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

STUDIES OF MAMMALIAN RIBONUCLEOTIDE REDUCTASE ACTIVITY IN I N T A C T PERMEABILIZED CELLS: A GENETIC APPROACH JIM A. WRIGHT, ROBERT G. HARDS and JOHN E. DICK Department of Microbiology, Universityof Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 INTRODUCTION

Ribonucleotide reductase is the enzyme solely responsible for the conversion of the four ribonucleotides to their corresponding deoxyribonucleotides required for DNA synthesis (l). In addition to ribonucleotide reductase two small proteins, thioredoxin through the thioredoxin reductase system, and glutaredoxin via glutathione and glutathione reductase also participate in the reduction process as hydrogen carriers (I, 2). Based upon substrate specificity two general types of ribonucleotide reductase have been described. The first type (EC !. 17.4. I) reduces ribonucleoside diphosphates, contains non-haem iron, and is best represented by the Escherichia coil enzyme, which has been purified to homogeneity and studied in detail (I). This activity is also found in mammals, higher plants and in some fungi (3-5). The second type (EC 1.17.4.2) reduces ribonucleoside triphosphates, has an absolute requirement for 5'-deoxyadenosylcobalamin as a cofactor and is represented by the Lactobacillus leichmanii enzyme, which has also been purified to homogeneity and examined in detail (6). It is interesting to note that Bacillus megaterium, Corvnebacteriurn nephridii and Rhizobium melilote contain enzyme activity intermediate between the two described above; enzymes from these organisms reduce ribonucleoside diphosphates but require 5'-deoxyadenosylcobalamin for activity (7-9). In keeping with the importance of this activity in the physiology of the cell, ribonucleotide reductase from all sources examined exhibits complex and strict allosteric control by a variety of positive and negative nucleotide effectors (I). Many independent investigations have uncovered a critical association between the mammalian reductase and cell growth. For example, the specific activity'of ribonucleotide reductase increases dramatically just prior to and during periods of DNA synthesis, and then returns to a low basal level near the end of S phase (10-12). Moreover, enhanced levels of ribonucleotide reductase are characteristic of rapidly proliferating tissues such as regenerating liver (13), embryonic organs (14) and growing tumors (15). This type of 105

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evidence has led to the suggestion that ribonucleotide reduction may be a ratelimiting step in eucaryotic DNA synthesis and therefore may play a key role in the regulation of cell division (15 18). However, it is obvious that much more information is required before there will be a clear understanding of the regulatory properties of the mammalian enzyme activity (18, 19). During the past decade development of selection procedures for isolating a large variety of stably altered mammalian cell variants in culture has been extremely rapid. The list now includes temperature sensitive, auxotrophic, drug resistant and lectin resistant cell lines, as well as cultures containing altered products of differentiation (20-22). Genetics has greatly contributed to our understanding of regulatory mechanisms in microorganisms and it is now clear that the field of somatic cell genetics is capable of making similar contributions to our understanding of mammalian cells (20). As a general class, the drug resistant mutants have been studied more often than any of the others because of the wide variety of specific mutants that can be isolated and the ease with which they can usually be obtained. Moreover, there are many cytotoxic drugs which specifically inhibit essential cellular functions and thus can be used as selective agents to isolate drug resistant mutants (20). Our laboratory has been involved in establishing a genetic approach to analyzing the complex properties of the mammalian ribonucleotide reductase. Cytotoxic drugs whose intracellular target is ribonucleotide reductase have been used as selective agents in cell culture to isolate drug resistant cell lines with specific alterations in the enzyme activity. The genetic and biochemical characteristics of these cell lines are being investigated. In order to study the enzyme properties in these mutants, in a situation resembling physiological conditions, we have developed a convenient assay system for the reductase in intact cells and have uncovered some interesting changes in enzyme activity. MATERIAI.

S AND

METHODS

Cell Cultures Chinese hamster ovary (CHO) cell lines, mouse k cell lines and normal human diploid fibroblasts (designated HSC 172; 3, 23) were cultured at 37 ° on the surface of glass bottles (Brockway Glass Co.) or on plastic tissue culture plates (Lux Scientific Ltd.) in t~-minimal essential medium (a-MEM: Flow Laboratories, Ltd.) supplemented with antibiotics and fetal calf serum (FCS; Gibco Ltd.) as described previously (3, 24, 25). To determine plating efficiencies, cells were added to culture plates at cell numbers ranging fron 102 to i 06 cells/100 m m plates. After a suitable period of incubation at 37 °, the plates were drained and a saturated solution of methylene blue in 50% ethanol was added to the plates. After about 10 rain the plates were rinsed in water and the stained colonies on each plate were counted. The plating efficiency was estimated by dividing the number of

RIBONUCLEOTIDE REDUCTASE: GENETIC APPROACH

107

colonies by the number of cells plated. The relative plating efficiency was defined as plating efficiency in the presence of the drug divided by that in the absence of the drug. The D~0 value of a drug is the concentration at which the relative plating efficiency is reduced to 10%. Cell Permeabilization and Ribonucleotide Reductase Assa.v with C H O Cells Cells were made permeable to nucleotides using the Tween 80 method developed in our laboratory (3, 12, 25, 26). Exponentially growing cells were plated at a density of 2 × l06 cells/100 m m or 5 × 106/150 mm plastic tissue culture plate containing a - M EM plus 10% FCS and incubated at 37 ° . After 40 hr the cells were removed from the surface of plates with a 0.05% buffered trypsin solution, centrifuged, washed once with a t~-M EM plus 10% FCS and counted by means of a Coulter particle counter (Coulter Electronics Co.). The cells were then resuspended at l07 cells/ml of permeabilizing buffer consisting of I% Tween 80 (J. T. Baker Co.), 0.25 M sucrose, 50mM N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid (Hepes) buffer, pH 7.2 and 2mM dithiothreitol (DTT). The cells were incubated at 22 ° for 30 min with occasional agitation and then centrifuged. The cell pellet was gently resuspended in fresh permeabilizing buffer at a concentration of 2.5 × l07 cells/ml. These permeabilized cells exhibited plating efficiencies ranging between 65 and 80%, indicating that most of them retain proliferative abilities after treatment. Aliquots of 200 ,ul, corresponding to 5 × 106 cells, were then added to assay tubes. For C D P reduction, the cell pellet was resuspended in the above specified permeabilizing buffer and made up to a final vol of 300 ~1. The standard reaction mixture contained 50ram Hepes pH 7.2, 2 mM ATP, 6mM DTT, 8 mM MgCI 2, 0.4 mM ~4C-CDP (5000 c p m / n m o l ) , 0.67% Tween 80 and 0. 167 M sucrose. The assays were then agitated with either a Dubnoff shaking water bath (Precision Scientific) or 2 × 7 mm stirring bars for 20-25 min at 37 °, after which the reaction was terminated by boiling. The nucleotides were converted to nucleosides by treatment for 2 hr at 37" with 2 rag/assay of Crotalus atrox venom dissolved in 0. I M Hepes pH 8.0 plus 10 mM MgC! 2. The reaction was again terminated by boiling for 4 min and 0.5 ml distilled H20 was added to each assay tube. The heat precipitated material was removed by centrifugation. The supernatant was kept and the deoxycytidine separated from the cytidine compounds on a 5 )< 80 m m D o w e x - l - b o r a t e or an A G I - X 8 borate column (Bio-Rad Labs) according to the Steeper and Steuart (27) method as modified by Cory and Whitford (17). Briefly, the separation depends upon the formation of a complex between ribonucleosides and borate ions in the column resin. Deoxyribonucleosides do not possess a cis-diol and hence cannot form a complex. Deoxycytidine was eluted from the column with 4 ml of distilled water. 10 ml of ACS (Amersham/Searle) or 16 ml of Scintiverse (Fisher Scientific) were then added. The mixture was shaken until a single translucent

