, M1 cDNA transfectant, M2 cDNA transfectant, M1/M2 cDNA transfectant, and HU-induced resista nt clone. The dCTP levels of the cells mimicked the total dNTP pools o n a smaller scale. The significant increases in the dCTP pool sizes of the M2-D, X-D, and HUR clones were proportional to their respective i ncreases in RR activity. Relative to all other transfectants, the M1-D clone demonstrated lower dCTP levels but increased dATP pools. The M1 -D clone demonstrated a significant resistance to dNTP inhibition of R R activity compared with the control KB wild-type cells. In contrast, a profound inhibition of dCTP and a decreased sensitivity to dATP inhi bition was observed in M2-D, X-D, and HUR clones. In summary, M2 cDNA transfectants and HUR clones had increased RR activity as well as expa nded dNTP pools, particularly dCTP, when compared with wild-type KB ce lls. These data provide evidence for the intertwined relationship betw een RR activity and dNTP pools. (C) 1998 Elsevier Science Inc.