Functional Regulation of Mammalian Ribonucleotide Reductase

FUNCTIONAL REGULATION OF MAMMALTAN RIBONUCLEOTIDE REDUCTASE HOWARD L. ELFORD Department of Medicine and Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina INTRODUCTION MAMMALIAN cell replication and the concomitant synthesis of new DNA requires the active formation of deoxyribonucleotides. The rapid synthesis of deoxyribonucleotides is necessitated by the fact that the pool size of deoxyribonucleotides in mammalian cells is inadequate to support DNA synthesis for vigorous cell replication (1, 2). Of the various reactions leading to the formation of deoxyribonucleotides, the reduction of ribonucleotides to deoxyribonucleotides is believed to be a crucial and rate-limiting step. Ribonucleotide reduction systems have been extensively studied in Escherichia coli and Lactobacillus leichmannii (3,4). Much less information is known concerning mammalian ribonucleotide reductase. The reason for this is that cell replication rates are higher in bacteria than in normal mammalian tissues. Additionally, the auxotroph of L. Zeichmannii appears to be in a naturally depressed state for ribonucleotide reductase. Deoxyribonucleotides are formed by direct reduction of ribonucleotides. The general scheme for ribonucleotide reduction is :

base-ribose di- or triphosphate

nucleotide effector Mg+ + ribonucleotide reductase

I

I

I

I

Thiorefoxin

base-deoxyribose dior triphosphate

t

z

(SH), +

Thioredoxin Sz I

N;DP+

4

Thioredoxin reductase

NADPH 4 H+

In all systems studied it has been shown that the reduction of the four substrates is catalyzed by a single enzyme. A low molecular weight disulfhydryl protein, thioredoxin, in the reduced state

possible ribonucleotide

A.E.R.-B

19

20

HOWARD L. ELFORD

serves as the physiological hydrogen donor. Tile resultant oxidized thioredoxin is regenerated by reduction with N A D P H in conjunction with a second protein, the enzyme thioredoxin reductase. The activity of the ribonucleotide reductase is modified by an elegant and complex regulatory system involving ATP and deoxyribonucleotides which serve as both positive and negative allosteric effectors. This reaction seems to play an important role as a regulatory switch which when activated channels the normal utilization of ribonucleotides away from normal cellular processes to serve as precursors for D N A synthesis. The aim of this report is to present experimental evidence obtained in my laboratory which substantiates the hypothesis that ribonucleotide reductase is intimately associated with the rate of proliferation of mammalian cells and the malignant state. It is also my intention to provide some information concerning the relative rate of synthesis and turnover of the enzyme in rapidly proliferating normal and neoplastic tissues. Evidence will be provided to support the contention that the large increments in enzyme activity noted in a series of hepatomas and in early neonatal development are a consequence of increased rates of de novo enzyme synthesis and not enzyme activation.

MATERIALS

AND

METHODS

Animals The Morris hepatomas were obtained from Dr. H. P. Morris of Howard University sometime after implantation. The growth properties and the biology of the Morris hepatomas have been previously described (5). The Novikoff hepatoma was perpetuated in my laboratory by transplantation bilaterally into the femoral muscle or by subcutaneous implants into male Sprague Dawley rats. The animals were maintained in my laboratory until the tumors reached a standard size ( ~ 2 . 0 cm). The animals were killed by decapitation, and the tumors were dissected and cleaned of necrotic material. The neonatal studies were done on Sprague Dawley rats obtained in early pregnancy from Holtzman Rat Co. or in a few experiments on Sprague Dawley rats which were mated in the author's laboratory. The young rats were removed from their mothers 21 days after birth. All rats were aUowed to eat and drink ad libitum. The killings of all animals took place when possible at approximately the same time in order to minimize diurnal variations.

Enzyme Assays The preparation of crude and ultracentrifuged tumor and liver extracts as well as the assay for ribonucleotide reductase which involves monitoring the conversion of CDP to dCDP have been previously described (6).

CONTROL OF ANIMALRIBONUCLEOTIDEREDUCTASE

21

100,000 50,000

40,000 30,000 ,:,, 20,000 E

v

10,000 5,000

_~

4,000 3,000

'% 2,000

1,000 IT9#B.

500 300 200 .~r

i

-5040 3'0 2'0

ib

I

I

I

54 3

i

GROWTHRATEIN WEEKS

FIG. 1. Relationship between tumor growth rate and ribonucleotide reductase specific activity. Activity was measured in supernatants from 104,000 × g for 1 hr centrifugation of the various hepatoma and liver extracts. Data and assay conditions are from Fig. 1 in Elford et al. (6) by permission of the Editor of Journal of Biological Chemistry.

