Hydroxyurea inhibition of greening, macromolecule synthesis and cell division in Euglena gracilis

395

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95515

H Y D R O X Y U R E A I N H I B I T I O N OF G R E E N I N G , MACROMOLECULE SYNT H E S I S AND CELL D I V I S I O N IN E U ( ; L E N A G R A C I L I S

D. E. B U E T O W * AND J. L. M E G O

Geronlology Branch, National Heart Instit~f/e, Nalional h~stitutes o/Health, Public Health Service, U.S. Deparh~ent o/ Health, F~ducation & Wel/are, Bethesda, and the Baltimore Cily Hospitals, Baltimore, Md., and The Nettrosurgery Research Laboralory, Baltimore City Hospilals, Baltimore, Md. (U.S.A.) (Received J u n e z8th, I966)

SUMMARY

Hydroxyurea inhibits chlorophyll synthesis and cell division in heat-bleached or dark-grown Euglena gracilis. 5o °i) inhibition of chlorophyll synthesis occurs at o.o2-o.o35 M hydroxyurea. Euglena multiplies with a normal generation time and can at least partly recover the ability to green following removal of hydroxyurea. The syntheses of chlorophyll, DNA, RNA and protein are inhibited by hydroxyurea, that of chlorophyll being the most sensitive, followed in order by DNA, RNA and protein.

INTRODUCTION

Hydroxyurea is active against a variety of leukemias 1,2, tissue-culture cells originally derived from tumors a G, certain animal viruses 7, and bacteria 8,9. In the case of Escherichia coli C600, hydroxyurea is bacteriostatic rather than bactericidal 9a°. FISrrBEL',T AND CAm~ON~;'t suggested that hydroxyurea inhibits oxidative phosphorylation. Evidence has accumulated, however, that the antitumor activity of hydroxyurea appears directed primarily at DNA synthesis. Hydroxyurea has a degradative effect on isolated DNA (ref. I2) and causes breakages and abnormalities in the chromosomes of mouse-embryo cells in culturO 2, of a Chinese-hamster cell line a, and of Pseudomonas aeruginosa s. Thymidine and/or a2P-labeled orthophosphate incorporation into DNA is inhibited by hydroxyurea in ascites cells 4-6 and E. coil TM. The incorporation of RNA and protein precursors is little affected, if at all, b y concentrations of hydroxyurea which are highly inhibitory to DNA synthesis 5,6,9,1°. * P r e s e n t a d d r e s s : D e p a r t m e n t of P h y s i o l o g y a n d Biophysics, U n i v e r s i t y of Illinois, U r b a n a , Ill. (U.S.A.)

t~'iochim. 14iophys. Acla, 134 (~967) 395-4o~

390

i). E. BUETOW, J. L. MEGO

In P. aeruginosa cultured in the presence of hydroxyurea, the ratio, D N A : R N A , is reduced b y 38-61 ~, (ref. 8). Further, hydroxyurea inhibits the conversion of cytidine to deoxycytidine in the bone marrow of rats la and YOUNC; aND HODAS5 concluded that hydroxyurea inhibits the reduction of ribonucleosides to deoxyribonuclosides in the H e L a cell. The photosynthetic apparatus of Ez~glena gracilis is exceptionally labile and can be bleached by a variety of chemical and physical agents. In the present study, hydroxyurea is shown to be inhibitory to greening in Euglena as well as to cell division and the synthesis of DNA, RNA and protein. A preliminary report of this study has appeared aa.

MATERIALS AND METHODS

Organism and growlh conditions E. gracilis, strain Z, was grown at room temperature in a medium ~5 containing the following per 1:2.5 g proteose-peptone, 2.5 g bacto-tryptone, o.I g yeast extract, o . I g MgSO4.7H20 and I . o g each of sodium a c e t a t e ' 3 H ~ O , glucose, KHePOa and K oHPO 4. Heat-bleached cells were prepared by growing cultures in a water bath at 34.5 ° under incandescent light (2oo W) for 3 days. Dark-grown organisms were produced ~6 b y wrapping freshly inoculated flasks with aluminum foil and placing them in a light-proof incubator at 25 ° for 5 days. Subsequent greening experiments were done at room temperature with incandescent light (total of 25o W) placed above the flasks. Cell counts were made with a Coulter counter.

