Mammalian Ribonucleotide Reductase Research Articles

Cell cycle-dependent regulation of mammalian ribonucleotide reductase. The S phase-correlated increase in subunit M2 is regulated by de novo protein synthesis.

Ribonucleotide reductase in mammalian cells is composed of two nonidentical subunits, proteins M1 and M2. Protein M2 contains a tyrosyl free radical, essential for activity, which can be quantified directly in frozen, packed cells by EPR spectroscopy. A 3-7-fold increase in the concentration of tyrosyl radical-containing M2 subunit was observed when mouse mammary tumor TA 3 cells passed from the G1 to the S phase of the cell cycle. Similar results were obtained with cells synchronized by isoleucine starvation or separated by centrifugal elutriation. Addition of deuterated tyrosine to cells give rise to a different EPR signal in newly synthesized protein M2. Pulse-chase experiments with deuterated tyrosine showed unequivocally that the S phase-correlated increase in radical-containing M2 subunit was due to de novo protein synthesis. Labeled M2 molecules disappeared with a half-life of 3 h, and therefore new molecules must be synthesized at a high rate during the S phase. In contrast, after hydroxyurea inactivation, cells rapidly regenerated the tyrosyl radical in already existing protein M2 molecules. This enzyme activation mechanism is clearly different from the one responsible for regulating protein M2 activity during the cell cycle.

Nature and properties of mammalian ribonucleoside diphosphate reductase

In mammalian cells, the synthesis of DNA requires a continuous supply of deoxyribonucleoside triphosphates (dNTPs). The formation of dNTPs can be either through the 'de novo' pathway using ribonucleotides as precursors, or utilizing preformed deoxyribonucleosides through the 'salvage' pathway (Fig. 1). The contribution of either pathway to overall dNTP formation depends on the type of cell, the amounts of the enzymes involved, the status of the metabolites and the environment of the cells. Synthesis of dNTPs via the de novo pathway requires the action of ribonucleotide reductase (EC 1.17.4.1). The activity of ribonucleotide reductase is a!losterically regulated. Ribonucleotide reductase from bacteria has been characterized extensively (for a review see Thelander and Reichard, 1979). Information regarding the regulation of the mammalian enzyme was summarized recently (Hunting and Henderson, 1983). In the present review, fluctuations of reductase activity with the cell cycle and the relativn of dNTP pools to this activity, and the regulation and structural components of mammalian ribonucleotide reductase will be discussed, and a new model (intercalating model) of reductase regulation is proposed. Finally, pertinent information regarding an altered reductase in virus infected mammalian cells is summarized.

Alterations in the components of ribonucleotide reductase in hydroxyurea-resistant hamster cells.

Two components of mammalian ribonucleotide reductase have been separated by blue dextran-Sepharose chromatography from a hydroxyurea-resistant cell line, NCR-30A2, and its parental wild type. Analysis of reductase activity in these cells and the enzyme components reveals that there are three alterations involving ribonucleotide reductase activity in NCR-30A2 cells. There is an elevation in the effector-binding (EB) component, an elevation in the non-heme-iron-containing (NHI) component, and an alteration in the NHI component that renders the enzyme less sensitive to inhibition by hydroxyurea. These findings easily account for the resistance of NCR-30A2 cells to the antitumor agent hydroxyurea, and to other drugs with a similar mode of action.

Mechanism of inhibition of mammalian ribonucleotide reductase by the iron chelate of 1-formylisoquinoline thiosemicarbazone. Destruction of the tyrosine free radical of the enzyme in an oxygen-requiring reaction.

The iron chelate of 1-formylisoquinoline thiosemicarbazone is one of the most potent inhibitors known for mammalian ribonucleotide reductase. In this study, we show that the target for the drug is the tyrosine free radical of the M2 subunit of the enzyme. The radical is destroyed by the drug in a reaction which requires oxygen. After removal of the drug, the tyrosine radical and ribonucleotide reductase activity can be regenerated by incubation of the enzyme with dithiothreitol. We propose that the iron chelate of the drug binds at the active site of the enzyme, and then the ferrous form of the chelate reacts with molecular oxygen in a redox process that, via a 1-electron reduction, leads to destruction of the M2 tyrosine radical.

