Effects of certain pyrimidines on cleavage and nucleic acid metabolism in sea urchin. Strongylocentrotus purpuratus, embryos

EFFECTS

OF

CERTAIN

NUCLEIC

ACID

PYRIMIDINES METABOLISM

STRONGEZOCENTROTUS L. ‘CC. STEARNS. Biology

W.

Department,

IN

SEA

W.

of Southern Received

B. J0LLE-f California,

September

AND

URCHIN.

PURPURATUS,

E. K~RTTI, Unitvrsity

ON CLEAF’AGE EMBRYOS’ and

J.

W.

Los Angeles,

BAMBERGER Calif.,

C~‘..S.d.

8. 1961

T\vo

pyrimidine derivatives, 2,4-diamino-5-%(p-chlorophenyl) (i-ethyl pyrimidine and 2,-l-diamino-.i-(3’,4’-dichlorophenyl) G-ethyl pyrimidine have been shown to inhibit growth, certain physiological processes, embryonic development, and/or the proliferation of cells of neoplastic tissue in certain organisms from bacteria to mammals. Hitchings [‘L?], Hitchings et nl. [23, 241, Greenberg and Richeson [18], Sumners [34], etc., hare shown that these inhibitory effects are apparentl! due to antagonism to the co-factor activity of folic acid or to interference with one-carbon-fragment transfer by its derivatives. Doctor [9], Hitchings [Z], M’ood and Hitchings [38], etc., have made progress in elucidating the general mechanisms of this antagonism, but the more detailed aspects of both the antagonism and its effects as well as the question of how thv antagonism may vary in different tissues and organisms are still subjects for inrestigation. Davidson and Freeman [Cc] haT-e found that the t\vo pyrimidines cause a decrease in uptake of 32P hv. r)h’A in mouse sarcoma I70 and Lepage and Greenlees [26] hare noted that the two co~npounc~s marltedl~ reduce the incorporation of glyine-2-‘“C into adenine and guanine c:f DN.1 and RN;\ in Ehrlich ascites tumors in mice. Fernandes et nl. [IS] have found that the 5(p-chlorophenyl) derirative inhibits the incorporation of formate14C into adenine and guanine of mixed nucleic acids in normal rat spleen. Heidelberger and Keller [‘LO] reported that the ,i-(3’,4’-dichlorophenyl) derivatire stimulates the incorporation of formate- lAC into adenine and guanine of RNA in the Flesner Jobling tumor. From in uifro studies, Lepage and Greenlees [2G] noted that both pyrimidines mildly inhibit the incorporation of glycine-2-l% into protein in Ehrlich ascites tumors in mice. The present study \vas performed to determine the effect of the t\\o corn1 This work Experimenlal

was supported Cell Research

by LISPHS 27

contract

CY-3,546.

Effects of pyrimidines

on cleavage and metabolism

in embryos

251

pounds on cleavage, on certain aspects of nucleic acid metabolism, and on the uptake of isotopic glycine by proteins in embryos of the sea urchin, Strongy-

locen h-0 tus purpura tus. MATERIALS

AND

METHODS

The antimetabolites, 2,4-diamino-5-(p-chlorophenyl) 6-ethyl pyrimidine (N.S.C. #3061); 2,4-diamino-5-(3’,4’-dichlorophenyl) g-ethyl pyrimidine (N.S.C. #3062); 2,4-diamino-5-(3’,4’-dichlorophenyl) 6-methyl pyrimidine (N.S.C. #19494) and 4-aminopyrazolo (3,4-d) pyrimidine (N.S.C. #1393) were received from the Cancer Chemotherapy National Service Center of the National Cancer Institute, Bethesda, Maryland. Techniques for culturing sea urchin embryos were essentially those described by Harding and Harding [I91 except that ovaries were not treated with potassium chloride to induce shedding. Embryos were cultured in glasstrays (culture volume 600 ml containing approximately forty million embryos) that were constantly rocked under conditions of high humidity. Embryos were maintained at 7°C for 104 hr after fertilization to increase synchrony of cleavage. Temperature was then raised to 15°C. Experiments were terminated when embryos in control cultures reached the late blastula stage (approximately 24 hr after fertilization). Six mg of the appropriate analogue was added to each 600 ml culture 30 min after fertilization. Control cultures received no analogue. Thirty-eight PC or 2000 pg of glycine-l-*X or glycine-2-W, depending on the experiment, was added to each tray 4 hr prior to the termination of the culture. Following culture, the embryos were concentrated by centrifugation; washed twice in cold, filtered sea water; and lyophilized. Four groups of experiments were performed. The first was a screening test to determine which of four analogues would show the greatest inhibitory effect on cleavage and nucleic acid metabolism. The other three groups each consisted of a control experiment and an experiment to test the effects of each of the analogues selected for study on the basis of the screening experiment. Those selected were 2,4diamino-5-(p-chlorophenyl) 6-ethyl pyrimidine and 2,-i-diamino-5-(3’,4’-dichlorophenyl) 6-ethyl pyrimidine. Chemical procedures were as follows. Lipids were extracted from lyophilized embryo powders by treatment with ethanol, ethanol-ether, and ether. Nucleotides and nucleic acids were extracted by the method of Schmidt and Thannhauser [31] with minor modifications. Purines were liberated from mixed nucleic acids, RNA, DNA, and an aliquot of the acid-soluble nucleotide fraction by acid hydrolysis (HCl for 1 hr at 100°C). Purine baseswere isolated by ion-exchange chromatography as described by Heinrich et al. [21] and identified and quantified by their respective ultraviolet absorption properties at 250, 260, and 280 my (Beckman Du Spectrophotometer). Total RNA was determined with orcinol by the method of Mejbaum [28]; total DNA was determined with diphenylamine by the method of Seibert [33]. Radioactivity was determined from aliquots plancheted at infinite thinness and counted with a windowless gas flow counter. hdenosine monophosphate (AMP) was obtained by ion-exchange chromatography of an aliquot of the acid-soluble Esperimenlnl

Cell

Research

27

nucleotide fraction by the method of Hurlbrrt et cd. (251 with slight Irlotlil’ications. The AUUP was hydrolyzed to atlcninc and purified l>y elution through a I)owes 30 colmnn. The radioactivity was determined as described above and recalculatctl t’ot XRIP. The protein fraction from the nucleic acid cstraction was dried with ;I solvent series similar to that used for lipid removal. It was then dissolved in formic acid to produce a solution of known strength and planchetetl. Radioactivity was determined as described above. RESULTS

Table I gives the results of the screening test. It sho\vs the effects of four pyrirnidines on the uptake of glyine-1-‘YI by adenine and guanine of misetl nucleic acids. The T!C ratio espresses inhibition in ternls of the result obtained by dividing any measurement rnatle on the activities or yielas of treated culture by the correspondin,. dnleasurement from the control culture and multiplying by 100. All four pyrimidines caused a pronounced cleavage delay. The inhibition of specific activity of mixed nucleic acid purines 1~) 2.4~diamino pyrimidines n-as much more niarked than that produced b\ the f-aminopyrazolo derivative. The two dichlorophenyl derivatives had similar inhibitory effects. Because they produced the greatest effect on the rate of cleavage, on the morphology of the blastolneres during cleavage, and on the activity of nucleic acid purines , 2,4-tliamino-S-(p-chlorophenyl) and 2,4-diamino 5-5-(3’,4’-dichlorophenvl) pyrirnidines were selected for further study. Since the two con~pounds differ structurally only in the number of chlorine atoms on the 3-aryl group, they shall be designated as the p-chloro ant1 the dichloro derivative respectively. I. Incorporntion

TABLE

of’ glycincj-1 -I% into prims of’ mixed nucleic rccirl 01’ fbw pyrimidines. Specificactivity in counts per min/~.ll.