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gel formed. Radioactivity was determined with a Beckman model LS230 liquid scintillation spectrophotometer. For A D P reduction, the cell pellet was resuspended in permeabilizing buffer containing 1% Tween 80, 0.25 M sucrose, 50 mM piperazine-N, N'-bis (2-ethanesulfonic acid)(Pipes) pH 6.8, and 2 mM DTT. The final reaction vol of 300 #1 standardly contained 50 mMPipes pH 6.8, 0.5 mM dGTP, 6 mM DTT, 0.4 mM ~4C-ADP (5000 cpm/nmol), 0.67% Tween 80 and 0.167 M sucrose. The assays were then agitated with a shaking water bath for 15 25 min and boiled for 4 rain to terminate the reaction; 0.3 #mol carrier d A M P was added and each assay was treated with snake venom as described above. Deoxyadenosine was separated from adenosine according to the method of Cory et al. (28). To the boiled reaction mixture was added 0.5 ml of I mM sodium borate. Assay tubes were centrifuged to remove heat precipitated material and the supernatant was added to a Dowex-l-borate or AGI-X8borate column previously equilibrated with 10 ml of I mM sodium borate. The deoxyadenosine was eluted with 20 ml of I mM sodium borate of which the first 4 ml were discarded and 4 of the remaining 16 ml being transferred to a scintillation vial. Sixteen ml of Scintiverse or 10 ml of ACS were added as before. Preliminary work using PEI thin layer chromatography (29) to identify labelled nucleosides and nucleotides in the assay mix after completion of reactions appears to indicate that other intracellular activities do not significantly affect estimates of ribonucleotide reduction in permeabilized cells treated by the procedure outlined in this report. These studies are still in progress and are being extended to include H PI,C procedures. One unit of enzyme activity is defined as the amount of enzyme that reduces I nmol of nucleotide per 5 × l0 b permeabilized cells in 1 hr. Source

of Chemicals

Radiochemicals were purchased from Amersham, Searle. Hydroxyurea, DTT, Hepes, Pipes, Crotalus atrox venom, and all nucleotides were obtained from the Sigma Chemical Company. Guanazole (NSC 1895)was provided by Dr. Harry B. Wood, Jr. as a gift from the Drug Development Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland. Ncarbamoyloxyurea (SQ @10,726) was obtained as a gift from Ms. B. Stearn, Research Evaluation, Squibb Institute for Medical Research, Princeton, New Jersey. R E S U I . TS A N D

DISCUSSION

Hydroxyurea, Guanazole and N-Carbamoylox.vurea as Selective Agents

Hydroxyurea was first synthesized over 100 years ago (30) and is a simple analog of urea in which a hydrogen atom is replaced by a hydroxyl group. The

RIBONUCLEOTIDE REDUCTASE: GENETIC APPROACH

109

drug contains an oxidized peptide bond and is usually classified as an hydroxamic acid. It is a potent and specific inhibitor of D N A synthesis and has been used clinically to treat a wide range of solid tumors as well as acute chronic leukemia; and recently it has been used in combination with other cancer chemotherapeutic agents to achieve a potentiated therapeutic response (31-33). Guanazole, an aryl diamino substituted triazole (3,5-diamino-1,2,4triazole), was also synthesized many years ago (34). The compound is an effective inhibitor of D N A synthesis and has antineoplastic properties (33, 35). Both hydroxyurea and guanazole are known to be potent inhibitors of ribonucleotide reductase activity (35, 36). N-carbamoyloxyurea has been isolated as an oxidation product from hydroxyurea incubated at 56 ° for 72 hr (37), The drug is cytotoxic for rapidly proliferating cells and effectively inhibits D N A synthesis (37, 38). Recently we have observed that the drug is an inhibitor of the mammalian ribonucleotide reductase as well (Table 5; reference 66). Figure ! shows the decrease in relative plating efficiency of wild type hamster cells cultured in the presence of increasing concentrations of the three drugs; on a molar basis hydroxyurea is the most effective inhibitor of colony formation and guanazole is the least potent. The intracellular action of these drugs is very quick since D N A synthesis is strongly inhibited within min of adding cytotoxic concentrations to cells in culture ( ! 8, 36, unpublished results). The properties of these drugs make them very efficient selective agents for isolating resistant cell lines from rapidly growing cells in culture. Figure I shows the relative plating efficiency of one of the cell lines selected in our laboratory for the ability to grow in the presence of normally cytotoxic concentrations of hydroxyurea. Note that the cells are cross-resistant to guanazole and N-carbamoyloxyurea supporting the view that all three drugs act at a c o m m o n intracellular site. Resistant cell lines like the one shown in Figure I can be isolated at a frequency of about ! X 10-5 in rodent cell cultures and they usually exhibit stable drug resistant phenotypes. Some cell lines have been in continuous culture for several years in the absence of a selective agent without showing a change in cellular properties. In Table I we compare the resistance of three hamster cell lines independently selected in the presence of cytotoxic concentrations of each of the three drugs. In agreement with the information in Figure I each mutant line is resistant to the selective and the nonselective drugs, suggesting that the selective pressure exerted by the three drugs is directed towards a c o m m o n intracellular site. In view of the concerns raised by some geneticists that altered mammalian cell phenotypes selected in culture may result from non-mutational, epigenetic events (see reviews, 20, 22), as opposed to true mutations of the type described for various bacterial systems, it is worth noting that we have carried out a detailed genetic study of the drug resistant cells isolated in our laboratory. The essential results of thse studies are presented in Table 2 and can be summarized as follows. It is possible to select in a single step, phenotypically

1 10

J I M A. W R I G H T

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F I G . I. Relative plating efficiencies o f wild type C H O cells ( O ) a n d a hydroxyurea resistant C H O cell line ( n ) in the presence of increasing concentrations of hydroxyurea (a), guanazole (b) and Nc a r b a m o y l o x y u r e a (c).

