Effect of thymidine on deoxyribonucleic acid synthesis and cytidine metabolism in rat-thymus cells

44

BIOCHIM1CA ET BIOPHYSICA ACTA

BE,A 95308

E F F E C T OF T H Y M I D I N E ON DEOXYRIBONUCLEIC ACID SYNTHESIS AND CYTIDINE METABOLISM IN RAT-THYMUS CELLS

E L I Z A B E T H D. W H I T T L E

Department o] Radiotkerapeutics, University o] Cambridge, Cambridge (Great Britain) (Received April 28th, 1965)

SUMMARY

I. The effect of thymidine on 3zp incorporation into the deoxyribonucleic acid of rat-thymus cells was inhibitory under the incubation conditions used: about a 50 % inhibition was produced b y 0.08 mM thymidine, which was completely prevented b y 0.04 mM deoxycytidine and partially prevented by o.ooi mM deoxycytidine. 2. Examination of the specific activities of the four constituent deoxyribonucleoside 5'-monophosphates of deoxyribonucleic acid, and of the total amount and specific activity of the labile phosphate of the acid-soluble nucleotide fraction, indicated that the changes in sap incorporation into deoxyribonucleic acid represented true changes in deoxyribonucleic acid synthesis. This was further confirmed by using another precursor, E8-14C]adenine. 3. The incorporation of [2-14C]cytidine into deoxyribonucleic acid-deoxycytidylate was found to be more sensitive to inhibition by thymidine than that of 32p or I8-14C]adenine into deoxyribonucleic acid. This result is consistent with the primary site of inhibition by thyrnidine for these cells being at the reductive step in which deoxycytidylate is formed. 4. The results on incorporation of isotopes into ribonucleic acid have suggested a further, though less sensitive, inhibitory effect of thyrnidine on cytidine metabolism, namely, on the formation of cytidine triphosphate from cytidine. 5. Degradation of thymidine by these cells was negligible.

INTRODUCTION

Results of studies carried out in recent years have suggested three possible effects that thymidine may have on DNA synthesis in whole cells. A relationship has been found to exist between the activities of the kinases which phosphorylate thymidine to T T P and the proliferative state of the tissuO -4. TMP kinase has been shown to be stabilized by its substrate and, to a lesser degree, by thymidine 5,6.

Biochim. Biophys. Acta, 114 (1966) 44-60

THYMIDINE AND NUCLEIC ACID METABOLISM

45

These results might suggest that DNA synthesis would be stimulated by thymidine. HIATT AND B O I A R S K I ~ have shown some stimulation of 14C incorporation from [14C]orotic acid into the DNA of liver slices from thymidine-infused rats. Also, GREULICH et al. 8 reported an increased mitotic activity in the duodenal epithelium of the mouse following subcutaneous injection of a small dose of thymidine. On the other hand, REICHARD et al. 9 using a chick-embryo extract showed that T T P inhibited deoxycytidylate formation from labelled CMP and suggested that, if this inhibitory-feedback mechanism were to operate in whole cells, then an increase in the intracellular level of T T P might be expected to lead to an inhibition of DNA synthesis due to lack of deoxycytidylate. Inhibition of cellular growth by thymidine has been shown to occur for several types of cultured mammalian cells 1°-~4. MORRIS et al. 15 have further shown that, for a murine neoplastic cell line, inhibition of growth by thymidine was correlated with an inhibition of formation of deoxycytidine phosphates from [SH]uridine, and indeed the latter was apparent long before inhibition of growth. Lastly, results have been reported in which thymidine supplied even in high (i.e. 2-5 mM) concentrations to cells appeared to have no effect on incorporation of labelled precursors into DNA 1e-is. In view of these different possibilities, a study has been made on the effect of thymidine on nucleic acid metabolism in rat-thymus cells. The effect on DNA synthesis was found to be inhibitory, and is discussed in relation to the possibility of thymidylate being a normal controlling factor in DNA biosynthesis. In addition, evidence is presented suggesting an inhibitory effect of thymidine on the utilization of cytidine for CTP formation. Preliminary accounts of this work have been publishedlg,~0. MATERIALS

F~sP]PI in isotonic solution (pH 7) was obtained from the Radiochemical Centre, Amersham, [8-1~C]adenine (22. 9 mC/mmole) from the California Corporation for Biochemical Research, and [2-14C]cytidine (21.4mC/mmole) from Schwarz BioResearch, Inc. Thymidine was purchased from the Sigma Chemical Co., and deoxycytidine and thymine from L. Light and Co. Deoxyribonuclease (EC 3.1.4.5) (I × crystallised) and snake-venom phosphodiesterase (EC 3.1.4.1) were obtained from the Worthington Biochemical Corporation. The snake-venom phosphodiesterase used in the earlier experiments was prepared sl from snake venom (Crotalus adamanteus) obtained from Ross Allen's Reptile Institute, Silver Springs, Florida. Charcoal (Norit " N K " ) from Hopkin and Williams was purified by refluxing 3 times with 2 N HC1 (ref. 22). Dowex-5o and Dowex-2 (lOO-4OO mesh) were converted into the H+ and HCO s- forms ss, respectively. Mixed Dowex (CO s treated) was prepared by mixing equal parts of Dowex-5o(H+) and Dowex-2(HCOs-) and bubbling CO s through the mixture before use. METHODS

Preparation o / t h y m u s cells Male albino rats weighing between IOO and 125 g were used. The animals were Biochim. Biophys. Mcta, 114 (i966) 44-6o

46

E. D. WHITTLE

exsanguinated under ether, anaesthesia, and the thymus glands were removed and placed in cold Krebs~Ringer solution 24. Suspensions of thymus cells were prepared b y the technique used by ITZHAK123, b y lightly squeezing the glands on a net of monel metal (zoo mesh) and washing the cells through with cold Krebs-Ringer bicarbonate solution 2~. The cells were packed b y centrifugation at 3 °.

Incubation procedure In order to test the effect of pyrimidine nucleosides (in one experiment thymine) on incorporation of labelled precursors into the nucleic acids, the cells were pre-incubated with the appropriate compound prior to the addition of the labelled precursor. The following was the standard procedure used: (a) Pre-incubation. The medium used was Krebs-Ringer bicarbonate solution, gassed with 02-CO 2 (95:5, v/v), to which glucose at a concentration of 5 rag/m1 was added. The packed cells obtained from 4 thymus glands were suspended in IO ml of medium. In each ice-cooled Ioo-ml conical flask were placed 2 ml of the cell suspension and 2 ml of medium containing an amount of the nucleoside to give the desired final concentration. In each experiment, four flasks were generally used, one of which was the control with no added nucleoside. The flasks were gassed with O2-CO2 (95:5, v/v), tightly stoppered, and shaken for 9 o min in a water bath at 37.4 °. (b) Incubation with labelled precursor. To each flask were then added 4 ml of medium containing the same concentration of nucleoside used for pre-incubation and to which had been added the required amount of labelled precursor, this solution having been brought to 37.4 ° prior to the addition. The concentration of cells during the incubation was about 6. IOv cells per ml. When the contents of each flask had been incubated with the labelled precursor for exactly 30 min, they were poured into ice-cold centrifuge tubes, spun at 3 °, and to the packed cells cold 0.6 N HC104 was added with stirring.

Extraction o/ nucleic acids The cells from each incubation flask were treated further as follows: The tissue was stirred in cold 0.6 N HC104 to extract the acid-soluble material, 0.05 M NaH2PO 4 being added to the 0.6 N HC104 for the asp experiments. The suspension was centrifuged at 3 ° , and the residue washed successively in the cold with 0.6 N HC1Q, 0.2 N HC104, 0.o6 N HCIO 4, 95 % ethanol, absolute ethanol and ether. The dry residue was refluxed for I h with 40 ml methanol-chloroform (I : I, v/v) to extract the fipids. The dried defatted tissue was treated with hot IO % NaC1 solution to extract the sodium nucleates25, z6. Before heating, the suspension of tissue in IO % NaC1 was brought to a constant p H of 6.5-7 b y addition of N a O H and occasional stirring at room temperature. Two extractions at IOO° of the tissue in 4.5 ml each time, for i h and 0.5 h, respectively, then gave at least a 95 % yield of DNA. The sodium nucleates were fractionated into RNA (as nucleotides) and DNA b y incubation in I ml of 0.3 N K O H at 37 ° for 18 h, with subsequent acidification with 3 N HC104 in the cold to precipitate the DNA (ref. 25). The mixture was cenBiochim. Biophys. Acta, 1i 4 (1966) 44-60

THYMIDINE AND NUCLEIC ACID METABOLISM

47

trifuged, and the supernatant containing the ribonucleotides was decanted, amL if required for later analysis, neutralised with K O H and stored at --20 °. DNA was recovered from the precipitate b y dissolving in dilute K O H and re-precipitating it a t p H 4 with ethanol. In general, this purified DNA was divided into two portions: one for the direct determination of the specific activity of the DNA, and the other for enzymic hydrolysis.

Enzymic hydrolysis o / D N A The DNA was degraded to deoxyribonucleoside 5'-monopkosphates b y the combined action of deoxyribonuclease and snake-venom phosphodiesterase 27. The DNA was suspended in 0.02 M MgSO4 (about 3 mg DNA in 0.2 ml solution), and dissolved b y adding I or 2 drops of dilute N H 4 0 H (final p H 7.6). A few crystals of deoxyribonuclease and a few drops of chloroform were added, and the mixture incubated at 37 ° for 5 h, the p H being kept at 7.6 by occasional addition of NH4OH. The partial digest was brought to p H 8. 4, 0.25 volume of o.I M glycine buffer (pH 8.4) and a small amount of phosphodiesterase were added, and it was further incubated overnight.

Separation o[ deoxyribonucleotides by dectrophoresis Paper electrophoresis was carried out on W h a t m a n paper No. 3MM, soaked in 0.05 M formate buffer (pH 3.5), using a voltage gradient of 20 V/cm for a 3-h run 2s. All the DNA digest was applied to the base-line as a band 12 cm wide. The separated deoxyribonucleotides were located on the paper by ultraviolet photog r a p h y 29 and eluted from their bands with water.