Esperinlent Control

.\denine

T/C “,

Guaninr

T/C (“,

2r45

3 I 89

2.4~diamino-5-(3’,4’-dichlorophengl)Gethyl

213

6.i

10.5

3.x

2.-l-diamino-5-(p-chlorophenyl) C-ethyl pyrimidine

l(l9

34

6s

2.5

2.4.diamino-5-(3’,4’-dichlorophenyl)&methyl pyrimidinc

‘41

75

158

5.8

1125

35.5

4.aminopyrazolo pgrimidine Erperimentul

pyrimidine

(3,-M)

Cell Research

27

124’

45.1

Effecfs of pyrimidines

on cleavage and metabolism

in embryos

253

The dichloro derivative produced a delay of one or two cleavages after 17 hr of culturing. Abnormal blastomeres were erident at 21.5 hr. -At 24 hr there was a delay of two or more cleavages, and some embryos were disorganized. Only a fe\v embryos blastulated, and those that hatched lacked TABLE

II.

I’ield NO.

1A 1B IC 2 .I 'LB 2c 3 *i 3B 3c

TARLE

III.

I’ield

of RS.4 Esperiment

RN;\

Control p-Chloro Dichloro Control p-Chloro Dichloro Control +Zhloro Dichloro

57

Experiment

1A IB IC 2 A 2B

Control p-Chloro Dichloro Control p-Chloro Dichloro Control p-Chloro Dichloro

3 A

3B 3c

60 57

11 39 37

of lipid-free

powder.

T/C O<,

10s 100 95 90

38

39 38

of DA\‘=1 in pglnzg

No.

2c

in ,ug/mg

DNA

102 1 no

of lipid-free

powder.

T/C “.

x.5 5.0

59

5.3

62

7.3 4.3

59

4.0

55

7.1 3.9 3.9

55 55

the gastrocoele. The results with the p-chloro derivative were similar, but the disorganization of embryos was greater at 24 hr. Table II shows the total RN.1 in each of three series of experiments performed on three different batches of embryos. The T/C percentage does not fall below 100 except in Series 2; even there the difference is not considered significant. Table III gives similar data for DNA. The T/C value is about the same for the two inhibitors within each series, and varies little among the three series. The specific activity of purine bases of RNA is shown in Table I\‘. The Experimental

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radioactirc precursor in Series 1 and Series ‘1 \vas gI\-cine-2-14t:. ant1 in Series 3 it \vas glvcine-l-14C. In almost all instances, the inhibitor!. clt’ect to the I)-chloro compound is somewhat greater than that of the tlichloro con~pound. This tendency has been notetl in all esperimcnts performetl to

No.

Esperitnent

1 r\ lB 1c 2A ‘B 2C 3A 3B

Conlrol p-Chloro Dichloro Control p-Chloro Dichloro Control [Khloro Dichloro

3c

TABLE

Guanine

V. Specific activity Esperiment

NO.

1A IB 2A ‘B 2c

Guanine

Control p-Chloro Control p-Chloro Dichloro Control p-Chloro Dichloro

3 A 3B 3c

of purine basesof DlV.1 in counts per min/pSI. r/c:

0”

9 38 39

Xclenine 1460 140 2590 1040 1175

T/C ‘1”

III 411 45

14Zl 35

516

:<6

39

656

46

date in this laboratory and has been found to apply to the incorporation of isotopic glycine into nucleic acid purines, acid-soluble purine nucleotides, and proteins. Table I’ sho\vs the specific activity of the DNA purines. These results exhibit greater uniformity than do those for the RKV,\ purines, \\‘ithin a given experiment, the depression of activity of adenine and guanine is approximately the same. The lowering of specific activity of DNA purines in the experimental cultures of Series 1 far exceeds that of the other two series, in which the inhibitory effects do not differ markedly from those of RNA from embryos of the same cultures. Experimental

Cell

Research

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Effects

of pyrimidines

on cleavage and metabolism

in embryos

255

Table 1’1 gives the specific activities of the proteins in Series 2 and 3. There is a definite depression of activity in all experimental cultures except the one containing the dichloro derivative in Series 3, where the incorporation of glycine probably is similar to that of the control culture. The inhibitory VI.

TABLE

Specific activity

No.