The subcellular fractionation of Novikoff tumor and 24-hr regenerating rat liver tissue was accomplished by the procedure outlined by Baril et al. (7, 8). RESULTS Ribonucleotide Reductase and Tumor Growth Rate In order to determine the relative importance of ribonucleotide reduction in the control of the rate of mammalian D N A synthesis and cell proliferation rates, ribonucleotide reductase activity was measured in soluble extracts of a series of rat hepatomas composed mainly of the Morris variety (6). Included in the study were thymidine kinase and thymidylate synthetase because these enzymes have been shown previously to be associated with increased mare-

22

HOWARD L. ELFORD

malian cell replication rates and to occupy a unique role because thymidylate has a special position in DNA synthesis. The relationship of ribonucleotide reductase activity with tumor growth rates, as measured by transplantation time, is shown in Fig. 1. An excellent correlation is found between enzyme activity and tumor growth rates in a series of hepatomas. Differences greater than 200-fold in enzyme specific activity exist between the very rapid hepatomas, Novikoff and 3683F, and the slow-growing tumors such as 7787. In addition, no enzyme activity could be detected in either normal rat liver or liver from a tumor-bearing animal. The assay was performed in such a manner that 1/500 of the activity of the fastgrowing tumor extracts or 1/1000 of a fetal (18-day) liver extract could have been detected. This represents one of the largest enhancements of any enzyme activity measured in these tumors. In contrast, the largest measurable differences in thymidylate synthetase and thymidine kinase activity between the fast-growing, poorly differentiated tumors and the slow-growing, weU-differentiated tumors were much less: only about 20-fold in the case of thymidylate synthetase and 50-fold in the case ofthymidine kinase. Several discrepancies were noted between the enzyme activity and tumor growth rate in a few of the tumors. The physiological reducing complex, thioredoxin-thioredoxin reductase, is not the rate-limiting factor accounting for the large differences in ribonucleotide reductase activity observed between the various hepatoma extracts. A lack of thioredoxin-thioredoxin reductase is also not responsible for the absence of detectable activity in adult rat liver. The evidence for this is twofold: firstly, the assays were conducted in the presence of a disulfhydryl compound, dithiothreitol, which would partially substitute for the natural hydrogen donor complex; secondly, it was possible to demonstrate that the thioredoxin-thioredoxin reductase complex is present at comparable levels in both tumor and normal adult liver (Table 1). This was accomplished by use of a purified Novikoff enzyme preparation. This ribonucleotide reductase is completely devoid of activity unless an auxiliary reducing system such as a chemical reducing agent, i.e. dithiothreitol, or the physiological reducing complex NADPH-thioredoxin-thioredoxin reductase is supplemented to the purified enzyme in the assay. Thus, the addition of a 70°-heated, 40-70~ ammonium sulfate fraction prepared in an identical manner from both Novikoff tumor and adult liver led to a comparable amount of ribonucleotide reductase activity. Treatments to remove nucleotides or other lower molecular weight substances by dialysis, Dowex and molecular gel chromatography or charcoal adsorption had no appreciable effect (< 20) on any of the tumor extracts. I interpret the results of this last series of procedures as evidence against the possibility that soluble nucleotides exerting negative allosteric effects on the ribonucleotide reductase are the reason for the large differences in activities

C O N T R O L OF A N I M A L R I B O N U C L E O T I D E R E D U C T A S E

23

TABLE ]. EVIDENCE FOR THE PRESENCE OF COMPARABLE LEVELS OF THIOREDOXIN--THIOREDOXIN REDUCTASE COMPLEX IN RAT LIVER AND TUMOR

Reducing system Dithiothreitol NADPH NADPH + Novikofftumor (NH4)2SO4 40-70 Novikoff tumor (NH4)2SO4 40-70 NADPH + rat liver (NH4)2SO4 40-70yo Rat liver (NH4)2SO4 40-70

Deoxyribonucleotide formed (m/zmoles) 19.4 0

6.8 0

7.7 0

The ribonucleotide reductase used for this experiment is a partially purified 0-40% (NH4)2SO4 fraction from Novikoff tumor extracts. The 40-70% (NH4)2SO4 fractions from normal rat liver and Novikoff tumor are preparations heated 2 min at 70°. 1.0 mg of rat liver and 1.3 mg of the Novikoff tumor 40-70~o (NH4)2SO4 fraction are added. Data from Elford et al. (6).

seen between the hepatomas and for the absence of enzymatic activity in liver. However, it is possible that a negative effector such as a deoxyribonucleotide is too tightly bound to the enzyme to be removed by the methods employed. Ribonucleotide Reductase and Development The profile of ribonucleotide reductase activity during neonatal development in several rat organs has been measured in my laboratory. The basis for this study is that we wanted to have background information about this crucial reaction in D N A synthesis during early development in order to possibly relate changes in ribonucleotide reductase levels with malignant transformation and stages of differentiation of the organ. It has been proposed that a block in differentiation may lead to neoplastic transformation. Potter (9) has recently reformulated this concept, that malignancy m a y arise from a block in differentiation or the reactivation of latent genes, in his "Oncology as Blocked Ontogeny" concept. Abundant evidence has appeared in the literature (10) that in m a n y hepatomas and other tumors there is an expression of isozymes and proteins resembling those of the fetal and newborn state. In addition, if our contention that one can equate ribonucleotide reductase activity with D N A synthesis and cell replication rates is valid, it then should be possible by measuring ribonucleotide reductase activity to see whether during a particular key stage in differentiation an intense period of cell replication ensues to achieve the differentiated state of the next plateau of maturation.

HOWARD L. ELFORD

24

::1 ~'°°°-~k Z LIJ I--

o Q.

d,

-

40,000 t_. ~ooo

o.

