Printed in Sweden Copyright © 1977 by Academic Press, lnc. All rights of reproduction in any form reserved ISSN 0014~t827

Experimental Cell Research 108 (1977) 87-93

CELL SURFACE CHANGES ASSOCIATED WITH AGING OF CHICK EMBRYO FIBROBLASTS IN CULTURE K. YAMAMOTO, M. YAMAMOTO and H. OOKA

Department of Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaeeho, Itabashi-ku, Tokyo-173, Japan

SUMMARY Using cultured chick embryo fibroblasts with a finite life span in vitro, the cellular agglutinability by concanavalin A (ConA) and the cellular capacity to bind [3H]ConA have been investigated in the course of serial subcultivations. The following results were obtained: 1. The sensitivity of the EDTA-dissociated cells to ConA-mediated agglutination was found to decrease with the advance of subcultivation. 2. The agglutinability of the "aged" cells was markedly enhanced by treating the cells with hyaluronidase, returning back to an agglutinability similar to that of the "young" cells. On the other hand, the hyaluronidase treatment of the "young" cells did not affect the sensitivity of the cells to ConA-mediated agglutination. 3. In contrast to hyaluronidase, trypsin treatment resulted in enhancement in the sensitivity of the cells to ConA-mediated agglutination without regard to in vitro cellular "aging". 4. The [3H]ConA-binding capacity of the cells was found to increase with "aging" when it was expressed in terms of per cell (or per cellular protein), while it remained nearly constant when expressed in terms of per surface area. These results together with the others seem to suggest that the "age"-dependent depression in the sensitivity to ConA-mediated agglutination of cultured chick embryo fibroblasts may at least partly be due to hyaluronic acid-protein conjugates likely to be enriched at the cell surface as in vitro cellular "aging" advances.

Chick embryo fibroblasts have a finite life span in vitro [1-4]. The growth potential of the cells declines progressively in the course of serial subcultivations. According to the hypothesis proposed by Hayflick & Moorhead [5], the system may be taken as a model for the cellular expression of aging. Changes of several biochemical parameters have been reported to occur in the course of in vitro aging of human fibroblasts [5-10] as well as of chick embryo fibroblasts [1-2]. Cell surface membranes seem to be relevant to some important biological phenomena or functions such as contact inhibition, cellular adhesion, intercellular com-

munications and so on [11-16]. However, changes in cell membranes associated with aging have been very little reported [3-4, 17-18]. Various plant lectins such as concanavalin A (ConA) [16, 19-29], wheat germ agglutinin (WGA) [22-23, 25-27, 30], and others [22-23, 31-33], which are known to bind specifically to certain sugar moieties of glycoproteins at the cell surface, have recently been used as a probe to analyse the structure and function of the cell surface. Differences in the lectin-mediated agglutination [16, 19-20, 22-23, 25-27, 30-34] and sometimes also in the number of lectinExp Cell Res 108 (1977)

Yamamoto, Yamamoto and Ooka

88

T a b l e 1.

Agglutination of chick embryo fibroblasts by ConA Degree of agglutination as conc. of ConA (/xg/ml)

Passage

5

10

25

50

100

250

500

3 6 11 15 19 23

---

+ +

+ +

--

+

+

--.

---

+ --

++ ++ ++ + +

+++ +++ ++ + + +

+++ +++ +++ ++ ++ +

++++ ++++ +++ ++ ++ +

.

.

.

Cells were dissociated with EDTA (10 min at 37°C) and examined for agglutination after 15-min incubation with ConA at 24°C.

b i n d i n g s i t e s [20--21, 24, 2 8 - 2 9 ] h a v e b e e n reported between

normal and transformed

cells. In the present

study,

we have investi-

gated the agglutinability of cultured chick embryo

fibroblasts

by

ConA,

and

found

depression of the cellular agglutinability by ConA

in t h e c o u r s e

tions. Moreover

of serial subcultiva-

it w a s f o u n d t h a t r e d u c e d

sensitivity of the aged cells can be enhanced

10% fetal bovine serum and kanamycin (60 /zg/ml), pH 7.4. Under these conditions, the whole life span throughout a series of subcultivations may be classified into the following 3 phases: Phase I (corresponding to the lst-5th passage); phase II (corresponding to the 6th16th passage), and phase III (corresponding to the 17th-24th passage), in according to the criteria reported by others [1-4]. Counting of cell numbers and measuring of cell volumes were carried out by means of a hemocytometer and a Model ZBI Coulter counter (Coulter Electronics, Inc.), respectively. The viability test of cells was done by staining with 1% eosin yellow in 0.9% NaCI solution.

up to a level of the young cells by treating the

cells

with

hyaluronidase,

suggesting

t h a t h y a l u r o n i c a c i d at t h e c e l l s u r f a c e m a y be responsible for the decreased ability

associated

agglutin-

with

in

vitro

AND

METHODS

cellular

aging.

MATERIAL

Cell culture Chick fibroblasts were obtained from leg muscle of 10-day-old embryos of White Leghorn chick according to the procedure of Hay & Strehler [1] with a little modification. The tissue minced with surgical knives was treated with 0.05 % trypsin (Difco 1 : 250) in Ca 2+and Mg2+-free Hanks' balanced salt solution at 37°C for 20 rain to dissociate the cells. The dissociated cells were then washed with the culture medium and plated in 60 mm plastic Petri dishes at the concentration of 3.6 × 106 cells/dish, followed by culturing at 37°C under 5 % CO2, 95 % air. Upon reaching the confluency, the cells were removed from the dish by treating with 0.05 % trypsin, and the dissociated cells thus obtained were equally divided into two parts, each of them being plated anew in a dish. The sequential plating with a 1:2 sprit ratio was continued throughout the following subcultivations. The culture medium used was Eagle's minimum essential medium supplemented with

ExpCellRes 108(1977)

Assay of cellular agglutinability by ConA Cultured cells at the confluency were washed with Ca 2+- and Mg2+-free phosphate-buffered saline (8.0 g NaCI, 0.2 g KC1, 1.15 g Na2HPO4, and 0.2 g KH2PO4 per 1 liter distilled water, pH 7.2) and then incubated with 0.1% EDTA in the same Ca ~+- and MgS+-free phosphate-buffered saline (PBS) at 37°C for 10 min, except when otherwise specified. The dissociated cells were washed three times with PBS and finally suspended in PBS at 1.4× 106 cells/ml. The cell suspension (0.5 ml) was mixed with 0.5 ml of PBS containing ConA of various concentrations (from 10/zg to 1000 /~g) in 35 mm Petri dishes. After 15 min of incubation at room temperature (24°C+2°C) with gentle shaking, cellular agglutination was observed under a microscope. The degree of agglutination was expressed in terms of either the serological scale (from - to + + + +) or the percentages of single cells in the total cells. ConA (grade IV) used was obtained from Sigma Chemical Co.

Assay of cellular capacity to bind [zH]ConA [aH]ConA (Radiochemical Centre) was co-chromatographed with an appropriate amount of unlabelled ConA on Sephadex G-75 according to the procedure of Noonan & Burger [28], and a major fraction corresponding to the tetrameric ConA was separated. The

Age-related cell surface changes [3H]ConA fraction thus obtained showed the specific activity of 8x l0 s cpm/mg protein, and was adequately diluted with PBS. To measure the cellular capacity to bind ConA, 0.5 ml of the [~H]ConA solution and 0.5 ml of the dissociated cell suspension (prepared as described earlier) were mixed in a small test tube. After 15 min of incubation at 0 or 24°C, the cells were collected by filtering through a glass fiber filter (Whatman GF/B) and washed with PBS. The radioactivity retained on the filter was counted in a liquid scintillation spectrometer using toluene containing 0.4 % PPO (Merck) as a scintillation fluid. The similar binding experiments were carried out in the presence of 0.1 M c~-methyl-D-mannoside, and the count thus obtained was subtracted from the count in the absence of amethyl-D-mannoside to get the specific binding of [3H]ConA.

Estimation of cellular surface areas The cellular surface area was estimated from the average cellular volume measured by a Coulter counter by assuming that the cells are smooth spheres.

