Effect of the immediate precursors of phenylalanine and tyrosine on growth and protein synthesis in phenylalanine- and tyrosine-deprived HeLa cells

Biochimica et Biophysica Acta, 1164(1993)209-214

209

© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00

BBAPRO 34517

Effect of the immediate precursors of phenylalanine and tyrosine on growth and protein synthesis in phenylalanine- and tyrosine-deprived HeLa cells Denys N. Wheatley a, Attila Miseta b, Eleanor M. Love a, Dawn Strickland and Ian Harris c

a

a Cell Pathology Laboratory, Department of Pathology, UniversityMedical Buildings, Aberdeen (UK), b Department of Clinical Chemistry, Medical University of P£cs, P£cs (Hungary) and c Rowett Research Institute, Aberdeen (UK)

(Received 8 October 1992) Key words: Amino acid deficiency;Protein synthesis; Precursor; Cell growth; Cell culture; (HeLa cell) Although Phe is an essential amino acid in mammalian cells, its immediate precursor,/3-phenylpyruvic acid (BPP), when present in Phe-deficient medium at 10 - 4 and 10 -3 M is converted at a sufficient rate to Phe to sustain growth at 60 and 100% of non-deficient control HeLa S-3 cells, respectively. In contrast, Tyr-deficient cells were unable to convert the immediate precursor of Tyr, OH-fl-phenylpyruvic acid (OHBPP), nor could BPP rescue Tyr-deficient cells. The results are considered in terms of the organization of intracellular pathways by which precursors are transaminated and made available for protein synthesis. Introduction Mammalian cells need about 10 essential amino acids in order to grow, including the aromatics Phe, Tyr and Trp. Sometimes cells are unable to make sufficient quantities of a particular amino acid for themselves, for example arginine in young rats [1] and histidine in human infants and parenterally-fed adults [2], with the result that their diets have to be supplemented with these 'semi-essentials'. Tyr is supposed to be a non-essential amino acid in organisms capable of hydroxylating Phe, which should according to Bender [1] include man, as in other organisms such as Tetrahymena [3,4]. However, Tyr is an essential requirement in cultured mammalian cells, with > 3" 10 -5 M being needed for optimal growth [5]. Recent studies with the immediate precursor of Phe, fl-phenylpyruvic acid (2-oxo-3-phenylpyruvic acid; BPP) indicate that a close relationship or 'coupling' exists between amino-acid synthesis and protein synthesis in auxotrophs such as Escherichia coli (Ref. 6 and Miseta, A. and Wheatley, D.N., data not shown). Since mammalian cells are incapable of de novo synthesis of Phe, their apparent inability to convert BPP to Phe and thereby reduce the incorporation of exogenously-supplied [3H]Phe into protein provides further Correspondence to: D.N. Wheatley, Cell PathologyLaboratory, Department of Pathology, University Medical Buildings, Foresterhill, Aberdeen AB9 2ZD, UK.

circumstantial evidence for the preference for the precursor route in wild-type E. coll. Subsequent studies reported here show for the f r s t time, however, that HeLa and other mammalian cells in culture can transaminate BPP to Phe, and therefore this precursor will rescue Phe-deficient cells. On the other hand, BPP failed to rescue Tyr-deficient cells, as did the Tyr precursor, OHBPP. Materials and Methods Culturing o f cells. H e L a S-3 cells were grown in basal Eagle's medium (BME) supplemented with 10% neonatal calf serum, streptomycin (100 /zg/ml) and penicillin G ( 6 0 / z g / m l ) under 5% CO 2 in air, usually within 25-cm 2 flasks (Nunc, Roskilde, Denmark) or 12-well plates (Corning, Coming, NY, USA) with areas of 1.520 cm 2 per well. Seeding densities were 6.103 cells per cm 2 in flasks (which tended to be used for experiments lasting more than 4 days), or about 10.103 per well in 12-well plates. Cell counting. Monolayers were washed briefly twice with prewarmed Dulbecco's phosphate buffered saline (DPBS) and the cells were removed from the flask or wells by treatment for 3 - 4 min with 0.15% tryspin in DPBS. After gentle dispersion, the cells were diluted 10-fold with Isoton II (Coulter Electronic, Luton, UK), and counted with Coulter Zm counter with an aperture of 100 ~m. At least four estimates were made per sample of the total number of cells per flask or well, usually in triplicate. In order to ensure that cell loss by

