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Effect of methionine deprivation on methylation and synthesis of macromolecules.

The growth of 4 tumour-cell lines (Walker rat mammary carcinoma (W-256), a mouse lymphoma (TLX5), a mouse bladder carcinoma (MB) and a human bladder carcinoma (EJ) was much reduced when methionine in the culture medium was substituted by homocysteine. In contrast, a human embryonic fibroblast line grew equally well under such conditions. Although homocysteine alone was unable to support growth of W-256 it stimulated growth at low methionine concentrations. When W-256 was cultured for 24 h in medium containing homocysteine only, the extent of methylation of nucleic acids and the acid-soluble pool of methionine were decreased. However, under such conditions there was an increased methylase activity towards both endogenous substrate and E. coli tRNA. The effect of methionine removal was to cause a large increase in the Vmax value for methylation of tRNA, without any change in the Km value towards S-adenosyl-L-methionine (SAM). For both W-256 and TLX5, methionine deprivation caused a rapid inhibition of RNA biosynthesis, followed by inhibition of DNA synthesis, while protein synthesis tended to increase. This suggests that the inability of W-256 and TLX5 to survive and grow in methionine-deficient, homocysteine-supplemented medium is not due to insufficient methionine for protein biosynthesis, but may be related to an enhanced methylating activity of some tumour-cell lines.

Summary.-The growth of 4 tumour-cell lines (Walker rat mammary carcinoma (W-256), a mouse lymphoma (TLX5), a mouse bladder carcinoma (MB) and a human bladder carcinoma (EJ)) was much reduced when methionine in the culture medium was substituted by homocysteine. In contrast, a human embryonic fibroblast line grew equally well under such conditions. Although homocysteine alone was unable to support growth of W-256 it stimulated growth at low methionine concentrations.
When W-256 was cultured for 24 h in medium containing homocysteine only, the extent of methylation of nucleic acids and the acid-soluble pool of methionine were decreased. However, under such conditions there was an increased methylase activity towards both endogenous substrate and E. coli tRNA. The effect of methionine removal was to cause a large increase in the Vmax value for methylation of tRNA, without any change in the Km value towards S-adenosyl-L-methionine (SAM). For both W-256 and TLX5, methionine deprivation caused a rapid inhibition of RNA biosynthesis, followed by inhibition of DNA synthesis, while protein synthesis tended to increase. This suggests that the inability of W-256 and TLX5 to survive and grow in methionine-deficient, homocysteine-supplemented medium is not due to insufficient methionine for protein biosynthesis, but may be related to an enhanced methylating activity of some tumour-cell lines.
UNLIKE NORMAL cells, the growth of many tumour and transformed cells of both animal (Halpern et al., 1975;HIoffman & Erbe, 1976) and human (Kreis & Goodenow, 1978) origin is severely restricted when methionine (Met) is replaced by its immediate precursor homocysteine. The original suggestion (Ashe et al., 1974) for this lack of growth in homocysteine-supplemented medium was diminished in vivo activity of 5-methyltetrahydropteroyl -Lglutamate: Lhomocysteine S-methyltransferase (EC 2.1.1.13) the enzyme which catalyses the terminal reaction in Met biosynthesis. However, in normal and SV40-transformed human fibroblasts, the latter of which show a growth requirement for Met, the activities of both 5,10-methylenetetrahydrofolate reductase and the transmethylase are similar (Kamely et al., 1977). Furthermore, 2 revertant SV40-transformed cell lines regained the ability to grow in homocysteine-supplemented medium without any substantial changes in homocysteine transmethylase, 5,10-methylenetetrahydrofolate reductase, or in their ability to take up 5-methyltetrahydrofolate (Hoffman et al., 1978). These observations indicate that the absolute growth requirement for Met observed in some transformed cells does not involve a deficiency in enzymes related to Met synthesis, but could be due to a higher Met requirement of some tumour cell lines.
The present experiments investigate the ability of 1 normal and 3 tumour-cell lines to proliferate in Met-depleted, homocysteine-supplemented medium and the effect of such culture conditions on nucleic acid and protein synthesis, methylation of nucleic acids and methylase activity towards both endogenous substrate and E. coli tRNA. Nucleic acid and protein synthesis-Incorporation of radioactivity into acid-insoluble material was determined after lh incubation of lml portions of the cell suspension at 37W, with either (methyl-3H) thymidine (5 [LCi/ ml), [5-3H] uridine (5 ttCi/ml) or [4,5-3H] lysine (10 [tCi/ml). At the end of the incubation the cells were washed onto glass-fibre filter discs (Whatman GF/C, 2-5 cm) with 0-90o NaCl. The cells on the filter were then washed with 10 ml ice-cold 500 trichloroacetic acid and 5 ml of absolute ethanol. After drying at 70°C for 2 h the radioactivity on the filters was determined using a toluene, PPO, POPOP scintillation mixture.
When the incorporation of radioactivity into nucleic acids and proteins was determined. cells (1-5 x 105/ml) were incubated for 24 h in the presence of 20mM sodium formate and l,uCi/ml [methyl-3H] Met. At the end of the incubation the cell suspension was sedimented by centrifugation at 300 g for 3 min, followed by washing in 0-900 NaCl and recentrifugation. The cell pellet was treated with 1 ml of ice-cold 0-5mi perchlor ic acid. anid the precipitate was washed x 4 by resuspension and centrifugation in 1 ml of 0-5ii perchloiic acid. An aliquot of the acid supernatant after neutralization with 5N KOH -was counted in PCS scintillation fluid (Hopkin ard WXilliams) to determine the acid-soluble radioactivity. A nucleic acid soluble fraction (DNA + RNA) was prepared by heating the acid precipitate at 70°C for 20 min in 1 ml of I-OM perchloric acid, cooling rapidly on ice and centrifuging at 600 g for 10 min at 4°C.
The 70°C perchlorate hydrolysis was repeatcd on the remaining residue, and after neutralization of a portion (1.6 ml) of the combined supernatant, the radioactivity was determined as above. The residue remaining after acid hydrolysis was dissolved in IN NaOH and the concentration of protein was determined by the methcd of Lowry using bovine serum albumin as a standard. The remaining residue was neutralized with IN HCI and the radioactivity determined in PCS scintillation fluid.
Methylatioan of tRNA. Cells wrere sedimented by centrifugation at 300 g for 3 min, washed with 0.900 NaCl and sonicated in 10mM Tris HCI (pH 7-4) containing 10mM NaCI, 1P5mM MgCl2 and 1mM 2-mercaptoethanol. The supernatant fraction obtained after centrifugation at 2000 g for 1 h was used as a source of methylase after dialysis overnight against 100 volumes of the reaction buffer. The reaction mixture (final volume 125 ,ul) contained 50mM Tris (pH 7-8) 0-2M NaCl, 4mM dithiothreitol, 50 ,ug E. coli tRNA and 40-332 pmol S-adenosyl-L-[imethyl-3H1] Met and enzyme extract. After incubation at 37°C for various times, the reaction was stopped by the addition of an equal volume of 10% trichloroacetic acid. The insoluble material was collected by filtration through glass-fibre filters (Whatman GF/C 2-5 cm) after 10 min at 4C. The filters were washed with ethanol and dried at 70WC for 2 h and the radioactivity was determined in a toluene: PPO scintillation mixture. To eliminate incorporation into endogenous substrate a blank without tRNA was subtracted from each assay. To measure the incorporation of The assay is based on the enzymatic transfer of a methyl group from SAM to an acceptor molecule [3H] dopamine, in the presence of COMT, as described by Yu (1978). An aliquot of SAM or tissue extract (225 1l borate buffer (pH 10). The labelled 3-methoxytyramine was extracted from the incubation mixture by shaking with 1 ml of toluene: isoamylalcohol (3:2 v/v). After centrifugation, 800 pi of the organic phase was transferred to a clean tube containing 600 ,ul of 0-4N HC1, vortex mixed and centrifuged. The radioactivity in the aqueous HCI phase (500 ,ul) was determined in 5 ml of PCS scintillation fluid. A standard curve was performed for each experiment.

Ability of cell lines to yrouw in the absence of methionine
The effect of substituting extracellular Met for homocysteine on the growth of 4 tumour cell lines (the Walker rat mammary carcinoma (W-256), a mouse bladder carcinoma (MB), a mouse lymphoma (TLX5) and a human bladder carcinoma (EJ)), and one normal cell line (a human embryonic fibroblast line (HE)) is shown in Table I. The results are expressed as a percentage of the control growth rate in medium containing 30 ,tg/ml of Met and are derived from the linear portions of the growth curves. Although there is some reduction in growth of the normal cell line at low homocysteine concentrations, optimal growth is observed in medium containing only 0-66mM homocysteine. In contrast none of the tumour lines show optimal growth in medium containing homocysteine only. The response to substitution of Met by homocysteine does vary, however, between the tumour lines from a 62% reduction of growth in EJ to no cell growth with W-256. There is also a loss of viability of W-256 cells in such depleted medium, which is halved within 72 h (Tisdale, 1980). This loss of proliferative ability in medium containing homocysteine only is not related to differences in homocysteine :transmethylase, as shown in Table II.   TABLE II acid into [methyl-14C] methionine as previously described (Tisdale, 1980).

Effect of methionine deprivation on the methylation of macromolecules
Although homocysteine alone cannot support growth of W-256 in medium lacking Met, it can stimulate growth when Met concentrations are limiting (Fig. 1). This suggests that the lack of growth of some cell lines in homocysteine-supplemented media may be due to their higher    ,ug/ml Met +01mM homocysteine, 01ImM folate and 7-5 iM OH-B12, and cell number was determined as (lescribe(l in Methods. Met requirement possibly due to enhanced methylation reactions. The results in Table III show the effect of 24h culture of W-256 in medium containing homocysteine only or low concentrations of Met, with or without homocysteine, as depicted in Fig. 1, on the subsequent incorporation of [methyl-3H] Met into the acid-soluble pool and into nucleic acids in whole cells, and the effect on the intracellular level of SAM. Incubation was carried out in the presence of 20mM sodium formate to reduce methyl incorporation into purines and thymine via the "one carbon" pool. Under these conditions incorporation of [methyl-3H] groups into the nucleotide pool is essentially eliminated (Caboche & Hatzfeld, 1978). There is an increase in the incorporation of [methyl-3H] groups into nucleic acids after 24 h in medium lacking Met. This suggests undermethylation of nucleic acids under such conditions, which is also supported by the decrease in the intracellular level of SAM and the increased Met accumulation into the acidsoluble pool. The effect of Met deprivation on the incorporation of methyl groups from S-* Cultures supplemented with 0-1mM folate+7-5 ,uM OH-B12. Cells were incubated with the indicate( coincentrations of Met or liomocysteine in Dulbecco's modified Eagle's medium for 24 h. The Met concentration of each culture was then made up to that of the control (30 1tg/ml) and [methyl-3H] Met (1 ,tCi/ml) and sodlium formate (to 20 mM final concentration) were added. The cells were then reincubated for a further 24 li at 37°C after which nucleic acids an(d proteins were extracted as described in Methods. Tlhe intracellular level of SAM was determinedl in cells cultured under the appropriate condlitions for 24 h as (lescribe(l in Methodls.

