16

Biochimica et Biophysica Acta, 476 (1977) 16--23 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 9 8 9 1 8

F U R T H E R STUDIES OF THE ACTION OF METHIONYL ADENYLATE ON CHICK EMBRYO FIBROBLASTS

a F R A N C O I S E L A W R E N C E , b D A V I D A. L A W R E N C E , a M A L K A R O B E R T o G E R O and a P I E R R E B L A N C H A R D

a Institut de Chimie des Substances Naturelles, 9 1 1 9 0 G i f s u r Yvette and b Fondation Curie, Institut du Radium, 9 1 4 0 0 Orsay (France) (Received O c t o b e r 2 9 t h , 1976)

Summary Methionyl adenylate (MetAMP) inhibits protein synthesis by interacting with methionyl-tRNA synthetase. Addition of 1--3 mM inhibitor to chick embryo fibroblasts rapidly stops protein synthesis and DNA synthesis but not RNA synthesis. These effects can be reversed by renewal of the medium. The extent and reversibility of protein and DNA syntheses depend on the concentration of MetAMP in the cultures, the length of exposure and the cellular density. MetAMP is recognised by several enzymes as substrate and/or as inhibitor. MetAMP is degraded to methionol plus 5'-adenylic acid by 5'-phosphodiesterase. Adenosine deaminase, adenylic acid deaminase and 3':5'-phosphodiesterase cannot use MetAMP as substrate but the last enzyme is inhibited. The presence of MetAMP in cultures provokes a small but reproducible increase in the level of methionyl-tRNA synthetase and 5'-phosphodiesterase.

Introduction Previous studies have shown that aminoalkyladenylates which are very p o t e n t and specific inhibitors of aminoacyl-tRNA synthetases in vitro [1] can also operate in vivo since the growth of procaryotic [2,3] as well as eucaryotic cells [4] is inhibited by L-methionyl adenylate (MetAMP). The purpose of the present work was to investigate some of the consequences on macromolecular syntheses and on certain enzymes of the presence of MetAMP and its stability in chick embryo fibroblasts.

Abbreviations: MetAMP, methionyl adenylate; cyclic AMP, 3':5'-cyclic adenylic acid; Met-tRNAsynthetase, methionyl-tRNA synthetase.