T A B L E I. D R U G S E N S I T I V I T Y E X H I B I T E D BY T H R E E INI)EPENDEN'I"I.Y ISOLATED MUTANTS AND THE PARENTAl. CELL LINE

(.'ell lines Wild t y p e C H O Hu R 2 G~ I NC a 30A

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0.21 3.10 0.71 0.35

1.21 14.52 4.51 1.77

0.32 2.88 0.77 0.84

* H U = hydroxyurea; G U = guanazole; N C U = N-carbamoyloxyurea. For more information on H u R 2 a n d G K I see references 39 41. Hards and Wright h a v e described the properties o f N C g 3 0 A cells in a report accepted for publication (66).

stable colonies of cultured cells that exhibit drug resistance and contain altered ribonucleotide reductase activity. Fluctuation analysis indicates that the appearance of resistant cells in wild type populations occurs spontaneously and at a reasonable rate. Mutagens like ethyl methane sulfonate significantly increase the frequency of resistant cells in a population. Somatic cell hybridization experiments reveal that drug resistance behaves as a dominant or codominant trait and that the drug resistant marker is useful for selecting hybrid cells. Furthermore, similar to many other drug resistance systems, cells

RIBONUCLEOTIDE REDUCTASE: GENETIC APPROACH

111

TABLE 2. A SUMMARY OF THE GENETIC PROPERTIES OF DRUG RESISTANT RODENT CELl. LINES I. At a frequency of approximately I X 10-~it is possible to select, in a single step, clones capable of proliferating in the presence of normally cytotoxic concentrations of drug. 2. The drug resistant cell lines are cross-resistant to a number of non-selective agents whose intracellular site of action appears to be ribonucleotide reductase. 3. The drug resistant phenotype is stable even after many years of continuous culture in the absence of a selective agent. 4. The frequency of occurrence of the resistant phenotype is increased by mutagen treatment 5. The altered phenotype appears in a spontaneous random fashion and at a reasonable rate as determined by Luria Delbrfick fluctuation analysis. 6. Expression of the drug resistant phenotype in somatic cell hybrids occurs in a dominant or codominant manner. selected for resistance to h y d r o x y u r e a , g u a n a z o l e or N - c a r b a m o y l o x y u r e a exhibit a n altered sensitivity to a n u m b e r of non-selective agents (e.g. formamidoxime, hydroxyurethane, hydroxyurea, guanazole and N-carbam o y l o x y u r e a ) . O u r genetic studies s u p p o r t the view that resistance to " h y d r o x y u r e a - l i k e " d r u g s is expressed as a n o r m a l genetic trait (20, 25, 39-42, 66, u n p u b l i s h e d d a t a ) since the results satisfy the m a j o r i t y o f criteria (22) for classification as a u t h e n t i c s o m a t i c cell m u t a n t s .

Mammalian Ribonucleotide Reductase in Intact Cells W e have e x a m i n e d the reductase activity in cell-free extracts and partially purified p r e p a r a t i o n s (e.g. 16, 25, 36) and have described a 50-fold purification p r o c e d u r e for e n z y m e activity f r o m the C H O cell line (40). Investigations using cell free e x t r a c t s can be c o m p l i c a t e d by several p r o b l e m s which include low enzyme activities, the presence of d A T P and other n a t u r a l l y o c c u r r i n g effectors which must be removed by such techniques as gel filtration or ion e x c h a n g e c h r o m a t o g r a p h y , a n d the lack of linearity of activity with increasing e n z y m e at low protein c o n c e n t r a t i o n . Unfortunately, the m a m m a l i a n r i b o n u c l e o t i d e reductase system has p r o v e n to be much m o r e difficult to purify to h o m o g e n e i t y t h a n the E. coli enzyme, and no l a b o r a t o r y to d a t e has r e p o r t e d a h o m o g e n e o u s p r e p a r a t i o n from cultured m a m m a l i a n cells. Recently, a highly purified, a l t h o u g h a p p a r e n t l y not h o m o g e n e o u s , m a m m a l i a n activity has been o b t a i n e d by using large quantities of calf t h y m u s tissue as starting m a t e r i a l (43, 44). O u r l a b o r a t o r y decided to develop an assay system for r i b o n u c l e o t i d e reductase in intact cells which we h o p e d would be c o n v e n i e n t to use a n d should m o r e closely resemble n o r m a l physiological c o n d i t i o n s for the enzyme. T h e value of this in vivo assay system becomes a p p a r e n t when the p r o c e d u r e is used to e x a m i n e the essential features of the m a m m a l i a n r i b o n u c l e o t i d e reductase activity. General properties o f ribonucleotide reduction in intact hamster cells. Since the e n z y m e catalyzes the r e d u c t i o n o f both purine and p y r i m i d i n e

112

JIM A. WRIGHT et al.

r i b o n u c l e o s i d e d i p h o s p h a t e s required for D N A synthesis, we e x a m i n e d the p r o p e r t i e s of the enzyme in the presence o f a purine ( A D P ) and a pyrimidine ( C D P ) substrate. The o p t i m u m reduction c o n d i t i o n s for the two substrates were very similar to those observed with cell-free p r e p a r a t i o n s from m a n y m a m m a l i a n sources (e.g., I i, 13, 40, 44, 45). The most favorable conditions for assaying these activities are listed in Table 3 and can be s u m m a r i z e d as follows. E n z y m e activity was largely d e p e n d e n t u p o n the e x o g e n o u s reducing agent, D T T , with m a x i m u m activity occurring at 6 mM followed by a slow decline in activity up to 20 mM. C D P r e d u c t i o n was greatly stimulated by 8mM MgCI 2 and showed an a b s o l u t e r e q u i r e m e n t for A T P with the o p t i m u m c o n c e n t r a t i o n being 2mM. A D P r e d u c t i o n had an a b s o l u t e requirement for d G T P with m a x i m u m activity t a k i n g place at 0.5 mM. Both C D P and A D P r e d u c t i o n o c c u r r e d within a b r o a d p H range with an o p t i m u m of pH 7.2 for C D P r e d u c t i o n a n d p H 6.9 for A D P reduction. Also a t e m p e r a t u r e of 37 ° was f o u n d to be most f a v o r a b l e for the r e d u c t i o n o f both C D P and A D P . Using the o p t i m u m assay c o n d i t i o n s described in this report, purine and pyrimidine r e d u c t i o n was linear with cell n u m b e r up to and including 107 cells/assay, and with time to at least 30 minutes. Linearity of activity at low enzyme c o n c e n t r a t i o n is a feature of the in v i v o assay system not observed for C D P , U D P , or G D P r e d u c t i o n in cell-free extracts (13, 40, 43, 45, 46). The presence o f iron has been used to stimulate C D P reduction in some m a m m a l i a n cell e x t r a c t s (13, 45, 46). H o p p e r (47) found the degree of s t i m u l a t i o n of b o n e m a r r o w C D P reductase varied c o n s i d e r a b l y from only a slight s t i m u l a t i o n to essentially c o m p l e t e d e p e n d e n c e u p o n a d d e d iron. In the present study, with p e r m e a b i l i z e d cells, we also found c o n s i d e r a b l e variation IABI.E 3. GENERAl. PROPERTIES OF CDP AND ADP REDUCTION IN INTACT HAMSTER CELLS Addition DTr AI'P dGTP MgCI, pH lemperature Omission None DTI" AIP dGTP MgCl2