Protein assay Protein was assayed by the method of Lowry et al. [35] with bovine serum albumin as a standard.

RESULTS

Change in the sensitivity of chick embryo fibroblasts to ConAmediated agglutination in the course of serial subcultivations Cultured chick embryo fibroblasts were examined for agglutinability by ConA at various stages of a series of subcultivations. As summarized in table 1, the sensitivity of cells to ConA-mediated agglutination seemed to decrease as the number of passages advanced. Young cells such as those harvested at the 3rd passage (phase I) or at the 6th passage (early phase II) showed a (+) agglutination-score at the ConA concentration as low as 25 /~g/ml, and the ( + + + + ) agglutination-score (maximal agglutination) at 500/.~g/ml ConA. On the other hand, aged cells such as those harvested at the 19th passage (phase III) were not agglutinated by ConA at 25 ~g/ml, and showed only the (+) agglutination-score at 100/.~g/ml ConA.

89

100 0.....0 ~

80

A 0 .______.._____ 0

e~ • -------______ 0

60

40

~ o ~ 2(3

170

5I

~

1

l 15

l

l

I l l l l

l 30

5

10

15

30

Fig. 1. Abscissa: time (rain); ordinate: % single cells. O - - O , Young cells (6th passage); O - - O , aged cells (19th passage). The kinetics of cellular agglutination of chick embryo fibroblasts by ConA. (A) 10 /xg/ml ConA; (B) 100/zg/ml ConA. Cells were dissociated with EDTA (10 rain, 37°C) and examined for agglutination at 24°C. Each point represents the average value of four different experiments.

It should be noted that the cells incubated with PBS only (blank) did not show any sign of cellular agglutination within an observation period of 30 rain without regard to the number of passages or in vitro cellular aging. The ConA-mediated agglutination of the cells was completely prevented or reversed by 0.1 M ~-methyl-D-mannoside added before or after the incubation with ConA, but not by 0.1 M N-acetyl-D-glucosamine. Moreover, it was confirmed that ConA-mediated agglutination of the cells is temperature-dependent, and does not take place at 0°C. Next, the dissociated cells harvested at the two different stages (passages) were incubated with ConA at 10 or 100/xg/ml at 24°C for various intervals of time, and the percentages of single cells in the total cells were counted. The agglutination kinetics shown in fig. 1 (A, B) indicate that the cellular agglutination may reach equilibrium at 15 min or so at 24°C. At the same time, it was found that the higher the concentrations of ConA or the smaller the number of passage, the smaller become the portions of single (unaggregated) cells at the time of Exp Cell Res 108 (1977)

90

Yamamoto, Yamamoto and Ooka

Table 2. Effects of trypsin and hyaluronidase treatments on agglutination of chick embryo fibroblasts by ConA Degree of agglutination as conc. of C o n A (/xg/ml) Passage treatment

19

5

10

25

50

100

250

500

EDTA Trypsin Hyase ~

_+ -

_+

+ +

+ +++ ++

++ ++++ ++

+++ ++++ +++

+++ ++++ +++

++++ ++++ ++++

EDTA Trypsin Hyase a

-

+ +

+++ ++

_+ .++++ ++

+ ++++ +++

++ ++++ +++

++ ++++ ++++

-

Cells were treated with 0 . 1 % E D T A (10 rain at 37°C), 0.05 % trypsin (5 or 10 rain at 37°C), or hyaluronidase (100 U/ml, 30 min at 37°C) and e x a m i n e d for agglutination after 15 min incubation with C o n A at 24°C. a H y a s e , abbreviation of hyaluronidase.

agglutination equilibrium, confirming the earlier results. In fig. 2 are summarized the results of cellular agglutination tests as assayed at 15 min of incubation with ConA of a wide concentration range (5-500/~g/ml). Here again it was clearly shown that the cells of the 6th passage are more sensitive to ConAmediated agglutination than the cells of the 19th passage.