17 Materials and Methods

Cells and media. Experiments were performed with secondary cultures of lymphomatosis-free Brown Leghorn chick embryo fibroblasts, prepared as previously described [5]. Falcon petri dishes were seeded with either 3 . 1 0 s 5 • l 0 s chick e m b r y o fibroblasts/35 mm dish or 5 • 106--10 • 106 chick e m b r y o fibroblasts/100 mm dish to obtain fast growing cultures, or 1 • 106--2 • 106 chick embryo fibroblasts/35 mm dish to obtain density-inhibited cultures. The medium was Ham F-10 [6] (Flow Laboratories Inc. Rockville, Md.) plus 5% calf serum and antibiotics (penicillin, streptomycin, kanamycin and fungisone). Incubation temperature was 37 ° C. Effect on macromolecular synthesis. Following different exposure times to various MetAMP concentrations, L-[4-3H]leucine (35 Ci/mM, 5 pCi/ml), [5-3H]uridine (25 Ci/mM, 1 pCi/ml) [7] or [Me-3H]thymidine (52 Ci/mM, 2 pCi/ml) were added to the cultures for 1 h at 37°C. Uptake of radioactive precursors into the acid-soluble pool and their incorporation into macromolecules were followed by one or other of two previously described methods [4--8]. Preparation of cell-free extracts. Extraction was carried out at 4°C as previously described [4], except that the extraction buffer was 20 mM potassium phosphate, pH 7.5, containing 20 mM 2-mercaptoethanol. Protein concentration was determined by the Lowry method [9] with crystalline bovine serum albumin as standard. Enzyme assays. Adenosine deaminase (EC 3.5.4.4) and adenylic acid deaminase (EC 3.5.4.6) activities were followed by measuring the change of absorbance at 265 nm resulting from the conversion of adenosine to inosine or 5'-AMP to 5'-IMP, respectively [10]. 5'-Phosphodiesterase activity (EC 3.1.4.1) was assayed according to the method of Razzel and Khorana [11] using bis-p-nitrophenyl phosphate and adenosine 5'-p-nitrophenyl phosphate as substrates, the latter one being preferred to thymidine-p-nitrophenyl phosphate because of its closer chemical structure to MetAMP. One unit of enzyme activity is defined as 1 pmol of substrate hydrolysed in 1 h at 25°C per mg of protein. Methionyl-tRNA synthetase (EC 6.1.1.10) was measured as described previously [4]. 3' : 5'-cyclic AMP phosphodiesterase (EC 3.1.4.17) and 3' : 5'-cyclic GMP phosphodiesterase (EC 3.1.4) were assayed as described by Brooker et al. [12]. One unit of enzyme activity is defined as 1 nmol of cyclic nucleotide hydrolysed in 20 min at 30°C per mg of protein. RNAase (EC 2.7.7.16) activity was estimated according to the method described by McDonald [13]. Cyclic AMP-dependent protein kinase was assayed using the method described by Miyamoto et al. [14] as modified by Gilman [15]. Cyclic AMP level was determined as previously described [16]. Chemicals. Adenosine deaminase from calf intestine 200 units/mg and brewer's yeast tRNA were products from Boehringer; adenylic acid deaminase from rabbit muscle, 5'-phosphodiesterase from snake venom, 3' : 5'-cyclic AMP phosphodiesterase from bovine heart, bovine heart protein kinase, ATP, cyclic

18 AMP and cyclic GMP were purchased from Sigma. Bis-p-nitrophenyl phosphate and adenosine 5'-p-nitrophenyl phosphate were from Merck. L-[4-3H] Leucine, [ 5-3H] uridine, [Me-3H]thymidine, sodium [ 32p] pyrophosphate, L-[Me-t 4C] methionine were obtained from the Commissariat ~ l'Energie Atomique (Saclay, France), cyclic [3H]AMP and cyclic [3H]GMP from the Radiochemical Centre (Amersham, U.K.). MetAMP was prepared in the laboratory as described earlier [ 1]. Results and Discussion

Effect of MetAMP on macromolecular biosyntheses Protein synthesis. Table I shows that when 2 mM MetAMP, was added at the same time as the labelled leucine, inhibition of the incorporation of the radioactivity into hot acid-insoluble material occurred very rapidly; thus, after 10 min there was already 33% inhibition of protein synthesis in normal non-confluent chicken embryo fibroblasts. Since protein synthesis inhibitors have been reported to decrease amino acid uptake [17--19], we checked what was happening to the acid-soluble pool. As shown in Table I, the rapid inhibition of protein synthesis was not due to a lower uptake of leucine into the treated cells, since acid-soluble material was not significantly affected, at least until 40 min by 2 mM MetAMP. Beyond this time, the soluble pool decreased. RNA synthesis. Confluent chicken embryo fibroblasts treated for 3 h with MetAMP up to 3 mM were not affected in their capacity to synthesize RNA, measured by uridine incorporation into acid-insoluble material. However, a slight inhibition (13%) was obtained with non-confluent chicken embryo fibroblasts treated with 3 mM MetAMP for 3 h. Furthermore, the uridine incorporated into acid-insoluble material of confluent cells was one-third that of nonconfluent cells. This decrease in uridine incorporation in confluent cells as corn-

TABLE

I

EFFECT OF MetAMP ON THE INCORPORATION OF LEUCINE INTO THE ACID-SOLUBLE POOL AND INTO HOT TRICHLOROACETIC ACID-INSOLUBLE MATERIAL OF NORMAL NON-CONFLUENT CHICKEN EMBRYO FIBROBLASTS Non-confluent cultures of chicken embryo fibroblasts, seeded with 3 - 105 cells per 35 mm dish were incubated 1 day after plating with 2 mM MetAMP and 2 pCi L-[3H]leucine/ml for various times. Determ i n a t i o n o f r a d i o a c t i v i t y w a s c a r r i e d o u t o n a s o d i u m d o d e c y l s u l f a t e - E D T A cell l y s a t e as d e s c r i b e d p r e v i o u s l y [ 4 ] . a, p e r c e n t o f i n c o r p o r a t i o n o f l e u c i n e in t r e a t e d c e i l s r e l a t i v e t o c o n t r o l cell. b , p e r c e n t o f l e u c i n e i n t h e a c i d - s o l u b l e p o o l o f t r e a t e d c e l l s r e l a t i v e t o c o n t r o l ceils. Time (min)

2 5 10 20 40 60 90

2 mM MetAMP a

b

110 93 67 66 64 46 38

(109) (103) (80) (91) (90) (79) (83)

19

100

c

00

i

x £

IQ-...o o

,"so o

~ i 50

u o

E u 4Z 0

i

I

i

2

L 0 3 ~M E4etAMP]

i

~

2

L

amM ~etAMP3

Fig. 1. Effect of MetAMP on the acid-soluble pool and incorporation of macromolecular precursor into normal non-confluent chicken embryo fibroblasts. Cells w e r e t r e a t e d for S h with various MetAMP concentrations a n d t h e n labelled 1 h with 2 pCi [3H]leucine, or 1/~Ci [3H]uridine or 2 ~uCi [3H]thymidine. Radioactivity was measured in the acid-soluble and acid-insoluble fractions as described previously [8]. Results are expressed in percent of specific radioactivity r e m a i n i n g a f t e r t r e a t m e n t . X X, uridine; o-~), thymidine; • e, leucine.

pared to non-confluent cells, is in agreement with the work of Weber and Rubin [20], who showed that chicken embryo fibroblasts reduce the rate at which they incorporate [3H]uridine into RNA as their growth becomes density inhibited. The decline in [3H]uridine incorporation was paralleled by a decline in the rate of uptake of the isotope into the acid-soluble pool. DNA synthesis. Thymidine incorporation into acid-insoluble material was strongly inhibited by MetAMP. Confluent cells were less sensitive than non-confluent chicken embryo fibroblasts. Thus, after 3 h treatment with 1 and 3 mM MetAMP the percentage of specific activity remaining in treated cells was 32 and 20% in non-confluent cells and 60 and 40% in confluent cells, respectively. Inhibition was concentration dependent (Fig. 1). The decrease in incorporation was not directly due to a lower uptake of thymidine into treated cells since even when the amount of [3H]thymidine in the acid-soluble pool of the cells was not affected (1 mM MetAMP, Fig. 1) there was a drastic inhibition of thymidine incorporation into the acid-insoluble material. Higher MetAMP concentrations lead to a strong decrease of thymidine in the acid-soluble pool (Fig. 1). This result could be explained either by inhibition of thymidine uptake analogous to that reported by high levels of heterologous nucleosides [22] and/or the involvement of protein synthesis for thymidine uptake. The reduction in DNA synthesis presumably results, from inhibition of protein synthesis by MetAMP and not from an interaction of the drug with elements of the replicative machinery, since treatment with cycloheximide [23] or with puromycin [24] produced a similar diminution in the rate of DNA synthesis in other eucaryotic cells. Furthermore, it has been shown that protein synthesis is required for both the initiation of DNA synthesis and for its elongation [25].