MSSayS

Cells were concentrated by centrifugation and assayed for chlorophyll by extraction with 8o % acetone for 2 h in the dark. The absorbance of the extract was measured at 663 and 645 m/~ with a Beckman DU spectrophotometer. Chlorophyll concentration was calculated according to ARNON1L Total protein was determined on triplicate samples by the method of Lowboy et al. 18 as described 19 on concentrated cells washed once with o. 9 o/,.o NaC1. Triplicate samples of saline-washed cells were assayed for RNA as previously described 19. Cell pellets remaining after RNA extraction were extracted for DNA by heating twice (15 min each extraction) at 9 °0 in I.O M perchloric acid (I.3 ml total). Insoluble material was centrifuged out and the supernatants were filtered through a medium-porosity flitted-glass disc filter and measured for DNA content by the diphenylamine procedure of BURTON2°.

RESULTS

H y d r o x y u r e a at 0.035 M inhibited both chlorophyll synthesis and cell division in Euglena when added to cultures placed in the light following either 5 days growth in the dark (Fig. I) or 3 days growth at high temperature (Fig. 2). Dark-grown cells were almost at the stationary phase of growth when placed in the light. During a 7o-h period in the light, the control culture increased from 520 ooo to 690 ooo cells Biochim. Biophys. Acta, 134 (~967) 395 4 ol

397

HYDROXYUREA INHIBITION OF EUGLENA

per ml, whereas no increase in cell density was observed in the presence of hydroxyurea. Control heat-bleached Euglena increased from x8o ooo to 71o ooo cells/ml when placed in the light at room temperature (Fig. e). In the presence of hydroxyurea, heat-bleached Euglena also increased in cell density (18o ooo to 34 ° ooo cells/ml), but less than control cells. Following an initial decline in chlorophyll content per cell at room temperature, heat-bleached Euglena began a net synthesis of chlorophyll

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Fig. I. H y d r o x y u r e a and d a r k - g r o w n EugIena. Cells were g r o w n in the dark at 25 ° for 5 days and then placed in the light (incandescent bulbs, 25 ° \V) at r o o m t e m p e r a t u r e with and w i t h o u t h y d r o x y u r e a . Ceils per ml and chlorophyll c o n t e n t are s h o w n for control cultures ( O - D ) and cultures with o.o35 M h y d r o x y u r e a (O - O ) during exposure to light. Fig. 2. H y d r o x y u r e a and heat-bleached Euglena. Cells were g r o w n for 3 days at 34.5 ° and t h e n placed m the light (incandescent bulbs, 25 ° W) at r o o m t e m p e r a t u r e with and w i t h o u t h y d r o x y urea, o.o35 M. S y m b o l s as in Fig. I.

by 48 h, as observed previously 21. When 0.035 hydroxyurea was present, the cells synthesized only . about 5o o, /o as much chh)rophyll as control cells (Fig. 2). Table I shows the effect of increasing concentrations of hydroxyurea on the amount of chlorophyll synthesized by dark-grown Euglena placed in the light at room temperature. Concentrations of hydroxyurea above I mM were inhibitory. Chlorophyll synthesis was not blocked completely, however, even at o.2o M hydroxyurea. Table II shows the recovery of chlorophyll synthesis following removal of o.o5 M hydroxyurea. At the end of 69 h in the presence of hydroxyurea, Euglena contained only 37 #g chlorophyll per xo7 cells compared with a control value of I54/*g. Cells were then washed free of hydroxyurea under sterile conditions and placed in fresh medium for 93 h. Although these Euglena synthesized chlorophyll, they were not able to recover completely and synthesized only 7o o~ ( i i 9 # g ) as much as the control (I69 #g). In the case of cell division, there was little "carryover" effect of hydroxyurea when the cells were placed in fresh medium. A lag Biochim. Biophys. Acta, I34 (1967) 395 4 °1

398

i). E. BUETOW, J. L. MEGO

TABLE

I

HYDROXYUREA

INHIBITION

OF

CHLOROPHYLL

SYNTHESIS

E u g l e n a were g r o w n in t h e d a r k for 5 days, t h e n p l a c e d in t h e l i g h t (25 ° W, i n c a n d e s c e n t bulbs, r o o m t e m p e r a t u r e ) for 45 h in t h e p r e s e n c e of i n c r e a s i n g a m o u n t s of h y d r o x y u r e a a n d t h e n m e a s u r e d for c h l o r o p h y l l c o n t e n t . I n i t i a l c h l o r o p h y l l pe r lO 7 cells w a s 1.9/*g in E x p t . I a n d 4.4/*g in E x p t . II.