Requirement for optimum activity CDP reduction ADP reduction 6 mM 2 mM

6 mM 0.5 mM

8 mM 7.2 37°

6.9 37°

Percent activity remaining CDP reduction ADP reduction 100q~ 42 0

IOOCki

42 0

14

113

R I B O N U C L E O T I D E REDUCTASE: G E N E T I C APPROACH

in C D P reduction in the presence of iron. For example in the presence of 0.4 mM ferric chloride the activity varied from slight inhibition to a stimulation as high as 16-fold. The reasons for this variation are not understood. Moreover, the reduction of A D P was not stimulated by ferric chloride concentrations up to 0.4 mM while 0.6 mM reduced the activity to 20%. Therefore iron was not included during the assay of either the purine or pyrimidine reductase activities. Kinetic studies o f ribonucleotide reduction in intact hamster cells. Figure 2 shows that double reciprocal plots of C D P and A D P reduction at varying substrate concentrations are linear for both ribonucleoside diphosphate substrates. For pyrimidine reduction the data yielded an apparent Km value of 0.09 + 0.02 mM C D P and a Vm~xvalue of 0.49 + 0.17 nmols d C D P formed/5 X 106 cells/hr. Experiments with the purine substrate yielded an apparent Km estimate of 0.22 + 0.04 mM A D P and a Vma~value of 1.0 + 0.13 nmols d A D P produced/5 X i06 cells/hr. It is worth noting that these experiments have been performed at least 10 times over the course of more than a year with similar results being obtained each time. Although estimates of substrate Km values vary from one laboratory to another depending upon assay conditions and enzyme source, it is interesting that the values obtained with intact hamster cells are similar to many of those reported for enzyme prepared from a

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FIG. 2. (a) Double reciprocal plots of the variation in the rate of CDP reduction with CDP concentration in intact CHO cells. Activity was expressed as nmols dCDP formed/5 X IIY' cells/hr. Inset: the velocity of C D P reduction in the presence of varying concentrations of CDP. (b) Double reciprocal plots of the variation in the rate of A DP reduction with ADP concentration in intact CHO cells. Activity was expressed as nmols d A D P formed/5 X 10~ cells/hr. Inset: the velocity of A D P reduction in the presence of varying concentrations of ADP.

1 14

JIM A. W R I G H T et al.

diversity of sources (e.g., II, 40, 44, 45) and resemble the apparent Km estimates of 0.04 mM C D P and 0.1 mM A D P we have recently observed with intact permeabilized human diploid fibroblasts (3). Using the V,~, data it is possible to make some general statements about the reductase activity determined in the cells in relation to that required to support in vivo D N A synthesis. If logarithmically growing C H O cells contain 9.6 pg DNA/nucleus (i 2) and they double every 16 hr, then it can be estimated that the C D P and A D P reductase activities at saturating substrate concentrations should account for about 30 and 50%, respectively, of the C D P and A D P reduction necessary for de n o v o D N A synthesis. "Fhese values compare well with an estimate for C D P reduction of 25% of the corresponding rate of DNA synthesis in toluene treated E. coli cells (48). in a study with a mutant hamster cell line auxotrophic for glycine, adenine and thymine we recently observed that C D P and G D P reductase activity in this particular cell line could be as high as 50 and 70%, respectively, of the in vivo levels needed for synthesizing D N A (12). Moreover, some drug resistant mouse and hamster cells isolated in our laboratory apparently have reductase activities that can account for more than 100% of the required rate of de novo deoxyribonucleotide synthesis. Substrate specificity and activity of ribonucleotide reductase are controlled by allosteric mechanisms involving nucleotide effectors, with d A T P acting as an overall negative effector in the reduction of all four ribonucleotide substrates (I). Figure 3 shows pyrimidine and purine reductase activity in intact hamster cells in the presence of varying concentrations of dATP. As with studies using cell-free preparations from a variety of mammalian sources (13, 44, 49-51) d A T P acts as a very potent inhibitor of both C D P and A D P reduction in intact hamster cells; a 50% inhibition of pyrimidine and purine reduction was observed at approximately 8 and 45 ~M dATP, respectively. These results with hamster cells are similar to the ones we have recently obtained with intact human diploid fibroblasts (3).

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RIBONUCLEOTIDE REDUCTASE: GENETIC APPROACH

1 15

The C H O system was examined further to determine whether other known effectors of pyrimidine and purine reduction operated as expected in intact cells. For example d G T P and A T P have been described as negative effectors in the complex regulation of C D P and A D P reductase activity (l). Figure 4 shows that in intact cells, C D P and A D P reductions are potently inhibited by the effectors with 0.25 mM d G T P and 0.60 mM ATP causing 50% inhibition of pyrimidine and purine reduction, respectively. Substrate specificity as well as reaction rates are strictly regulated in an allosteric fashion by the presence of nucleoside triphosphates. Some experiments have been carried out with a variety of these compounds to get some preliminary information about intracellular enzyme activity in their presence (Table 4). Optimum concentrations were not determined for all nucleotides and for some of them the concentrations used were those previously found to be favorable for enzyme activation in other assay systems (e.g. 49, 5l). With this limitation in mind it is worth noting that the results generally agree with studies in cell-free preparations. CDP reduction was greatly enhanced by ATP and d G T P was an effective activator of ADP reduction. These experiments do not allow us to evaluate the importance of low level activation of C D P and A D P reduction by several of the nucleotides, hut the observation (49, 52; Table 4) that G T P can partially replace (30 to 38% in intact hamster cells) d G T P as a positive effector of A D P reduction at least