~o,ooo to~oo

i

I -20 0

I

40

t I~,

80

120

160

"

°

200

240

, JI,

280

~

"11 1

520 ll3WKS. 4WKS. 5WKSI. MATURE

POSTNATAL AGE IN HOURS

FIG. 2. Ribonucleotide reductase activity in developing liver. Activity was measured in supernatants prepared by centrifuging at 40,000 x g for 30 min. Assay conditions are described in Fig. 1 of reference (6). ~,800,000 I,S00,000 1,400,OO0

Z LIJ I--

o

t,2oo,ooo

t,000,000 aO0,O00

E O_

s00,ooo 400,O00 200,000

/ I

40

I

80

120

I$0

200

240

280

320

5WKS. 4WKS. 5WKS. MATURE

POSTNATAL AGE IN HOURS

FIG. 3. Ribonucleotide reductase activity in developing spleen. Conditions are the same as in Fig. 2.

CONTROL OF ANIMAL RIBONUCLEOTIDE REDUCTASE

25

The profile of ribonucleotide reductase activity in the developing liver is shown in Fig. 2. There is a considerable change in enzyme activity within a relatively short time span of three weeks. The fetal liver extract has a very high level of enzyme activity, about two-fold higher than the fast-growing, poorly differentiated hepatomas such as Novikoff and 3683F. At the time of birth, there is a sharp decrease in activity and then progressive decreases in activity until at one week after birth there is only a very small amount of activity remaining, 1/20 of the newborn liver activity and only 1/200 of the 18-19-day fetal liver extract activity. By 3 to 4 weeks after birth there is no activity discernible, although 1/1000 of fetal liver activity could have been detected. In contrast, the developing spleen has a much different chronological profile of ribonucleotide reductase activity which is shown in Fig. 3. Enzyme activity is low at birth and then dramatically increases, reaching a peak in activity approximately six days after birth. This peak in activity is about fivefold higher than in any other rat tissue tested, including Novikoff hepatomas. This maximum activity occurs at a time when the onset of white cell production is initiated in the rat spleen; therefore, a higher mitotic activity is occurring. This burst in lymphocyte production may be a response to the recent exposure of the newborn to antigens of the extra-maternal environment. Activity then declines rather abruptly until by three weeks after birth there is less than 15~ of the peak activity. Activity continues to decline until in the adult spleen there is only about 1/50 of the maximum activity seen during the neonatal development. The pattern of ribonucleotide reductase activity in the developing thymus, another lymphopoietic organ, is similar to that just described for the spleen (Fig. 4). Enzyme activity is very low at the time of birth and then rapidly increases, although not as steeply as in the case of the spleen. The maximum peak of activity in the thymus is much broader than in the spleen and is not quite as high in specific activity. This maximum activity occurs at about the time that the colonization of the lymphoid system by the thymus and the ability to synthesize a new pattern of immunoglobulins or the so-called delayed hypersensitivity are thought to occur. Activity then declines, although not as abruptly as with the spleen, and significant activity is retained throughout maturity. De novo Enzyme Synthesis Although ribonucleotide reduction is under stringent allosteric control by ATP and deoxyribonucleotides, it is my contention that the large differences of ribonucleotide reductase activity seen between the tumors of different growth rates and the large fluctuations of enzyme activity observed in the early stages of neonatal development of several rat organs are a consequence

26

HOWARD L. ELFORD

30~000 250~00

z I.d I--200,000 0 no.. c~ 150,000 E =E o..

I00,000

50,000

40

80

120

160

200

240

280

320

5WKS 4WK$. 5WK$.

MATURE

POSTNATAL AGE IN HOURS

FIo. 4. Ribonucleotide reductase activity in developing t h y m u s . C o n d i t i o n s are t h e s a m e as in Fig. 2.

of varying rates of de novo enzyme synthesis and degradation and not the result of enzyme activation mediated by aUosteric agents. To test this hypothesis, a protein synthesis inhibitor, cycloheximide, was injected (i.p.) into a neonatal rat at a time when large increases in ribonucleotide reductase activity are occurring in the developing spleen and thymus. The effect of cycloheximide on ribonucleotide reductase activity in a typical experiment is shown in Fig. 5. In this particular experiment, cycloheximide was injected (i.p.) into neonatal rats at 55 hr after birth; the rats were then sacrificed 24 hr later at 79 hr after birth. In the second part of the experiment, cycloheximide was injected at both 55 and 79 hr after birth; again the rats were sacrificed 24 hr later. The specific activities of the extracts prepared after these two treatments were compared to spleen extracts of control animals at the same time intervals. As is shown in Fig. 5, there is not only a prevention of increased enzyme activity expected with increase in age of the animal, at this stage in development for these organs, but the level of activity diminishes below the level found at the time of the cycloheximide treatment. In comparable experiments with developing thymus, the effect of cycloheximide was also determined (Fig. 6). As in the previously described experiments with spleen, cycloheximide prevented the expected increases in enzyme activity and the enzyme level fell to values below the amount observed at the time of the

CONTROLOF ANIMALRIBONUCLEOTIDEREDUCTASE 800

~J

SPLEEN

700

xZ

~

V///A YIIII~

c~ CYCL(~EXlMIDE

600

W//A I

I).uecT~AT

o~500

IP'///A / ~//~ 1

CY~E~MI~ INJECTEDAT

n.(1. E 400

27

300

20O I00

55

79 103 79 103 AGE IN HOURS AT TIME OF SACRIFICE

FIG. 5. Effect of cycloheximide on ribonucleotide reductase activity in the developing spleen. Cycloheximide (2.5/~g) was injected intraperitoneally 24 hr prior to decapitation. Saline was injected (i.p.) into control rats 24 hr prior to killing. Preparation and assay of extracts are outlined in Fig. 2.