20

Enzymatic modifications of MetAMP In preliminary experiments on entry of MetAMP into chicken e m b r y o fibroblasts, we measured the MetAMP c o n c e n t r a t i o n in culture media as a function of time. Since radioactive MetAMP was not available, the c o n c e n t r a t i o n was estimated indirectly by the inhibition of the ATP ~ PPi exchange catalysed by an Escherichia coli m e t h i o n y l - t R N A synthetase. We f o u n d that MetAMP disappeared from the culture media during growth and th at its disappearance was t e m p e r a t u r e and c o n c e n t r a t i o n dependent. For example, assays at 6 h and 24 h revealed that the c o n c e n t r a t i o n dropped from 0.5 mM MetAMP to 0.35 mM and to 0.20 mM, respectively, at 37°C, but more rapidly at 41°C, where the respective values were 0.29 and 0.14 raM, whereas at 3 mM MetAMP, the values were 2.7 and 1.98 mM (37°C) and 2.25 and 2.07 mM (41°C). If this diminution was solely due to the penet rat i on of MetAMP into cells, and on the basis o f the relationships, 106 cells ~ 2.55 gl [5] or 106 cells - 1.3 pl [26], then the intracellular c o n c e n t r a t i o n of MetAMP would be above 100 mM after 6 h c o n t a c t with exogenous 0.5 mM MetAMP. This concent rat i on seemed to us very high bearing in mind the decreased rate of protein synthesis and the inhibition constant of MetAMP for m e t h i o n y l - t R N A synthetase in chicken e m b r y o fibroblasts [4]. Since previous studies showed that adenosine was a weaker inhibitor of E. coli Met-tRNA synthetase [27] than MetAMP, and that modifications at positions 1, 2 and 6 of the purine ring of adenosine largely eliminate the inhition of the ATP ~ PPi exchange reaction [28], we assumed th at if MetAMP was modified, the Met-tRNA synthetase inhibition would be smaller and this could than a c c o u n t for an overestimation o f the penetration of MetAMP into cells. As a first step towards testing for a possible degradation of MetAMP, we studied ~he action of variods commercially purified enzymes potentially able to m o d i f y MetAMP, and then studied the stability of MetAMP in a crude e x t ract from chicken e m b r y o fibroblasts. Adenosine deaminase from calf intestine was unable to deaminate MetAMP to m e t h i o n i n y l inosylate, since no change in absorbance at 265 nm was observed even after 18 h incubation at 25°C of 0.033 mM MetAMP with 4 pg of protein. Th e e n z y m e was still fully active since addition of adenosine after 18 h to the incubation mixture was immediately followed by a decrease in absorbanee. The reaction with adenosine is complete in less than 30 s. As expected f r om these results a crude extract from normal chicken e m b r y o fibroblasts, was unable to deamn, ate MetAMP. Adenylic acid deaminase from rabbit muscle was also unable to deaminate MetAMP. 5'-Phosphodiesterase from snake venom degrades MetAMP to methioninol plus 5'-AMP (ehromatogram not shown). Similarly, MetAMP incubated with a crude ex tr act from chicken e m b r y o fibroblasts is converted to methioninol and 5'-AMP, of which the latter does not accumulate. MetAMP is also a good inhibitor of 5'-phosphodiesterase activity in chicken e m b r y o fibroblasts. The inhibition is competitive with respect to both substrates used; kinetic parameters measured according to Lineweaver and Burk [30] being Km 19 gm and Ki 145 pM with 5'-AMP-p-nitrophenyl esters, and K m 2100 pM and Ki 440 gM with bis-(4-nitrophenyl phosphate).

21 3' : 5'-cyclic AMP phosphodiesterase from bovine heart is unable to m o d i f y MetAMP. Although it is not a substrate for 3' : 5'-cyclic AMP phosphodiesterase, MetAMP is recognized by this enzyme since it inhibits, albeit weakly, the formation of 5'-AMP from 3' : 5'-cyclic AMP: thus at 2.4 ~M cyclic AMP there was 5 and 30% inhibition using 2 and 4 mM MetAMP respectively. This inhibition led us to test the effect of MetAMP on cyclic AMP levels. However, in control experiments we found that MetAMP inhibits ~,.32p transfer from ATP to histone by the cyclic AMP-dependent protein kinase which forms the basis of the commercial cyclic AMP assay kit used, and mimics cyclic AMP in this type of assay. Thus 26 nmol MetAMP decreased cyclic [3H]AMP bound to the cyclic AMP binding protein to the same extent as did 6.6 pmol cyclic AMP.

