Effects of perturbations of pools of deoxyribonucleoside triphosphates on expression of ribonucleotide reductase, a G1/S transition state enzyme, in p53-mutated cells

Scott Wadler, Robert Horowitz, Hong Yang Zhang, Edward L. Schwartz

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Effects of drug treatment with antimetabolites on a human colon cancer cell line, SW480, were studied. Cells were treated with 10 μM of 5- fluorouracil (5FU), an inhibitor of pyrimidine synthesis, or 1000 μM of hydroxyurea (HU), an inhibitor of both purine and pyrimidine syntheses, or the combination. Recombinant α-2a-interferon (IFN), 500 U/mL, also was employed, as this augments the effects of both antimetabolites in vitro and in vivo. The predominant effect of this combination was to block cells in early S phase as measured by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation. By 24 hr, 86% of the cells had accumulated in S phase, but failed to progress to G2/M. This was accompanied by an early, rapid decline in all four deoxyribonucleoside triphosphates (dNTPs) by 38-86% at 4-24 hr. Despite these effects, expression of the G1/S transition state enzyme, ribonucleotide reductase (RR), increased at 24 hr as measured by a 3 to 5-fold increase in mRNA levels for the M2 subunit, in the absence of a measurable effect on protein levels. The rise in levels of RR mRNA and the continued progression of cells into S phase were associated with a synergistic inhibition of cell cycle proliferation resulting from treatment with the three-drug combination. This suggests that in the presence of antimetabolite-induced depletion of dNTPs, SW480 cells, which lack a normal p53 gene, will proceed into S phase, and that this is associated with a rise in expression of the G1/S transition state enzyme, RR. Cells arrested in S phase by a p53-independent mechanism will undergo a synergistic enhancement of cell death.

Original languageEnglish (US)
Pages (from-to)1353-1360
Number of pages8
JournalBiochemical Pharmacology
Volume55
Issue number9
DOIs
StatePublished - May 1 1998

Fingerprint

Deoxyribonucleosides
Ribonucleotide Reductases
Antimetabolites
S Phase
Cells
Enzymes
Drug therapy
Messenger RNA
Hydroxyurea
Cell death
Drug Combinations
Bromodeoxyuridine
Fluorouracil
Interferons
Genes
p53 Genes
Colonic Neoplasms
triphosphoric acid
Cell Cycle
Proteins

Keywords

  • 5- fluorouracil
  • Cytokinesis
  • Deoxyribonucleoside triphosphates
  • Hydroxyurea
  • p53
  • Ribonucleotide reductase

ASJC Scopus subject areas

  • Pharmacology

Cite this

Effects of perturbations of pools of deoxyribonucleoside triphosphates on expression of ribonucleotide reductase, a G1/S transition state enzyme, in p53-mutated cells. / Wadler, Scott; Horowitz, Robert; Zhang, Hong Yang; Schwartz, Edward L.

In: Biochemical Pharmacology, Vol. 55, No. 9, 01.05.1998, p. 1353-1360.

Research output: Contribution to journalArticle

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abstract = "Effects of drug treatment with antimetabolites on a human colon cancer cell line, SW480, were studied. Cells were treated with 10 μM of 5- fluorouracil (5FU), an inhibitor of pyrimidine synthesis, or 1000 μM of hydroxyurea (HU), an inhibitor of both purine and pyrimidine syntheses, or the combination. Recombinant α-2a-interferon (IFN), 500 U/mL, also was employed, as this augments the effects of both antimetabolites in vitro and in vivo. The predominant effect of this combination was to block cells in early S phase as measured by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation. By 24 hr, 86{\%} of the cells had accumulated in S phase, but failed to progress to G2/M. This was accompanied by an early, rapid decline in all four deoxyribonucleoside triphosphates (dNTPs) by 38-86{\%} at 4-24 hr. Despite these effects, expression of the G1/S transition state enzyme, ribonucleotide reductase (RR), increased at 24 hr as measured by a 3 to 5-fold increase in mRNA levels for the M2 subunit, in the absence of a measurable effect on protein levels. The rise in levels of RR mRNA and the continued progression of cells into S phase were associated with a synergistic inhibition of cell cycle proliferation resulting from treatment with the three-drug combination. This suggests that in the presence of antimetabolite-induced depletion of dNTPs, SW480 cells, which lack a normal p53 gene, will proceed into S phase, and that this is associated with a rise in expression of the G1/S transition state enzyme, RR. Cells arrested in S phase by a p53-independent mechanism will undergo a synergistic enhancement of cell death.",
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AU - Horowitz, Robert

AU - Zhang, Hong Yang

AU - Schwartz, Edward L.

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N2 - Effects of drug treatment with antimetabolites on a human colon cancer cell line, SW480, were studied. Cells were treated with 10 μM of 5- fluorouracil (5FU), an inhibitor of pyrimidine synthesis, or 1000 μM of hydroxyurea (HU), an inhibitor of both purine and pyrimidine syntheses, or the combination. Recombinant α-2a-interferon (IFN), 500 U/mL, also was employed, as this augments the effects of both antimetabolites in vitro and in vivo. The predominant effect of this combination was to block cells in early S phase as measured by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation. By 24 hr, 86% of the cells had accumulated in S phase, but failed to progress to G2/M. This was accompanied by an early, rapid decline in all four deoxyribonucleoside triphosphates (dNTPs) by 38-86% at 4-24 hr. Despite these effects, expression of the G1/S transition state enzyme, ribonucleotide reductase (RR), increased at 24 hr as measured by a 3 to 5-fold increase in mRNA levels for the M2 subunit, in the absence of a measurable effect on protein levels. The rise in levels of RR mRNA and the continued progression of cells into S phase were associated with a synergistic inhibition of cell cycle proliferation resulting from treatment with the three-drug combination. This suggests that in the presence of antimetabolite-induced depletion of dNTPs, SW480 cells, which lack a normal p53 gene, will proceed into S phase, and that this is associated with a rise in expression of the G1/S transition state enzyme, RR. Cells arrested in S phase by a p53-independent mechanism will undergo a synergistic enhancement of cell death.

AB - Effects of drug treatment with antimetabolites on a human colon cancer cell line, SW480, were studied. Cells were treated with 10 μM of 5- fluorouracil (5FU), an inhibitor of pyrimidine synthesis, or 1000 μM of hydroxyurea (HU), an inhibitor of both purine and pyrimidine syntheses, or the combination. Recombinant α-2a-interferon (IFN), 500 U/mL, also was employed, as this augments the effects of both antimetabolites in vitro and in vivo. The predominant effect of this combination was to block cells in early S phase as measured by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation. By 24 hr, 86% of the cells had accumulated in S phase, but failed to progress to G2/M. This was accompanied by an early, rapid decline in all four deoxyribonucleoside triphosphates (dNTPs) by 38-86% at 4-24 hr. Despite these effects, expression of the G1/S transition state enzyme, ribonucleotide reductase (RR), increased at 24 hr as measured by a 3 to 5-fold increase in mRNA levels for the M2 subunit, in the absence of a measurable effect on protein levels. The rise in levels of RR mRNA and the continued progression of cells into S phase were associated with a synergistic inhibition of cell cycle proliferation resulting from treatment with the three-drug combination. This suggests that in the presence of antimetabolite-induced depletion of dNTPs, SW480 cells, which lack a normal p53 gene, will proceed into S phase, and that this is associated with a rise in expression of the G1/S transition state enzyme, RR. Cells arrested in S phase by a p53-independent mechanism will undergo a synergistic enhancement of cell death.

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