210 damage did not unduly bias counting by this method, cells were also counted in the same area (measuring 300 × 200 ~m) of each flask or well each day. The total number of cells per flask or well was recorded. This latter method allowed sequential growth to be followed within the same population each day, whereas the first method relies only on collection of three of more flasks chosen at random from each group per time. Estimates on any single flask were almost invariably better than 5% different from each other, and the difference within any group rarely exceeded 10%. Special media. BME deficient in Phe or Tyr (or both) was carefully prepared in the laboratory. It was supplemented with 5% freshly dialyzed neonatal calf serum [7], the free Phe and Tyr content of which was below computer-detection of peaks at the appropriate positions on the traces from the Locarte amino-acid analyzer (Locarte, London, UK), but some deviations from the base-line could be detected by inspection (i.e., the levels would have been lower than < 5 • 10-10 M). The medium with this serum added to 5% was further checked by means of the analyzer for the presence of Phe and Tyr, but here even the suggestions of base-line deviations had disappeared. Deprivation of cultures. Monolayers of cells were washed twice with Dulbecco's phosphate buffered saline (DPBS) at 37°C, and deficient medium added, or as subsequently proved more effective, the monolayers were removed as quickly as possible in 0.15% trypsin in DPBS and seeded directly into the required deficient medium at the start of the experimentation period. Over a 5-h period of observation, cells placed into three-amino-acid-deficient medium (Phe, Tyr and Leu), and in other deficiency conditions showed no difference in percentage cell attachment from controls, except in the case where all amino acids were omitted (Table I). This second method frees cells of some contaminating amino acids which are otherwise held under the monolayer, and could sustain cells at low density ( < 6000 cm 2) for up to two generations. It was therefore invaluable to use this method in most highly sensitive experiments where low concentrations of amino acids or precursors were to be added. Chemicals. BPP and OHBPP were obtained from Sigma (Poole, UK) and checked for purity of > 99% (see Discussion). BPP was water-soluble, but OHBPP was first dissolved in 0.2 M NaOH at high concentration, an equal volume of serum-free medium was added and the mixture adjusted to pH 7.2 with 1 M HC1, and then used immediately at the correct dilution to remain in solution. Phe (lot no. 99C 022B), and Tyr (lot no. 81F 0198) were selected for use because of their freedom from contamination with each other by amino-acid analysis, which was absolutely essential in work on amino-acid deprivation. Radiochemicals of the highest

TABLE I

Percentage of cells attaching to 12-well plates in different media deficient in amino acids relative to controls seeded at 2.10 4 cells per well - 3 ess, BME minus Phe, Tyr and Leu; - a l l ess, BME without any essential amino acids; Glu only, BME with no amino acids except Glu; aa-free, BME without any amino acids. min after seeding

control

- 3 ess

- all ess

Glu only

aafree

90

100 (6250) *

90

95

94

97

180

100 (10500) *

102

114

81

80

300

100 (13150) *

107

116

107

79

* Actual number of cells attached out of the seeded 2.104.

available specific activity for tritium of Val, Tyr, Phe and Leu were obtained from Amersham International. Analysis of pools and incorporation into protein. Incorporation into pools and protein was measured after appropriate incubation of cells in the desired medium at 37°C, followed by washing of the cells twice in ice-cold saline, precipitating macromolecules with 0.6 M perchloric acid (PCA), collecting the precipitate and washing once again PCA. This was followed by dissolution of the pellet in 1 M NaOH, from which aliquots were taken for protein estimation by a modified Lowry method [8], and radioactivity was measured using Supersolve scintillant (Koch-Light, Colnbrook, UK) to allow specific activity to be determined when necessary [9]. In some experiments, extracts of the pools were made after the initial saline washes in sulphosalicyclic acid to obtain free amino-acid pools for analysis on an amino-acid analyzer. Protein residues from the above were washed once with 100% ethanol, precipitated by centrifugation and hydrolyzed overnight in 6 M HCI in sealed tubes at 150°C. A Locarte (London, UK) single column amino-acid analyzer was used, basic amino acids being ignored in this work. The elution buffers were of lithium citrate, the first at pH 2.7 and the second at pH 4.2. Norleucine and /3-alanine (25 nmol) standards were also run for calibration purposes. Integration of the areas under the peaks was carried out by triangulation using an appropriate soft-ware programme, and amounts of amino-acid present were finally expressed in molar concentrations after correction for the dilution of the solutions from which the original samples were taken. Results

Cell growth after deprivation of Phe and Tyr HeLa S-3 cells required Phe and Tyr at > 10 -6 M to sustain normal growth. In the absence of either or

211

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800

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30

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200

o

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Fig. 1. (a), Growth of HeLa cells in BME containing 5% dialysed serum (11), compared with cells deprived of Phe (13), Tyr (zx) or both ( • ) . (b), Protein increase corresponding to (a), given for controis and - P h e cultures, the other two being identical to the latter.

both amino acids, cell number increased by just over one doubling following the first procedure outline in Materials and Methods, and less than one doubling by the second method. Protein estimates were in good agreement (Fig. la,b). Protein synthesis, estimated by 30-min pulses with [3H]leucine at daily intervals, showed a progressive fall to 55% of control values by 4 days of deprivation, based on specific acitivity measure-

4

o

| 1

2

3

4

days

Fig. 2. (a), [3H]leucine incorporation into cells in BME containing 5% dialysed serum (solid bars) compared with Phe-deficient cultures (open bars). The value are given as averages of triplicate estimates of the incorporation of 30-min pulses at daily intervals, expressed as a specific activity (dpm/izg protein) (b), As for (a), but giving the total incorporation into each culture well.

ments. This hides the true suppression in synthesis, which can be seen for each culture well in Fig. 2b, as opposed to Fig. 2a. Deprived cells assumed different shapes after 1 day or more in deficient medium, and became progressively more ragged by 4 days when many cells were moribund (Fig. 3).

Recovery of deprived cells with Phe and Tyr replacement Restoration of either Phe or Tyr failed to rescue cells starved of both amino acids, or the opposite one (Fig. 1), confirming that Phe cannot be hydroxylated to

Fig. 3. (a), Photomicrograph of control HeLa cell culture ( × 250). (b), HeLa cells after 1 day deprivation in Phe-deficient medium, showing much detachment and many cells with ragged edges. By 3 days only the flatter, spread cells persisted ( × 250). (c), HeLa cell culture exposed for 24 h to 10 -2 M OHBPP, showing changes due to some general toxic effect ( × 250).

212

t

3O

data, but contrasts with P. aeruginosa which can carry out this interconversion (Miseta, A. and Wheatley, D.N., data not shown).

2s

Recovery of Phe-deprived cells treated with BPP, but not with OHBPP

ii ,°15 |

lO

E

0

Q. 13

10.5 10.4 Phe©oncn(M)

10.3

Fig. 4. Effect of increasing concentrations of Phe on pools (solid bars) and protein incorporation (open bars) of [3H]Tyr (5.10 -5 M; 37 kBq/ml). Tyr and Tyr cannot be dehydroxylated to Phe by H e L a cells. We confirmed the total absence of interconver° sions by analyzing protein on the amino-acid analyzer from cultures treated with [3H]Phe, which gave no tritium labelling in the Tyr peak, and vice-versa. A further set of experiments in which supported this conclusion involved the addition of Phe at increasing concentrations to previously Phe-deprived cells given [3H]Tyr. The added Phe did not alter the incorporation of the labelled Tyr into proteins, which would have been expected if it had provided extra Tyr by interconversion (Fig. 4). This agrees with our E. coli

a

In keeping with our work on bacteria, the originally series of short-term experiments with H e L a cells lasted a maximum of 2 days, and gave no indication of BPP utilization by H e L a cells. However, in one experiment, a series of H e L a cell flasks deprived of Phe for 24 h were given 10 -4 M BPP and inadvertently left unexamined for 7 days. These cultures had grown to confluency and were capable of continued growth in similar (fresh) medium upon subculturing, and indicating that Phe per se is not absolutely essential for H e L a growth. A detailed follow-up showed that 10 -5 M BPP was unable to rescue Phe-deprived cells, 10 -4 M allowed growth to resume after 2 days, accelerating to 60% of control rate by 3 days (Fig. 5a). Withdrawal of BPP after growth had been resumed quickly led to cells re-entering the deprived state (Fig. 5b). Initially, the delay in growth after BPP had been added to Phe-deprived cells was thought to be due the time of induction of the appropriate glutamate-dependent aminotransferase. However, the delay in response of H e L a cells to 10 -3 M BPP was less than 90 rain when measured in terms of stimulation in protein synthesis (Fig. 5c), even in cultures rescued with BPP after 7 days as opposed to Phe (Fig. 6). Cells grew in 10 -3 M BPP at equivalent rates to normal Phe controis. Significant levels of Phe were found after 24 h incubation in the otherwise Phe-deficient medium containing 10 -3 M BPP (1.2 n m o l / m l ) , but was inde-

300

800 b 700

250 200

600 500

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i 150

400 300 200 100 0

i~ 400 300 200 100 0 5 5 7 0

|

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~

800r c ! 700 i 600 500

Ip f

0 1 234 days

1

2 hours

3

Fig. 5. (a), Hela cell growth in coltrol medium (11) Phe-deficient medium (n), and in medium containing 10-4 M BPP (o). (b), As for (a) but with the following treatments: (It), controls; (n) Phe-deficient throughout; (e), 10-4 M BPP given on day 3 (arrow up) and removed on day 5 (arrow down); (o), 10-4 M BPP from day 3 onwards; (A), 10-4 M Phe from day 3 onwards. (c), HeLa cells deprived of the Phe for 24 h were given 5' 10-s M [3H]Leu (37 kBq/ml) in the presence of the following treatments and incorporation into protein measured over 3 h; (~2), continued Phe-delarivation;(0). 10 - 4 M BPP; (zx), 10 - 3 M BPP; (v), 10-2 M BPP; (me), 10-4 M Phe; (v), 10 - 7 M Phe.

213 tectable when 10 -4 M BPP was present, indicating generation of a small surplus to requirements at the higher concentration. Phe-deprived cells could not be rescued with the Tyr precursor OHBPP, even when present at the highest concentration attainable in culture medium (10 -2 M).

a

b 400

~

100 i300

--" 80

o 200

Effect of BPP on Phe pools and incorporation Since BPP can be transaminated to Phe, it should affect the incorporation of exogenously-supplied (tritiated) Phe into protein and intracellar pools. The evidence indicates a slow conversion rate of BPP to Phe, which explains why 10 -3 M BPP is required to generate sufficient Phe for protein synthesis (equivalent to a medium concentration of about 10 -6 M Phe). This has been verified by analysis of the rate at which [3H]Phe incorporation from concentrations of 10 -8 to 10 -3 M is affected by BPP at from 10 -4 to 10 -2 M (Fig. 7). When no Phe was present, BPP competition was evident as a lowering of incorporation of [3H]Phe to a greater extent than when Phe was at low levels (10 -5 to 10 -6 M). However, no competition could be detected when the normal BME concentration of Phe (10 -4 M) was present. BPP was effective in reducing pools of Phe when these were formed from the lowest concentrations mentioned above (Fig. 7b), but not at higher than 10 -5 M level of Phe. At 10 -2 M BPP, a sudden reduction in pool size of Phe was seen, but this was in register with falls in the pools of Val and Leu, indicative of some adverse effects of high levels of precursors on ceils. Having confirmed that neither Phe nor BPP acts as a precursor for Tyr, Fig. 4 also con-