Assay of ribonucleotide reductase activity in intact permeabilized hamster cells: An evaluation

An intact cell assay system, based on Tween-80 permeabilization can be used to investigate ribonucleotide reductase activity in a variety of mammalian cell lines. An important consideration in the use of intact cells is the presence of other nucleotide metabolizing activities. The influence of these activities on estimates of pyrimidine (CDP) and purine (ADP) reductase in permeabilized hamster cells has been examined. Studies on the incorporation of label from CDP and ADP into RNA indicated that a very small proportion of the reductase substrates was eventually incorporated into RNA during routine enzyme assays, and would have no detectable effect on activity estimates. The possibility that the products of the reaction (dCDP and dADP) were eventually phosphorylated and incorporated into DNA was also examined, and it was found that proper permeabilization of the cells eliminated or greatly reduced loss of deoxyribonucleotides to DNA. An analysis by HPLC of nucleotides present during CDP and ADP reductase reactions showed that various kinases and phosphatases were active in permeabilized cells, as all levels of phosphorylation of nucleotide substrates and allosteric effectors were detected. The base composition of the nucleotides added to the assay systems were not altered. Although movement of phosphates occurred during the assay, the concentrations of substrates quickly reached equilibrium (within 1 min) with their respective nucleosides and nucleotides, resulting in a relatively constant although reduced concentration of CDP or ADP substrates during the 20-min assay. Similarly the levels of allosteric effectors, ATP for pyrimidine and dGTP for purine reductase activities, declined within the first minute of the assays and quickly reached an equilibrium with their respective adenine or guanine containing nucleotides during most of the reaction time. Although useful approximations of intracellular reductase activity can be obtained without correcting for modified nucleotide concentrations, precise determinations can be calculated when these alterations are taken into consideration. For example, estimates of intracellular K m values for CDP closely resembled those reported with highly purified mammalian enzyme preparations in other studies. Clearly, the intact cell assay system provides worthwhile information about mammalian ribonucleotide reductase in its physiologically relevant environment.

A series of N-carbamoyloxyurea resistant cell lines with alterations in ribonucleotide reductase: lack of coordination in pyrimidine and purine reductase activity.

N-Carbamoyloxyurea is cytotoxic for cells in culture and, like hydroxyurea and guanazole, the drug is an effective inhibitor of mammalian ribonucleotide reductase and thus DNA synthesis. In addition to ribonucleotide reductase, N-carbamoyloxyurea has a second site of action which also appears to be in the pathway of DNA synthesis. A series of drug-resistant cell lines, which contain alterations in ribonucleotide reduction, have been sequentially selected in the presence of increasing concentrations of N-carbamoyloxyurea. CDP and ADP reductase activities in these drug-resistant lines have been investigated and two types of alterations have been identified: elevated levels of enzyme activity with wild-type sensitivity to drug and altered levels of reductase with reduced drug sensitivity, probably owing to structural modification of the enzyme. Furthermore, N-carbamoyloxyurea resistant lines contain another alteration as well, presumably at a second site of drug action. They are also cross-resistant to hydroxyurea and guanazole, and studies on enzyme activity levels support our previous findings with cells selected for resistance to hydroxyurea, which showed changes in CDP reductase activity are not always coordinated with changes in ADP reductase. Although several possibilities exist, these observations are most easily explained by the existence of independent enzyme substrate binding subunits which are regulated by different mechanisms. Moreover, increases in cellular resistance were accompanied by significant increases in CDP but not ADP reductase, suggesting that an ability to maintain an adequate level of CDP reductase activity is especially important to achieve resistance to DNA synthesis inhibitors like N-carbamoyloxyurea, hydroxyurea, and guanazole.

