Changes of enzymic activities in human diploid cell line WI-38 at various passages

Experimental Cell Research 61 (1970) 357-364

CHANGES OF ENZYMIC

ACTIVITIES

IN HUMAN

DIPLOID

CELL LINE

WI-38 AT VARIOUS PASSAGES K.-M. WANG’*,

N. R. ROSE2, E. A. BARTHOLOMEW: K. BERDEl and M. FOLDVARY’

M. BALZERI,

IAging Research Laboratory, Veterans Administration Hospital, and Departments of Biochemical Pharmacology and of 2Microbiology, State University of New York at Buffalo, Buffalo, N.Y. 14215, USA

SUMMARY Three groups of enzymes, namely, phosphatases, dehydrogenases and transaminases have been studied in the develonment of human didoid cells. WI-38, from nassage 20 to nassage 52. There was a general decrease in the activities of-these three groups of e&ymesafter passage 42 and some increases of enzymic activities in the 3142 passagerange. The total cellular proteins were generally constant from the 20th to the 48th passage and increased after passage 48. However, an increase of protein in a specific cellular fraction at certain passages was observed. From pH and inhibitor studies on WI-38 diploid cells, malate dehydrogenaseshowed a shift to a high pH optimum at later passages, while acid phosphatase has shown no change to inhibitors from early to later passages. However, sodium tartrate showed an increased inhibitory effect on the soluble Zn2+ -activated acid phosphatase as passages progressed. Decreased inhibitory effect by cysteine and cyanide on alkaline phosphatase has been observed in the later passages.

Alteration of environmental conditions can change Amoeba proteus from the “immortal” to the “spanned” state [5, 141. The transformed “spanning” characteristics in Amoeba proteus have been shown to be associated with both nucleus and cytoplasm [15]. Hayflick [8] has shown that cell cultures of the WI-38 human diploid cell line have a defined life span of 50+ 10 passages. Some chromosome anomalies have been observed in phase III. Such anomalies may be expected to be reflected in the cytoplasm. One of the approaches to examine this problem is through the study of enzymic activities. Any changes in cytoplasmic enzymes would indicate possible alteration of the rate of metabolic reaction or blockage of one or more reactions. Investiga* Mailing address: Veterans Administration Hospital, 3495 Bailey Avenue Buffalo, N. Y. 14215, USA.

tion of some cytoplasmic enzyme activities in the development of WI-38 diploid cells has been reported [3]. The study showed progressive increase in acid phosphatase activity from 20-40 passages and no change in lactate dehydrogenase and alkaline phosphatase activities from 1545 passages. Since the localization and distribution of these enzymes in cellular compartments are different, the changes of their activities in a specific cellular fraction may not be the same as that in the total homogenate. The present report is a study of the changes of three classes of enzymes, namely, phosphatases, dehydrogenases and transaminases, in cellular fractions of WI-38 diploid cells in order to relate the changes of enzymic activities in their specific cellular compartments with various passages of cell culture. Furthermore, the use of various pH Exptl Cell Res 61

350 K.-M. Wang et al. and inhibitors in the assay system allows us to test whether there are any characteristic changes of these enzymes in WI-38 diploid cells from early to later development.

MATERIALS

AND METHODS

Cell culture WI-38 diploid cell line deriving from female human fetal lung obtained originally from Dr Hayflick was employed throughout all the experiments. The medium, culture conditions and subcultivation of confluent cultures were based on a previous report [l]. Mycoplasma determinations were made at every other passage. All cultures in the present work were free of mycoplasma.