220

180 160

~. 140 120 IO0 so 4o

e~

20

"0

o

0 0

10-4 10"3 Conc BPP (M)

10"2

0

10.4 10.3 10.2 Conc BPP (M)

Fig. 7. (a), Uptake of [3H]Phe into the intracellular acid-extractable pool of HeLa cells, normalised to the control levels (absence of BPP) for each of the Phe concentrations. Phe concentrations were: (1:3), [3H]Phe alone; ( • ) , [3H]Phe+10-6 M unlabelled Phe; ([]), [3H]Phe+ 10 -5 M unlabelled Phe. The data indicate that reduction is greatest where Phe level is lowest. (b), As for (a), but for incorporation into protein, expressed as actual values, not normalized to the non-BPP-treated controls. Only at very low levels of Phe was a concentration-dependent inhibition of [3H]Phe observed.

firms that BPP has no effect on a Tyr pool, except at 10 -2 M, as for Val and Leu.

Failure of OHBPP to rescue Tyr-deficient cells O H B P P failed to reverse Tyr deficiency in all attempts with HeLa cells. Similarly, BPP had no ability to substitute for the absence of Tyr. O H B P P had no effect on pools of labelled Phe, Tyr or Val (data not shown), except following the injurious effects of its addition at 10 -2 M, which was poorly tolerated by cells.

Discussion

200 •=

c 100

!

1

2

3 4 days Fig. 6. Incorporation of [3HlLeu given as 30-min daily pulses into protein per culture well in HeLa cells treated as follows: (t3), controls; ( • ) , Phe-deprived; ([]), Phe-deprived plus (10-4) M BPP; (rq), Phe-deprived plus 10-3 M BPP.

The evidence presented here shows mammalian cells are capable of converting BPP to Phe, and therefore the appropriate glutamate-dependent aminotransferase must be present. The same enzyme, according to Bender [1], is reponsible for O H B P P transamination to Tyr, and therefore it was surprising to find that this substrate was not handled by cells. In the absence of interconversion of Phe to Tyr, these substrates are very useful in dissecting the 'remnants' of the pathways in amino-acid synthesis in mammalian cells. Some bacteria are capable of making the conversion of Phe to Tyr, but not all. Therefore, another series of experiments to be reported shortly (Miseta, A. and Wheatley, D.N., data not shown) will deal with this problem, and the relative abilities of E. coli and P. aeruginosa to utilize the two precursors of interest here. Since the latter organism is quite capable of converting O H B P P to Tyr, the precursor appears to have no problems accessing the internum of cells where it is metabolised in this

