Isotope-dilution studies of the effects of 5-fluorodeoxyuridine and hydroxyurea on the incorporation of deoxycytidine and thymidine by cultured thymus cells

Abstract

From experimentally induced changes in the slope (Vmax) and intercept (pool) of isotope-dilution plots inferences may be drawn on the position and regulation of rate-limiting steps affecting the incorporation of pyrimidine deoxynucleosides by intact cells. 5-Fluorodeoxyuridine (FdUrd; 1 μM) reduced the Vmax of radioactive labelling with deoxy[5-3H]cytidine; this was reversed by thymidine (19 μM) suggesting that FdUrd makes the concentration of deoxythymidine triphosphate (dTTP) rate-limiting for DNA synthesis. With deoxy[U-14C]cytidine the reversal of FdUrd inhibition by thymidine was only partial; this was in keeping with (i) deoxy[U-14C]cytidine labelling both cytosine and thymine in DNA, and (ii) a continuing inhibition of thymidylate synthetase by FdUrd in the presence of thymidine (19 μM).The deoxycytidine competitor pool was increased by cytidine (10–50 μM) and decreased by (i) thymidine (19 μM), (ii) hydroxyurea (50 μM) and (iii) deoxycytidine (12 μM, in the presence of FdUrd). It is suggested that these pool-decreasing agents, or their derivatives (e.g., dTTP), inhibit ribonucleotide reductase and hence prevent the entry of pyrimidine ribonucleotide derivatives into the deoxycytidine competitor pool; because of this pool decrease, radioactive labelling with deoxy[5-3H]cytidine was enhanced by thymidine (19 μM) and hydroxyurea (50 μM). However, at the latter hydroxyurea concentration, labelling with [Me-3H]thymidine was inhibited, due to a decrease in the Vmax of the rate-limiting step for thymidine incorporation (probably thymidine kinase). This sensitivity of labelling with [Me-3H]thymidine to inhibition by hydroxyurea (50 μM) was reduced by adding FdUrd to prevent the accumulation of dTTP. At high hydroxyurea concentrations (0.1–1.0 mM), labelling with deoxy[5-3H]cytidine was also inhibited, due to a decrease in Vmax of the rate-limiting step, which was probably at the level of DNA polymerase.The results suggest that hydroxyurea inhibits DNA synthesis by making the concentration of purine deoxynucleotides rate-limiting. Pyrimidine deoxynucleotides are formed in sufficient quantities from deoxycytidine by way of salvage pathways. Indeed, dTTP accumulates and inhibits thymidine kinase, thus amplifying the inhibitory effect of hydroxyurea on labelling with [Me-3 H]thymidine.

Isotope-dilution analysis of the effects of deoxyguanosine and deoxyadenosine on the incorpoŕation of thymidine and deoxycytidine by hydroxyurea-treated thymus cells

“…Since thymus cells have highly active purine deoxyribonucleoside kinases (Durham & Ives, 1969;Carson et al, 1977), this lymphoid tissue would appear most suitable for testing the ability of purine deoxyribonucleosides to prevent inhibition by hydroxyurea. The present paper extends previous studies of the inhibition by hydroxyurea of the incorporation of [Me-3Hlthymidine and deoxy[5-3Hlcytidine by cultured thymus cells (Scott & Forsdyke, 1976). In the first part of the paper we report the effects of various concentrations and combinations of hydroxyurea and purine deoxyribonucleosides.…”

“…4 shows changes in the incorporation of [3Hlthymidine and deoxy[ 3Hlcytidine produced by different concentrations of hydroxyurea. As reported previously (Scott & Forsdyke, 1976), the curve for inhibition of [3Hlthymidine incorporation was to the left of the curve for inhibition of deoxy[3Hlcytidine incorporation. Deoxyguanosine (400,UM) was found to decrease the increased sensitivity of PH1thymidine incorporation to inhibition by hydroxyurea (Fig.…”

“…200g, were from Biobreeding Laboratories, Ottawa, Ontario, Canada. S Cell culture and determination ofradioactivity These were as described previously (Forsdyke, 1971;Sjostrom & Forsdyke, 1974;Scott & Forsdyke, 1976). Rat thymus cells were suspended at a concentration of 2 x 106/ml in medium 199 (90%) and fresh homologous serum (10%).…”

“…The theoretical basis of the isotope-dilution model has been discussed in detail elsewhere (Forsdyke, 1971;Sjostrom & Forsdyke, 1974;Scott & Forsdyke, 1976, 1978a. A constant quantity of a radioactively labelled DNA precursor is added to cultured cells together with various quantities of the corresponding unlabelled precursor.…”

Validity of the tritiated thymidine method for estimating bacterial growth rates: measurement of isotope dilution during DNA synthesis

“…A necessary condition is that the rate-limiting step for the incorporation of thymidine be the final one, i.e., the DNA polymerase step. The following explanation of the concepts of the isotope dilution methodology has been adapted from the work of Forsdyke and his colleagues (9,10,(35)(36)(37)(38). In the simplest model, the incorporation of a labeled precursor into a macromolecule may be represented as a one-step transfer from a pool into a stable product; two assumptions were made.…”