WI-38 diploid cells fractions WI-38 diploid cells obtained at various pasages were processed as described by Bartholomew et al. [l]. The differential centrifugation procedure was based on a previous report on developing chick embryonic liver [26].

ing to the method of Lowry & Lopez [lo]. Acid and alkaline phosphatase (AcPase, E.C. 3.1.3.2 and AlPase, E.C. 3.1.3.1) activities were determined by the method of Lowry et al. [13] using p-nitrophenyl phosphate as substrate in 0.05 M sodium acetate buffer, pH 5.2, and in 2-amino-2-methyl-l-propanol buffer, pH 10.0, respectively. Zn2+-activated acid phosphatase (Zn-AcPase) activity was assayed by a method described previously [24]. The phosphatase activities are expressed as pmoles substrates liberated/ h/mg protein. Lactate dehydrogenase (LDH, E.C. 1.1.1.27) activity [12], malate dehydrogenase (MDH E.C. 1.1.1.37), glutamate dehydrogenase (GDH, E.C. 1.4.1.2), glutamic-oxalacetic transaminase (GOT, E.C. 2.6.1.1), glutamic-pyruvic transaminase (GPT, EC. 2.6.1.2) [ll, 211 and isocitrate dehydrogenase (ICDH, E.C. 1.1.1.42) [20] activities were determined as previously described [23, 25, 271. LDH, GDH, GPT, GOT activities are expressed as pmoles NADH* oxidized/h/mg protein. MDH activity is expressed as pmoles NAD reduced/h/mg protein. ICDH activity is expressed as pmoles NADP reduced/h/mg protein. The protein concentration in various cellular fractions of WI-38 diploid cells was determined by the method of Lowry et al. [14] with versatol as the standard. All buffers, substrates and other reagents were made with demineralized double glass-distilled water.

RESULTS Chemicals Substrates, enzymes and coenzymes were purchased from Sigma Chemical Company, St Louis, MO. Versatol (freeze-dried human serum) was purchased from Warner-Chilcott. Morris Plains, N. J. Other chemicals were purchased from Fisher Scientific Company, Pittsburg, Pa.

Enzyme assays Glucose-6-phosphatase (G-6-Pase, E.C. 3.1.3.9) activity was determined by Pi release from the substrate, glucose-6-phosphate [22]. Pi was measured accord-

The results of the studies of phosphatases, dehydrogenases, and transaminases are shown in tables I, 2,3. Among the ten enzymes studied AcPase activity (table I), LDH activity (table 2) and GOT activity (table 3) showed significant decreases after passages 42 and 48. Some increases in G-6-Pase, AcPase, MDH, GPT, and GDH activities have also been noted between passages 31 and 42.

Table 1. Phosphatase activities of WI-38 diploid cells Phosphatase activities Passage range 22-30 31-36 37-42 43-48 49-54

aG-6-Pase b0.49riro.03 (2) 0.85 k 0.30 (2) 0.40 (1) 0.45 kO.10 (2)

aAcPase

aZn-AcPase

aAIPase

3.75 + 0.22 (5) 3.15 (1) 3.92 i 0.49 (2) 3.01& 0.32 (2) c3.00* 0.04 (2)

2.38 +0.29 (3)

0.47 (1) 0.32 (1) 0.34 (1) 0.45 (1) 0.55 (1)

1.88+0.30 (2) 1.59 (1) 1.85+0.14 (2)

a G-6-Pase, glucose-6-phosphatase. AcPase, acid phosphatase. Zn-AcPase, Zn 2+-activated acid phosphatase. AlPase, alkaline phosphatase. The phosphatase activities are expressed as pmoles substrates liberated/h/mg protein. b Mean values f S.E.M. and number of determinations in parenthesis. ’ The decrease was significant p < 0.05. ExptI Cell Res 61

Enzymes in human diploid cells, WI-38

359

Table 2. Dehydrogenase activities of WI-38 diploid cells Dehydrogenase activities Passage range

aLDH

aMDH

aICDH

22-30 31-36 37-42 4348 49-54

b24.13t-3.69 (6) 23.73 t4.26 (4) 20.86 k 1.73 (3) 11.25 (1) ‘10.68 +O.SO(2)

