Determination of deoxyribonucleoside triphosphate pool sizes in ribonucleotide reductase cDNA transfected human KB cells.
Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis, which is responsible for controlling deoxyribonucleoside triphosphate (dNTP) pool size. It has been shown that transfection of RR M2 cDNA in human KB cells (M2-D clone) results in overexpression for the M2 subunit and resistance to hydroxyurea (HU). In this study, dNTP pool assays were performed to measure the pool sizes in six cell lines: two controls, three transfectants, and drug-induced HU-resistant (HUR) cells. Total dNTP levels among the six cell lines rose in the following order: KB wild-type, KB vector-only transfectant, M1 cDNA transfectant, M2 cDNA transfectant, M1/M2 cDNA transfectant, and HU-induced resistant clone. The dCTP levels of the cells mimicked the total dNTP pools on 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 increases 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 RR activity compared with the control KB wild-type cells. In contrast, a profound inhibition of dCTP and a decreased sensitivity to dATP inhibition 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 expanded dNTP pools, particularly dCTP, when compared with wild-type KB cells. These data provide evidence for the intertwined relationship between RR activity and dNTP pools.
Zhou
B S
BS
City of Hope National Medical Center, Duarte, CA 91010-3000, USA.
Ker
R
R
Ho
R
R
Yu
J
J
Zhao
Y R
YR
Shih
J
J
Yen
Y
Y
D002999:Clone Cells; D018076:DNA, Complementary; D003854:Deoxyribonucleotides / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D003854:Deoxyribonucleotides; D012333:RNA, Messenger; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea