140

Biochimica et Biophysica Acta, 1097 ( 19911 14(1 144 ~'~ 1991 Elsevier Science Publishers B.V. All rights reserved 0925-4439/91/$113.5fl A D O N I S I19254439910ill 128

BBAD1S 61(163

Methionine adenosyltransferase activity in cultured cells and in human tissues Karl A k e r m a n i, Kari Karkola 2 and Olavi Kajander i Departments of" 1 Biochemistry and Biotechnology and 2 Forensic Medicine, Unit'ersity q[" Kuopio. Kuopio (Finland) (Received 19 February 199l) (Revised manuscript received 16 May 1991 )

Key words: Methionine adenosyltransferase; S-Adenosylmethionine; Cell line: (Human)

We have investigated methionine adenosyltransferase activity (MAT) in extracts of a variety of normal and malignant human tissues and cultured cell lines. MAT activity assayed from 17 different cultured cell lines varied to a great extent. Ramos (human, Burkitt's lymphoma) and EIA (mouse, T cell lymphoma) cells showed MAT activity near 300 p m o l / m g per min. Daudi (human, Burkitt's lymphoma) and almost all monolayer cells had MAT activity below 100 p m o l / m g per min. Human peripheral blood lymphocytes had MAT activity of 36 p m o l / m g per min. The MAT activity of the cell lines can be related to doubling time: cell lines with short doubling times have much higher MAT activity than other cell lines. A large variation in MAT activity in different human tissues was observed. In autopsy samples MAT activity was highest in the brain and in the colon. Malignant tissue samples gave much higher MAT activity than normal tissues. Lung cancer (carcinoma squamocellulare pulmonis) had MAT activity of 30.7 p m o l / m g per min, while in normal lung it was 2.4 p m o l / m g per rain.

Introduction Methionine is an essential amino acid with several special functions [1]. All mammalian cell lines need either methionine or its precursors for growth. Usually, cultured normal mammalian cells can convert homocysteine to methionine. Another source is methylthioribose derived from methionine via methylthioadenosine. However, several malignant cell lines are methionine-dependent and grow poorly or not at all in the absence of methionine [2]. Apart from its role in protein synthesis, methionine per se is not needed. Methionine is converted to S-adenosylmethionine (AdoMet) in a complex reaction with adenosyltriphosphate catalysed by methionine adenosyltransferase [3]. AdoMet is the major methyl group donor in biological

Abbreviations: EDTA, ethylenediaminetetraacetate (Merck); PMSF, phenylmethylsulfonyl fluoride (Sigma); AdoMet, S-adenosylmethionine; MAT, methionine adenosyltransferase. Correspondence: K. ,~kerman, Department of Biochemistry and Biotechnology, University of Kuopio, P.O. Box 6, 7(1211 Kuopio, Finland.

methylation reactions and after decarboxylation it takes part in the synthesis of spermidine and spermine [4]. In many diseases with changes in the morphology of tissues (e.g., cancer), alterations in the levels of AdoMet and polyamines take place [5]. Metabolic and nutritional factors regulate levels of AdoMet. Imbalances in transmethylation may lead to liver tumour formation and to transformation of liver cells in culture [6]. Recently, AdoMet has been used as a drug with reputed anti-inflammatory and central nervous system

effects [7,8]. Methionine adenosyltransferase (MAT, EC 2.5.1.61 synthesises AdoMet, and the enzyme activity has been determined or the enzyme partially purified from different animal tissues and cell lines [9-22]. To our knowledge, no comprehensive studies of enzyme activities in human tissues and in cell lines has been published before. Previous studies have demonstrated that there are three different isozymes of MAT, two present in hepatic tissues (alpha and beta form) and the third, gamma in all tissues [23-24]. In this report MAT activities of several human tissues are compared to those in turnouts and in cultured malignant cells.

141 TABLE !

Cultured cell lines used Cell lines were obtained from American Type Culture Collection (Rockville, MD), or they orginate from Hankinson (Hepa, [25]) or from Thompson (HTC, [26]). Code cell line a b c d e f g h i j k I m n o p r

Origin

Ramos human, Burkitt's lymphoma,B lymphocytecell human, Burkitt's lymphoma,lymphoblast-likecell Raii Daudi human, Burkitt's lymphoma,B lymphoblastcell WIL2 human, B-lymphocyte CCRF-CEMhuman, lymphoblastoid cell Molt-4 human, acute lymphoblasticleukemia HL-60 human, promyelocyticleukemia cell human, chronic myelogenousleukemia K-562 L1210 mouse, lymphocyticleukemia EL4 mouse, T cell lymphoma mouse, T cell lymphoma Rl.l BHK syrian or golden hamster, kidney chinese hamster, ovary CHO human, placenta, choriocarcinoma JAR HepG2 human, hepatoma HEPA * mouse, hepatoma HTC - + rat. hepatoma

E x p e r i m e n t a l procedures

Materials L-[methyl-14C]methionine (58 m C i / m m o l ) was obtained from A m e r s h a m International (Amersham, U.K.). Sephadex G-25 was from Pharmacia (Uppsala, Sweden). All other reagents were of analytical grade from E. Merck (Darmstadt, F.R.G.) or Sigma (St.Louis,

MO). Cell culture and harvesting of cells for enzyme analysis Cell culture vials were from Nunc (Roskilde, Denmark). The origin and properties of the established cell lines used are shown in Table I. Cell lines a to k were maintained in suspension culture in R P M I 1640 medium (Gibco, Uxbridge, Middlesex, U.K.) supplemented with 10% heat-inactivated fetal bovine serum (Cibco, lot No. 10G7572F), penicillin (100 u n i t s / m l ) and streptomycin ( 1 0 0 / z g / m l ) in a humid incubator at 37°C equilibrated with 5% C O 2 / 9 5 % air. Cells at late logarithmic growth phase were collected by centrifugation and washed three times with phosphate-buffered saline (30 ml per wash for l0 s cells). Cell lines I to r were grown in D M E M medium (Gibco) supplemented with 10% fetal bovine serum containing penicillin and streptomycin at 37°C in an atmosphere of 10% C O 2 / 9 0 % humidified air. When cells were near confluence they were trypsinized and collected as above. Cell pellets were stored at - 8 0 ° C until used for enzyme analysis.

Assay of methionine adenosyltransferase actit'ity M A T activity was measured as described by Kajander et al. [27]. The assay mixture contained 76 mM

Tris-HC1 (pH 7.4) (at 21°C), 40 mM KC1, 25 mM MgC12, 10 mM ATP, 2.5 mM fl-mercaptoethanol and 150 p~M methyl-14C-labelled c-methionine (4.9 p, C i / /~mol) and enzyme extract in a final vol. of 50 /~1. Incubations were started by the addition of enzyme preparation and incubations were carried out for 45 min at 37°C. Incubation was terminated by transferring 25 /~1 of the reaction mixture on a P-81 phosphocellulose paper disc (diameter 2 cm), which was then wetted with water and washed three times with 600 ml distilled water, dried and the radioactivity was counted in 2 ml of a toluene-based scintillation fluid. Enzyme activities from human liver were measured both in the presence and absence of 10% dimethylsulphoxide.

Protein determination Protein was determined by the method of Spector [28] with bovine serum albumin as a standard.

Assay conditions for methionine adenosyltransferase and stability of the enzyme Crude tissue homogenates were concentrated, when necessary, with a Centriflow c* concentrator. Linearity of the enzyme activity assay was examined varying the enzyme concentrations and the incubation times. The stability of the enzyme was examined using liver and placental homogenates. Liver and placental homogenates were freeze-thawed 1 to 15 times and the enzyme activity was determined.

Preparation of extracts from cultured cells Cell pellets (about l0 s cells) were resuspended in 1 ml of water containing 2 mM dithiothreitol and were subjected to 3 rapid cycles of freeze-thawing at - 7 0 ° C and 20°C, respectively. The cell suspensions were centrifuged at 12000 × g for 5 min at 4°C and supernatant fractions were separated and stored at - 8 0 ° C until assayed.

Preparation of tissue extracts Samples of normal and malignant human tissues removed at surgical operations were obtained via the department of Pathology, Kuopio University Hospital. These were immediately frozen and stored at - 8 0 ° C . Autopsy material was obtained from forensic autopsies (sudden accidents as the cause of death) performed within 2 days from death and the samples were stored at -80°C. Homogenization was carried out in 3 - 4 vol. of 0.25 M sucrose containing 0.1 mM E D T A , 0.2 mM PMSF and 2 mM /3-mercaptoethanol by using an O m n i - m i x e r h o m o g e n i z e r (Sorvall, Wilmington, U.S.A.). The homogenates were centrifuged for 45 min at 100000 x g (4°C). The supernatant fractions were filtered through glass wool, applied to a Sephadex G-25 column to remove excess salts, and eluted by 25 mM Tris-HC1 buffer (pH 7.4) containing 2 mM /3-mer-

142 400

protein (rag) 300C []

200C

DJ ~

~

~

2.34

E

300

~-

200

× o) 1 72

114

5

ta

0 1000

100

O.48 0.34

0

017 __

0

.

I

10

,

I

i

I

,

|

20 30 40 Incubotion time (rain)

I

I

50

I

l

60

Fig. ]. Assay of methionine adenosyltransferase by varying protein

concentration and incubation time. Values are means of duplicate measurements.

o

b

c

d

e

f

g

h

i

j

k

1

m

n

o

p

r

s

Fig. 2. Methionine adenosyltransferase activity in cultured cells and isolated peripheral blood lymphocytes. All assays were performed in duplicate and repeated twice and the results are expressed as the average of these determinations. Cell lines: a to r, see Table I, s, human peripheral blood lymphocytes (four samples).

Methionine adenosyltransferase in cultured cell fines captoethanol. The eluted fractions were used for the enzyme assays. Results

Assay of human placental methionine adenosyltransferase Linearity of MAT assay was examined using enzyme concentrations 0.17-2.34 rag/assay testing at 15-60 min incubation times (Fig. 1). All protein concentrations and incubation times produced enzyme activity curves that were linear ( r = 0.99-1.00). These tests were carried out using human placental crude supernatant fractions. Similar results were obtained using lymphocyte preparations as enzyme source. Partially purified placental enzyme also showed linear assay kinetics. The placental enzyme showed a K m value of 3.8 IzM for L-methionine. No substrate inhibition was observed at 150 /xM L-methionine and routinely enzyme activity assays were carried out using this saturating e-methionine level.

Fig. 2 shows MAT activity in 17 different cultured cell lines. Large variation in this enzyme activity was observed. Non-adherent cells (cell lines a to k) contained higher enzyme activity than monolayer cells (cell lines i to r). Ramos (human, B lymphocyte cell) and EL4 (mouse, T cell lymphoma) cell lines had MAT activity near 300 p m o l / m g per min. Daudi (human, B lymphoblast cell), peripheral blood lymphocytes and almost all monolayer cells contained MAT activity below 100 p m o l / m g per rain. When comparing MAT activity and doubling time (Fig. 3), it is observed that there is a correlation between short doubling time and high MAT activity (r = 0.86).