Effects of trypsin and hyaluronidase treatments on ConA-mediated agglutination of the cultured fibroblasts Cultured chick fibroblasts harvested at various passages were treated either with 0.05 % trypsin at 37°C for 5 or 10 rain or with hyaluronidase (Tokyo Seikagaku Kogyo Co.) at 100 U/ml at 37°C for 30 min, followed by washing with PBS. The cells thus obtained were examined for ConA-mediated agglutination. The results summarized in table 2 seem to show that trypsinization of cells results in a marked enhancement of cellular sensitivity to ConA-mediated agglutination also for cells harvested at the 3rd and 19th passages, and seemingly up to a level of maximal sensitivity, thus removing the ageExp Cell Res 108 (1977)

dependent difference in the sensitivity to ConA-mediated agglutination. In contrast to trypsinization, treatment of the cells with Streptomyces hyaluronidase resulted in a marked sensitization only in the case of cells of the 19th passage, while the sensitivity of the 3rd passage cells to ConA-mediated agglutination was not affected by the hyaluronidase treatment. Moreover, it was found that the sensitivity of the 19th passage cells treated with hyaluronidase is almost similar to that of the {00 0 ~ 0

80

\

°~

60

°~

40 20 0

~o I

I I IIill 5 10

I

I

I I illll 50 100

I

I

I I 500

Fig. 2. Abscissa: C o n A conc. (/~g/ml); ordinate: % single cells, e - - O , y o u n g cells (6th passage); O - - O , aged cells (19th passage). Effect of C o n A concentration on cellular agglutination of chick e m b r y o fibroblasts. Cells were dissociated w i t l ' / E D T A (10 min, 37°C) and e x a m i n e d for agglutination after 15 rain incubation with C o n A at 24°C. E a c h point r e p r e s e n t s the average value of four different experiments.

Age-related cell surface changes K

I

A

,

i

B

A ::

10 o

O ~O

6

o~o

~o/O/°~°

/~

~

o

B .o~o_o~O--~--~

---~

o

_ ~ ~-2---,~ ~ - _0~0~0--0--0

4

i~

/o j

8

o

91

--£

-°-

~/~

/ /

A/A 2

0 ,0

2o

,o

,5

2'°

I

I

10

15

[ 20

10

1

20

Figs 3, 4. Abscissa: passage; ordinate: (fig. 3) cpm × 103/cell; (fig. 4) cpm × 106//z2 surface area. 0--(3, EDTA-dissociated cells; Z~--L~, trypsinized cells. Age-dependence of binding of [3H]ConA to chick embryo fibroblasts. Cells were dissociated with EDTA

or trypsin and examined for their binding of [3H]ConA after 15 min incubation with ConA (100/xg/ml) at (A) 0°C or (B) 24°C. Each point represents the average value of four different experiments.

3rd passage cells with or without hyaluronidase treatment. Since the hyaluronidase used in the present study has been confirmed not to contain protease or esterase to any significant extents, the results described here may suggest that hyaluronic acid at the cell surface may somehow be responsible for the age-dependent desensitization of cultured fibroblasts to ConAmediated agglutination. It should be noted here that the viability of cells after treatment with trypsin or hyaluronidase is retained during assay of cellular agglutination by ConA.

in the following experiments to measure the specific ConA-binding capacity of cells, the cells were incubated with [3H]ConA at 100 /zg/ml at 0 or 24°C for 15 min. The EDTA-dissociated cells or trypsinized cells were prepared at various passages, and assayed for the [~H]ConA-binding capacities. As shown in figs 3 and 4, the [3H]ConA-binding capacity of the cells was always larger in the EDTA-dissociated cells as compared with trypsinized cells. At the same time it was found that the [3H]ConA-binding capacity of the cells measured at 24°C is larger than that of the corresponding cells measured at 0°C. As for the effect of in vitro cellular aging on the ConA-binding capacity of the cells, it was found that the binding capacity expressed in terms of per cell (or per cellular protein) increased significantly as aging of cultured cells advanced in the cases of both the EDTA-dissociated and trypsinized cells without regard to whether the assay was carried out at 0 or 24°C (see fig. 3). On the other hand, when the binding capacity was expressed on the basis of per unit area of cell surface it was found that the binding capacity remained unaltered in the course