800 TO0 :"

THYMUS

I ~ V/lllA

600

I

W///A ~//,/~

- 1

~/,/~

~(3f3

0 ----fiE4 0 0

I

--

CYCLOHEXIMIDE

N I JECTED AT 55AND79HOURS

N N~/~

zoo

1

F~//~J

CONTROL CYCLOHEXIMIDE [INJECTEDAT 55 .ouRs

~

0 55

0

~

79 103 79 103 AGE IN HOURS AT TIMEOF SACRIFICE

Fio. 6. Effect of cycloheximide on ribonucleotide reductase activity in the developing thymus. Injection and experimental conditions are the same as in Fig. 5. cycloheximide injection. When actinomycin D, a DNA-directed R N A synthesis inhibitor, was tested similar results to those found with cycloheximide were observed (Fig. 7). It is possible to detect a decrease in enzyme activity as early as 90 min after injection o f cycloheximide (Fig. 8). For this particular experiment the decrease in ribonucleotide reductase activity at 90 min was not significant with actinomycin D, but a similar decrease as is found with cycloheximide was seen at 3 hr.

28

HOWARD L. ELFORD

40/SPLEEN

[•]CONTROL /

ACTINOMYCIND ~TREATED AT

~24 I-

Q_ O

8

102 102 78 AGE IN HOURS AT TIME OF SACRIFICE

FIQ. 7. Effect of actinomycin D on ribonucleotide reductase activity in the developing spleen. Actinomycin D (50/~g) was injected intraperitoneally 24 hr prior to sacrifice. Control animals sacrificed at 102 hr after birth received saline injection (i.p.) at 78 hr of age. Preparation of extract and assay conditions are the same as in Fig. 2.

These findings on the effect of cycloheximide and actinomycin D in the developing spleen and thymus support the contentions that de novo enzyme synthesis is required for increased enzymatic activity, there is a rapid turnover of the enzyme and the messenger for ribonucleotide reductase has a short half-life.

,<-

0

75 --

5 DAY NEONATAL RAT SPLEEN

D

x E :E o.

60

~ 45 II.-

3o

u 3

15

a W UNTREATED

CYCLOHEXIMIDE I ~ hours

CYCLOHEX IMIDE 3 hours

W

ACTINOMYCIN D :3houri

FIG. 8. Effect of short term treatment of cycloheximide and actinomycin D in the developing spleen. Cycloheximide (2.5/~g) was injected intraperitoneally 1.5 hr and 3 hr prior to sacrifice. Actinomycin D (2.0 t~g) was administered (i.p.) 3 hr prior to decapitation. All other conditions are described in Figs. 2 and 5.

29

C O N T R O L OF A N I M A L R I B O N U C L E O T 1 D E R E D U C T A S E I-U l-X

w I00 0 I--

w 75 I-Z

~,

5o

I> I'--

'~ 2 5 Z

W U W Q.

untreoted

I

2 4 8 16 HOURS AFTER TREATMENT

24

Effect of cycloheximide on ribonucleotide reductase activity in Novikoff tumor. Cycloheximide (1.5 mg/kg body wt) was injected intraperitoneally. Assays of activity were performed on supematants from centrifugation at 40,000 × g f o r 1 hr. Experimental conditions and assay are described in reference (6). The values shown are averages from several experiments. FIG. 9.

To obtain information about the rate of de novo enzyme synthesis and enzyme turnover in neoplastic tissue in order to be able to compare it with normal rapid cell proliferation, a series of experiments was performed on a rapidly growing hepatoma, Novikoff, utilizing cycloheximide and actinomycin D. The results of a single i.p. injection of cycloheximide into a Novikoff tumor-bearing animal at a time of rapid tumor growth are shown in Fig. 9. In a time span of only 1 hr, there is a sharp decline in enzyme activity, reaching a minimum at 2 hr in soluble extracts (40,000 × g for 30 min) of the tumor as compared with the uninjected tumor extract. The activity remains less than 40% of the uninjected tumor extract until 8 hr after the cycloheximide injection, when a significant upturn in enzyme activity is noted. There is a continued increase in activity until by 24 hr after the cycloheximide injection activity has returned to approximately the uninjected control level. In contrast to the finding that actinomycin D prevented an increase and also led to a decrease in reductase activity in early development in the spleen and thymus, actinomycin D had no appreciable effect on ribonucleotide

30

HOWARD

RIBONUCLEOTIDE REDUCTASE ACTIVITY

h

L. ELFORD

URIDINE-3H INCORPORATION INTO RNA

,oo

v

> 0

z a w I'--

80

h,

a: 6 0 I.-Z 2~ I.Z

40

U

~. 2O

UNTREATED

(+) ACTINOMYCIN D

UNTREATED

(+)ACTINOMYCIN D

FIG. 10. Effect of actinomycin D on ribonucleotide reductase activity in Novikoff tumor. Actinomycin D was injected intraperitoneally in two dosages of 200/~g each 3 hr apart. The tumor-bearing animal was killed 7 hr after the first injection of actinomycin D. Uridine-3H (75 t~c) was injected 1 hr prior to sacrifice. Assay procedure and extract preparation as outlined in Fig. 2 and reference (6). reductase levels in Novikoff tumor extracts even when injected in two doses of 200/zg each over a 7-hr period (Fig. 10). It does not appear that the decrease in enzymatic activity obtained with cycloheximide is a consequence of the production of some inhibitory substance because when the cycloheximide-treated extract is mixed with the uninjected extract in the assay the total activity for the mixture is the sum of the activities of each assayed separately (Table 2).