Regulation of ribonucleotide reductase activity in mammalian cells.

Mammalian ribonucleotide reductase catalyzes the rate-limiting for the de novo synthesis 2'-deoxyribonucleoside 5'-triphosphates. There is some suggestion that this step may also be the rate-limiting step of DNA synthesis. It is apparent that the level of the enzyme, ribonucleotide reductase, varies through the cell cycle and is highest in those tissues with the greatest proliferation rate. This increase in activity is associated with increased protein synthesis. The purified enzyme has been shown to be subject to strict allosteric regulation by the various nucleoside triphosphates and it has been proposed that allosteric regulation plays an important role in the level of ribonucleotide reductase activity which is expressed. All experimental data relating to this point, however, do not support the role of deoxyribonucleoside triphosphates as a major factor in determining cellular reductase activity during normal cell division. Several naturally occurring factors have been isolated from cells which lower ribonucleotide reductase activity in vitro. These factors have been found in tissues of low growth fraction and appear to be absent or low in tissues or high growth fraction such as tumor, regenerating liver and embryonic tissues. The expression of intracellular ribonucleotide reductase activity is therefore controlled at various levels and by various factors and the prevailing mode of regulation may vary throughout the cell cycle transverse and also in the various types of cells.

An iron-dependent 5'-nucleotide phosphoribohydrolase activity in the venom of Crotalus atrox and its effect upon the determination of mammalian ribonucleotide reductase.

There is an iron-dependent activity in the venom of Crotalus atrox which appears to hydrolyze the N-glycosidic sugar-base bond of pyrimidine and purine nucleotides. Maximal activation of this activity occurred at 1.0 mmol/l and 0.8 mmol/l FeCl3 for cytidine diphosphate (CDP) and ADP substrates, respectively. The release of free base affects the background values of control experiments carried out during nucleotide-labelled assays of ribonucleotide reductase which require the conversion of nucleotides to nucleosides by snake venom treatment. When the reductase is examined in intact cells, a situation closely resembling normal physiological conditions for the enzyme, FeCl3 was found to be an inhibitor of ADP reductions and varied from a mild stimulator to a significant inhibitor of CDP reductions depending upon FeCl3 concentration. Possible explanations for previously observed variability in ribonucleotide reductase activity in the presence of iron are suggested.

Involvement of ribonucleotide reductase activity in the senescence of normal human diploid fibroblasts

The levels of intracellular ribonucleotide reductase activity, a highly regulated ratelimiting step in DNA synthesis, were investigated during serial subculture of normal human diploid fibroblasts in vitro. This key enzyme activity was found to decline significantly during cellular senescence. This observation along with previous findings of a mutator gene associated with mammalian ribonucleotide reductase suggests a possible mutation mechanism for aging which involves changes in reductase activity during cellular senescence. Furthermore, in keeping with the decrease in enzyme activity, we show that cell resistance to the antitumor agent hydroxyurea, whose site of action is ribonucleotide reductase, decreases progressively with increasing passage numbers. This indicates that an important factor to be considered in drug therapy aimed at the reductase is the increased sensitivity of normal cells to drug with cell age, due to a decline in enzyme activity. Much remains to be determined about age-dependent factors involved in drug therapy; cultured normal human diploid fibroblasts provide a useful system in which to investigate these important parameters.

Effects of azidocytidine on DNA synthesis and deoxynucleotide pools of mouse fibroblast cell lines.

