Effects of Deoxycytidine and Thymidine Kinase Deficiency on Substrate Cycles Between Deoxyribonucleosides and Their 5'-Phosphates1
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[CANCER RESEARCH 48, 3681-3687, July I, 1988] Effects of Deoxycytidine and Thymidine Kinase Deficiency on Substrate Cycles between Deoxyribonucleosides and Their 5'-Phosphates1 Lars Höglund,Elisabet Pontis, and Peter Reichard2 Department of Biochemistry, Medical Nobel Institute, Karolinska Institute!, S-I04 01 Stockholm, Sweden ABSTRACT as witnessed by excretion of deoxyribonucleosides into the medium is significant and appeared to have a regulatory func Substrate cycles constructed from a deoxyribonucleoside kinase and a deoxyribonucleotidase contribute to the metabolism of deoxyribonucleo- tion. In cycling 3T6 mouse fibroblasts as much as 28% of newly tides in cultured cells. The two enzymes catalyze in opposite directions synthesized dCDP was excreted as deoxynucleoside, mostly the irreversible interconversion between a deoxyribonucleoside and its deoxyuridine (6). When DNA synthesis was blocked, dNTP S'-phosphate. Depending on the balance between the two reactions the synthesis continued, albeit at a reduced rate, with massive net result of the cycle's activity will be synthesis or degradation of the excretion of deoxynucleosides. In contrast, a block of de novo deoxyribonucleotide, and favor import or export of the deoxyribonucleo synthesis of dNTPs led to a decrease in the excretion with a side. With genetically changed hamster cells (V79 and (I IO) deficient concomitant influx of deoxyribonucleosides from the medium in either deoxycytidine or thymidine kinase we now quantify by kinetic (7). isotope flow experiments the contributions of the two kinases to the To explain these and other results we proposed the model function of the respective cycles. For each, loss of the relevant kinase involving a substrate cycle, depicted in Fig. 1. The cycle is made was accompanied by an increased degradation of the deoxynucleotide, a slower rate of DNA synthesis, and a longer generation time for the up from a deoxyribonucleoside kinase (3, 4) and a deoxyribo mutant cells. The size of the corresponding deoxyribonucleoside triphos- nucleotidase (8) that catalyze in opposite direction the irrevers ible interconversion between a pyrimidine deoxyribonucleoside phate pool was apparently not decreased. and its 5'-phosphate. The kinase is regulated by allosteric effects exerted by dNTPs; the activity of the deoxynucleotidase INTRODUCTION largely depends on the concentration of its substrate. Together, Analogues of pyrimidine nucleosides are used to treat malig the relative activities of the two enzymes set the intracellular nancies and virus infections and have recently gained added level of the deoxyribonucleoside and thereby determine its flux interest in connection with attempts to treat patients infected in or out from the cell. This in turn affects the level of the with human immunodeficiency virus (1). The analogues inter dNTP via the activities of deoxynucleoside mono- and diphos- fere more or less specifically with nucleic acid synthesis in phate kinases. The model also indicates the relation between malignant or virus-infected cells. To this purpose they must the de novo synthetic pathway and the substrate cycle. In the first be phosphorylated by nucleoside and nucleotide kinases. present context it is of interest to point out that a decrease in These enzymes are often named "salvage" enzymes (2) to ribonucleotide reduction may shift the balance of the cycle indicate that they function in the reutilization of material towards synthesis, both via diminished allosteric inhibition of resulting from the degradation of nucleic acids. Considering the kinase and a decrease in the concentration of the deoxyri such a "cleaning up" function, it was surprising to find that the bonucleoside 5'-phosphate. Such an approach, reviewed in Ref two deoxynucleoside kinases phosphorylating thymidine and erence 9, has been used to increase the effectiveness of nucleo deoxycytidine are tightly controlled enzymes, subject to both side analogues in therapy. allosteric inhibition and stimulation (3,4). Understanding these In this communication we evaluate how the loss of deoxycy controls in intact cells and how they may be manipulated to tidine or thymidine kinase affects the metabolism of dNTPs in phosphorv late nucleoside analogues used in the clinic becomes cycling hamster cells. Earlier work with mouse fibroblasts (10) an important concern. and human B- and T-lymphoblasts (11) indicated that loss of Work in this laboratory, summarized in (5), aims at an either of the two deoxynucleoside kinases led to a large increase understanding of the regulation of deoxyribonucleotide synthe in the