Evidence for Natural Inhibitor in Liver Although the preceding portion of this paper has been directed toward establishing that enzyme synthesis and not enzyme activation is the principal mechanism to achieve large increments of ribonucleotide reductase activity in rapid cell proliferation, some preliminary experiments in my laboratory suggest that there may be a natural material in adult rat liver that inhibits ribonucleotide reductase. The inclusion of supernatant fractions of rat liver obtained by centrifugation results in a significant inhibition of activity on a partially purified Novikoff tumor ribonucleotide reductase (Table 3). This factor is distinct from nucleotides or other low molecular weight substances because dialysis, charcoal treatment or molecular gel chromatography had no

CONTROL OF ANIMAL RIBONUCLEOTIDE REDUCTASE TABLE 2. EVIDENCE AGAINST INHIBITOR PRODUCTION BY CYCLOHEXIMIDE TREATMENT

Extract

Deoxynucleotide formed (cpm)

Untreated Novikoff CXM treated Novikoff Untreated Novikoff + CXM treated Novikoff Untreated neonatal spleen CXM treated neonatal spleen Untreated neonatal spleen + CXM treated neonatal spleen

7,225 2,775 9,980 173,500 83,500 286,000

The assay was performed as reported in Elford et aL (6) except that specific activity of the CDP is one-third as high and incubation time was 40 min. In all instances, 0.5 mg of protein extract was added. Cycloheximide = CXM (350/~g) was injected intraperitoneally (i.p.) into Novikoff tumor-bearing rats 6 hr prior to decapitation. CXM (5/zg) was administered i.p. 4 hr prior to decapitation to 6-day-old rats. Untreated animals were injected (i.p.) with equivalent vol. of 0.9 ~o NaC1.

TABLE 3. EVIDENCE FOR A NATURAL INHIBITOR OF RIBONUCLEOTIDE REDUCTASE IN LIVER

Enzyme

Rat liver extract addition

Purified Novikoff reductase Crude Crude charcoal treated Crude dialyzed Crude heated 60 ° Crude heated 100 ° 32-hr regenerating liver Newborn 3-week-old Fetal

Activity ~o of control 100 30 35 35 35 80 55 50 35 >100

The ribonucleotide reductase used for this experiment is a partially purified 0--40yo (NH4)2SO,, fraction from Novikoff tumor extracts. All rat liver extracts were prepared by centrifugation at 40,000 x g for 30 min. Dialyzed extract was dialyzed against 0.025 M phosphate buffer, pH 7.4 for 16 hr. Heat treatments were for 2 rain, followed by centrifugation. The assay procedure is as described in reference (6).

31

32

H O W A R D L. E L F O R D

effect in decreasing the inhibitory effect. The inhibitory substance is destroyed by heating at 100° for 2 min but not by heating at 60 ° for 2 min. The amount of inhibitory materialin the liver appears to be a function of the physiological state of the liver because there is less inhibition by extracts of 24-hr and 32-hr regenerating liver and early neonatal liver. Also fetal live extracts are not inhibitory and yield additional activity. On the other hand, there is appreciable inhibitory substance in 5-hr regenerating liver and in 3-week-old liver extracts. These results are only preliminary and it remains to be determined whether the inhibitory material in liver is not destroying the substrate or product of the reaction or affecting the reaction in some other indirect way. Subcellular Localization o f Ribonucleotide Reductase

Close scrutiny of the subceilular localization of ribonucleotide reductase in mammalian ceils was initiated by the reports of Baril et aL (7, 8) that there is a D N A polymerase associated with smooth membrane elements from rat liver and hepatoma cytoplasm. The subcellular fractionation procedure of Baril et al. (7, 8) was followed which employs differential centrifugation and sucrose density gradient separation techniques to yield preparations of isolated nuclei, microsomes, mitochondria, ribosomes, smooth membrane elements of the cytoplasm and a soluble fraction (S-4) obtained after centrifugation at 78,000 × g for 15 hr of the post-microsomal supernatant from Novikoff hepatoma and 24-hr regenerating rat liver cells. The survey of ribonucleotide reductase activity in the various subceilular fractions of Novikoff tumor revealed (Table 4) that activity is found only in the soluble TABLE4. INTRACELLULAR LOCALIZATION OF RIBONUCLEOTLDE REDUCTASE ACTIVITY

Fraction 600 × g supt. (S-l) Nuclear extract (P-l) Post-mitochondrial supt. (S-2) Mitochondrial extract (P-2) Post-microsomal supt. (S-3) Microsomal extract (P-3) Centrifugation of S-3

Supernatant (S-4) Pellet (P-4)

Total units

Specificactivity (units/mg)

2055 0 3270 0 3190 0

2.21 0 3.65 0

1950 1070

5.21 10.53

5.44 0

The subcellular fractions were obtained by the procedure as outlined by Baril et al. (7, 8). The assay followed the procedure of Elford et al. (6). A unit of activity is the formation of 10,000cpm of deoxynucleotide in 1 hr.