Experiment

2A

Control p-Chloro Dichloro Control p-Chloro Dichloro

2B 2C 3 .I 3B 3c

TABLE

VII.

of proteins in counts per min/,uJI. Precursor

Cpm/mg

Glycine-2-W Glycine-2-11C Glycine-2-W Glycine-1-W Glycine-l-l% Glycine-I -1%

T/C ?b

728 505 639

69 7-l

368

290

78 91

335

Specific activity of acid-soluble adenine and gurrnine from the screening test in counts per minlpuill. Experiment

Control p-Chloro Dichloro

TABLE

VIII. Glycine-2-W

Experiment Control p-Chloro Dichloro

Adenine

T/C ?;

Guanine

170

16

4950 28

565

68

216

1122

Specific activity

1

6 44

of AMP in counts per nzin/pudl.

was used in series Series

T/C Ob

1 and glycine-1-W T/C ”

11200

in series 3.

Series 3

T/C 00

4550

2500

22

1662

37

3200

29

1810

40

effects produced in proteins by the two pprimidines are less serere than those produced in nucleotides and nucleic acids. Table VII shows the effect of the two analogues on specific activity of the total acid-soluble nucleotide fraction from the screening test. The inhibition is not as great as in the mixed nucleic acids of the same screening test. Guanine in control embryos has a much higher specific activity than does adenine, Esperimenlcd

Cell Research

27

and in the esperimcntai cultures the inc~c)rl~or~~tion of latwl into gualiinc i*; more severely inhil)itecl than incorporation into aclt~ninc. Table I’ll1 sho\vs the s[wcitic activitv . of A\II on Series 1 ancl ;$. The ‘I’/(: ptwcntagcs arc very close to those for RX;\ atlenine in the same series. In Series 3 the T:‘(: percentages for guanosine mono-, (Ii-, and tril~hosl~hates and for adenosine mono-,di-, ant1 triphosphates all lie betlveen 36 anal 40 per cent for the p-chloro derivative and bet\veen 40 and 30 per cent for the dichloro derivative. The size of the acid-soluble nucleoticle pools of mono-, di-, and triphosphates of both adenosinc and guanosine has been tlrtcrmined in Series 1 and 3, and there is no evidence that either of the analogues causes any decrease in the size of these pools.

DISCUSSION

hlirsky and Ris [?$I], Thompson et nl. [XI], Elson et rrl. [12], etc., haw shown that the amount of DNA per cell is constant for many organisms including sea urchin embryos. AIcRIaster [Zi], Daoust et al. [i], etc., have shown that this constancy is maintained by doubling of DNb prior to each cell division. Since the T/C percentage for DNA yield is about the same for all experimental cultures and since the yield represents approximately the amount of DNA produced after fertilization, it is likely that the T/C percentage for the number of cell divisions is roughly the same in all esperimental cultures. The marked difference in inhibition of specific activity of DN.1 purines bet\veen embryos of Series 1 and Series 3 in cultures containing the p-chloro derivative suggests that cells of one culture must hare been synthesizing DNA and diriding more rapidly than those of the other at some time during culture. The work of Fresco et trl. [11], Cohn and Yolkin [3] and Pelt [SO] supports this assumption. \\‘here inhibition involves almost exclusively DNAI, there is commonly a retardation or cessation of cleavage. Barner and Cohen [J] and Dunn and Smith [lO] hare observed this in Escherichin coli, and \Yelch [37] mentioned similar results in a variety of bacterial and mammalian cells. The analogues used in the present study hare been reasonably well established as being folic acid antagonists. Folic acid inhibition is kno\vn to depress DA’;\ synthesis by interference with purine production and with methylation of thyminc precursors. Cleavage retardation probably is due largely to decrease in DN.1 synthesis. In Series 1 the T/C percentages for specific activity of DNA4 purines in cultures esposed to the II-chloro derivative are much lower than those fo1 Erperimentnl

