Ribonucleotide reduction in intact human diploid fibroblasts

Abstract

There have been very few studies on ribonucleotide reductase activity in human tissue. In this report we describe a rapid and convenient procedure for determining purine and pyrimidine ribonucleotide reduction in normal human diploid fibroblasts and use the method to examine some general properties of the activity in these cells. ADP and CDP reductase was characterized for its response to the positive effectors, ATP and dGTP, the negative effector dATP, and the reducing agent dithiothreitol. Apparent Km values for ADP and CDP were determined to be 0.1 mM and 0.04 mM respectively. THe antitumor agent hydroxyurea inhibited both purine and pyrimidine reductase in a noncompetitive fashion, giving Ki value of 0.40 mM and 0.41 mM for ADP and CDP respectively. These Ki estimates are about four to five times higher than those reported for some permanent cell lines. An examination of the cytotoxic effects of hydroxyurea indicated a close correlation between the concentration of drug which inhibited enzyme activity and decreased colony-forming ability. Clearly the ability to investigate ribonucleotide reduction in low numbers of normal human diploid cells will be useful for genetic and biochemical studies.

The ribonucleotide reductases — A unique group of metalloenzymes essential for cell proliferation

“…The in vitro reduction of CDP, ADP, and GDP by different mammalian enzymes is as sensitive to hydroxyurea (I50 = 2 -4 x 10 -4 M) as with E. coli enzyme 62, 76,88,171,172,182) The tyrosine radical of mouse fibroblast ribonucleotide reductase disappears upon addi-tion of the inhibitor in vitro as well as in vivo indicating an analogous mode of action as with the bacterial enzyme 59'~°). Comparison of the inhibitory potency of a series of polyhydroxybenzohydroxamic acids towards rat tumor ribonucleotide reductase showed the most effective radical scavengers to be the best inhibitors 19s), and radical destruction was directly established with the most potent substance, 3,4-dihydroxybenzohydroxamic acid (Fig.…”

Effects of deoxyadenosine on ribonucleotide reductase in adenosine deaminase‐deficient lymphocytes

“…It has not been demonstrated whether ribonucleotide reductase activity in ADA-deficient lymphocytes is in fact inhibited. To answer this question, we prepared intact permeabilized cells for the measurement of ribonucleotide reductase activity, since the activity in a homogenate did not reflect the intracellular activity (Dick and Wright, 1980;Reddy and Pardee, 1982). Entry of the nucleotide into the cells takes place in the presence of DEAE-dextran (Figure 1), a polycationic polysaccharide derivative which has been used to potentiate the uptake of nucleic acids into cultured animal cells (Pagono, 1970).…”

Glycopeptide hormone production by cultured human diploid fibroblasts

“…Those strains that constitutively produced hCG exhibited a statistically significant increase in hor-mone levels when cultured in medium containing 1 mM HU. The mechanisms responsible for HU effects are not known, although this drug blocks cells in early S phase of the cell cycle, apparently by inhibiting ribonucleotide reductase and thereby limiting the availability of deoxyribonucleotides for DNA synthesis (Dick and Wright, 1980). Cells accumulate in early S phase where chromatin is maximally decondensed and presumably available for transcription.…”