2'-Deoxy-2'-azidocytidine inhibits DNA synthesis of cells in tissue culture. In vitro, the diphosphate of azidocytidine was earlier shown to inhibit mammalian ribonucleotide reductase, while the triphosphate did not inhibit DNA replication in isolated cell nuclei. In intact cells, however, deoxyribonucleoside triphosphate pools were not decreased by azidocytidine in a manner expected from an inhibitor of ribonucleotide reductase. In order to differentiate between inhibition of precursor synthesis and DNA replication we studied in the present investigation the effects of azidocytidine on pool sizes of deoxynucleotides as well as on the incorporation of labeled cytidine or deoxycytidine into deoxycytidine triphosphate pools and DNA of cultured growing mouse fibroblasts. Azidocytidine was transformed to an active metabolite by phosphorylation with deoxycytidine kinase. Cell lines deficient in this enzyme showed an increased resistance against the analogue. Azidocytidine had a dual effect on the incorporation of labeled nucleosides. At low concentrations, the incorporation of cytidine into DNA was severely inhibited without a decrease of the specific activity of the dCTP pool or the size of the other three dNTP pools, suggesting a direct effect on DNA replication. At high concentrations of azidocytidine, incorporation into dCTP was also affected, demonstrating inhibition of CDP reduction. Our data also show that reduction of CDP is not necessarily tightly coupled to DNA replication since a rapid turnover of the dCTP pool occurred at the low concentrations of azidocytidine which severely inhibited DNA synthesis.

Characterization of the free radical of mammalian ribonucleotide reductase.

Mouse fibroblast 3T6 cells, selected for resistance to hydroxyurea, were shown to overproduce protein M2, one of the two nonidentical subunits of mammalian ribonucleotide reductase. Packed resistant cells gave an EPR signal at 77 K very much resembling the signal given by the tyrosine-free radical of the B2 subunit of Escherichia coli ribonucleotide reductase. Also, the M2-specific free radical was shown to be located at a tyrosine residue. Of the known tyrosine-free radicals of ribonucleotide reductases from E. coli, bacteriophage T4 infected E. coli and pseudorabies virus infected mouse L cells, the M2-specific EPR signal is most closely similar to the signal of the T4 radical. The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures can be explained by slightly different angles of the beta-methylene group in relation to the plane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around the tyrosines. The pronounced difference in microwave saturation between the E. coli B2 tyrosine radical EPR signal and the M2 signal could be due to their different interactions with unspecific paramagnetic ions or with the antiferromagnetically coupled iron pair, shown to be present in the E. coli enzyme and postulated also for the mammalian enzyme. A difference in the iron-radical center between the bacterial and mammalian ribonucleotide reductase is also observed in the ability to regenerate the free radical structure. In contrast to the B2 radical, the M2 tyrosine free radical could be regenerated by merely adding dithiothreitol in the presence of O2 to a cell extract where the radical had previously been destroyed by hydroxyurea treatment.

A degradation product of fenitrothion, 3-methyl-4-nitrophenol, is an inhibitor of mammalian ribonucleotide reductase.

A degradation product of fenitrothion, 3-methyl-4-nitrophenol, is an inhibitor of mammalian ribonucleotide reductase.

Rat liver ribonucleotide reductase: separation, purification, and properties of two nonidentical subunits.

Two nonidentical subunits of mammalian ribonucleotide reductase, L1 and L2, from regenerating rat liver have been extensively purified for the first time. They were separated by dATP-Sepharose affinity chromatography. Subunit L1, which bound to dATP-Sepharose, was eluted with 50 mM ATP and purified to homogeneity (as demonstrated by sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis) by molecular exclusion high-pressure liquid chromatography (HPLC). This subunit had an apparent relative mass (Mr) of 45 000 and a Km of 0.9 X 10(-4) for CDP. Subunit L2, which did not bind to dATP-Sepharose, was purified by pH 5.2 precipitation followed by chromatography on CM-Sephadex, molecular exclusion HPLC, and DEAE-cellulose. This subunit contained iron and had an apparent Mr of 120 000 by HPLC molecular exclusion chromatography, but showed two bands (Mr 75 000 and Mr 47 000) on SDS-polyacrylamide gel electrophoresis. Neither L1 nor L2 separately had any enzyme activity but when combined they reduced CDP to dCDP.

Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus.