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1 16

JIM A. WRIGHT et al. TABLE 4. PRELIMINARY STUDIES OF THE EFFECTS OF VARIOUS NUCLEOSIDE TRIPHOSPHATES AS ACTIVATORS OF CDP AND ADP REDUCTION IN INTACT HAMSTER CELLS Nucleoside triphosphate ATP dGTP GTP dATP CTP dCI'P TTP UTP none

Concentration (mM) 2.0 0.5 1.5 0.05 1.0 1.0 1.0 1.0

% of activity* CDP reduction ADP reduction 100 4,8 6,8 < I,< 1 1,2 14,18 <1,3 <1,<1

<1,<1

4.9 100 30,38 < I,< I <1.3 1.3 12.15 8.11 <1.<1

*% activity when compared to CDP reduction in the presence of ATP and ADP reduction in the presence of dGTP. Results are from two independent trials. AT P. dGTP and GTP were added at concentrations optimum for activation of either CDP or ADP reduction. ]he d A T P concentration inhibits ADP reduction by about 50e~in a standard assay. CTP, dCTP, TTP, UTP were tested at a concentration within the range shown to be effective in other systems (49, 51).

suggests that G T P may play a regulatory role u n d e r n o r m a l physiological conditions. We have investigated the i n h i b i t i o n of intracellular ribonucleotide reductase by h y d r o x y u r e a , g u a n a z o l e and N - c a r b a m o y l o x y u r e a , the three drugs used as selective agents to isolate resistant cell lines. Figure 5 shows d o u b l e reciprocal plots of r i b o n u c l e o t i d e reduction at several fixed c o n c e n t r a t i o n s of h y d r o x y u r e a . The plots were linear for both nucleoside d i p h o s p h a t e substrates. C D P reduction appears to be inhibited in an u n c o m p e t i t i v e m a n n e r by the drug whereas the pattern is a p p a r e n t l y a m i x t u r e of n o n c o m p e t i t i v e and u n c o m p e t i t i v e i n h i b i t i o n when the substrate is A D P . Replots of velocity intercepts against drug c o n c e n t r a t i o n s were also linear (inset, Figure 5) a n d gave K, values of 0 . 2 0 m M and 0.16mM h y d r o x y u r e a for C D P and A D P reduction, respectively. N - c a r b a m o y i o x y u r e a and g u a n a z o l e i n h i b i t i o n of enzyme activity has also been analyzed and the essential results of kinetic experiments have been s u m m a r i z e d and presented in T a b l e 5. It is interesting to note that the i n h i b i t i o n patterns for hydroxyurea and g u a n a z o l e are identical but are not the same as the ones observed with Nc a r b a m o y l o x y u r e a , suggesting that there are i m p o r t a n t differences in the way N - c a r b a m o y l o x y u r e a and the other two inhibitors interact with the enzyme. Other differences between the effects of N - c a r b a m o y l o x y u r e a and h y d r o x y u r e a or g u a n a z o l e on C H O cells a n d nucleotide reduction were also noticed. If the ratio of K, to D~0 estimates are c o m p a r e d for each drug, values of 0.86, 0.95 a n d 7.2 are f o u n d for h y d r o x y u r e a , guanazole and Nc a r b a m o y l o x y u r e a , respectively. F o r h y d r o x y u r e a a n d guanazole it would

RIBONUCLEOTIDE REDUCTASE: GENETIC APPROACH

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118

JIM A. WRIGHT et al. TABLE 5. INHIBITION O F CDP AND ADP REDUCTION IN INTACT H A M S T E R C E L L S WITH N-CARBAMOYLOXYUREA AND GUANAZOLE

Inhibitor N-carbamoyloxyurea N-carbamoyloxyurea Guanazole Guanazole

Varied substrate

Type of inhibition

K, values

CDP ADP CDP ADP

mixed noncompetitive uncompetitive mixed

2.3 mM 2.3 mM I. 15 mM I. 15 mM

hamster cells fail to support the view previously presented (37, 38), that the antitumor agent, hydroxyurea, owes its cytotoxic action to an intracellular conversion of the drug to N-carbamoyloxyurea. If this hypothesis was correct one would expect the K~ and D~0 values for N-carbamoyloxyurea to be significantly less than those for hydroxyurea. However, on a molar basis hydroxyurea is 12 times more potent as an inhibitor of intracellular ribonucleotide reductase and 1.5 times more potent as a cytotoxic agent than N-carbamoyloxyurea. C o m m e n t s on ribonucleotide reduction in other intact m a m m a l i a n ('ells.

The general procedure outlined in this report, for determining intracellular enzyme activity in hamster cells, has been successfully used to examine the activity in a variety of other cell types as well, including mouse L cells (25, 26), 3T3 and SV40 transformed 3T3 cells (12, unpublished results), mouse T i y m p h o m a cells (53), and normal human diploid fibroblasts (3). It should be noted that the optimum assay conditions for each cell line or strain are usually different and have to be worked out in each case. Some differences in o p t i m u m conditions are occasionally observed even between different clones of the same cell line. This point is particularly important when comparing activities in mutant cells with wild type cells. Care should be taken to be sure that comparisons are made between mutants and the exact parental wild type line from which they were selected. The ability to analyze ribonucleotide reductase activity in normal human cells is particularly interesting because very few detailed studies have been carried out with the enzyme from this source, although it has been detected in abnormal malignant cells (50, 52) and in normal cells such as peripheral leukocytes which contain very low activity (54). Normal human fibroblasts have a limited lifetime in culture, exhibit anchorage dependent growth and a high degree of contact inhibition; properties which often make detailed biochemical studies more difficult to perform than with the permanent cell lines. However, these cells are potentially very valuable to study since their physiology and cytogenetic properties closely resemble the in vivo situation (55, 56). These considerations, and the recent findings (53) indicating that some immunodeficiency diseases in man appear to be caused by an imbalance

RIBONUCLEOTIDE REDUCTASE: GENETIC APPROACH

119

of the allosteric control of ribonucleotide reductase, prompted us to examine the intracellular properties of the normal human enzyme. In Figure 6 we show the C D P and ADP reductase activities in normal human diploid fibroblasts determined by a modification (3) of the assay procedure described for hamster cells. Unlike studies in cell-free extracts, high enzyme activity and linearity of activity with enzyme concentration are important characteristics of ribonucleotide reductase in intact cells. Since it is often difficult to obtain large batch cultures of normal diploid cells this property is particularly valuable because the reductase can be accurately assayed at low cell numbers conveniently grown on a single tissue culture plate. Furthermore, the enzyme in whole cells is inhibited by specific drugs like hydroxyurea (Fig. 7) and is amenable to kinetic analysis (3) similar to those described for hamster cells in this report. These observations suggest that it would be worthwhile selecting hydroxyurea resistant human diploid fibroblasts, and we are now in the process of searching for these mutants. Our investigations with hydroxyurea resistant rodent cells show that resistance is expressed in a dominant or codominant fashion in cell-cell hybrids (39, 42). It should be possible, therefore, to use existing methods (20, 57) to map the human chromosome(s) involved in hydroxyurea resistance and presumably necessary for human ribonucleotide reductase activity. Ribonucleotide Reductase in Drug Resistant Cell Lines Ribonucleotide reductase activity in cell lines resistant to hydroxyurea, guanazole and N-carbamoyioxyurea has been investigated in partially purified enzyme preparations and by the intact cell assay system described in