Effect of MetAMP in vivo Methionyl-tRNA synthetase. A decrease in the in vivo acylation level of methionyl-tRNA induced by MetAMP leads to a specific derepression of methionyl-tRNA synthetase formation in procaryotes [31--34]. The regulation of aminoacyl-tRNA synthetases in eucaryotes is not as well documented as in procaryote cells [32]. However, valyl- and methionyl-tRNA synthetases are reported to be regulated at least in yeast [33--35]. Since methionyl-tRNA synthetase activity is strongly inhibited by MetAMP in vitro [4] we measured the level of this enzyme throughout growth of chicken embryo fibroblasts, in the absence or in the presence of various MetAMP concentrations. From 3 h up to 48 h of growth in the presence of 0.1 mM MetAMP, the level of Met-tRNA synthetase is 110--117% of that in the cells grown in the absence of MetAMP. No such difference is observed when cells are grown in the presence of 0.3 mM MetAMP, a concentration at which protein synthesis is 50% inhibited after 3 h. Further controls to check if these results could be significant showed that the 5'-phosphodiesterase and RNAase activities were increased in extracts from cells grown for 48 h in the presence of 0.1 mM MetAMP; thus it is possible that the level of methionyl-tRNA synthetase measured by the aminoacylation reaction is underestimated. This problem is under investigation. If this small increase of methionyl-tRNA synthetase activity in treated chicken embryo fibroblasts is truly significant its extent is lower but of the same order as that reported for procaryotic cells treated with MetAMP [31]. Further experiments are needed to see if regulation of aminoacyl-tRNA synthetases in chicken embryo fibroblasts occurs. 5'-Phosphodiesterase. Since MetAMP was recognised by 5'-phosphodiesterase we investigated what was happening to the level of this enzyme when cells were cultivated in the presence of 0.1 mM MetAMP for 48 h. The reason for this phodiesterase activity increased from 0.142 to 0.253 unit when cells were cultivated in the presence of 0.1 M MetAMP for 48 h. The reason for this increase is not yet known but it could be due to a higher stability of this enzyme in vivo in the presence of MetAMP or to a differential sedimentation behaviour or to an u n k n o w n regulation. Increases in 5'-phosphodiesterase [36] and RNAase [37] activities have previously been reported in diseases of h u m a n muscle but their significance was unknown. 3': 5'-Cyclic phosphodiesterases. -The activities of 3' : 5'-cyclic AMP and 3' : 5'-cyclic GMP phosphodiesterases {measured at 2.4 pM cyclic AMP and 2.0

22 pM cyclic GMP, respectively) were not significantly changed after 48 h culture in the presence of 0.1 mM MetAMP {mean values at 48 h 2.56 and 2.61 units, respectively). In conclusion, we note firstly that in chicken embryo fibroblasts, MetAMP leads to a rapid cessation of protein and DNA syntheses without affecting RNA synthesis. Secondly, over prolonged periods of contact with chicken embryo fibroblasts, there is a significant degradation of MetAMP to methioninol and 5'-AMP. Finally, we were unable to show any large variations in the activities of several enzymes in chicken embryo fibroblasts treated with MetAMP. Our results suggest that MetAMP may be a valuable inhibitor for investigating the interrelationship between protein and DNA syntheses on the one hand and RNA synthesis on the other, particularly in chicken embryo fibroblasts where transformation by Rous Sarcoma virus requires the RNA viral genome to be transcribed into a DNA copy which is then integrated into the cellular genome.

Acknowledgements We are grateful to Dr. P. Vigier for a critical reading of the manuscript. The authors are indebted to Mrs. Y. Nouvian for her c o m p e t e n t assistance. Support for this investigation was received from the Commissariat ~ l'Energie Atomique for the purchase of labelled compounds.

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