Mutation Research, 256 (1991) 169-175
169
© 1991 Elsevier Science Publishers B.V. All rights reserved 0921-8734/91/$03.50
MUTAG1 00159
Changes in the cell surface of human diploid fibroblasts during cellular aging Kiyotaka Yamamoto and Mari Yamamoto Department of Cell Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173 (Japan) (Accepted 10 June 1991)
Keywords: Cell surface change; Human diploid fibroblasts; Cellular ageing
Summary The electrophoretic mobility of 13 human diploid cell strains, TIG-1, TIG-2, TIG-3, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, TIG-1H, TIG-1L, TIG-2M, TIG-2B, and TIG-3S, which were established from different tissues of human embryos, was studied at different passages. The net negative surface charge of the cells was characteristic for each cell strain and decreased significantly during the in vitro aging of the cells. The decrease in the net negative charge of the cells correlated well with the decrease in cell density throughout the life span of the cells. A strict linear correlation between the electrophoretic mobility and the number of cells harvested at each passage was obtained for all the human diploid cell strains. Moreover, almost the same linear regression coefficient of the cells was obtained among these cell strains. Therefore, the net negative surface charge of human diploid cell strains could serve as a cell surface marker for in vitro cellular aging.
The replicative capacity of normal human diploid fibroblasts decreases with advancing subcultivation; fibroblasts thus have a finite doubling potential (Hayflick and Moorhead, 1961; Jacobs et al., 1970, 1979; Nichols et al., 1977, 1983; Ohashi et al., 1980; Yamamoto et al., 1991). This progressive decrease might be regarded as a manifestation of senescence at the cellular level and therefore diploid cell cultures are widely used as model systems to study cellular aging (Cristofalo, 1972; Goldstein et al., 1969; Hayflick, 1965; Holli-
Correspondence: Dr. Kiyotaka Yamamoto, Department of Cell Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173 (Japan). Tel.: 03-3964-3241 ex. 3023; Fax: 03-3579-4776.
day and Tarrant, 1972; Martin et al., 1970; Macieira-Coelho et al., 1974; Schneider and Mitsui, 1976). During the past three decades, extensive investigations have revealed age-related changes in various properties (morphology, division pattern, cytogenetics and biochemistry) of these cells (Hayflick, 1980a, b; Yamamoto et al., 1990). Changes in cell-substratum adhesion and spreading (Chandrasekhar and Millis, 1980; Crusberg et al., 1979; Hall et al., 1988; Yamamoto et al., 1991), cell-cell adhesion (Azencoott and Courtois, 1974), agglutination (Kelley et al., 1978; Yamamoto et al., 1977), cell-ligand adhesion and transport (Aizawa and Mitsui, 1979; Press and Pitha, 1974), surface charge (Bosmann et al., 1976), surface proteins, glycoproteins, gangliosides, and glycosaminoglycans (Blondal et al.,
; Courtois and Hughes, 1974; Matuoka and Ji, 1981a; Ohsawa and Nagai, 1982; Palumbo, ; Schachtschabel and Wever, 1978; Sluke et .981; Stein and Atkins, 1986; Vogel et al., a), fibronectin (Edick and Millis, 1984; Sorno and Millis, 1984; Vogel et al., 1981b), ce antigens (Moley and Engelhardt, 1981), keleton (Kelley et al., 1980, 1985), and mem~ structure and function (Boak et al., 1983; man and Daniel, 1975; Kelley, 1976; 9eder et al., 1984) during the in vitro cellular ~of human diploid fibroblasts and chick emfibroblasts have been reported. Reviews deing aspects of membrane aging are already able (Macieira-Coelho, 1983; Zs.-Nagy, ). e have determined the electrophoretic moin human diploid fibroblasts (TIG-1 cells) in M phosphate buffer containing 5.4% glu(pH 7.3) at 25 + 0.5 ° C, and found that the lity of the cells decreased with the number ~ssages (Yamamoto et al., 1988). the present study, we investigated the ges in electrophoretic mobility of 13 human .id cell strains, which were established from lung, heart, liver, brain, muscle, and skin ~s, during in vitro aging in phosphate buffer, found that the mobility decreased with the ne in proliferative capacity. ~rials and m e t h o d s
:ulture firteen human diploid cell strains, TIG-1, 2, TIG-3, TIG-7, WI-38, IMR-90, MRC-5, 3-9, TIG-1H, TIG-1L, TIG-2M, TIG-3B, and 3S, were used in the present study. TIG-1 ~shi et al., 1980), TIG-3 (Matsuo et al., 1982), 7 (Yamamoto et al., 1991), IMR-90 (Nichols ., 1977), MRC-5 (Jacobs et al., 1970), and 3-9 (Jacobs et al., 1979) cells were estab:t from fetal lung tissues. TIG-2 cells were established from the lungs of a human fefetus obtained from a therapeutic abortion ~rmed on a Japanese female, as described ously (Ohashi et al., 1980). TIG-1L, TIG-1H, 2M, TIG-3B, and TIG-3S cells were estab:1 from fetal liver, heart, muscle, brain, and respectively, at the Tokyo Metropolitan In-
stitute of Gerontology. All cells were cultured in 5 ml of Eagle's minimal essential medium (MEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco, lot No. 34N1204) in plastic dishes (Falcon, 3002) at 37°C under humidified 5%-95% air. The cells were removed from dishes by treatment with 0.25% trypsin (Difco, 1:250)-0.02% EDTA in Ca2+-Mg2÷-free phosphate-buffered saline (PBS-) at 37°C for 10 min, and subcultured weekly at a 1 : 4 split ratio. The cells were counted with a hemocytometer after trypsin treatment. The cells were tested for viability by staining with 0.2% nigrosin in 0.9% NaCI solution. The cells were determined to be mycoplasma-free in early (or middle) and late passages by the method of Kihara et al. (1981).
Measurement of cell electrophoretic mobility Cell electrophoretic mobility was measured according to the method described previously (Yamamoto et al., 1988). Briefly, cells on the seventh day after subcultivation (at confluence) were harvested by EDTA treatment (10 min at 37°C) from dishes and suspended in 1/15 M phosphate buffer with 5.4% glucose (pH 7.30; ionic strength, 0.167) at approximately 1 × 106 cells/ml. The electrophoretic mobility of the cells was measured at 25 + 0.5°C with a Cell Electophoresis Microscope System (Model II V, Sugiura Laboratory Inc., Tokyo). The mobility of cells was calculated i n / x m / s / V / c m . Each cellular mobility value was obtained by timing the movements of at least 100 cells with reversal of polarity after each measurement. For determination of the reproducibility of the system, the electrophoretic mobility of the erythrocytes of normal rats (Wistar, 4 weeks) was measured whenever the mobility of human diploid fibroblasts was measured. Under these conditions, the mean mobility of rat erythrocytes was - 1.099 _+ 0.028/xm/s/V/cm. Results
Changes in the net negative surface charge of human diploid embryonic fibroblasts during the in vitro life span Fig. 1 shows the changes in cell density of 13 human diploid embryonic cell strains on the sev-
171 Cell
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e n t h day a f t e r s u b c u l t i v a t i o n d u r i n g t h e life s p a n o f t h e culture. T h e cell strains h a d d i f f e r e n t cell growth characteristics. T a b l e I shows t h e life s p a n of e a c h cell strain. M o s t o f the cells in t h e 7-day c u l t u r e w e r e a r r e s t e d in t h e G l p h a s e o f t h e cell cycle t h r o u g h o u t t h e i r life s p a n in c u l t u r e ( d a t a n o t shown). Fig. 2 shows t h e c h a n g e s in the n e t n e g a t i v e s u r f a c e c h a r g e of h u m a n d i p l o i d cells d u r i n g in vitro c e l l u l a r aging. A d e c r e a s e in m e a n elec-
TABLE 1 DESCRIPTION OF THE 13 HUMAN DIPLOID CELL STRAINS Race a
Tissue
Sex
Life span (PDL)
M M M M C C C C M M M M M
lung lung lung lung lung lung lung lung heart liver brain muscle skin
F F M M F F M F F F F F M
67 71 83 77 51 57 62 60 51 39 45 79 75
a M, Mongoloid; C, Caucasian.
-_
• .~
Fig. 1. Changes in cell density during the in vitro life span of 13 human diploid cell strains. Cells were subcultured weekly at a 1:4 split ratio, and cells were counted with a hemocytometer.
TIG- 1 TIG-2 TIG-3 TIG-7 WI-38 IMR-90 MRC-5 MRC-9 TIG-1H TIG-1L TIG-2B TIG-2M TIG-3S
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Fig. 2. Changes in e]ectrophoretic mobility of 13 human diploid cell strains during the in vitro life s~an. Electrophoretic mobility of the cells on day 7 after subcultivation (at confluence)were measured in 1/15 M phosphate buffer at 25~C as described in Materials and methods.
t r o p h o r e t i c m o b i l i t y was a c h a r a c t e r i s t i c of e a c h cell strain. T h e m e a n m o b i l i t y o f T I G - 1 , T I G - 7 , I M R - 9 0 , T I G - 1 H , T I G - 1 L , a n d T I G - 2 B cells g r a d u a l l y d e c r e a s e d in the m i d d l e p a s s a g e s ( d u r ing t h e p e r i o d of p h a s e II), a n d r a p i d l y d e c r e a s e d in s e n e s c e n t cells. T h e m e a n m o b i l i t y of T I G - 2 a n d T I G - 2 M was stable d u r i n g the p e r i o d o f p h a s e II b u t d e c r e a s e d in early a n d late passages. T h e m e a n m o b i l i t y o f T I G - 3 , WI-38, M R C - 5 , a n d M R C - 9 cells was stable d u r i n g t h e p e r i o d o f p h a s e II a n d r a p i d l y d e c r e a s e d in s e n e s c e n t cells. T h e m e a n m o b i l i t y of T I G - 3 S cells c h a n g e d over f r a c t u a t i o n d u r i n g the p e r i o d o f p h a s e II a n d r a p i d l y d e c r e a s e d in s e n e s c e n t cells. T h e viability of the cells was h e l d c o n s t a n t at a b o u t 92% d u r i n g the assay of e l e c t r o p h o r e t i c m o b i l i t y in all cell strains. C h a n g e s in t h e m e a n m o b i l i t y o f the cell strains r e s e m b l e d t h o s e in t h e i r cell d e n s i t y t h r o u g h o u t the life s p a n in culture.
Relationship between trophoretic mobility
cell density
and
elec-
T h e r e l a t i o n s h i p b e t w e e n t h e shift to lower m o b i l i t y a n d t h e d e c r e a s e in cell d e n s i t y t h r o u g h o u t the life s p a n of h u m a n d i p l o i d f i b r o b l a s t s was e x a m i n e d . T h e e l e c t r o p h o r e t i c m o b i l i t y of T I G - 1 cells c o r r e l a t e d well with t h e cell d e n s i t y at e a c h p a s s a g e of t h e cells, a n d t h e r e l a t i o n s h i p b e t w e e n
172 107-
TABLE 2
o/
/
REGRESSION COEFFICIENTS AND CORRELATION COEFFICIENTS OF THE 13 HUMAN DIPLOID CELL STRAINS
~o
~°
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.
,
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/
2.0
(/J,m/sec/V/cm)
Fig. 3. Relationship between electrophoretic mobility and cell number of TIG-1 cells at each passage. Electrophoretic mobility and cell number at harvesting time of each passage decreased throughout the life span of the cells. A linear correlation was found between the mobility and the cell number.
t he mobility an d t h e cell density f o l l o w e d a strict p o w e r law (Fig. 3). T h e l i n e a r r e g r e s s i o n coeffic ie n t o f t h e mobility on t h e cell density was 2.85, and the c o r r e l a t i o n coefficient, 0.987, was highly significant ( P < 0.001). This r e l a t i o n s h i p b e t w e e n mobility and cell density was o b s e r v e d in all the h u m a n e m b r y o n i c cell strains that we used in the
CeLL
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I0 ~
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TIG-3 TIG-7 W1-38
/
o o
6
~,~ o
10 -0.5
. . . . . . . . . . . . . . -1.0 -1.5 EPM
5 -2.0
Fig. 4. Linear correlation between electrophoretic mobility and cell number of 13 human diploid cell strains based on the data given in Figs. 1 and 2. A linear correlation was obtained for all the cell strains examined.
TIG-1 TIG-2 TIG-3 TIG-7 WI-38 IMR-90 MRC-5 MRC-9 TIG-1H TIG- 1L TIG-2B TIG-2M T1G-3S
Regression coefficient
Regression line
Correlation coefficient
2.38 2.27 1.90 2.02 1.81 1.80 2.08 2.07 2.30 2.09 2.35 2.12 1.99
y= y= y= y= y= y= y= y= y= y= y= y: y=
0.987 0.983 0.982 0.993 0.988 0.997 0.994 0.993 0.994 0.957 0.994 0.988 0.997
2.85 + 2.38x 3.07 + 2.27x 3.61 + 1.90x 3.48 + 2.02x 3.69+ 1.81x 3.73 + 1.80x 3.33 + 2.08x 3.35 + 2.07x 3.12+ 2.30x 3.26 + 2.09x 3.05 + 2.35x 3.33 + 2.12x 3.48 + 1.99x
p r e s e n t study, and m o r e o v e r the l i n e a r r e g r e s s i o n c o e f f i c i e n t of e a c h cell strain was similar to that o f T I G - 1 cells (Fig. 4). T h e c o r r e l a t i o n coefficients o f t h ese cells w e r e highly significant as shown in T a b l e 2.
Discussion M o s t functions of the p l a s m a m e m b r a n e dep e n d primarily on the a d h e s i o n o f t h ei r constituents to external molecules. Migration, cellto-cell c o m m u n i c a t i o n , r e s p o n s e to h o r m o n e s and g r o w t h factors, and cell division are all e x a m p l e s of m e m b r a n e functions d e p e n d e n t on i n t e r m o l e c ular forces of a t t r a c t i o n at the cell surface ( M a c i e i r a - C o e l h o , 1983). A g e d fibroblasts in vitro show d e c r e a s e d cell a t t a c h m e n t ( C h a n d r a s e k h a r and Millis, 1980; K o n d o and Y a m a m o t o , 1981). Cell a t t a c h m e n t d e p e n d s on t h e electric ch ar g es at the cell surface. In the p r e s e n t study, e x a m i n a tion of the n et n e g a t i v e surface c h a r g e o f h u m a n diploid cell strains d e r i v e d f r o m various embryonic tissues r e v e a l e d an a g e - r e l a t e d d e c r e a s e in all the cell strains e x a m i n e d (Fig. 2). I n t e r e s t ingly, t h e n et n e g a t i v e surface c h a r g e a p p e a r e d to d e c r e a s e with t h e d e c r e a s e in the rate of ent r a n c e into D N A synthesis that occurs t h r o u g h o u t the in vitro aging o f h u m a n diploid fibroblasts (Hill et al., 1978; M a c i e i r a - C o e h l o and A z z a r o n e ,
173
1982). The extent of flattening of the cells needed to enter the division cycle seems to be controlled in normal cells by a protein component of the cell surface (Wells and Mallucci, 1978). Cell spreading has been reported to be closely correlated with the initiation of DNA synthesis (Folkman and Moscona, 1978). Therefore, changes in surface charge may increase the time needed to reach the right amount of spreading necessary to initiate DNA synthesis. The decrease in the net negative surface charge during the in vitro aging of human diploid cells (WI-38) was first reported by Bosmann et al. (1976). They speculated that the age-dependent decrease is related to a loss of external surface N-acetyl-neuraminic acid on the basis of their work on other mammalian cells. This is in agreement with the decrease in senescent WI-38 cells of membrane-bound sialic acid (Milo and Hart, 1976). Indeed, the molecule is mainly responsible for the negative charge of mammalian red blood