Methionine adenosyltransferase in normal and malignant human tissues Results in Fig. 4 show MAT activities in 12 normal human tissues. Large differences in enzyme activities were observed. From autopsy samples brain, colon and prostate had the highest MAT activity. Malignant tissue samples exhibited a much higher MAT activity than normal tissues. Lung cancer of the type carcinoma squamocellulore pulmonis had MAT activity of 30.7 p m o l / m g per min, which is over ten-times higher than

Stabifity of methionine adenosyltransferase activity Stability of MAT activity was measured from 100000 x g supernatant fractions of human placenta and liver (filtered with Sephadex G-25). The enzyme activity in human placenta was decreased by 35% after 10 freeze-thawings (by 29% after 5 and by 35% after 15 freeze-thawings) and that of human liver was decreased by 38% (by 25% after 5 and by 48% after 15 freeze-thawings). Liver tissue samples stored at + 4 °C lost the MAT activity rapidly, only 20% of total MAT activity in rat liver was left after 24 h storing at + 4°C and thus MAT could not be reliably assayed from liver obtained from autopsies. Lung tissue samples stored for 24 h at + 4°C lost < 5% of MAT activity. MAT was fairly stable in placentas with 95% activity remaining after storage at - 80°C up to 2 weeks (data not shown).

012

0.10

x

.c

x

O.Oe 0.06

O.Oz

8 ~.

0.02

O,OO 0

)( i

i 100

i

I 200

i

i 300

i 400

Sp. o. ( p m o l / Emg x min-~ )

Fig. 3. Relationship between MAT activity and doubling time in cultured mammalian cell lines (r = 0.86). Doubling times are from catalog of American Type Culture Collection or from our own measurements.

143 3O

:< o~

,E,

2(3

o_ v

1C

low e-methionine concentration ( < 1300 mM) in the assay mixture the presence of MAT/3-isozyme can be measured reliably only with dimethylsulphoxide activation. We tested all liver samples in the presence and absence of 10% dimethylsulphoxide. MAT activities in liver autopsy samples were activated by dimethylsulphoxide as follows: 5 h after death 2.3-fold from 239 to 541 pmol/mg per min, 10 h after death 1.6-fold (from 63 to 103 pmol/mg per min) and in all later autopsy samples (6) MAT activity was slightly inhibited (about 25%). Because both liver isozymes a and /3 are activable with dimethylsulphoxide [5,33,34] there seems to be only MAT y-isozyme activity in autopsy samples obtained 24 h after death.

o

C

2

3

4

5

6

7

8

9

10

11

12

Fig. 4. Methionine adenosy]transferase activity in human tissue sampies obtained from autopsy. A l l values are means of duplicate assays. (1) liver (n = 6); (2) spleen (n = 6); (3) pancreas (n = 6); (4) lugn (n = 6) (5) colon; (n = 6); (6) bone marrow (n = 6); (7) brain cortex, frontal lobe (n = 6); (8) kidney (n = 6); (9) prostate (n = 4); (10) uterus ( , = 1); (11) full-term placenta (n = 6); and (12) skin (n = 2) (n = number of assayed samples).

Discussion

that of normal lungs (2.4 pmol/mg per min). Normal mammary gland had MAT activity of 10.6 pmol/mg per min and breast carcinoma 41.4 pmol/mg per min. In our tests using 150 izM n-methionine, fresh rat liver had MAT activity of 640 pmol/mg per min. MAT /3-isozyme activity has also been measured with 25/~M n-methionine concentration [17,20,33]. When using a TABLE

As we see from Table II it is very difficult or impossible to compare published methionine adenosyltransferase activities in tissues. Although we calculate published activities using the same unit, the assay conditions are still different and the results are not comparable to each other. The kinetics of this enzyme is

11

Methionine adenosyhransferase activities published for mammalian cells and tissues All reported

activities are expressed

as pmol/mg

Tissue or

Enzyme

cell line

(pmol/mg

p e r rain. F o r c o m p a r i s o n ,

activity per min)

conditions of assay are given.

c

c

c

c

Met.

K

Mg

ATP

Ref.