[3H]ConA-binding capacity of chick embryo fibroblasts in the course of serial subcultivations In the preliminary experiments, the EDTAdissociated cells were incubated with various concentrations of [3H]ConA at 0°C for 15 min. The specific binding of [~H]ConA to the cells was found to increase almost linearly as the concentration of ConA increased within a range of 0-50 /zg/ml, gradually leveling off at the ConA concentrations beyond 50/xg/ml, and finally reaching a plateau level at 100/~g/ml. Therefore,

Exp Cell Res 108 (1977)

92

Yamamoto, Yamamoto and Ooka

of serial subcultivations or in vitro cellular aging (see fig. 4). It should be noted that the cellular sizes tended to increase as aging of cultured cells advanced.

DISCUSSION In the present paper we have shown that the sensitivity of cultured fibroblasts of a chick-embryo origin to ConA-mediated agglutination significantly decreases in the course of serial subcultivations or in vitro cellular aging, suggesting possible alterations of the cell surface associated with cellular aging. This phenomenon, however, does not seem to be ascribed to either (1) the gradual loss of ConA-binding sites or receptors of (2) the relative increase in the ConA receptors of "cryptic" form [30], which might occur at the cell surface with the advance of cellular aging. The cellular capacity to bind specifically [~H]ConA does not decrease but rather increases in the course of serial subcultivations on the per cell basis, or remains unaltered on the per surface area basis. It was also found that the binding decreases after trypsinization of the cells in accordance with the results of embryonic neural retina cells [36]. Trypsinization of the cells has been found to result in a marked enhancement of the cellular sensitivity to ConA-mediated agglutination in accordance with the results of other investigators [1920, 25, 30-31], leading to identical values in both the aged and young cells. These resuits seem to suggest that the reason for the change in sensitivity to ConA-mediated agglutination in the course of serial subcultivations should be sought for in cell surface components other than the lectin receptors. In contrast to trypsin, the Streptomyces hyaluronidase, which is known to specifExp Cell Res 108 (1977)

ically attack hyaluronic acid (but not chondroitin sulfates), has been shown to enhance the sensitivity to ConA-mediated agglutination of aged cells, but not that of young ones. Thus the sensitivity of hyaluronidase-treated aged cells to ConAmediated agglutination becomes equal to the sensitivity of young cells with or without hyaluronidase treatment. These results seem to suggest that hyaluronic acid probably associated with the cell surface, especially of aged cells, may somehow be responsible for modulating the cellular sensitivity to ConA-mediated agglutination. Courtois & Hughes [4] have already reported that the cell surface membranes of chick embryo fibroblasts in the late phase III are associated with larger amounts of mucopolysaccharides as compared with the surface membranes of corresponding cells in early phase I. We have found (unpublished data) by means of scanning electron microscopy that the surface of young cells is smooth, whereas that of aged cells is rough, and rich in the "pits" supposed to be the openings of pinocytotic vesicles. The cell coat like structure of EDTA-dissociated cells was found to be much thicker in aged cells than in young ones. Moreover, the cell surface of aged cells after treatment with hyaluronidase becomes identical with that of young cells with or without hyaluronidase treatment. Burger & Martin [27] have reported that the lectin-mediated agglutination of chick embryo fibroblasts transformed by Rous sarcoma virus becomes sensitized by pretreatment with hyaluronidase in contrast to untransformed ones, which are not affected by hyaluronidase treatment. The latter finding seems to be in accord with our present result, inasmuch as the untransformed cells should be "young" cells. The possible involvement of cell coat

A g e - r e l a t e d cell surface changes acid m u c o p o l y s a c c h a r i d e s in the m o d u l a t i o n of l e c t i n - m e d i a t e d cellular a g g l u t i n a tion or i n t e r c e l l u l a r a d h e s i o n h a v e first b e e n p r e s e n t e d b y T e r a y a m a a n d his a s s o c i a t e s

13. 14. 15. 16. 17.