Estimations DNA, and RNA [faction: For a comparison of specific activities in the same experiment, the absorbancy at 260 m/, of a suitable dilution of the DNA sample in o.I N K O H was determined. Similarly, the RNA fraction (mixed ribonucleotides) was determined b y the absorbancy at 260 m# of a sample of it in o.I N HC1. Deoxyribonucleotides: These compounds were estimated in o.I N HC1 b y ultraviolet spectrophotometry using the published extinction coefficients 3°. Each reading of an eluate was corrected for that of an eluate from a corresponding paper blank. Phosphate: For the 32p-labelling experiments, PI in the medium was determined by the method of Fiske and SubbaRow (in ref. 31). Labile phosphate from the acid-soluble fraction was estimated as PI using the method of WEIL-MALHERBE AND GREEN 32.

Isolation o/ labile phosphate [rom the acid-soluble nucleotide /raction When this fraction was required for analysis, 5 ml of cold 0.6 N HC104 were added to the packed cells after incubation to extract the acid-soluble material. The suspension was centrifuged at 3 °, and the supernatant and one washing of the tissue

Biochim. Biophys. Acta, ii 4 (x966) 44-60

48

E. D. WHITTLE

residue with o.6 N HCIO 4 were brought to p H 2-3 with KOH. After centrifugation, the supernatant was shaken for I h with o.I g purified charcoaP s. The charcoal residue was washed 3 times with 5 ml of cold o.06 N HC104. To release the labile phosphate of the nucleotide fraction as PI, the charcoal was suspended in 2 ml of I N HC1 and heated in a boiling-water b a t h for exactly IO min. The supernatant and washings of the charcoal residue with water were collected.

Measurement o/ radioactivity In general, 8,p_ and 1*C-labelled samples were plated on aluminium planchets, dried under a lamp and counted in a windowless gas-flow counter. In the earlier experiments using [32PIP1, radioactivity of the DNA sample in solution was assayed w i t h a liquid-dipping counter.

RESULTS

Suitability o/ pre-incubation period In the experiments to be described on the effect of thymidine and deoxycytidine on nucleic acid metabolism, the time chosen for pre-incubation of the thymus cells before addition of the labelled precursor was 9 ° min. It was therefore considered necessary to determine first the effect of length of pre-incubation on the metabolism of cells without any nucleoside in the medium. The results on incorporation of 82p into DNA and on total labile phosphate (Table I) indicated that the 9o-min preincubation period did not have a seriously deleterious effect under the conditions used. TABLE

I

EFFECT OF LENGTH OF PRE-INCUBATION ON INCORPORATION OF 3*p INTO D N A AND ON TOTAL

LABILE

PHOSPHATE

I n E x p t s . I a n d 2, t h e cells were p r e - i n c u b a t e d for t h e t i m e s s how n, a n d t h e n i n c u b a t e d w i t h [~*P]Pt (4.5 ~uC/ml) for 3 ° rnin. I n E x p t . 3, t h e cells were i n c u b a t e d w i t h o u t i s o t o p e for t h e t o t a l t i m e s shown. I n t h e s e e x p e r i m e n t s , no a d d i t i o n of n u c l e o s i d e w a s ma de . D i f f e r e n t gas-flow count e r s were u s e d for a s s a y i n g 32p in E x p t s . i a n d 2.

Expt. No.

Pre-inc.bation time (rain)

Speci[ic activity o/DNA (counts/rain per zo absorbancy units)

i

15 90

909 912

2

15 9°

1542 132o

Totalincubation ~me (rain) 3

Totallabile phosphate (#atoms P per flash)

45 12o

Biochim. Biophys. Acta, 95 (1966) 4 4 - 6 o

o.44o o.447

49

THYMIDINE AND NUCLEIC ACID METABOLISM

Experiments with 3,p A series of experiments was carried out to determine how thymidine in the medium affected the incorporation of 82p into the DNA of rat-thymus cells (Table II). With thymidine concentrations higher than 0.02 mM the effect was inhibitory, the degree of inhibition being dependent on concentration. About a 50 % inhibition of 32p incorporation into DNA was obtained with 0.08 mM thymidine. TABLE II EFFECT

OF THYMIDINE

ON INCORPORATION

O F $21~ I N T O

DNA

4.5/zC of [82PIP1 were p r e s e n t per ml in the m e d i u m for the i n c u b a t i o n period. W h e n more t h a n one d e t e r m i n a t i o n was made, the specific a c t i v i t y of D N A (as % of control w i t h o u t t h y m i d i n e ) is given as the m e a n i S . E . of the mean, w i t h the n u m b e r of e x p e r i m e n t s in brackets.

Concn. o] thymidine (mM)

Specific activity o[ DNA (% o] control)

0.005 0.02 0.04 0.08 o.1 0.2 I.O 2.0 IO.O

98 93 68 52~5(6 ) 4o:t=2(8) 23 12 lO. 4 5.9+o.1(2)

The results of experiments to determine the effect of two concentrations of deoxycytidine both alone and with an inhibitory concentration of thymidine on 3,p incorporation into DNA are shown in Table III. The effect of deoxycytidine alone was stimulatory, a situation also found by KLEI~OWTM with Ehrlich ascites-tumour TABLE III EFFECT OF DEOXYCYTIDINE ALONE AND WITH THYMIDINE ON INCORPORATION OF 321) INTO D N A 4-5/zC of [32P]Pt was p r e s e n t per ml in the m e d i u m for the i n c u b a t i o n period. E a c h value for specific a c t i v i t y of D N A (as % of control w i t h o u t nucleoside) is given as the m e a n 4-S.E. of the mean, w i t h the n u m b e r of e x p e r i m e n t s in brackets.