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RIBONUCLEOTIDE REDUCTASE: GENETIC APPROACH

121

reductase activity in a drug resistant C H O cell line belonging to this mutant class is compared to its parental wild type. The mutant cells exhibit approximately six times the normal level of C D P reductase activity. In addition, when the resistant cells are cultured in the presence of hydroxyurea prior to enzyme analysis (Figure 8c) enzyme activity can be elevated even further. Figure 8d shows a drug sensitive revertant cell line isolated from a mutant population containing elevated enzyme activity levels (42); these revertant cells contain reductase activity levels and cellular drug resistance properties similar to parental wild type cells. The intact cell assay for ribonucleotide reductase is very useful for measuring activity in cell lines with altered enzyme levels because the assay gives reproducible results, whereas the activity in cell-free extracts can vary considerably from one preparation to the next (12). It will be interesting to determine the mechanism responsible for increased enzyme activity in the various overproducing mutants. For example it is possible that ribonucleotide reductase or its m R N A is more stable in drug resistant than in wild type cells or that the mutants possess an altered regulatory gene involved in controlling intracellular levels of the reductase, in this regard it is worth noting that Sinensky and his colleagues (59) have recently described very interesting regulatory mutants in the pathway of cholesterol biosynthesis. Another exciting possibility is that these mutants may possess a gene amplification at the ribonucleotide reductase locus (loci)

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JIM A. WRIGHT et al.

122

and thus may resemble the methotrexate resistant cells which contain multiple copies of dihydrofolate reductase genes (60) or N-(phosphonacetyl)-Laspartate resistant lines which overproduce a multienzym¢ complex (CAD) associated with U M P biosynthesis through amplification of the C A D gene (61). Mutants of this type are extremely useful for investigating regulatory mechanisms of gen¢ expression in mammalian cells (20, 60-63). The third class of drug resistant mutants contain a combination of the first two alterations described. These mutants appear to contain elevated levels of a ribonucleotide reductase enzyme which has a structural alteration rendering it less sensitive to inhibition by drug (25, 26). For example, two resistant mouse L cell lines, LI H2 and LIH3, have recently been isolated by a single step selection procedure and shown to contain about 13 times more C D P reductase activity, and 4- to 5-fold more A D P reductase activity than parental wild type cells (26). It is interesting to note that mutants which overproduce enzyme activity often exhibit unequal increases in pyrimidine and purin¢ reducing ability (12, 25, 26, 42). The inhibitory effect of hydroxyurea on intracellular ribonucleotide reduction in these two resistant lines and their parental wild type population is shown in Figure 9. C D P and A D P reductase activities were inhibited by 50% in parental cells at 0.34 mM and 0.30 mM hydroxyurea, respectively. C D P and A D P reductions in LI H2 cells were

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RIBONUCLEOTIDE REDUCTASE: GENETIC APPROACH

123

inhibited by 50% at 1.4 mM and 1.2 mM hydroxyurea, respectively; these estimates are about four times higher than the wild type values. Similarly CDP and A D P reductions in LI H3 cells were inhibited by 50% at 1.8 and 1.6 mM hydroxyurea, respectively, and these values are about five times the concentrations required to bring about the same inhibition in wild type cells. It is not known whether the two changes in reductase activity in the third class of mutants is due to a single pleiotropic mutation, as has been observed in other mutants lines (e.g., 24, 64), or is due to two independent events which give rise to an apparent structural enzyme alteration and to enhanced levels of activity. Further analysis of various drug resistant cell lines could turn up other interesting examples of mutant classes. For example, we noted earlier that cell lines containing elevated levels of enzyme activity often exhibit unequal increases in their pyrimidine and purine reductase activities, in this regard it is worth mentioning that some preliminary work suggests that it may be possible to select resistant cell lines containing increased CDP reductase activity while maintaining wild type levels of A D P reductase activity. Although further work is required, mutants of this type would be valuable for investigating important questions on the interrelationship between the regulation of purine and pyrimidine reductions in mammalian cells.

SUMMARY

We are using a genetic approach to study the complex properties of mammalian ribonucleotide reductase activity. Cytotoxic drugs (hydroxyurea, guanazole and N-carbamoyloxyurea) whose intracellular site of action is ribonucleotide 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 106 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 variety of cell types, and since it allows a direct analysis of the reductase 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 intact cell assay procedure, we have characterized the reductase activity in CHO cells with respect to pyrimidine

124

JIM A. WRIGHT et

al.

(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 Ncarbamoyloxyurea 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, i. 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.

ACKNOWLEDGEMENTS Research funds were provided to J. A. Wright by the Natural Sciences and Engineering Research Council of Canada and the Muscular Dystrophy Association of Canada. J. A. Wright thanks his former graduate students, W. H. Lewis, B. A. Kuzik and S. E. Koropatnick for their valuable contributions to the work on ribonucleotide reductase. R. G. Hards and J. E. Dick acknowledge receipt of graduate scholarships from the Natural Sciences and Engineering Research Council of Canada.

REFERENCES I.

L. THELANDER and P. REICHARD. Reduction ofribonucleotides, Ann. Rev. Biochem. 48, 133 158(1979). 2. M. LUTHMAN, S. ERIKSSON, A. HOLMGREN and L. THELANDER, Glutathionedependent hydrogen donor system for calf thymus ribonucleoside-diphosphate reductase, Pros'. Natl. Acad. Sci. U.S.A. 76, 2158-2162 (1979). 3. J. E. DICK and J. A. WRIGHT, Ribonucleotid¢ reduction in intact human diploid fibroblasts, 105, 63-72 (1980). 4. B. HOVEMANN and H. FOLLMANN,Thedeterminationoflowribonucleotidereductase activity in plant extracts, Anal. Biochem. 79, 119-120 (1977). 5. W.H. LEWIS, D. R. MCNAUGHTON, H. G. LEJOHN and J. A. WRIGHT, Regulation of fungal ribonucleotide reductase by unusual dinucleotides, Biochem. Bioph.vs. Res. Cornmun. 7l, 128-135 (1976).

RIBONUCLEOTIDE REDUCTASE: GENETIC APPROACH 6.

7. 8. 9. 10.

I I. 12. 13. 14. 15.