214 case. The finding that no OHBPP is converted to Tyr at all by HeLa cells indicates either that a different aminotransferase is involved from that acting on BPP, or the same one might be used, except OHBPP cannot access it whereas BPP can. This is not an idiosyncrasy of HeLa because more recent experiments with primary skin fibroblast cultures show that these cells behave in a similar manner to HeLa cells in relation to Phe and Tyr deprivation, and rescue with BPP. The possibility has been raised, however, that differences could occur between normal and malignant cells of the same origin, and these are presently being investigated (data not shown). The implication in HeLa cells is that the organization of the Phe and Tyr biosynthetic pathways are separate and do not have a common terminal stage. This evidence would also be incompatible with the supposition that the law of mass action applies to this aminotransferase activity within the cell internum. The possibility that a non-specific 'soluble' aminotransferase is responsible cannot, however, be entirely ruled out, but it would have to have a low affinity for BPP considering the high concentration required to generate sufficient Phe for protein synthesis. And it follows that its affinity for OHBPP would be very much lower, which would seem to rule out the possibility that OHBPP produced endogenously is converted to Tyr by the same enzyme in this way. The delivery of substrates to enzymes within the biosynthetic pathways leading to amino-acid production is probably carefully regulated, and the question now arises as to whether exogenously supplied molecules of precursors have the same accessibility to these pathways as their endogenously synthesised counterparts. Resolution of this problem leads to a bigger issue raised by Miseta [6] that a coupling probably exists between the pathways of amino-acid biosynthesis in auxotrophs and the synthesis of proteins. Whether this also applies to heterotrophic cells, of which mammalian ceils and Tetrahymena [10] are good experimental subjects, remains to be seen. This work is being extended in bacteria and Tetrahymena, as discussed above, and to other amino acids precursors, e.g., the immediate precursors of Pro, His, lie and Leu. The immediate precursor of His - which we have already investigated, as have others [11] appears to be a special case since L-histidinol is an inhibitor of protein synthesis. Thus, a cell cannot release this intermediate into the 'cytosol', otherwise it would adversely affect protein synthesis - unless this is a natural feedback mechanism for protein synthesis, which is worth further investigation in its own right.

This adds further circumstantial evidence in favour of pathways of amino-acid synthesis being highly organized and tightly coupled. One problem relating to work on mammalian cell cultures is that serum is necessary in experiments lasting several days. This problem is overcome with Tetrahymena cultures (Ref. 10 and Rasmussen, L. and Wheatley, D.N., data not shown), since at least two excellent defined media are available for their cultivation, and these cells require the same 'essential' amino acids as mammalian ceils [3,4]. Auxotrophic mutants of E. coli also provide excellent cells for further work, and in this connection we have already found that strain 23785, a Phe-requiring auxotroph, grows normally on BPP (Miseta, A. and Wheatley, D.N., data not shown).

Acknowledgements We thank Professor Miklos Kellermayer (Department of Clinical Chemistry, Medical University of P6cs, Hungary) for his encouragement and constructive criticisms of this work. We are grateful to Mr. Joe Shaw (McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI, USA) for a new supply of HeLa S-3 cells. The British Council has funded travel between the Unversity of P6cs and the University of Aberdeen, and we are also grateful for funds from The Medical Endowments of the University of Aberdeen towards the cost of this preliminary work, and the EEC (ESF) for supporting the retraining programmes of E.M.L. and D.S. during their involvement with this work.

References 1 Bender, D.A. (1985) Amino acid metabolism, 2nd Edn., Wiley, Chichester. 2 Kopple, J.D. and Swendseid, M.E. (1975) J. Clin. Invest. 55, 881-891. 3 Rasmussen, L. and Modweg-Hansen, L. (1973) J. Cell Sci. 12, 275 -286. 4 Szablewki, L., Hove Andreasen, P., Tiedtke, A., Florin-Christensen, J., Florin-Christensen, M. and Rasmussen, L.J. Protozoology 38, 62-65. 5 Eagle, H. (1955) Science 122, 501-504. 6 Miseta, A. (1989) Physiol. Chem. Phys. 21,237-241. 7 Wheatley, D.N. and Henderson, J.Y. (1975) Exp. Cell Res. 92, 211 - 220. 8 Oyama, V.I. and Eagle, H. (1956) Proc. Soc. Exp. Biol. Proc. Soc. Exp. Biol. Med. 91, 305-307. 9 Inglis, M.S. and Wheatley, D.N. (1982) Cytobios 33, 15-28. 10 Wheatley, D.N. and Walker, E. (1980) J. Comp. Physiol. 140, 267-274. 11 Hansen, B.S., Vaughan, M.N. and Wang, L. (1972) J. Biol. Chem. 247, 3854-3858.