INVOLVEMENT OF SUBSTRATE CYCLES IN THE REGULATION OF PYRIMIDINE dNTP POOLS IN 3T6 MOUSE FIBROBLASTS: 168

Abstract

The in situ activities of some enzymes involved in the synthesis of pyrimidine dNTPs and DNA as well as the turnover of dNTP pools were measured in rapidly growing 3T6 cells from the flow of isotope from labeled nucleosides via nucleotides into DNA and excretion products in the medium during steady state conditions. The effects of inhibitors of ribonucleotide reductase, thymidylate synthetase or DNA-strand elongation were also determined. In non-inhibited cells as much as 28% of dCDP synthesized was degraded and excreted (mostly deoxyuridine) into the medium. Hydroxyurea, but not amethopterin or aphidicolin stopped the turnover of dNTP pools. In the latter case all dNTPs were excreted as deoxynucleosides, while with hydroxyurea there was a stimulation of uptake of deoxynucleosides from the medium. This stimulation also occurs in the presence of dipyridamole, an inhibitor of nucleoside transport, and therefore appears to involve an intracellular event. We propose that substrate cycles between deoxyribonucleosides and their monophosphates, involving the activities of kinases and phosphatases, normally participate in the regulation of pyrimidine dNTP pool levels. Inhibition of ribonucleotide reductase stimulates their anabolic activity while inhibition of DNA-strand elongation or thymidylate synthetase stimulates catabolism.