Cdl

Rrsenrch

27

Effects of pyrimidines

on cleavage and mefabolism

in embryos

257

RNA purines and ,UlP. This map hare been due to a sudden drop in DN.4 synthesis, possibly just before the isotopic glycine was added. Perhaps the analogues may hay-e caused more drastic interference with the methylation of thynine precursors than with purine synthesis so that thymine suddenly became limiting and DNA synthesis dropped to a low level. There is some likelihood that the pgrimidines may hare diminished inosine monophosphate, which is a metabolic fountainhead for purine nucleotide and nucleic acid purine production, at a time when this subtance was needed in large amounts for the synthesis of some compound(s) other than DN-1. The equal depression of specific. activity of DNA4 adenine and guanine in embryos of Series 1 exposed to the p-chloro derivative and the very high specific activity of ,IhlP in the control esperiment appears to harmonize with this explanation. I’illee et nl. [36], Schmidt et al. [32], and Elson and Chargaff [ll] have shown that in sea urchin embryos, there is no synthesis of RNA during the period estending from fertilization through blastulation. This finding offers a logical explanation for the failure of the two pgrimidines to effect RNA yield. Lack of uniformity in specific activities of RN=\ might be ascribed to the fact that the RN&\ is likely a mixture of nuclear, soluble and ribosomal, all having different specific activities. Possibly some variations in RNA could arise because of the attachment of purine or pyrimidine to side chains by esterification as reported by Crestfield and Allen [6], etc. The lowering of specific activity in the proteins can be said only to reflect a decrease in the incorporation of amino acids. The effect is mild compared with most of the others. This effect might be the result of decreased DNA synthesis as has been suggested for Staphylococcus aureus by Gale and Folkes [15, 16, 171 and calf thgmus nuclei by Allfrey [ 1 ] and Allfrey et trl. [2, 31. Much of this protein probably is directly associated with DN.1 since it must be separated from DN-4 by a somewhat drastic hydrolysis. The inhibition of label incorporation into proteins is greater with the glycine-2-W precursor, which can produce a labeled one-carbon-fragment. The results from the screening test show extremely severe inhibitions which are more pronounced in the mixed nucleic acids than in the acid-soluble nucleotides. hlost of the analogue effects observed in this study are probably largely due, directly or indirectly, to interference with the formation of formylglycinamide ribotide and/or 5-formamido-4-imidazole-carbosamide ribotide m the process of purine synthesis. Interference by analogues with the methylation of thynine precursors might play a prominent role in inhibition. Experimenlnl

Cell Reseurch

27

SUMMARY

The clfects of ‘,4-diamino-.~-( IJ-chtorol’l’ct~~t) ti-ethyl pyrimitlintB :tt~tI ‘L,4-diamino-.i-(5’,4’-tlichl~~ropl~e~~~l) (i-ethyl pyrimidine o11 the tlevelopllwnt of embryos of the sea urchin, Stronyy1ocentrottz.s purpuvttlu, \I-ere studied. These two w~up~u”ds are k~lowr~ to be folic acid antagonists. 2. Cleavage \\-as retarded in all cultures exposed to the antimetabolites. Cells of odd shapes and sizes were frequently produced in such cultures. 3. The specific activity of I)h’.\ purines was sharply reduced in embryos esposed to the t\vo antimetaholites with either glycine-l-14C or glycincb-2-1%Z The amount of TIN-1 synthesis was only 33-60 per cent of as precursors. the normal yield. 4. The specific activity of IAN.1 purines \vas also markedly reduced, although in some instances less seriously than that of the DN;\ purines. The yield \vas not affected by the antimetabolites. 3. In one set of experiments the specific activity of adenine and guanine from the total acid-soluble fraction was greatly reduced in the cultures containing the analogies. In t\vo other sets of experiments, the specific activity of adenosine monophosphate was depressed to a degree commensurate with that sustained by purines of RN,% in the same experimental cultures. 6. The proteins isolated suffered a moderate depression of incorporation of labeled amino acid in some of the cultures exposed to the antimetaholites. 7. The decrease in incorporation of label by nucleic acid purines, nucleotides, anct proteins was almost invariably more in embryos exposed to 2,4diamino-5-(p-chlorophenyl) B-ethyl pyrimidine than in those exposed to 2,4-diamino-3-(3’,4’-dichlorophenyl) G-ethyl pyrimidine. 1.

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on cleavage and metabolism

in embryos

259

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Experimental

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