The mammalian ribonucleotide reductase consists of two nonidentical subunits, protein M1 and M2. M1 binds nucleoside triphosphate allosteric effectors, whereas M2 contains a tyrosine free radical essential for activity. The activity of ribonucleotide reductase increased 10-fold in extracts of mouse L cells 6 h after infection with pseudorabies virus. The new activity was not influenced by antibodies against subunit M1 of calf thymus ribonucleotide reductase, whereas the reductase activity in uninfected cells was completely neutralized. Furthermore, packed infected cells (but not mock-infected cells) showed an electron paramagnetic resonance spectrum of the tyrosine free radical of subunit M2 of the cellular ribonucleotide reductase. These data given conclusive evidence that on infection, herpesvirus induces a new or modified ribonucleotide reductase. The virus-induced enzyme showed the same sensitivity to inhibition by hydroxyurea as the cellular reductase. The allosteric regulation of the virus enzyme was completely different from the regulation of the cellular reductase. Thus, CDP reduction catalyzed by the virus enzyme showed no requirement for ATP as a positive effector, and no feedback inhibition was observed by dTTP or dATP. The virus reductase did not even bind to a dATP-Sepharose column which bound the cellular enzyme with high affinity.

Monoclonal antibodies against mammalian ribonucleotide reductase.

Monoclonal antibodies against mammalian ribonucleotide reductase.

Effect of iron-chelating agents on inhibitors of ribonucleotide reductase

Ribonucleotide reductase from Ehrlich tumor cells is inhibited by hydroxyurea (HU), guanazole (GZ), pyrazolo-imidazole (IMPY), 1-formylisoquinoline thiosemicarbazone (IQ), 4-methyl-5-aminoisoquinoline thiosemicarbazone (MAIQ). and the dialdehydes of inosine and 5′-deoxyinosine. In the presence of EDTA, desferal, or 8-hydroxyquinoline (8-HQ) at a concentration that did not affect reductase activity, HU, GZ and IMPY more effectively inhibited the reductase. EDTA, desferal, or 8-HQ in combination with the nucleoside dialdehyde derivatives did not affect the inhibition caused by these compounds. The combination of these iron-chelating agents with MAIQ and IQ, however, reversed the inhibition caused by MAIQ and IQ. These data indicate that the inhibitors of the nonheme iron-containing component of mammalian ribonucleotide reductase can be subclassified, based on their response to the presence of iron-chelating agents.

Studies of mammalian ribonucleotide reductase activity in intact permeabilized cells: A genetic approach

We are using a genetic approach to study the complex properties of mammalian ribonucleotide reductase activity. Cytotoxic drugs (hydroxyurea, guanzole and N-carbamoyloxyurea) whose intracellular site of action is ribnonucleotide reductase have been used as selective agents in culture to isolate drug resistant cell lines with specific alterations in reductase activity. In general, cell lines resistant to “hydroxyurea-like” drugs exhibit genetic properties satisfying the majority of the criteria for classification as authentic somatic cell mutants. To study the mammalian enzyme properties, in a situation resembling physiological conditions, we have developed a convenient assay system for ribonucleotide reductase activity in whole cells. The procedure is relatively easy to perform, can accurately determine enzyme activity in as few as 10 6 cells grown conveniently on the surface of a tissue culture plate, and unlike cell-free enzyme preparations, activity is linear at low enzyme concentrations. The procedure can be adapted to investigate activity in a relatively small number of intact cells, it is very useful for studying cultures like human diploid fibroblasts, where large quantities of cells containing high enzyme activity may be difficult to obtain. Using this inact cell assay procedure, we have characterized the reductase activity in CHO cells with respect to pyrimidine (CDP) and purine (ADP) substrates, negative and positive effectors, and drug inhibition with the three antitumor agents employed as selective agents for isolating drug resistant cell lines. Some differences in the mode of action of N-carbamoyloxyurea when compared to hydroxyurea or guanazole were noticed and indicate the potential usefulness of the intact cell assay system for examining the action of specific drugs (e.g., 65) on intracellular enzyme activity. Intracellular ribonucleotide reductase activity was examined in the drug resistant cell lines and three different mutant classes were identified. 1. Cell lines containing an apparent structural alteration to the enzyme which results in reductase activity being less sensitive to drug inhibition. 2. Mutants containing elevated levels of reductase activity with a wild type sensitivity to the drug. 3. Cell lines containing a combination of the first two alterations described above; these mutants apparently contain elevated levels of a ribonucleotide reductase enzyme which has a structural alteration rendering it less sensitive to drug inhibition. Furthermore, some recent preliminary work suggests that additional mutant types with alterations in reductase activity can be isolated. The significance of these mutations in providing a better understanding of the regulation of mammalian ribonucleotide reductase was discussed.

