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

Biochem Pharmacol. 1998 May 1;55(9):1353-60. doi: 10.1016/s0006-2952(97)00641-2.

Abstract

Effects of drug treatment with antimetabolites on a human colon cancer cell line, SW480, were studied. Cells were treated with 10 microM of 5-fluorouracil (5FU), an inhibitor of pyrimidine synthesis, or 1000 microM of hydroxyurea (HU), an inhibitor of both purine and pyrimidine syntheses, or the combination. Recombinant alpha-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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Cycle / drug effects
  • Cell Cycle / physiology*
  • Cell Division / drug effects
  • Colonic Neoplasms
  • Deoxyribonucleotides / metabolism*
  • Fluorouracil / pharmacology*
  • G1 Phase
  • Gene Expression Regulation, Neoplastic* / drug effects
  • Genes, p53*
  • Humans
  • Hydroxyurea / pharmacology*
  • Interferon alpha-2
  • Interferon-alpha / pharmacology
  • Macromolecular Substances
  • Mutation*
  • RNA, Messenger / genetics
  • Recombinant Proteins
  • Ribonucleotide Reductases / genetics*
  • S Phase
  • Transcription, Genetic / drug effects
  • Tumor Cells, Cultured

Substances

  • Deoxyribonucleotides
  • Interferon alpha-2
  • Interferon-alpha
  • Macromolecular Substances
  • RNA, Messenger
  • Recombinant Proteins
  • Ribonucleotide Reductases
  • Fluorouracil
  • Hydroxyurea