Conch. o/deoxyribonucleoside Deoxycytidine (raM)

Thymidine (raM)

0.04 0.04 o.ool o.ooi

o 0.08 o 0.08

Specific activity o/DNA (% o/control)

I524-4(4) 163:l:3(3) I22~6(2) 874-3(2 )

cells. Nevertheless, the stimulation by 0.04 mM deoxycytidine was maintained in the presence of 0.08 mM thymidine. Even o.ooi mM deoxycytidine partially prevented the expected inhibition by thymidine alone. Two types of experiment were next performed to indicate whether the changes in 32p incorporation into DNA caused by the presence of thymidine and deoxycytidine Biochim. Biophys. Acta, 1i 4 (1966) 44-60

5°

E.D.

WHITTLE

in the medium represented true changes in DNA synthesis. If this were the case, a change in the specific activity of the DNA would be expected to result from somewhat similar changes imthe specific activities of each of its four constituent deoxyribonucleoside 5'-monophosphates. For each of the conditions in each experiment (Table IV), TMP had the highest specific activity of the four deoxyribonucleotides. It is seen from Expts. 4-6 that variations from the control of the specific activities of TMP, deAMP and deGMP with the different additions were similar. The specific activity of deCMP relative to the control was not so depressed by thymidine, and its value was higher than those of the other three deoxyribonucleotides when the deoxycytidine and deoxycytidine plus thymidine additions were made. The reason for this is not evident. However, it should be noted in Expts. 4 and 5 that the corresponding specific activities of each deoxyribo~:ucleotide, including deCMP, were similar when deoxycytidine plus thymidine were added and when deoxycytidine was added alone. This is strong evidence that in these experiments deoxycytidine was preventing entirely the inhibitory effect of thymidine. Assuming that the base ratio for rat DNA is given b y ( A + T ) / ( G + C ) -- 1.3 (ref. 34), a value was calculated for the specific activity of DNA from those of its constituent deoxyribonucleotides in each case. As seen in the last two columns of Table IV, the values of these calculated specific activities relative to the control are in good agreement with those obtained b y direct measurements on whole DNA, and confirm the radiochemical purity of the DNA in each experiment. The results of Expt. 7 (Table IV) show that I mM thymine had no significant effect on the incorporation of a2p into DNA and its constituent deoxyribonucleotides. I t would be possible for the inhibition by thymidine of s~p incorporation into DNA to be caused, not b y an inhibition of DNA synthesis, but by a general reduction in the specific activities of the e-phosphate groups of the four deoxyribonucleoside triphosphates, the immediate precursors of DNA. From the known pathways for formation of purine and pyrimidine deoxyribonucleotides, it can be seen that, in synthesis de novo, the e-phosphate group is derived, via the 5-phosphate group of 5-phosphoribosylpyrophosphate, from the phosphate group of glucose 6-phosphate. The latter arises through a transfer of the y-phosphate group of ATP to glucose in the hexokinase reaction. The e-phosphate groups of deoxyribonucleotides arising b y phosphorylation at the nucleoside level are also derived from the y-phosphate group of ATP. Thus, a general reduction in the specific activities of the ~-phosphate groups of the four deoxyribonucleoside triphosphates might result from a reduction in the labelling of the ),-phosphate group of ATP. To investigate this possibility, the effect of different concentrations of thymidine on both the total amount and specific activity of the labile phosphate of the acid-soluble nucleotide fraction was determined. This labile phosphate is derived mainly from the y- and/~-phosphate groups of A T P and the /5-phosphate group of ADP. In Expts. 8 and 9 (Table V), it is seen that, although concentrations of o.I mM and I mM thymidine caused the expected reductions in specific activity of DNA, the corresponding values obtained for total labile phosphate and its specific activity were very similar for both test and control cells. With IO mM thymidine, there appeared to be a slight reduction in the specific activity of the labile phosphate but this was small, 13 ~o, compared with the 94 % reduction in specific activity of the DNA. From these experiments it is concluded that thymidine in concentrations from o.I mM to IO mM had little or no effect on the Biochim. Biophys. Acta, 114 (1966) 44-60

O

[

v 4~

4~

~0

IV

7

6

-o.I

5

-o.ooi o.ooi --

-0-04 0.04 --

-0.04 0.04 --

Deoxycytidine (mM)

2730 2560

216o I7o7 2706 92o

2365 3455 2890 74 °

2420 2900 3320 913

IO95 lO45

982 I265 I3o2 738

io58 3085 2580 686

1178 2820 2790 794

752 796

613 379 813 189

648 958 1233 199

683 1145 III2 19o

(A+T)/(G+C)