[37-39]. I n fact, t h e y h a v e s h o w n that the p a t t e r n of acid m u c o p o l y s a c c h a r i d e s ass o c i a t e d with p l a s m a m e m b r a n e s differ bet w e e n l i v e r a n d a s c i t e s h e p a t o m a cells o r

18. 19. 20.

even a m o n g various strains of ascites hepatoma depending on intercellular adhesive-

21. 22.

ness. We are grateful to Dr H. Terayama of Zoological Institute, Faculty of Science, University of Tokyo, Tokyo, Japan, for his helpful suggestion and criticism throughout this study.

23. 24. 25. 26. 27.

REFERENCES 1. Hay, R J & Strehler, B L, Exp gerontol 2 (1967) 123. 2. Lima, L & Macieira-Coelho, A, Exp cell res 70 (1972) 279. 3. Azencott, R & Courtois, Y, Exp cell res 86 (1974) 69. 4. Courtois, Y & Hughes, R C, Eur j biochem 44 (1974) 131. 5. Hayflick, L & Moorhead, P S, Exp cell res 25 (1961) 585. 6. Macieira-Coelho, A, Pont6n, J & Philipson, L, Exp cell res 42 (1966) 673. 7. Cristofalo, V J & Kritchevsky, D, Med exp 19 (1969) 313. 8. Cristofalo, V J & Sharf, B B, Exp cell res 76 (1973) 419. 9. Sun, A S, Aggarwall, B B & Packer, L, Arch biochem biophys 170 (1975) 1. 10. Mitsui, Y & Schneider, E L, Mech age dev 5 (1976) 45. 11. Abercrombie, M & Ambrose, E J, Cancer res 22 (1962) 525. 12. Armstrong, PB, J cell biol 47 (1970) 197.

28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

93

Pessac, B & Defendi, V, Science 175 (1972) 898. Martinoz-Palomo, A, Int rev cytol 29 (1970) 29. Winzler, R J, Int rev cytol 29 (1970) 77. Burger, M M & Noonan, K D, Nature 228 (1970) 512. Brautbar, C, Payne, R & Hayflick, L, Exp cell res 75 (1972) 31. Kutchai, H, Barenholz, Y, Ross, T F & Wermer, D E, Biochim biophys acta 436 (1976) 101. Inbar, M & Sachs, L, Proc natl acad sci US 63 (1969) 1418. Ben-Bassat, H, Inbar, M & Sachs, L, J membrane biol 6 (1971) 183. Inbar, M & Sachs, L, Nature 223 (1969) 710. Inbar, M, Ben-Bassat, H & Sachs, L, Proc nail acad sci US 68 (1971) 2748. - - Nature new hiol 236 (1972) 3. Cline, M J & Livingston, D C, Nature new bio1232 (1971) 155. Moscona, A A, Science 171 (1971) 905. Kapeller, M & Doljanski, F, Nature new biol 235 (1972) 184. Burger, M M & Martin, G S, Nature new biol 237 (1972) 9. Noonan, K D & Burger, M M, J biol chem 248 (1973) 4286. Collard, J G & Temmink, J H M, J cell sci 19 (1975) 21. Burger, M M, Proc natl acad sci US 62 (1969) 994. Sela, B, Lis, H, Sharon, N & Sachs, L, J membrane biol 3 (1970) 267. Tomita, M, Osawa, T, Sakurai, Y & Ukita, T, Intj cancer 6 (1970) 283. Sela0 B, Lis, H, Sharon, N & Sachs, L, Biochim biophys acta 310 (1973) 273. Diizgfines, N, Biosystem 6 (1975) 209. Lowry, O H, Rosebrough, N J, Farr, A L & Randall, R J, J biol chem 193 (1951) 265. Martinozzi, M & Moscona, A A, Exp cell res 94 (1975) 253. Terayama, H, Membrane 1 (1976)27. In Japanese. Nakamura, J & Terayama, H, Proc natl acad sci US 72 (1975) 498. Yamamoto, K & Terayama, H, Cancer res 33 (1973) 2257.

Received February 7, 1977 Accepted March 11, 1977

Exp CellRes 108 (1977)

Cell surface changes associated with aging of chick embryo fibroblasts in culture.

Printed in Sweden Copyright © 1977 by Academic Press, lnc. All rights of reproduction in any form reserved ISSN 0014~t827 Experimental Cell Research...
509KB Sizes 0 Downloads 0 Views