(/~M)

(mM)

(mM)

(mM)

Liver human

2167

4000

100

115

10

9

Liver, human

1433

-"-

-"-

-"-

-"-

-"-

20

26

30

1

10 11

Eryth., human

0.05-0.2

Lymph., human

33-66

20

50

10

1

W I - 3 8 , cell l i n e

40.9

20

26

20

1

12

111.5

-"-

-"-

-"-

-"-

-"-

R - 5 , cell l i n e

132.6

-"-

-"-

-"-

-"-

-"-

B H K , cell l i n e

150.2

60

100

50

10

13

P-5, cell l i n e

MGF390,

cell line

57-70

100

133

67

13.3

36

MGF786,

cell l i n e

33-160

-"-

-"-

-"-

-"-

-"-

W1-38

93

- "-

- "-

- "-

- "-

- "-

Lymph. leukemia, human

71.7

10

50

40

5

14

0.3

20

25

20

2.5

15

Lens, rat Liver, rat

10

60

250

9

5

16

Liver, rat

66.7

25

150

7.5

5

17

1.2

20

150

20

10

18

Hepat., rat Liver, rat

7700

2000

250

150

10

19

Brain, rat

43

- "-

- "-

- "-

-"-

- "-

192

- "-

- "-

- "-

- "-

38

-"-

-"-

-"-

-"-

-"-

Pancreas, rat Spleen, rat

558 65

- "- "-

- "- "-

. . - "-

- "-

" - "-

Prostate, rat

141

Kidney, rat Lung, rat

- ".

.

-"-

-"-

-"-

-"-

-"-

Liver, mouse Lung, mouse

47 4.3

25 - "-

150 - "-

20 - "-

10 - "-

20 - "-

Kidney, mouse

12.3

- "-

- "-

- "-

- "-

- "-

Spleen, mouse

7.0

- "-

- "-

- "-

- "-

- "-

Brain, bovine

4.0

25

150

20

10

21

144

complex [14] and especially MAT/3-isoenzyme (present in liver) loses its activity during sample preparation and sample storage. Therefore, it is not acceptable to compare results with different assay conditions or sample preparation methods. The results show the difference of MAT activity between normal human tissues and cultured cell lines. It has been reported before that carcinogenesis changes enzyme levels in tissues [5] and Liau et al. [29] have advanced the opinion that the gamma-form of MAT is closely related to cell growth. These results give support to that, because all cell lines which have a short doubling time had very high enzyme activity and cell lines with long doubling times had low enzyme activity. Malignant tissue extracts produced for greater enzyme activity than normal tissues. Most malignacies grow rapidly and utilization of AdoMet is larger in them than in normal tissues [35]. Cell culture media contain 100-200/xM levels methionine, enough to saturate the gamma enzyme. Higher MAT activities may be needed to provide enough AdoMet for the cells. Caboche and Mulsant [30] have announced that AdoMet (or a derivative of AdoMet) is a regulator of MAT biosynthesis. Liau et al. [31-32] have reported that in the liver the total MAT activity decreased during carcinogenesis and c~- and /3-isoenzyme activity disappeared but yisoenzyme activity was slightly activated in both primary and transplantable hepatocellular carcinomas. Hepatoma cell lines have quite a low MAT activity, lower than cultured lymphocyte and thymocyte cell lines. It could be that there is no MAT /3-isoenzyme activity in malignant hepatocytes and that they are quite similar in this respect to other cultured cancer cell lines. Probably cancer cells 'shut down' enzyme activities which are not needed for their growth. Acknowledgements We thank Professor Y. Collan for providing tissue samples from malignancies. The skillful technical assistance of Miss Heli Martikainen is gratefully acknowledged. References 1 Meister, A. 11965) Biochemistry of the Amino Acids, 2nd Edn., Vol. I, 202-209, Academic Press, New York.