Synthesis of hydroxy- and amino-substituted benzohydroxamic acids: inhibition of ribonucleotide reductase and antitumor activity

Benzohydroxamic acids inhibit mammalian ribonucleotide reductase and exhibit antineoplastic activity in L1210 leukemic mice. Five new hydroxy- and amino-substituted benzohydroxamic acids (3,4- and 3,5-OH, 3,4-NH2, 2,3,4- and, 3,4,5-OH) were prepared and tested along with 12 previously reported benzohydroxamic acids (BHA) for enzyme inhibition and antitumor activity. The most potent enzyme inhibitor in this series was 2,3,4-OH-BHA (ID50 = 3.5 microM), which is 140 times more potent than hydroxyurea, but its toxicity limited the antitumor activity to a 30% increase in life span of L1210 bearing mice at 125 (mg/kg)/day ip for 8 days. The most effective antitumor agent in this series was 3,4-OH-BHA which prolonged the life span of L1210 bearing mice 103% at 600 (mg/kg)/day ip for 8 days.

Regulation of ribonucleotide reductase activity and its possible exploitation in chemotherapy

Although 2′-deoxyadenosine inhibits the growth of many organisms it is not inhibitory for Lactobacillus leichmannii. This is consistent with the presence of a powerful trans-N-deoxyribosylase in this organism, as well as the absence of any inhibitory effects of dATP on its ribonucleotide reductase. Specific activating effects of deoxyribonucleoside triphosphates on the reduction of ribonucleotides catalyzed by the L. leichmannii reductase did not act to equalize the production of pyrimidine and purine deoxyribonucleotides when GTP, ATP, CTP and UTP were reduced simultaneously in vitro. Under all conditions investigated dGTP and dATP were formed at a much greater rate than dCTP and dUTP. Deoxytubercidin has an ED 50 for CCRF-CEM cells considerably lower than that for deoxyadenosine and only slightly higher than that for deoxyadenosine in the presence of an inhibitor of adenosine deaminase. This observation suggests that other analogs of deoxyadenosine may efficiently inhibit mammalian cell proliferation via conversion to dATP analogs which inhibit mammalian ribonucleotide reductase.

Deoxyguanosine triphosphate as a possible toxic metabolite in the immunodeficiency associated with purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase (PNP) deficiency is associated with a severe defect in thymus-derived (T)-lymphocyte function combined with normal bone marrow-derived (B)-lymphocyte function. To investigate the role of this enzyme deficiency in the resulting immune dysfunction, we measured the levels of ribonucleoside and deoxyribonucleoside triphosphates in erythrocytes from two unrelated PNP-deficient, T-lymphocyte-deficient patients. Both PNP-deficient patients have abnormally high levels of deoxyguanosine triphosphate (deoxy-GTP) in their erythrocytes (5 and 8 nmol/ml packed erythrocytes). In contrast, normal controls and adenosine deaminase-deficient, immunodeficient patients do not have detectable amounts of deoxyGTP (<0.5 nmol/ml packed erythrocytes). We propose that deoxyguanosine, a substrate of PNP, is the potentially lymphotoxic metabolite in PNP deficiency. The mechanism of toxicity involves phosphorylation of deoxyguanosine to deoxyGTP, which acts as a potent inhibitor of mammalian ribonucleotide reductase.