excretion of deoxynucleosides. While these studies sup sis in relation to DNA replication and repair. We use intact port the concept of the model of Fig. 1 they were not designed cells in culture to measure the flow of isotope from trace to give a quantitative evaluation of the importance of the kinases amounts of labeled pyrimidine nucleosides into various prod for dNTP metabolism. To achieve this, we now use isotope ucts of deoxyribonucleotide metabolism. During steady state kinetic experiments and compare V79 and CHO hamster cell conditions, the specific activity of dNTPs3 reaches a plateau lines and mutants of the same lines selected for a deficiency in value and the accumulation of isotope in end products (DNA, either deoxycytidine or thymidine kinase with respect to size catabolic deoxyribonucleosides in the medium) can then be used and turnover of dNTP pools as well as rates of DNA synthesis to measure absolute rates of various processes. It became ap and deoxyribonucleoside excretion. parent that in 3T6 cells the degradation of pyrimidine dNTPs Received 11/10/87; revised 3/7/88; accepted 3/31/88. MATERIALS AND METHODS The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in Materials. [5-3H]cytidine, [5-3H]uridine, [6-3H]uridine, and [methyl- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3H]thymidine were purchased from Amersham. The labeled nucleosides 1This work was supported by grants from the Swedish Medical Research Council and the Medical Faculty of Karolinska Institute. had specific activities between 20 and 25 Ci/mmol and were purified 2To whom requests for reprints should be addressed, at Department of by HPLC immediately before use. Ara-C, 4-(2-hydroxyethyl)-l-piper- Biochemistry, Medical Nobel Institute, Karolinska Institute!, P. O. Box 60400, azineethanesulfonic acid, and EMS were from Sigma; cell media were S-104 01 Stockholm, Sweden. 3The abbreviations used are: dNTP, deoxynucleoside triphosphate; ara-C, 1- from Flow Laboratories. 0-D-arabinofuranosylcytosine; EMS, ethylmethanesulfonate; HPLC, high-per Cells. Two earlier characterized (12) hamster lung cell lines (V79 formance liquid chromatography; CHO, Chinese hamster ovary; FCS, fetal calf and V79/dC) were gifts from Dr. Vera Bianchi, University of Padova, serum; tk, thymidine kinase activity. Padova, Italy. V79/dC cells lack the enzyme dCMP deaminase and 3681 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1988 American Association for Cancer Research. DEOXYNUCLEOSIDE KINASES AND SUBSTRATE CYCLES dNTP and pool sizes and specific activities of the pyrimidine ribo- and deoxyNTPs were determined by HPLC (15) and the enzymatic DNA rNDP *** novo. dNDP polymerase assay (16-18), respectively. The cells remaining on the dish It were dissolved in 0.3 M NaOH, incubated for 16 h at room temperature /NMP.,ADP and incorporation of isotope into DNA was determined. Incorporation \ y If of radioactivity into deoxyribonucleosides of the medium was deter cell \ PjA J\ mined by HPLC that completely separated deoxyribonucleosides from membrane deoxyriboside AT free pyrimidines (6). No radioactivity was recovered in pyrimidines. Incorporation of isotope into water was determined after removal of deoxyribonucleosides from the medium by adsorption to charcoal (14). deoxyriboside In experiments with deoxyuridine or cytidine labeled in the 5 position (extracellular) of the pyrimidine ring incorporation of isotope into water was a measure Fig. I. Model for the interrelation between the de novo synthesis of a dNTP of thymidylate synthase activity (19, 20). All analyses were made on and a substrate cycle. De novo synthesis occurs by reduction of a ribonucleoside duplicate dishes, usually with results varying by less than 10%. The diphosphate to the corresponding deoxyribonucleotide. The substrate cycle in volves the interconversion of a deoxyribonucleoside and its S'-phosphate by a reproducibility of data is also exemplified by Tables 3 to 5 that give deoxyribonucleoside kinase and a deoxyribonucleotidase. The cycle is coupled to results of measurements of DNA synthesis from the incorporation of de novo synthesis via a reversible deoxyribonucleoside monophosphate kinase. different labeled nucleosides. Where appropriate, all values in Tables The intracellular deoxyribonucleoside is in rapid equilibrium with extracellular or Figures are normalized to 10'' cells. deoxyribonucleoside. The synthetic arm of the cycle is responsible for the import of nucleoside analogues. The relative rates of the kinase and the deoxyribonucleo tidase reactions decide whether the cycle operates towards synthesis or degrada tion. This balance is affected by the supply of and demand for the dNTP. The RESULTS kinase is inhibited by the dNTP by feedback inhibition, the rate of the deoxyri bonucleotidase