419 496

35 ° 213 391 I22

367 607 674 122

373 542 513 I38

IOO 94

IOO 79 125 43

IOO 146 122 31

IOO 12o 137 38

= 1. 3 (ref. 34).

deGMP

IOO 95

IOO I28 133 75

IOO 292 244 65

IOO 240 237 67

deCMP

TMP

deAMP

TMP

deCMP

Specilic activity (% ot control)

ioo ii8

IOO 61 112 35

ioo I65 184 33

ioo 145 138 37

deAMP

ioo io6

IOO 62 133 31

ioo 148 19o 31

ioo i68 163 28

deGMP

ld~ 98

IOO 85 126 47

IOO 177 16o 38

ioo 153 161 43

Calc." DNA

p e r /z-

ioo io2

IOO 89 127 48

IOO I72 156 38

IOO 156 151 42

Whole DNA

4, 5, 6 a n d 7 w a s 6 . 6 6 - io*, 5 . 8 5 . io*, 5 . 8 7 - I o 6, a n d 6 . 6 4 - i o ~ c o u n t s / m i n

Speci/ic activity (counts/rain per Izmole)

for Expts.

* Calculated value assuming that, for rat DNA, ** I n E x p t . 7 t h e a d d i t i o n w a s t h y m i n e .

-I.O'*

o.08

--

-0.08

o.I

--

-o.i -o.I

Thymidine (raM)

Additions

4

Expt. No.

The specific activity of [3zP]Pt in the medium atom P, respectively.

INCORPORATION OF 3~p INTO INDIVIDUAL DEOXYRIBONUCLI~OTIDES OF D ~ A

TABLE

H

¢dt

t2

t~

52

E.D.

WHITTLE

TABLE V EFFECT

OF

THYMIDINE

ON

AMOUNT

AND

SPECIFIC

ACTIVITY

OF

LABILE-PHOSPHATE

FRACTION

T h e specific a c t i v i t y of [32P]Pi i n t h e m e d i u m w a s 5.45' lO6 c o u n t s / m i n pe r p a t o m P i n E x p t . 8, a n d 5.28- IOe c o u n t s / m i n p e r #*atom P in E x p t . 9.

Expt.

Conch. o] thymidine Specific activity of (raM) DNA (counts~rain per zo absorbancy units)

No.

8

o o.I

9

o I IO

Total labile phosphate Specific activity of (l~moles per inculabile phosphate bation flash) (counts/min per mpatom P)

1278 543

0.594 o.58o

lO9O lO7O

1124 134 66

o.477 0.493 0.462

ilOO lO7O 960

total amount and specific activity of the labile phosphate of the acid-soluble nucleotide fraction, and therefore, presumably, on the formation and utilization of ATP.

Experiments with [8-14C]adenine As further confirmation that changes in incorporation of 32p into DNA caused b y thymidine and deoxycytidine can justifiably be considered to represent corresponding changes in DNA synthesis, the effect of these nucleosides on the incorporation into DNA of another labelled precursor, the purine [8-a4C]adenine, was determined. Thymidine was found to inhibit the incorporation of [8-14CJadenine into DNA (Table VI). For each concentration, it inhibited to the same degree the incorporation TABLE VI EFFECT

OF T H Y M I D I N E

ON I N C O R P O R A T I O N

OF

[8-1~CJADENINE

INTO

DNA

E x p t . io. T h e specific a c t i v i t y of E8-14C]adenine a d d e d t o t h e m e d i u m w a s 3.o8- lO 7 c o u n t s / m i n p e r /*mole.

Concn. of thymidine (raM)

-o.oi o.I i.o

Specific activity of DNA (counts/rain per Io absorbancy units)

Specific activity (counts]rain per pmole) deA M P

deGMP

Whole DNA

deAMP

deGMP

426o 4o6o 2o2o 402

12 72o

81o 766 356 68

ioo 95 47 9.4

ioo 97 47 9 .1

i oo 95 44 8.4

12 3oo 60oo 116o

Specific activity (% o/ control)

of 14C into both deAMP and deGMP of DNA, as can be noted from the values for specific activity expressed as a percentage of the control. In Fig. I, the results of the effect of thymidine on incorporation of isotope into DNA using L8-14C~adenine are shown compared with those in which [32p]pt was used. The points for the incorporation of both isotopes are seen to lie on the same curve. Biochim. Biophys. Acta, 114 (1966) 4 4 - 6 0

THYMIDINE AND NUCLEIC ACID METABOLISM

53

lOC

"6 o

4(

.y,

~ u

2c

o

0001

(101

(11

1.0

10

rhymidine (raM)

Fig. I. E f f e c t of t h y m i d i n e o n t h e i n c o r p o r a t i o n i n t o D N A of labelled precursors. O , [3zP]PI; O , [8-1~C]adenine; a n d /x, [2-1~C]cytidine.

cytidine

CMP

pathway de novo

• > CTP •

1 CDP

deoxycytidine

deCMP

1 > deCDP

> deCTP

) DNA-cytosine

RNA-cytosine Fig. 2. P a t h w a y s for t h e s y n t h e s i s of D N A - a n d RNA--cytosine. E v i d e n c e h a s been g i v e n t h a t t h e c y t i d y l a t e r e d u c t a s e r e a c t i o n occurs a t t h e d i p h o s p h a t e ]eve135, 3~.