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I

Expt. II

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i63

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0.02 0.03 0.04 0.05

lO8 61 60 48

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29

0.20

23

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133 i3i 142 lO 3

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TABLE

2

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RECOVERY

OF

CHLOROPHYLL

SYNTHESIS

FOLLOV¢ING

REMOVAL

OF

HYDROXYUREA

E u g l e n a were g r o w n in t h e d a r k for 5 days, t h e n p l a c e d in t h e l i g h t (25o W, i n c a n d e s c e n t bul bs , r o o m t e m p e r a t u r e ) . One flask was t r e a t e d w i t h h y d r o x y u r e a , o.o 5 M, for 69 h as s h o w n be l ow in t h e p r o c e d u r e . A second flask, t h e control, w a s m a i n t a i n e d in t h e a b s e n c e of h y d r o x y u r e a throughout the experiment.

Procedure I 2 3 4 5

Hydroxy~rea Control (ttg chlorophyll per lO 7 cells)

Removed from dark, h y d r o x y u r e a added I 69 h in p resence h y d r o x y u r e a ; cells w a s h e d free of h y d r o x y u r e a 37 24 h a b s e n c e of h y d r o x y u r e a 64 44 h a b s e n c e of h y d r o x y n r e a ii8 93 11 a b s e n c e of h y d r o x y u r e a 119

I 154 147 108 I69

phase of zz h was observed, but the cells subsequently multiplied with a normal generation time of ie h. The effect of o.o5 M hydroxyurea on macromolecule synthesis is shown in Figs. 3 and 4. Euglena were dark-grown and then diluted 5 fold into fresh medium. Although this condition allowed cell division to occur, cells grew more slowly in the presence of hydroxyurea than in its absence. The rates of synthesis of chlorophyll, DNA, RNA and total protein by the cell population (plotted as/ag per ml of culture) were all inhibited in the presence of hydroxyurea. Chlorophyll synthesis was the most inhibited. Bv~ 96.5 h, the culture with hydroxyurea contained only 30 O//o as much chlorophyll per ml as did the control. Further, in contrast to the control, chlorophyll synthesis in the hydroxyurea-treated culture appeared to be linear in time after 24 h. Net RNA and DNA synthesis ceased at about 5o h in the hydroxyurea-treated culture but continued past 6o h in the control. Protein synthesis was the least inBiochim. Biophys. Acla, i34 (1967) 3 9 5 - 4 o l

399

HYDROXYUREA INHIBITION OF EUGLENA

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HOURSIN LIGHT Fig. 3. H y d r o x y u r e a and chlorophyll synthesis. Cells were g r o w n for 5 days in the dark at 25 °, diluted 5 fold into fresh m e d i u m and placed in the light (incandescent bulbs, 25 ° \V) at r o o m t e m p e r a t u r e w i t h and w i t h o u t h y d r o x y u r e a . D a t a s h o w n are cells per 1111 in control cultures ( 0 - 0 ) , and in cultures w i t h 0.035 M h y d r o x y u r e a (@ - - ( 2 ) ) , and chlorophyll per ml in control c u l t u r e s ( • - • ) , and in cultures with 0.035 M h y d r o x y u r e a ( A - - - / x ) . Fig. 4. H y d r o x y u r e a and macromoleeule synthesis. D a t a are from the sanle cultures as s h o w n in Fig. 3. O - O , control; © - - - © , 0.05 M h y d r o x y u r e a - t r e a t e d . TABLE III HYDROKYUREA

INHIBITION

OF

RATE

OF

PROTEIN

AND

NUCLEIC

ACID

SYNTHESIS

D a t a are t a k e n from the linear p o r t i o n (up to 5 ° 7° h) of the curves s h o w n in Fig. 4.