CONTROL OF ANIMAL RIBONUCLEOTIDE REDUCTASE

33

fraction (S-4) obtained after 15 hr of centrifugation of the post-microsomal supernatant and in the pellet (P-4) also obtained by this extended centrifugation. The principal activity is associated with the pellet because the specific activity of the pellet is twice that of the soluble fraction. It also indicates that the activity found in the cell sap probably originated in the pellet material. No activity was discerned in either the soluble (S-4) or the pellet (P-4) fraction obtained by subcellular fractionation of normal rat liver. The pellet material does not absorb ribonucleotide reductase activity because the mixing of an active 24-hr regenerating rat liver S-4 supernatant and inactive P-4 pellet material of normal rat liver and subsequent re-isolation of the two fractions do not lead to any activity in the pellet (P-4) fraction. The pellet fraction (P-4) of Novikoff hepatoma was further purified by a discontinuous sucrose density gradient centrifugation by the method of Murray e t a L (11) as modified by Baril e t al. (7, 8) which yields free ribosomes, membrane fractions and a soluble fraction. Over 95~o of the activity is found associated with the membrane fractions and the remainder with the ribosome pellets at the bottom of the tube (Table 5). TABLE 5. SUCROSE DENSITY GRADIENT SUBFRACTIONATION

OFP-4 Sucrose density subfraction of P-4 Soluble (supematant) Membranes Free ribosomes

Specific activity (units/rag) 0

5.50 0.17

The discontinuous sucrose density gradient (0.8-2.0M) sucrose-Tris buffer (0.05 MTris-HCl, pH 7.5, 0.025KC1,0.005M MgC12, 0.001 M mercaptoethanol) separation of P-4 was done according to the procedure of Murray et al. (11) as adapted by Baril et al. (7, 8). Centrifugation of gradient was for 24 hr at 30,000 rpm (Spinco rotor). The membrane fraction banded at the interface of the 0.8-1.3 M sucrose layers and ribosomes are in a pellet at the bottom. A unit of activity is 10,000cpm of dCDP formed in 1 hr. The small amount of activity found with the free ribosomes is attributed to a small amount of contamination by membrane material adhering to the ribosomes. Subcellular fractionation of 24-hr regenerating liver yields identical results but with much lower specific activities in the membrane (P-4) and cell sap (S-4). We have also found a similar pattern for thymidine kinase, the membrane fraction having the highest specific activity.

34

HOWARD L. ELFORD

DISCUSSION

The contention that ribonucleotide reduction is a crucial and rate-controlling step in DNA synthesis and cell division and is closely correlated with mammalian cell proliferation rates is supported by the evidence reported in this paper. Whether increased cell replication is a primary, secondary or even a tertiary event of the neoplastic transformation process, it is an obligatory event for virulent malignancy and a usual consequence of the neoplastic state. This increase in cell replication rates results in a large enhancement of activities of several enzymes concerned with DNA synthesis. There appears to be an activation of genes normally in a depressed state in the differentiated nondividing cell. There is a strong possibility that several of these DNA synthetic enzymes are co-ordinately regulated and have a temporal relationship. This increase in activity can result from an increase in enzyme synthesis, decreased catabolism or enzyme activation. The dramatic increase of ribonucleotide reductase activity seen in fastgrowing hepatomas represents the most striking increase in enzyme activity observed in these hepatomas, including other enzymes of DNA synthesis and glycolysis. The high degree of correlation of ribonucleotide reductase with tumor growth rates in a series of hepatomas is an additional piece of evidence in supporf, of the "molecular correlation concept" expressed by Weber (12, 13) that predicts that certain key enzymes and metabolic pathways are correlated with tumor growth rate. He further proposes that the resulting metabolic imbalance caused by neoplastic transformation will reveal a meaningful biochemical pattern. The elucidation of the mechanism by which ribonucleotide reductase achieves its increase in activity is important because of the apparent pivotal and rate-limiting role ribonucleotide reductase plays in DNA synthesis. I envision the allosteric control of the reaction exerted by the nucleotide effectors, ATP and deoxyribonucleotide triphosphates to play an important role in the fine tuning of the reaction--that is, maintaining a balanced supply of deoxyribonucleotides and preventing overproduction of any single deoxyribonucleotides. However, to meet the challenge of supplying adequate quantities of deoxyribonucleotides for DNA synthesis in rapid cell proliferation, additional quantities of enzyme must be synthesized. Enzyme synthesis and not enzyme activation plays the dominant role. This is supported by experimental evidence discussed earlier that showed that a protein synthesis inhibitor prevented expected increases in enzyme activity, seen during the neonatal development of spleen and thymus in the rat, and also caused a marked decrease in activity in a neoplastic tissue that has a high capacity for ribonucleotide reduction.