The effect of o.I mM thymidine and o.o4 mM deoxycytidine, both separately and together, on the incorporation of 14C from [8JaC]adenine into DNA and its constituent purine deoxyribonucleotides was investigated in Expt. I I (Table VII). Again, the specific activities relative to the control of deAMP and deGMP are similar for each addition. The addition of 0.04 mM deoxycytidine had a stimulatory effect on the incorporation of [8J4C]adenine into DNA, and this concentration of deoxycytidine completely prevented the inhibition produced by o.I mM thymidine alone. The similarity of effect on the specific activity of DNA under conditions of the same concentration of thymidine and deoxycytidine when either [8-1aC]adenine or [3~P]P1 was used as the labelled precursor is evident from a comparison of Tables V I I and IV. These results with [8-14C]adenine thus supported those with 3~p, and together Biochim. Biophys. Acta, 114 (1966) 4 4 - 6 o

0

2I

C~

H

tv

OF [8-11C]ADENINE

65oo

--

o.o 4

o.I

-

0.04

o.I

-

6660

_

_

1741

489 °

Deoxycytidine (mM)

Thymidine (raM)

Additions

Specific activity o/ DNA (counts[rain per IO absorbancy units)

513 °

18 8 5 0

18 8 7 0

13 7 2 0

deA M P

261

115o

11o 7

768

deGMP

Specific activity (counts/rain per #mole)

138

137 37

136 133 36

34

15o

144

650

2 9 95 °

33 2 5 0

33

32 75 °

IO0

I00

IO0

Counts/min deAMP deGMP per xo absorbancy units

91

lO2

1°3

I00

% o/ control

Specific activity o] RNA /raction

Whole DNA

Specific activity (% o] control)

per/zmole.

ON INCORPORATION

E x p t . 1 i . T h e s p e c i f i c a c t i v i t y o f [ 8 - 1 * C ] a d e n i n e a d d e d t o t h e m e d i u m w a s 3 . 2 2 . lO ~ c o u n t s / m i n

AND THYMIDINE

AND RNA

VII

EFFECT OF DEOXYCYTIDINE

INTO DNA

TABLE

THYMIDINE AND NUCLEIC ACID METABOLISM

55

they enable the conclusion to be drawn that thymidine in the concentrations used inhibited DNA synthesis in the rat-thymus cells, and that this inhibition could be prevented by a smaller concentration of deoxycytidine. The specific activities of the RNA fractions in Expt. I i were determined and the results included in Table VII. The specific activity of the RNA fraction varied little with the different additions, in marked contrast to the corresponding value for DNA. It was concluded that these concentrations of thymidine and deoxycytidine had no significant effect on RNA synthesis.

Experiments with E2-1aCJcytidine The fact that deoxycytidine prevented the inhibition of DNA synthesis caused by thymidine alone, when RNA synthesis was unaffected by either deoxyribonucleoside, suggested that the primary site of inhibition by thymidine was likely to be at the reductive step in which deoxycytidylate is formed from cytidylate (see Fig. 2). This has been shown for the murine neoplastic cells, L5178Y (ref. 15). If it were also the case for the rat-thymus cells, then it might be expected that the incorporation of E2-1*CJcytidine into DNA-deCMP would be more sensitive to inhibition by thymidine than DNA synthesis itself. The effect of concentration of thymidine on incorporation of [2-1aCJcytidine into DNA was therefore studied (Table viii). It is seen that incorporation of [2-1aC]cytidine into DNA-deCMP was very sensitive to inhibition by thymidine, considerably more so than that of 32p or [8-1*C]adenine into DNA (c/. with Tables II and VI). It was to be expected, of course, that thymidine would considerably reduce the incorporation of 14C into DNA-TMP by a dilution effect. However, the labelling of DNA-TMP as compared with DNA-deCMP was not very high under the conditions of the experiment, 8.2 % for Expt. 12 and 15.7 % for Expt. 13. Thus in Expt. 13, when thymidine was present, the specific activity of deCMP relative to the control was only slightly higher than that obtained for whole DNA. In Fig. I, the results of the effect of thymidine concentration on incorporation into DNA of 32p, [8_14C]_ adenine, and E2-1*CJcytidine are shown together. In order that the effect of thymidine on incorporation of [8-~aC~adenine and E2-~4CJcytidine into RNA might be compared, the RNA fractions from Expts. io, 12 and 13 were estimated for specific activity (Table IX). Thymidine at concentrations of o.oi mM and o.I mM had no significant effect on the incorporation of [8-~*CJadenine into the RNA fraction, although I mM thymidine appeared to have some inhibitory effect. With [2-~4C]cytidine as the labelled precursor, o.oi mM thymidine was not significantly inhibitory, but 0.08 raM, o.I mM and I mM thymidine increasingly inhibited incorporation of [2-1*C~cytidine into the RNA fraction. Thus, the incorporation of E2-~aC~cytidine into RNA was more sensitive to inhibition by thymidine than the incorporation of [8-~*C~adenine. In view of the previous results, it would seem that thymidine has no specific effect on adenine metabolism, at least up to a concentration of o.I mM, and thus that incorporation of [8-14C1adenine into RNA represented true RNA synthesis. Then the greater sensitivity to inhibition by thymidine of incorporation of [2-1aC]cytidine into RNA must have been due to an inhibition, not of RNA synthesis, but of the utilization of cytidine to form CTP (Fig. 2). Thus it is suggested that thymidine inhibits either the entry of Biochim. Biophys. Acta, 114 (1966) 44 6o

G~ 0

I

G~

~D

~r

VIII

-o.o8

-o.oi o.I I.O

12

13

3o4 ° 1835 349 5°

211o 445 1o48o 673 ° I34°** .