2 3 4 5 6

Hoffman, R.M. (1984) Biochim. Biophys. Acta 738, 49-87. Cantoni, G.L. 11953) J. Biol. Chem. 21}4, 4(13-416. Raina, A. and JS.nne. J. (19751 Med. Biol, 53, 121-147. Tabor, C.W. and Tabor, H. (1984) Adv. [;nzymol. 5~, 251-256, Poirier, L,A., Wilson, M.J. and Shivapurkar N. (19861 Biological Methylation and Drug Design, pp. 151-161, The Humana Press Clifton. 7 Kagan, B.L., Sultzer, D.L., Rosenlicht, N. and Gerner, R.H. 119901 Am. J. Psychiatry 147, 591-595. 8 Stramentinoli, G. 119861 Biological Methylation and Drug Design, pp. 315 326, The H u m a n a Press Clifton. 9 Gaull, G.E., Tallan H.H., Lonsdale, D., Przyrembel, tt., Schaffner, F. and Von Bassewitz, D.B. (19811 J. Pediatr. 98, 734-741. 10 Kotb, M., Dale, J.B. and Beachey, E.H. 119871 J. Immunol. 139, 202-2(16. 11 ()den, K.L. and Clarke, S. (1983) Biochem. 22, 2978-298& 12 Oden, K.L., Carson K., Mecham, J.O., Hoffman, R.M. and Clarke. S. (1983) Biochim. Biophys. Acta 7611, 2711 277. 13 Caboche, M. 119751 J. ('ell Physiol. 87, 321-336. 14 Kotb, M. and Kredich, N.M. (1985) J. Biol. Chem. 2611, 3923 3930. 15 Geller, M.A., Kotb. M.Y.S., Jernigan, H.M., Jr. and Kredich, N.M. 11986) Exp. Eye. Res. 43, 997-11108. 16 Cabrero, C., Puerta, J. and Alcmany, S. (1987) Eur. J. Biochem. 1711, 299-304. 17 Hoffman, J.L. (1983) Methods Enzymol. 94, 223-228. 18 Sawai, Y., Suma, Y. and Tsukada, K. (19861 Life Sci. 38, 197519811. 19 Eloranta, T.O.(1977) Biochem. J. 166, 521 529. 2/I Cox, R. and Goorha, S. 11984) Cancer Res. 44, 4938-4941. 21 Mitsui, K., Teraoka, H. and Tsukada K. (19881 J. Biol. Chem. 263, 11211-11216. 22 Suma, Y., Shimizu, K. and Tsukada, K. 119861 J. Biochem. 100, 67 75. 23 Okada, G., Watanabe, Y. and Tsukada, K. (19811)Cancer Res. 40, 2895 2897. 24 Okada, G., Teraoka, [t. and Tsukada, K. 119811 Biochem. 211, 934 940. 25 llankinson, O. (1979) Proc. Natl. Acad. Sci. USA 76, 373-376. 26 Thompson, E.B., Tomkins, G.M. and Curran. J.F. 11966) Proc. Natl. Acad. Sci. USA 56, 296-3[)3. 27 Kajander, E.O., Kubota, M., Carrera, C.J., Montgomery, J.A. and Carson, D.A. (1986) Cancer Res. 46, 2866-28711. 28 Spector, T. (1978) Anal. Biochem. 86, 142 146. 29 Liau, M.C., Chang, C.F., Belanger, L. and Grenier, A. (19791 Cancer Res. 39. 162-169. 311 Caboche, M. and Mulsant. P. 11978) Somat. Cell Gen. 4, 4/17-421. 31 Liau, M.C., Lin, G.W. and Hurlbert, R.B. (1977) Cancer Res. 37, 427 435. 32 Liau, M.C., Chert, F.C. and Becker, F.F. (1979) Cancer Res. 39, 2113 2119. 33 Matsumoto, C., Suma, Y. and Tsukada, K. (1984) J. Biochem. 95, 287 2911. 34 Hoffman, J.L. and Kunz, G.L. (1977) Biochem. Biophys Res. Commun. 77, 1231-1236. 35 Stern, P.H. and Hoffman, R.M. (1984) In Vitro 20, 663-669. 36 Jacobsen, S.J., Hoffman, R.M. and Erbe, R.W. (19811) J. Natl. Cancer Inst. 65, 1237-1244.