55.67 k 4.07 (3) 46.38 + 14.63 (2) 56.90& 3.25 (2) 41.68 (1) 30.58t- 15.98 (2)

2.43 +0.71 (2) 1.21&0.04 (2) 1.91 (1) 1.69-tO.35 (2)

a LDH, lactate dehydrogenase; its activity is expressed as ,umoles NADH, oxidized/h/mg protein. MDH, malate dehydrogenase; activity expressed as pmoles NAD reduced/h/mg protein. ICDH, isocitrate dehydrogenase; activity expressed as pmoles NADP reduced/h/mg protein. ?JMean values k S.E.M. and number of determinations in parentheses. ’ The decrease was significant p < 0.01.

The total cellular proteins (fig. 1) were generally constant from the 20th to the 48th passages and increased after passage 48. However, an increase of protein in a specific cellular fraction at certain passages was observed. A peak increase of protein was first observed in the mitochondrial-lysosomal fraction at about passage 25, then in the microsomal fraction at about passage 31, thirdly, in the soluble fraction at about passage 48, and finally in the nuclear fraction at about passage 52. A decrease of these cellular proteins was noted after each increase. The localization of the above studied enzymes in various cellular fractions varied. In order to relate the changes of enzymic activi-

ties in their specific cellular compartments with various passages of cell culture, further study of AcPase, LDH, MDH and GOT activities in cellular fractions was carried out. Results on the distribution of these enzymic activities are shown in figs 2, 3. AcPase activity (fig. 2) was abundant in microsomal, soluble and mitochondrial-lysosomal fractions, whereas GOT activity was rich in mitochondrial-lysosomal fraction. The highest GOT activity was observed between 31-42 passage range. A significant decrease in the soluble fraction and total homogenate was observed after 42 passages. The highest MDH activity (fig. 3) in the mitochondrial-lysosomal and soluble fractions was observed

Table 3. Enzymes involved in amino acids metabolism of WI-38 diploid cells Enzymic activities Passage range 22-30 31-36 37-42 43-48 49-54

aGPT

aGOT

=GDH

b0.21+0.01 (3) 0.27 +0.07 (3) 0.17*0.01 (2)

5.08 kO.33 (6) 5.80+0.86 (4) 4.12 f 0.09 (2) 3.95 (1) ‘3.22 kO.40 (2)

0.94kO.11 (5) 1.19kO.11 (3)

a GPT, glutamic-pyruvic transaminase. GOT, glutamic-oxalacetic transaminase. GDH, glutamate dehydrogenase. The activities are expressed as pmoles NADH, oxidized/h/mg protein. b Mean values k S.E.M. and number of determinations in parentheses. ’ The decrease was significant p < 0.01. Exptl Cell Res 61

360

K.-M. Wang et al. H

N

M

P

S

H

N

M

P

s

80 01

I 30

I 40

I 50

Fig. 1. Abscissa: passage; ordinate:

% of total cellular protein. Protein content in WI-38 diploid cells showing the % distribution of total protein in nuclear (N), mitochondrial-lysosomal (M), microsomal (P) and soluble fractions. The inserted graph indicates the protein content of total homogenate at various passages.

*

40 20 0

/

b

Fig. 3. Abscissa: enzymic activities in WI-38 cells; ordinate: (tap) LDH; (bottom) MDH.

between 31-42 and 20-30 passage ranges, respectively. MDH is known as a mitochondrial and soluble enzyme, but we have also found it in the nuclear fraction. LDH. a soluble 5 4

H

N

M

P

S

3 2 1

a

0 H

M

P

s

81

Fig. 2. Ordinate: (tap) AcPase; (bottom) scissa: enzymic activities in WI-38 cells;

GOT; Ab-

Acid phosphatase activity, expressed as pmoles pnitrophenol liberated/h/mg protein and glutamicoxalacetic transaminase activity, expressed as pmoles NADHz oxidized/h/mg protein in various cellular fractions. H, total homogenate; N, nuclear; M, mitochondrial-lysosomal; P, microsomal and S, soluble fractions. 22-30, 3142 and 43-52 indicate passage ranges. Asterisks indicate significant decrease or increase of enzymic activity. The standard error of the mean is shown by the vertical bars. ExptI Cell Res 61

Lactate dehydrogenase activity, expressed as pmoles NADH, oxidized/h/mg protein and malate dehydrogenase activity, expressed as NAD reduced/ h/mg protein in various cellular fractions. H, total homogenate; N, nuclear; M, mitochondrial-lysosomal; P, microsomal and S, soluble fractions. 20-30, 3142 and 43-52 indicate passage ranges. Asterisks indicate significant decrease of enzymic activity. The standard error of the mean is shown by the vertical bars.

enzyme, which was found to be abundant in the soluble fraction, maintained a fairly high level at the passage ranges similar to the soluble MDH. A significant decrease of these two soluble enzymic activities was observed after passage 42. The fluctuation of enzymic activities observed at certain passages may be the result of many factors, of which one might be a shift of pH. Such a shift of pH could activate or inhibit certain enzymic activities. Table 4 shows the pH effect on two enzymes. In the early passages studied at 22 to 25 passages, only AcPase was more active at a lower pH. At 48 to 50 passages the activity of MDH was changed in favor of higher pH. We further studied the possible characteristic changes in enzymes from the early passages to the later passages by examining the

Enzymes in human diploid cells, WI-38

361

Table 4. Influence of pH on enzymic activities of WI-38 diploid cells Enzymic activity at passage: 22 25 PH Malate

10.3 “10.5 10.7

aH

48

50

%

H

S

H

18.64 72.52 82.88

52.99 61.96 56.84

68.16 63.13 67.61

55.29 55.29 66.50

2.81 2.60 1.92

3.59 3.31 2.53

2.67 2.21 1.68

3.31 3.33 2.70

H

S

15.43 79.62 103.92

52.15 49.88 51.59

51.03 52.60 53.23

2.41 2.32 1.84

3.12 3.26 2.63

2.29 2.19 1.I0

s

dehydrogenase

55.64 59.51 61.21

Acidphosphatase

4.8 b5.2 5.6

3.38 3.14 2.14

a H, homogenate. S, soluble fraction of WI-38 diploid cells. b Italicised pH were those pH values used for each enzymic assay. Malate dehydrogenase activity is expressed as /AmolesNAD reduced/h/mg protein. Acid phosphatase activity is expressed as pmolesp-nitrophenol liberated/ h/mg protein.

inhibitory effect on AcPase, Zn-AcPase and AlPase by fluoride, cysteine, sodium L( +)tartrate and cyanide. The results are shown in figs 4, 5, 6. There was no difference in the inhibitory effect on AcPase by fluoride, cysteine and tartrate in the three passage ranges studied, nor in various cellular fractions, except that the soluble AcPase was more inhibited by tartrate (fig. 4). The inhibitory effect on soluble Zn-AcPase (fig. 5) by tartrate was consistently greater as the passage range increased. As shown in fig. 6, fluoride has no effect on AlPase at the early passage range 20-31, but inhibition increased at later passage range. Cysteine and cyanide inhibited AlPase of all cellular fractions but the inhibition was decreased in the later passage (4352), especially of the microsomal fraction.

been indicated as the passage level reached prior to the cessation of mitoses [8], the decrease in enzymic activity after passage 42 is likely related to the cessation of mitoses and reflects a reduction of metabolic function. The usual metabolic function of phospha1,

201.

M

P

S 1

I

a

b iH

M

P

DISCUSSION One general phenomenon observed in the 3 groups of enzyme studied, namely, phosphatases, dehydrogenases and transaminases, is the decrease in enzymic activity after passage 42. Since the passage range 39-58 has

Fig. 4. Abscissa: inhibition of AcPase (a) fluoride (0.05 M); (b) cysteine (0.05 M); (c) Na-tartrate (0.05 M); ordinate: % of control activity. Effect of inhibitors on acid phosphatase activity. IT, homogenate; M, mitochondrial-lysosomal; P, microsomal and S, soluble fractions. 25-31, 32-42 and 43-52 indicate passage ranges. Each column represents the average value from 2 to 3 experiments. Exptl Cell Res 61

362 K.-M. Wang et al.

a

b -. H

N

M

P

S

70 50 30

Fig. 5. Abscissa: inhibition of Zn-AcPase (a) fluoride (0.05 M); (b) cysteine (0.05 M); (c) Na-tartrate (0.05 M); ordinate: % of control activity. Effect of inhibitors on Zn2+-activated acid phosphatase activity. H, homogenate; IV, mitochondriallysosomal; P, microsomal, S, soluble fractions. 2031, 32-42 and 43-52 indicate passage ranges. Each column represents the average value from 2 to 3 experiments.

a

3

M

P

S

Fig. 6. Abscissa: inhibition of AlPase (a) fluoride (0.02 M); (b) cysteine (0.02 M); (c) cyanide (0.02 M); ordinate: % of control activity. Effect of inhibitors on alkaline phosphatase activity. H, homogenate; M, mitochondrial-lysosomal; P, microsomal and S, soluble fractions. 20-31, 32-42 and 43-52 indicate passage ranges. Each column represents the average value from 2 to 3 experiments. Exptl Cell Res 61

tases is their hydrolytic action in liberating inorganic phosphates from phosphomonoesters. The high and low activity observed in various passage ranges of WI-38 diploid cells may reflect the metabolic need of inorganic phosphates. The high AcPase and AlPase activities and low activity of LDH observed in the present study in comparison to the results obtained by Cristofalo et al. [3] are due to different buffer systems and extraction media used. However, a similar developmental pattern of AlPase was obtained between the present study and the results obtained by Cristofalo et al. [3]. The fluctuating developmental pattern of AlPase activity in relation to certain metabolic function is not clear. AcPase in general is recognized as one of the lysosomal hydrolases @glycerophosphate as substrate) which can be released by treatment with Triton X-100 [6]. It has also been found in lysosomal fractions of WI-38 diploid cells [4] and rat liver [ 191;in large granules and microsomal fractions of livers of guinea pig, mouse [17,18] and chick [26]. In the present study there was no specific separation of the lysosomal fraction. Thus some of the lysosomal AcPase activity might have been combined with microsomal AcPase activity. Cristofalo et al. [3] have shown a significant increase in AcPase activity of total homogenate from passage 20 to 40. In the present study, some increase of AcPase activity in all cellular fractions at passage range 31-42 (fig. 2) and a significant decrease thereafter were observed. Since the passage range used in the present study is an average of several passages in comparison to three individual pasages shown by Cristofalo et al. [3], no clearcut significant increase would have resulted. Franklin [7] has shown a significant decrease of AcPase activity (with or without Triton X-100 treatment) in aged rat kidney but not in similarly aged rat liver. Such results indicate that a decrease in AcPase activity occurs