782o 16o3 1651 313 53 .

645 45

Speci/ic activity Speci/ic activity o/ DNA (counts~rain per (counts/rain per IO ab- ttmole) sorbancy units) deCMP T M P

.

IOO 64 I3"* .

IOO 2o.5

deCMP

.

IOO 19 3

IOO 7

TMP

--

57

ioo

ioo 19

Calc. ~ DNA

IOO 6o II 1.6

ioo 21

15. 7 4-7 4.o --

8.2 2.8

Specific activity of deCMP

Speci/ic activity o~ T M P X I00

12, a n d 2 . 4 1 - lO T c o u n t s / m i n p e r

Whole DNA

per #mole in Expt.

Speci/ic activity (% o[ control)

" Calculated value assuming that, for rat DNA, (A+T)/(G+C) = 1. 3 (ref. 34). "" T u b e b r o k e n . V a l u e s c a l c u l a t e d f r o m t h e s p e c i f i c a c t i v i t i e s o f D N A a n d T M P .

Concn. o~ thymidine (raM)

Expt. No.

T h e s p e c i f i c a c t i v i t y o f [ 2 - 1 4 C ] c y t i d i n e a d d e d t o t h e m e d i u m w a s 2 . 4 5 . lO 7 c o u r t t s / m i n # m o l e i n E x p t . 13.

E F F E C T OF T H Y M I D I N E ON INCORPORATION OF [ 2 - 1 4 C ~ c Y T I D I N E I N T O D I ~ A

TABLE

~n

57

THYMIDINE AND NUCLEIC ACIDXMETABOLISM

cytidine into the cell or the kinases of cytidine, CMP or CDP. If the site of action of thymidine is on the p a t h w a y of cytidine to CDP, it is seen from Fig. 2 that incorporation of [2A4C]cytidine into DNA-deCMP would thereby be reduced by the same amount as that into RNA. However, the inhibition of the latter required a higher concentration of thymidine (about IO times higher) than for a similar degree of inhibition of incorporation into DNA--deCMP; thus an inhibition b y thymidine of formation of labelled cytidine phosphates could only partially explain the greater sensitivity to inhibition of incorporation of [2-14CJcytidine into DNA-deCMP as compared with that of 32p or [8-HC]adenine into DNA. The results of the experiments with [2-14C~cytidine are therefore consistent with a primary inhibitory effect b y thymidine on the formation of deCDP from CDP, and suggest that higher concentrations of thymidine have a further inhibitory effect on the utilization of cytidine for CTP formation. TABLE

IX

EFFECT OF THYMIDINE ON INCORPORATION INTO THE 1RNA FRACTION OF [8-t*C]ADENINE AND

[2-14C~cYTIDINE The specific activities of labelled precursors in Expts. 10, I 2 a n d 13 a r e g i v e n i n T a b l e s V I a n d

VIII. Expt. No.

Labelled precursor

Concn. o/ thymidine

(raM)

Specific activity Specific activity o/ R N A * o/ R N A (counts~rain per (% o/ control) • o absorbancy units)

IO

[8-14C]Adenine

-o.oi o.i i.o

26 28 28 19

25 ° 95 ° ioo 62o

ioo iio lO 7 75

12

[2-14C]Cytidine

-0.08

16 42o I I 880

ioo 75

13

E2-14C]Cy t i d i n e

16 19 ° 15 09 ° IO 19o 614o

IOO 93 63 38

-

-

o.oi o.I I.O

* T h i s f r a c t i o n of h y d r o l y s e d R N A w a s s h o w n t o b e r a d i o c h e m i c a l l y p u r e b y e l e c t r o p h o r e t i c s e p a r a t i o n of i t s c o m p o n e n t s o n p a p e r , f o l l o w e d b y a u t o r a d i o g r a p h y .