Control

Hydroxyurea (l~g synthesized/ml/h )

DNA RNA Protein

o.o 16 0.229 2.42

0.006 o. 132 1.62

Hydroxyurea Control o.38 0.58 o.67

hibited and continued linearly throughout the course of measurement. Slopes of the linear portion of the protein and nucleic acid curves (up to 50--70 h) in Fig. 4 were' determined in order to evaluate rates of synthesis of these macromolecules b y the celi JBiochim. Biophys. Acta, I34 (1967) 395 4 °x

40o

D.E.

BUETOW, J. L. MEGO

population. D a t a are given in Table I I I a s / , g synthesized per ml of culture per h in the presence and absence of hydroxyurea (o.o5 M). When compared with control rates, the rate of synthesis of DNA was the most inhibited followed in order by RNA and protein.

DISCUSSION

Chlorophyll synthesis in Euglena is inhibited by hydroxyurea to an extent which depends on the amount of hydroxyurea present (Table I). However, Euglena cells can at least partially recover the ability to green following removal of hydroxyurea (Table II). This result is in contrast to the permanent effect of some bleaching agents, such as streptomycin 22, on Euglena. The high concentrations of hydroxyurea required for inhibition of chlorophyll synthesis in Euglena are reminiscent of the high concentrations required to inhibit the growth of E. coli (o.o35 M for 5o rJ~) inhibition) 9a°. The growth of P. aerugb~osa was depressed by 50 % at 0.0o25 M hydroxyurea, but complete inhibition was not observed even when the concentration was raised to o.oI M (ref. 8). The growth of Euglena was inhibited under conditions allowing exponential growth (Figs. 2, 3) or under conditions allowing only a small amount of residual growth in the culture (Fig. I). However, the extent of inhibition of chlorophyll synthesis by a given concentration of hydroxyurea did not increase as the number of cell divisions in its presence increased, a result in contrast to that previously observed in the case of streptomycin ~6 and heat 21 intfibition of greening in Euglena. Figs. I and 2 both show a m a x i m u m 5o ~, inlfibition of greening by o.o35 M hydroxyurea. The action of hydroxyurea appears to be more like that of 5-fluorouracil "a or hadacidin 1~ wtfich inhibit chloroplast formation in non-dividing cells perhaps by interfering with ribosome formation. In the presence of o.oI o,2o M hydroxyurea, the usually ellipsoidal Euglena became thin and elongated. Hydroxyurea also caused a marked elongation of P. aerztgi,wsa 8 and E. coli TM. In spite of the morphological changes, hydroxyurea at o.o5 M did not kill Euglena since, when washed free of the inhibitor, the cells resumed exponential growth with a normal generation time after an initial lag period of I I h. Related to these observations is the report that hydroxyurea is bacteriostatic rather than bactericidal for E. coli%~o. H y d r o x y u r e a inhibition of the rate of DNA synthesis is more marked than RNA or protein (Table I I I ) . H y d r o x v u r e a inhibition appears directed primarily at DNA synthesis in a number of cell systems. In ascites cells 5,6 and E. coli 9'~° hydroxyurea at concentrations which are highly inhibitory to DNA synthesis has little effect, if any, on the incorporation of RNA and protein precursors. In contrast however, in the present Euglena, the rates of RNA and protein synthesis by the population are also significantly reduced by hydroxyurea (Table I I I ) . Chlorophyll synthesis is the most inhibited bv hydroxyurea, a result which focuses attention on the chloroplast. Euglena chloroplasts contain a unique species of DNA (refs. e4-e6 ) and evidence has been presented for distinct chloroplastassociated RNA (refs. 27, 28). Further work inw)lving the subfractionation of Euglena is necessary to determine whether hydroxyurea preferentially affects nucleic acid and protein synthesis in the chloroplasts of these cells. Biochim. Biophys..4c/a, 134 (1967) 395-4o~

HYDROXYUREA INHIBITION OF EUGLENA

401

ACKNOWLEDGEMENTS

The authors thank Mrs. M. L. ROBINSONand Mr. P. J. BUCHANANfor technical assistance. I~EFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20

21 22 23 24 25 26 27 28

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Biochim. Biophys. Acta, 134 (1967) 395-4Ol