Enzymes in human diploid cells, WI-38 363 in some tissues (rat kidney, WI-38 diploid cells), but not in others (rat liver, etc.) A decrease of LDH activity has been observed in 74-week-old rat heart and brain [9], and in year-old mouse liver [2]. Kanungo & Singh [9] suggested that the total decrease in LDH activity was due to the decrease in cell number in heart and brain tissues with increasing age. The significant decrease of LDH activity in human diploid cells after passage 42 also appears to relate to the decrease in cell number and cellular activity, since this is the passage level coincident with cassation of mitoses [8]. Soluble MDH activity showed a similar decrease as that observed with LDH activity. The decrease of LDH and MDH activities, involved in glycolysis and citric acid cycle, may be the result of a declining rate of the carbohydrate metabolism due either to the decrease of the number of cells or to a reduced efficiency of the catalytic ability of the enzymes. The direct or indirect control mechanism of such decreases in enzymic activities, however, is unknown. Some increases in mitochondrial-lysosomal AcPase, GOT, LDH and soluble LDH activities occurred in the 31-42 passage range. This passage range falls within phase III during which the WI-38 diploid cells cannot carry on active multiplication. It appears that the enzymes in a specific cellular fraction would have to work harder in order to compensate for the loss of catalytic efficiency of enzymes in other cellular fractions and to maintain a pace similar to metabolic reactions of phase II. The decrease in enzymic activities might be caused by many factors. One appears to be the cessation of mitoses and consequently a reduction of cellular metabolic activity. In the present work some other factors have been tested. The data indicate that the decrease in enzymic activity might be due to a shift of pH as that seen in MDH activity. Further24-701816

more, some enzymic activities, even though decreasing in activity during later developmental passages do not change their characteristics as judged by the inhibitor results and pH studies, e.g. AcPase. Others, soluble ZnAcPase and AlPase, however, have changed gradually or abruptly. The changes could be the result of gradual unfolding of the enzyme molecule and an alteration in the requirement of cofactor and substrate. Certain fine differences in enzymic content of cells could be overlooked by the use of total enzyme quantitation. For, although total activity may be similar for cells of various generations, a shift in the isoenzyme picture could occur and remain obscured [l]. It would be important, therefore, to examine the zymograms of extract under study to obtain another parameter of enzyme content and distribution. The authors wish to thank Mrs J. Hermann for culturing the WI-38 diploid cells and for examining the mycoplasma. The work of N.R.R. and E.A.B. was supported by a NIH researchgrant CA 05203from the National CancerInstitute. REFERENCES 1. Bartholomew, E A, Bartholomew, W R & Rose N R, J immunology 103(1969)787. 2. Burich, R L & Ewing, K L, J gerontol 23 (1968) 180. 3. Cristofalo, V J, Parris, N & Kritchevsky, D, J cell physiol 69 (1967)263. 4. Cristofalo, V J, Kabakjian, J R & Kritchevsky D, Proc sot exptl biol med 126(1967)649. 5. Danielli, J F & Muggleton, A, Gerontologia 3 (1959)76. 6. de Duve, C, Pressman,B C, Gianetto. R, Wattiaux, R & Appelmans, F, Biochem j 60 (1955) 604.

Franklin, T, Biozhemj 82 (1962) 118. Hayflick, L, Exptl cell res 37 (1965)614. Kanungo, M S & Singh, S N, Biochem biophys res comm 21 (1965)454. 10. Lowry, 0 H & Lopez, J A, J biol them 162(1946) I. 8. 9.

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14. 15. 16. 17.

W S & Crawford, E J, J biol them 207 (1954) 19. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. Muggleton, A & Danielli, J F, Nature 181 (1958) 1378. 1 Exptl cell res 49 (1968) 116. Neil, M W & Horner, M W, Biochem j 92 (1964) 217.

18. - Ibid 93 (1964) 220. 19. Nelson, D, Proc sot exptl biol med NY 121 (1966) 998. 20. Roberts, N R, Codho, R R, Lowry, 0 H & Crawford, E J, J neurochem 3 (1958) 109.

Exptl Cell Res 61

21. Rosen, F, Roberts, N R & Nichol, C A, J biol them 234 (1959) 476. 22. Segal, H L & Washko, M E, J biol them 234 (1959) 1937. 23. Wang, K-M, Life sci 5 (1966) 2209. 24. - Experientia 24 (1968) 424. 25. - Comp biochem physiol 27 (1968) 33. 26. - Biochem j 115 (1969) 191. 27. Wang, K-M & Lin, A H, Europ j pharmacol 1 (1967) 347.

Received January 7, 1970