CONTROL OF ANIMAL RIBONUCLEOTIDE REDUCTASE

35

The decrease of approximately 50~o of ribonucleotide reductase activity in Novikoff tumor extract within 1 hr after cycloheximide administration as well as an apparent half-life of less than 2 hr in the developing spleen and thymus represents one of the shortest half-lives measured in mammalian tissue (14). Actinomycin D, a DNA-directed RNA synthesis inhibitor, has the same effect on ribonucleotide reductase activity in the neonatal development situation but has no effect on the tumor synthesis of enzyme. This might imply that neoplastic transformation confers increased stability to the RNA messenger for ribonucleotide reductase. Pitot (15) has advanced the theory of altered template stability as an explanation for the defective mechanisms regulating the control of genetic expression in neoplastic tissue. A selective growth advantage results in those cells whose templates for enzymes important to DNA synthesis are more stable than the normal cells. Additional evidence for the rapid turnover of ribonucleotide reductase has been noted by Turner et al. (16) who found that ribonucleotide reductase activity detected only in the S-phase in synchronized cultures of mouse L-cells decreased rapidly with a half-life of 2 hr when cycloheximide was added to the cultures. Also, cycloheximide and actinomycin D prevent an increase in ribonucleotide reductase activity as Chinese hamster fibroblast cells enter the S-phase (17). Furthermore, King and van Lancker (18)showed that x-irradiation and actinomycin D inhibit increases in ribonucleotide reductase in regenerating liver. The repressor, if one exists, for ribonucleotide reductase remains to be elucidated. It may be one of the deoxyribonucleotide triphosphates, especially dTTP, as is suggested by the bacterial and tissue culture data (19), DNA, or some component of the mitotic apparatus. Increase in ribonucleotide reductase upon liver regeneration has already been reported by King and van Lancker (18) and Larsson (20). However, I would like to briefly comment on some of our data (21) in order that a comparison in ribonucleotide reductase activities can be drawn between controlled rapid cell replication as exemplified by regenerating liver and malignant cell proliferation illustrated by the fast-growing hepatomas. We find that the peak in specific activity occurs between 32 and 40 hr after hepatectomy and that this maximum activity (15,000-20,000 cpm/mg/hr) in regenerating liver is much lower than the specific activity seen in the more rapidly growing tumors and falls in the range of moderately growing, welldifferentiated hepatomas. It has been noted a number of times that normal liver may undergo rapid cell proliferation without the concomitant severe imbalance in metabolic pathways observed in malignant transformation. The regulatory mechanism to halt cell replication at a prescribed point in time still is maintained. The enzyme profile in the developing liver fits the postulated enzyme pattern that Potter (9) envisions for crucial enzymes involved in DNA synthesis and

36

HOWARDL. ELFORD

cell division in the neoplastic cell. In the neoplastic state the cell expression would resemble some early stage in development and becomes "locked in" at this immature state, preventing it from differentiating into a mature cell that no longer divides. The question whether this change occurs during differentiation or involves the reaction of repressed genes is problematical. The enzyme pattern observed in the developing spleen and thymus reflects the functional development of the organs, that of cellular lymphocyte production, and not only the structural development of the organ. The data presented here that ribonucleotide reductase as well as thymidine kinase is associated with a smooth membrane organelle in proliferating cells, in conjunction with the finding of Baril et aL (7, 8) that there is a DNA polymerase associated with this smooth membrane fraction from hepatomas and rat liver cytoplasm, suggest that there may exist a complex of several DNA synthetic enzymes in a functional unit. SUMMARY Evidence is presented to substantiate the contention that the reduction of ribonucleotides to deoxyribonucleotides is a critical and rate-controlling step in the pathway leading to DNA synthesis and cell replication. One main piece of evidence is the demonstration of a close correlation between ribonucleotide reductase and tumor growth rates in a spectrum of rat hepatomas of varying growth rates. The physiological hydrogen donor for this reaction, thioredoxin-thioredoxin reductase, is found at comparable levels in both fast-growing tumor and normal liver. The modulation of ribonucleotide reductase activity was also examined during the structural and functional development of several rat organs. The various organs have different profiles of activity which are probably related to functional differentiation. For example, during liver development, activity is at a high level in the late fetal stage, but drops sharply at birth and becomes non-detectable in 3-4 weeks, while in the developing spleen and thymus, activity is low at birth and reaches a maximum about one week after birth and then declines. However, activity is still maintained in the adult state. It is suggested that heightened activity may be associated with an intense period of cellular replication related to the process of differentiation. The dramatic fluctuations in ribonucleotide reductase activity observed in tumors and during neonatal development are shown to be due to de novo enzyme synthesis and not to enzyme activation. A protein synthesis inhibitor, cycloheximide, prevents increases in enzyme activity in developing organs and causes a rapid decrease in enzyme activity in Novikoff hepatoma. The inhibition of DNA-directed RNA synthesis by actinomycin D also prevents increases in enzymatic activity in developing organs but has no effect on Novikoff tumor activity.

CONTROL OF ANIMAL RIBONUCLEOTIDE REDUCTASE

37

Subcellular f r a c t i o n a t i o n o f N o v i k o f f h e p a t o m a a n d 24-hr r e g e n e r a t i n g liver reveals t h a t r i b o n u c l e o t i d e r e d u c t a s e ' is a s s o c i a t e d with a s m o o t h m e m b r a n e c o m p o n e n t o f the c y t o p l a s m . T h y m i d i n e kinase activity is also f o u n d with this subceUular m e m b r a n e fraction. These data, in c o n j u n c t i o n with d a t a o f Baril et aL (7, 8) t h a t a D N A p o l y m e r a s e is a s s o c i a t e d with this s m o o t h m e m b r a n e organelle, suggest t h a t there m a y exist a c o m p l e x o f several D N A synthetic enzymes in a f u n c t i o n a l unit.