16. 17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

125

D. H. SINGH, Y. T A M A O and R. L. BLAKLEY, Allosterism, regulation and cooperativity: the case of ribonucleotide reductase of Lactobacillus leichmannii. Advances in Enzyme Regulation 15, 81-100 (1977). S. YAU and J. T. W A C H S M A N , The Bacillus megaterium ribonucleotide reductase: evidence for a BI2 coenzyme requirement, Mol. Cell. Biochem. 1, 101-105 (1973). J . R . COWLES and H. J. EVANS, Some properties of the ribonucleotide reductase from Rhizobium meliloti. Arch. Biochem. Bioph.vs. 127, 770-778 (1968). P. K. KUO and P. C. H O G E N K A M P , The purification and characterization of an adenosylcobalamin-dependent ribonucleoside diphosphate reductase from Corynebacterium nephridii, J. Biol. Chem. 255, 1273-1278 (1980). S. M U R P H R E E , E. S T U B B L E F I E L D a n d E. C. MOORE, Synchronized mammaliancell cultures. Iil Variation of ribonucleotide reductase activity during the replication cycle of Chinese hamster fibroblasts, Exptl. Cell Res. 55, 118-124 (1969). D.M. PETERSON and E. C. MOORE, Independent fluctuations ofcytidine and adenosine diphosphate reductase activities in cultured Chinese hamster fibroblasts, Biochim. Bioph.vs. Acla 432, 80-91 (1976). W . H . LEWIS, B. A. KUZIK and J. A. WRIGHT, Assay of ribonucleotide reduction in nucleotide-permeable hamster cells, J. Cell Physiol. 94, 287-298 (1978). A. LARSSON, Ribonucleotide reductas¢ from regenerating rat liver, Europ. J. Biochem. 11, 113-121 (1969). H.L. ELFORD, Functional regulation of mammalian ribonucleotide reductase, Advances in Enzyme Regulation 10, 19-38 (1972). H. L. ELFORD, M. FREESE, E. PASSAMANI and H. P. MORRIS, Ribonucleotide reductase and cell proliferation. I. Variation of ribonucleotide reductase activity with tumor growth rate in a series of rat hepatomas, J. Biol. Chem. 245, 5228-5233 (1970). W . H . LEWIS, D.R. MCNAUGHTON, S.H. GOH, H.B. Lf=JOHNandJ.A. WRIGHT, Inhibition of mammalian ribonucleotide reductas¢ by a dinucleotide produced in eucaryotic cells, J. Cell. Physiol. 93, 345-353 (1977). J.G. CORY and T. W. WHITFORD, JR., Ribonucleotide reductase and DNA synthesis in Ehrlich ascites tumor cells, Cancer Res. 32, 1301 - 1306 (1972). P. R E I C H A R D , From deoxynucleotides to DNA synthesis, Federation Proc. 37, 9 14 (1978). J . G . CORY, Properties of ribonucleotid¢ reductase from Ehrlich tumor cells: multiple nucleoside diphosphate activities and reconstitution of activity from components, Advances in Enzyme Regulation 17, 115-131 (1979). J . A . WRIGHT, W. H. LEWIS and C. L.J. PARFETT, Somatic cell genetics: a reviewof drug resistance, lectin resistance and gene transfer in mammalian cells in culture, Can. J. Genet. CvtoL 22, 443-496 (1980). J . A . WRIGHT, Minireview. Membrane variants of mammalian cells resistant tocytotoxic lectins, Int. J. Biochem. 10, 951-956 (]979). L. SI M INOVITCH, On the nature of hereditable variation in cultured somaticcells, Cell7, I-II (1976). M. B U C H W A L D and C. J. INGLES, Humandiploid fibroblast mutants with altered RNA polymerase II, Somat. Cell Genet. 2, 225-233 (1976). J . A . WRIGHT, Evidence for pleiotropic changes in lines of Chinese hamster cells resistant to concanavalin A and phytohemagglutinin-P, J. Cell Biol. 56, 660-675 (1973). B.A. KUZIK and J. A. WRIGHT, Hydroxyurea resistant mouse L cells with elevated levels of drug resistant ribonucleotide reductase activity, Biochem. Genet. lg, 311-331 (1980). S . E . KOROPATNICK and J. A. WRIGHT, Altered ribonucleotide reductas¢ activity in drug-resistant, mammalian ceils detected by an assay procedure for intact cells, Enzyme 25, 220-227 (1980). J. R. S T E E P E R and C. D. STEUART, A rapid assay for C D P reductase activity in mammalian cell extracts, Anal. Biochem. 34, 123-130 (1970). J . G . CORY, F. RUSSELL and M. M. MANSELL, A convenient assay for A D P reductas¢ activity using Dowex- I-borate columns, Anal. Biochem. 55, 449-456 (1973). Y.-C. YEH, A simple and sensitive assay procedure for ribonucleotide reductase system, Anal. Biochem. $6, 175-183 (1978).

126 30. 31. 32.

33.

34. 35.

36.

37. 38. 39. 40. 41. 42. 43.

44. 45. 46. 47. 48. 49.

50. 51.

52. 53.