adenosyl-L-[methyl-3H] methionine (SAM)
into endogenous macromolecules of W-256, TLX5 and HE is shown in Fig. 2. The basal level of methylation for the 2 tumour cell lines is 3 times that for the normal cell line. Furthermore, although there is an increase in the acceptance ability for all 3 cell lines, the extent of induction for TLX5 (12-fold) and W-256 (7-fold) is greater than for HE (5-fold) over the 24h period studied. This increase in the incorporation of [methyl-3H] groups from SAM may be due to either an increase in methylase activity or to an undermethylation of macromolecules under conditions of Met deprivation.
The former possibility was studied by investigating the effect of Met-deficient, homocysteine-supplemented medium on Eagles's medium lacking Met and supplemented with 01ImM homocysteine, 01mM folate and 7 5,uM OH-B12, and at intervals cells were sedimented by centrifugation, washed in 099% NaCl and sonicated in 10mM Tris HC1 (pH 7 4) containing 10mM NaCl, 1 5mM MgCl2 and 1mM 2mercaptoethanol. The incorporation of [methyl 3H] groups into endogenous material obtained after centrifugation was measured as for methylation of tRNA, using 83 pmol S-adenosyl-L-[methyl-3H] methionine per assay. The insoluble material obtained after addition of 10% trichloroacetic acid was collected by filtration through glass-fibre filters, and the radioactivity was determined in a toluene PPO scintillation mixture. the activity of tRNA methylase from cytosol extracts of all 3 cell lines, using "methyl deficient" E. coli MRE 600 tRNA as substrate. The Lineweaver-Burk plots for methylation of such tRNA by cytosol extracts of TLX5 after 24 h in either normal medium, or in medium lacking Met and supplemented with OI mm homocysteine, is shown in Fig. 3. Both extracts were dialysed for 24 h before the assay, since this has been shown to increase methylase activity, possibly by removing S-adenosyl-L-homocysteine (SAH) a potent inhibitor of transmethylation reactions (Kredich & Martin, 1977). Met re- 1-67+0-12 1-75+0-08 5 00+0 4 1-67+0-13 1-92+0-1 7-14+0-5 1-43+0-14 4-65+0-2 7-7+0-8 Values were derived from Lineweaver-Burk plots of the kinetic assays carried out in triplicate. (Means + s.e. of 2 experiments.) moval had no effect on the Km value towards SAM (1P67 ,uM) for methylation of tRNA, but caused an increase in the Vmax value from 1x92 to 7-14 pmol/min/mg protein. A similar increase in tRNA methylase resulting from an increase in Vmax with no change in Km was also seen with W-256 and HE after 24h culture in medium containing homocysteine only, as shown in Table IV. This shows that Met deprivation leads to an increased tRNA methylase activity. Effect of methionine deprivation on the biosynthesis of macromolecules The effect of Met-deprivation and homocysteine supplementation on nucleic acid and protein biosynthesis was studied by pulse labelling cells after various times in deficient medium. Alterations in the precursor pools could produce differences in the rate of incorporation of the radioactive isotopes. However, Kuebbing & Werner (1975) have shown that when [3H]-TdR is added to cells grown in TdRfree medium, it is incorporated into DNA almost immediately, at full specific activity, blocking any further incorporation of de novo synthesized TdR nucleotides. Thus for TdR the de novo nucleotide pools and salvage nucleotide pools are compartmentalized, and the pulse label will be incorporated directly into DNA. Like thymidine, uridine is also taken up by a salvage pathway, but no information is available on mixing of the 2 compartments. However, since neither Met nor SAM are involved in the biosynthesis of uridylic acid, incorporation of precursor has been used as a measure of RNA synthesis. A similar situation exists for lysine, as little information is available on shown to give a measure of macromolecular synthesis in the presence of cycloleucine (Caboche & Hatzfeld, 1978) which produces an effect on the intracellular level of SAM similar to that of Met deprivation. The results using these labels as an indication of macromolecular synthesis for W-256 and TLX5 are shown in Figs 4 and 5. This indicates that RNA biosynthesis is rapidly affected, followed by DNA synthesis, whereas protein synthesis tends to increase, at least initially, in the case of W-256. This shows that the effect of such deficient media on growth is not due to a lack of Met for protein biosynthesis, which could possibly arise from the rapid doubling time of some tumour cell lines. As shown in Table I, there is no correlation between the doubling time of the cell lines and the ability to grow in Met-deficient, homocysteine-supplemented media.