ACKNOWLEDGEMENTS T h e a u t h o r w o u l d like to express his a p p r e c i a t i o n to his colleague, D r . E a r l Baril, for his c o o p e r a t i o n o n the subcellular f r a c t i o n a t i o n w o r k in this r e p o r t a n d his critical review o f this m a n u s c r i p t . The excellent technical assistance o f Miss S h a r o n H a r p e r a n d M i c h a e l Freese is gratefully a c k n o w ledged. A l s o the a u t h o r w o u l d like to state his g r a t i t u d e to D r . H a r o l d M o r r i s for p r o v i d i n g us with the " M o r r i s " h e p a t o m a s . The assistance o f Mrs. R o b e r t a E l f o r d a n d Miss Bonnie H i n k l e in the p r e p a r a t i o n o f this m a n u s c r i p t is greatly a p p r e c i a t e d . T h e research was s u p p o r t e d b y grants f r o m the A m e r i c a n C a n c e r Society (P-407) a n d U n i t e d States Public H e a l t h Service (CA-11978).

REFERENCES 1. R. M. BEHKI and W. C. SCHNEIDER,Intracellular distribution of deoxyribosidic compounds in normal and regenerating liver and in Novikoff hepatoma, Biochim. Biophys. Acta 61, 663-667 (1962). 2. B. A. NORDENSKJrLD,L. SKeet, N. C. BROWNand P. REICHARD,Deoxyribonucleotide pools and deoxyribonucleic acid synthesis in cultured mouse embryo cells, J. Biol. Chem. 245, 5360-5368 (1970). 3. P. REICHARD, The biosynthesis of deoxyribonucleotides, European J. Biochem. 3, 259-266 (1968). 4. A. LARSSONand P. REICHARD,Enzymatic reduction of ribonucleotides, pp. 303-347 in Progress in Nucleic Acid Research 7 (J. N. DAVlDSONand W. E. COHN, eds.), Academic Press, New York (1967). 5. H. P. MORRISand B. P. WAGNER,Induction and transplantation of rat hepatomas with different growth rate (including "minimal deviation" hepatomas), pp. 125-152 in Methods in Cancer Research 4 (H. BUSCH, ed.), Academic Press, New York (1967). 6. H. L. ELFORD,M. FREESE,E. PASSAMANIand H. P. MORRIS,Ribonucleotide reductase and cell proliferation I. Variations of ribonucleotide reductase activity with tumor growth rate in a series of rat hepatomas, J. BIoL Chem. 245, 5228-5233 (1970). 7. E. F. BARIL,M. D. JENKINS,O. E. BROWNand J. LASZLO,DNA polymerase activities associated with smooth membranes and ribosomes from rat liver and hepatoma cytoplasm, Science, 169, 87-89 (1970). 8. E. F. BARIL, O. E. BROWN, M. D. JENIONS and J. LASZLO,Deoxyribonucleic acid polymerase associated with rat liver ribosomes and smooth membranes. Purification and properties of the enzyme, Biochemistry, 10, 1981-1992 (1971). 9. V. R. POTTER, Recent advances in cancer biochemistry" The importance of studies on fetal tissue, Canadian Cancer Conference 8, 9-30 (1969). 10. Isozymes and enzyme regulation in cancer, Gann Monograph (1972), in press.

38

HOWARD L. ELFORD

11. R.K. MURRAY,R. SUSSand H. C. PITOT)Isolation and characterization of cytoplasmic components of cancer cells, pp. 239-286 in Methods in Cancer Research 2 (H. BUSCH, ed.), Academic Press, New York (1967). 12. G. WEBER, The molecular correlation concept: Studies on the metabolic pattern of hepatomas, Gann Monograph 1, 151-178 (1966). 13. G. WEBERand M. A. LEA) The molecular correlation concept: An experimental and conceptual method in cancer research, pp. 523-578 in Methods in Cancer Research 2 (H. BUSCH,ed.), Academic Press, New York (1967). 14. R. T. SCH~KE and D. DOYLE, Control of enzyme levels in animal tissues, Ann. Rev. Biochem. 39, 929-976 (1970). 15. H. C. PITOT,Altered template stability: The molecular mask of malignance? Perspectives Biol. Med. 8, 50-70 (1964). 16. M. K. TURNER, R. ABRAMSand I. LIEBERMAN,Levels of ribonucleotide reductase activity during the division cycle of the L cell, J. Biol. Chem. 243, 3725-3728 (1968). 17. S. MURPHREE,E. STUBBLEHELDand E. C. MOORE, Synchronized mammalian cell cultures. HI. Variation of ribonucleotide reductase activity during the replication cycle of Chinese hamster fibroblasts, Exptl. Cell. Res. 58, 118-124 (1969). 18. C. D. K ~ o and J. L. VANLANCKER,Molecular mechanisms of liver regeneration. VII. Conversion of cytidine to deoxycytidine in rat regenerating liver, Arch. Biochem. Biophys. 129, 603-608 (1969). 19. R. L. BLArnEYand E. VITOL,The control of nucleotide biosynthesis, Ann. Rev. Biochem. 37, 201-224 (1968). 20. A. LARSSON,Ribonucleotidereductase from regenerating rat liver, EuropeanJ. Biochem. 11, 113-121 (1969). 21. H. L. ELFORD, Mammalian ribonucleotide reductase and cell proliferation, Gann Monograph (1972), in press.