JIM A. W R I G H T et al. W. D R E S L E R and R. STEIN, 0 b e t den Hydroxylharnstoff, Justus Liebig's Ann. Chem. Pharmacol. 150, 242- 252 (1869). B. BOLTON, D. K A U N G , R. L A W T O N and L. W O O D S , Hydroxyurea (NSC-32065): a phase I study, Cancer Chemother. Rep. 39, 47-51 (1964). D. J O H N S O N , L. R O D R I Q U E Z , P. HOLOYE and M. S A M U E L S , Combination vincristine (NSC-67574) and hydroxyurea (NSC-32065) for metastatic renal carcinoma, Cancer Chemother. Rep. 59, 1159-1160 (1975). G. R. GALE, L. M. ATKINS, S. J. M E I S C H E N and P. S C H W A R T Z , Scheduledependency assessments of ribonucleoside diphosphate reductase inhibitors when used in combination with platinum c o m p o u n d s plus cyclophosphamide in the treatment of advance LI210 leukemia, Cancer Treatment Rep. 63, 449-465 (1979). K . A . H O F F M A N and O. E H R H A R T , Einwirkung von Hydrazin a u f Dicyandiamid, Ber. Deutsch. Chem. Ges. 45, 2731-2740 (1912). R . W . B R O C K M A N , S. S H A D D I X . W. R. L A S T E R , JR. and F. M. SCHABEL, JR. Inhibition of ribonucleotide reductase, D N A synthesis and LI210 leukemia by guanazole. Cancer Res. 30, 2358-2368 (1970). W . H . LEWIS and 3. A. W R I G H T , Altered ribonucleotide reductase in mammalian tissue culture cells resistant to hydroxyurea. Biochem. Biophys. Res. Commun. 60, 926 933 (1974). S . J . J A C O B S and H. S. R O S E N K R A N Z , Detection of a reactive intermediate in the reaction between D N A and hydroxyurea, Cancer Res. 30, 1084-1094 (1970). J . L . C A M E R O N and J. R. J E E T E R , Action o f h y d r o x y u r e a a n d N-carbamoyioxyureaon the cell cycle of Tetrahymena. Cell Tissue Kinet. 6, 289-301 (1973). W . H . LEWIS and J. A. W R I G H T . Genetic characterization of hydroxyurea-resistance in Chinese hamster ovary cells, J. Cell. Physiol. 97, 73-86 (1978). W. H. LEWIS and J. A. W R I G H T , Ribonucleotide reductase from wild type and hydroxyurea-resistant Chinese hamster ovary cells, J. Cell. Phrsiol. 97, 87 9 8 (1978). J . A . W R I G H T a n d W. H. LEWIS, Evidence o f a c o m m o n site ofaction for t h e a n t i t u m o r agents, hydroxyurea and guanazole, J. Cell. Phrsiol. 83, 437-400 (1974). W. H. LEWIS and J. A. W R I G H T , Isolation of hydroxyurea-resistant C H O cells with altered levels of ribonucleotide reductase, Somat. Cell Genet. 5, 83-96 (1979). Y. E N G S T R O M , S. E R I K S S O N , L. T H E L A N D E R a n d M. A K E R M A N , Ribonucleotide reductase from calf thymus. Purification and properties. Biochemistry lg, 2941 2948 (1979). S. E R I K S S O N . L. T H E L A N D E R and M. A K E R M A N , Allosteric regulation of calf thymus ribonucleoside diphosphate reductase, Biochemistrr Ig, 2948-2952 (1979). B . A . KUZIK and J. A. W R I G H T , Characterization of ribonucleotide reductase activity from mouse L cells, Enzyme 24, 285 293 (1979). S. H O P P E R , Ribonucleotide reductase of rabbit bone marrow. I. Purification properties and separation into two protein fractions, J. Biol. Chem. 247, 113 121 (1972). S. H O P P E R , Ribonucleotide reductase of bone marrow, Methods in En:ymologr 51, 237-247 (1978). H. R. W A R N E R , Properties of ribonucleoside diphosphate reductase in nucleotidepermeable cells. J. Bacteriol. 115, 18-22 (1973). S. M U R P H R E E , E. C. M O O R E and P. T. BEALL, Regulation by nucleotides of the activity of partially purified ribonucleotide reductase from rat embryos, Cancer Res. 28, 8 6 0 863 (1968). C.-H. C H A N G and Y.-C. C H E N G , Effects of nucleoside triphosphates on human ribonucleotide reductase from Molt-4F cells, Cancer Res. 39, 5087 5092 (1979). E.C. M O O R E a n d R. B. H U R L B E R T , T h e r e g u l a t i o n o f m a m m a l i a n d e o x y r i b o n u c l e o t i d e biosynthesis by nucleotides as activators and inhibitors. J. Biol. Chem. 241, 4802-4809 (1966). C.-H. C H A N G and Y.-C. C H E N G , Substrate specificity of h u m a n ribonucleotide reductase from Molt-4F cells, Cancer Res. 39. 5081 5086 (1979). B. U L L M A N , L. J. G U D A S , S. M. C L I F T and D. W. M A R T I N , JR., Isolation and characterization of purine-nucleoside phosphorylase--deficient T-lymphoma cells and secondary mutants with altered ribonucleotide reductase: genetic model for immunodeficiency disease. Proc. Natl. Acad. Sci. U.S.A. 76, 1074-1078 (1979).

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54. 55. 56. 57. 58. 59.

60. 61. 62.

63.

64. 65. 66.

127

G. TYRSTED and V. G A M U L I N , Cytidine 5'-diphosphate reductase activity in phytohemagglutinin stimulated human lymphocytes, Nucleic Acids Res. 6, 305-319(1979). L. H A Y F L I C K , The limited in vitro lifetime of human diploid cell strains, Exptl. Cell Res. 37, 614-636 (1965). M. ABERCROMBIE, Contact inhibition and malignancy, Nature 281,259-262 (1979). S.J. GOSS, Gene mapping by cell fusion, Internat. Rev. o f Cytology 8, 127-169 (1978). R.G. HARDS, J. E. DICK and J. A. WRIGHT, Altered levels ofribonucleotide reductasc activity in drug resistant mammalian cell lines, Internat. Congress o f 8iochem. I !, 317A (1979). M. S I N E N S K Y , G. D U W E and F. P I N K E R T O N , Defective regulation of 3-hydroxy-3methylglutaryl coenzyme A reductase in a somatic cell mutant, J. Biol. Chem. 254, 4482-4486 (1979). R.T. SCHIMKE, R. J. K A U F M A N , F. W. ALTand R. F. KELLEMS, Geneamplification and drug resistance in cultured routine cells, Science 202, 1051 1055 (1978). G. M. WAHL, R. A. PADGETT and G. R. STARK, Gene amplification causes overproduction of the first three enzymes of U M P synthesis in N-(phosphonacetyl)-Laspartate-resistant hamster cells, J. Biol. Chem. 254, 8679-8689 (1979). R.T. S C H I M K E , P. C. BROWN, R. J. K A U F M A N and J. H. NUNBERG, Structure and localization of dihydrofolate reductase genes in methotrexate-resistant cultured cells, pp. 499-510 in Eucar~'otic Gene Regulation (R. AXEL, T. MANIATIS and C. F. FOX, eds.), Academic Press, New York (1979). P . W . MELERA, J. A. LEWIS, J. L. BIEDLER and C. HESSION, Antifolate-resistant Chinese hamster cells. Evidence for dihydrofolate reductase gene amplification among independently derived sublines overproducing different dihydrofolate reductases, J. Biol. Chem. 255, 7024-7028 (1980). J . A . WRIGHT, J. C. J A M I E S O N and H. CERI, Studies ofglycoprotein biosynthesis in concanavalin A-resistant cell lines. Defective formation of mannose-linked lipid intermediates, Exptl. Cell Res. 121, I-8 (1979). B. VAN'T RIET, G. L. W A M P L E R and H. L. ELFORD, Synthesis ofhydroxy-and amino substituted benzohydroxamic acids: inhibition of ribonucleotide reductase and antitumor activity, J. Med. Chem. 22, 589-592 (1979). R.G. H A R D S and J. A. WRIGHT, N-Carhamoyloxyurea resistant Chinese hamsterovary cells with elevated levels of ribonucleotide reductase activity, J. Cell. Physiol. (in press).