DISCUSSION
The growth results with the cell lines under study confirm the reported capability of normal mammalian cells to substitute homocysteine for Met and the absolute requirement for preformed Met by some tumour cells (Hoffman & Erbe, 1976). There is, however, a variability in the ability of the tumour lines to proliferate under such nutritional conditions. It has been shown previously (Tisdale, 1980) that even in cells with an absolute Met requirement, conversion of homocysteine to Met is increased when homocysteine is substituted for methionine. This accords with the enhanced proliferation of W-256 cells in the presence of homocysteine at concentrations of Met which are limiting for growth.
An important role of Met in cellular metabolism is the formation of S-adenosyl-L-methionine (SAM). SAM plays a central role as the biological methyl-group donor (Mudd & Cantoni, 1969;Cantoni, 1975) and as the source of aminopropyl groups in polyamine biosynthesis (Janne et al., 1978). An increased tRNA methylase activity has been found in several experimental and human tumours (Baguley & Stahelin, 1968). Also an increase in polyamine biosynthesis is associated with the neoplastic state (Janne et al., 1978). This increased requirement presumably accounts for the high levels of SAM in whiteblood-cell preparations from patients with chronic myeloid leukaemia compared with normal peripheral white cells or tboracicduct lymphocytes (Baldessarini & Carbone, 1965).
The present experiments show that replacement of Met by homocysteine causes a reduction in the methylation of nucleic acids, suggesting that the lack of growth under such conditions may be due to a deficiency of the methyl donor SAM.
Indeed under such culture conditions the intracellular level-of SAM has been shown to be reduced, possibly due to a high utilization rate. That the inability to grow under conditions of Met insufficiency is related to a low level of SAM is shown by the similarity of the effect of such culture conditions on nucleic acid and protein synthesis to that produced by cycloleucine (I -aminocyclopentane carboxylic acid) a competitive inhibitor of methionine adenosyltransferase, an enzyme involved in SAM biosynthesis (Caboche & Hatzfeld, 1.978). In both cases the rate of RNA biosynthesis is rapidly and significantly reduced, with a slower effect on DNA synthesis. This depression in the rate of RNA biosynthesis in the presence of cycloleucine was mainly attributable to a reduction in the rates of processing ribosomal and transfer RNA molecules, inducing a slow down of transcription of the corresponding precursor molecules (Caboche & Bachellerie, 1977). Also, when mouse 3T3 or SV40-transformed 3T3 cells are deprived of Met, DNA synthesis continues for several hours, but is eventually inhibited (Culp & Black, 1971). These results suggest that cell growth stops in Met-depleted media due to inhibition of nucleic acid synthesis by the accumulation of methyl-deficient nucleic acids.
Substitution of homocysteine for Met also causes an increase in the Vmax Of tRNA methyltransferases towards E. coli tRNA and an increase in methyltransferase activity towards endogenous substrate. It appears that the fall in the level of SAM causes an increase in methyltransferase activity. Administration of the Met antagonist ethionine also produces an increase in rat liver tRNA methylating enzymes (Wainfan et al., 1975). A similar increase in 5-methyltetrahydrofolate: homocysteine methyltransferase has been observed when homocysteine was substituted for Met in the growth medium of cultured baby hamster kidney cells (Kamely et al., 1973) and Walker carcinoma cells (Tisdale, 1980). In both cases a fall in the level of substrate appears to cause a depression in the synthesis of the corresponding enzyme.
These results support the conclusion that the inability of some tumour lines to proliferate in Met-depleted medium is not due to any intrinsic biochemical defect, but may be due to the higher Met requirement of some cell lines.
This work has been supported by a grant from the Cancer Research Campaign.