Determination of deoxyribonucleoside triphosphate pool sizes in ribonucleotide reductase cDNA transfected human KB cells

Bing Sen Zhou, Rhonda Ker, Roger Ho, Jonathan Yu, Yong Ren Zhao, Jennifer Shih, Yun Yen

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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 dCIT 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.

Original languageEnglish
Pages (from-to)1657-1665
Number of pages9
JournalBiochemical Pharmacology
Volume55
Issue number10
DOIs
Publication statusPublished - May 15 1998
Externally publishedYes

Fingerprint

Deoxyribonucleosides
KB Cells
Ribonucleotide Reductases
Complementary DNA
Clone Cells
Hydroxyurea
Cells
Cell Line
triphosphoric acid
Transfection
Assays
2'-deoxycytidine 5'-triphosphate
DNA
Enzymes

Keywords

  • Deoxyribonucleoside triphosphate pools
  • Drug resistance
  • Hydroxyurea
  • Ribonucleotide reductase

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

Cite this

Determination of deoxyribonucleoside triphosphate pool sizes in ribonucleotide reductase cDNA transfected human KB cells. / Zhou, Bing Sen; Ker, Rhonda; Ho, Roger; Yu, Jonathan; Zhao, Yong Ren; Shih, Jennifer; Yen, Yun.

In: Biochemical Pharmacology, Vol. 55, No. 10, 15.05.1998, p. 1657-1665.

Research output: Contribution to journalArticle

Zhou, Bing Sen ; Ker, Rhonda ; Ho, Roger ; Yu, Jonathan ; Zhao, Yong Ren ; Shih, Jennifer ; Yen, Yun. / Determination of deoxyribonucleoside triphosphate pool sizes in ribonucleotide reductase cDNA transfected human KB cells. In: Biochemical Pharmacology. 1998 ; Vol. 55, No. 10. pp. 1657-1665.
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abstract = "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 dCIT 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.",
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