Degradation o~ thymidine An experiment was carried out to assess the degree of breakdown of thymidine b y the cells under the conditions used for pre-incubation (Table X). With o.I mM thymidine, 8 7 % of it was recovered from the medium after the 9o-min period of preincubation. If the same amount of thymidine was not added to the mixture till after pre-incubation and cooling, but before separation of the medium from the cells, only slightly more, i.e. 89 %, was recovered. Some of this loss with the latter conditions must represent thymidine that had penetrated the cells (the cells were not washed), and some slight losses probably occurred in the recovery procedure. However, the small difference suggests that degradation of thymidine during pre-incubation was negligible. COOPER AND MILTONss have recently reported a similar lack of degradation of thymidine by mouse thymocytes. Biochim. Biophys. Acta,

114 (1966) 4 4 - 6 o

5~

E. 1), WHITTLJL

TABLE X RECOVERY OF THYMIDINE FROM THE PRE-1NCUBATION MEDIUM P r e - i n c u b a t i o n of t h y m u s cells was carried out for 9o m i n as described in METHODS. The c o n t e n t s of each flask (4 ml) were t h e n cooled in ice, combined in pairs and centrifuged. The m e d i u m t h u s recovered w a s b r o u g h t to p H 5-6, heated at ioo ° for 5 rain, cooled and centrifuged. The supern a t a n t w a s desalted w i t h Dowex resins: drops of D o w e x - 5 o ( H +) and I)owex-2(HCO3- ) were added a l t e r n a t e l y till no f u r t h e r p H change or evolution of gas occurred, the s o l u t i o n w a s passed t h r o u g h a s h o r t c o l u m n of mixed Dowex, and the resins were washed w i t h water. The eluate (about 75 ml) w a s c o n c e n t r a t e d at 5 °° to a b o u t o.2 ml, and c h r o m a t o g r a p h e d on W h a t m a n No. i paper, u s i n g the descending t e c h n i q u e w i t h the u p p e r layer of an equilibrated m i x t u r e of e t h y l a c e t a t e - w a t e r - f o r m i c acid (12:7:1, v/v) av as solvent. T h y m i d i n e was eluted w i t h w a t e r f r o m its b a n d and e s t i m a t e d s p e c t r o p h o t o m e t r i c a l l y , a c o r r e c t i o n being applied for t h e control.

Conditions

Thymidine recovered ]rom medium (2/lasks) (ttmoles)

¢~Jo thymidine recovered

Pre-incubation with thymidine (o. lO 3/zmole/ml) Pre-incubation without thymidine; t h y m i d i n e t h e n added to cooled i n c u b a t i o n m i x t u r e (o. Io 3/zmole/ml)

o.718

87

0.732

89

In view also of the other finding that I mM thymine in the medium caused little effect on incorporation of 3~p into DNA and its constituent deoxyribonucleotides (Expt. 7, Table IV), the inhibitory effect of thymidine could not have been due to its possible breakdown products.

DISCUSSION

Thymidine in concentrations greater than o.o2 mM has been shown to inhibit DNA synthesis in rat-thymus cells, and inhibition of deoxycytidylate formation has been implicated as the primary cause. No stimulation of DNA synthesis by thymldine was found down to a concentration of 0.005 mM, although the 9o-min pre-incubation period should have been sufficient for any possible effect on TMP kinase to become apparent (see ref. 3). These findings thus agree with those of MORRIS el al. 1~ using L5178Y cells. It would seem probable that the same mechanism is responsible for the inhibition of growth by thymidine found by various workers for other cultured mammalian cells, namely, human monocytic leukaemic (J-III) 1°, Chang appendix la, Novikoff 13, and HeLa cells 14, especially where the inhibition was shown to be reversible by deoxycytidine12, is. However, from available evidence it appears that resultant inhibition by thymidine of DNA synthesis may not hold for all types of cells. Thus, with Ehrlich ascites cells in vitro, PRUSOFFle obtained no inhibition with 4 mM thymidine of incorporation of [8-14Cladenine into DNA, and KLENOW18 showed no effect of 2 mM thymidine on 32p incorporation into DNA. The possibility that the actual inhibitory derivative of thymidine for the thymus cells could be a breakdown product has been excluded, but its nature was not further investigated. MORRIS AND FISCHER~9 obtained evidence suggesting tha~ it was not thymidine itself but a phosphorylated derivative of thymidine. If the inhibition by thymidylate is a normal controlling factor of DNA synthesis for Biochim. Biophys. Acta, 114 (1966) 44-6o

THYMIDINE AND NUCLEIC ACID METABOLISM

59

some mammalian cells in vivo, then two conditions must be realised for its operation: it must be possible that inhibitory concentrations of the thymidine derivative are produced under physiological conditions, and extracellular deoxycytidine would then not have to be available to the cells. Although deoxycytidine is normally present in the blood 4°, it is possible to speculate that its availability might be capable of restriction by local factors. A further inhibitory effect of thymidine on the formation of CTP from cytidine is suggested; this effect required a concentration of thymidine of the order of IO times higher than that causing the same degree of inhibition of incorporation of [2-14CJcytidine into DNA-deCMP. A similar inhibition by thymidine of the incorporation of IaHJcytidine into the RNA of HeLa cells has been described 41, and could be similarly explained. It is tempting to suggest that this might be due to a specific inhibition by TTP of cytidine kinase, in analogy with the known inhibitory action of TTP on thymidine kinase42m, but further studies are required to define the site of inhibition more precisely.

ACKNOWLEDGEMENTS

I wish to thank Professor J. S. MITCHELL for his interest, and Dr. ]3. E. HOLMES and Dr. S. ITZHAKI for helpful discussion. This work has been supported by grants from the British Empire Cancer Campaign which are gratefully acknowledged.

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E.D.

WHITTLE

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Biochim. Biophys. Acta, 114 (1966) 44-60