cells expressed at their surface (Cook et al., 1961; Eylar et al., 1962). However, we found that the net negative surface charge in human diploid fibroblasts contributes to cell surface glycosaminoglycans rather than sialic acid and that a decreased negative surface charge in aged cells is ascribed to decreases in the hyaluronic acid and chondroitin sulfates at the cell surface (Mitsui et al., 1985). It is known that cell surface glycosaminoglycans change in association with the cellular aging of human diploid fibroblasts (Matuoka and Mitsui, 1981a; Schachtschabel and Wever, 1978; Sluke et al., 1981; Vogel et al., 1981a) and that heparan sulfate on the cell surface is involved in the density-dependent inhibition of cell proliferation (Matuoka and Mitsui, 1981b). Therefore, cell surface glycosaminoglycans, characteristic of each cell strain, probably contribute to the age-related changes in cell surface charge and regulate the substratum adhesion and growth of aged cells. A strict linear relationship between the electrophoretic mobility and the cell number at confluence in several passages was found in all the human diploid cell strains (even TIG-3S cells which showed a fractually proliferative capacity) examined in the present study. Moreover, the regression coefficient of each cell strain (change
in cell number per change in electrophoretic mobility) was similar among the cell strains regardless of differences in genotype, sex, and original tissue. The higher the electrophoretic mobility, the higher the cell density, and the higher the proliferative activity, that is, the electrophoretic mobility implies the proliferative potential of the cells. Increased cell volume during in vitro aging has been reported to be directly related to a concomitant decline in cell replication rate (Mitsui and Schneider, 1976a) and reversion from large to small size seems possible (Mitsui and Schneider, 1976b). We found that the mean value of the negative surface charge is distinct for the passage number regardless of cell size, and that the negative surface charge is lower in the growing phase than in the stationary phase (Yamamoto et al., 1988). The electrophoretic mobility ( / z m / s / V / c m ) reflects the amount of negative charge per unit area of cell surface and decreases when cells are enlarged by hypotonic treatment. Therefore, determination of the mobility of different-sized cells in young and senescent cultures confirmed that the decrease in the negative surface charge in senescent cultures occurred even in the small cell populations and in the rapidly dividing cell populations of heterogeneous senescent cultures. This is in agreement with the findings reported by Aizawa et al. (1980), who found that small cells (even rapidly dividing cells) in late passage populations adsorbed concanavalin Amediated red blood cells just as well as did large cells of this population, while small cells in early passage populations did not. These findings indicate that small cells in late passage populations suffer age-dependent alterations in their surface structure, and differ from the cells in early passage populations. During cellular aging of human diploid fibroblasts, continuous changes in cell surface charge start early and progress throughout the life span of the populations and events at the levels of the membrane that lead to changes in the surface charge could be responsible for the retardation of the initiation of cell division through a delay in cell adhesion. Therefore, the cell electrophoretic mobility could serve as a cell surface marker for the population doubling levels of human diploid cell strains.
174
Acknowledgements We thank Drs. H. Okumura National Institute of Health,
and M. Kihara, Tokyo, for my-
coplasma testing of the cultures.
References Aizawa, S., and Y. Mitsui (1979) A new cell surface marker of aging in human diploid fibroblasts, J. Cell. Physiol., 100, 383-387. Aizawa, S., Y. Mitsui, F. Kurimoto and K. Matsuoka (1980) Cell surface changes accompanying aging in human diploid fibroblasts. III. Division age and senescence revealed by concanavalin A-mediated red blood cell adsorption, Exp. Cell Res., 125, 297-303. Azencott, R. and Y. Courtois (1974) Age-related differences in intercellular adhesion for chick fibroblasts cultured in vitro, Exp. Cell Res., 86, 69-74. Blondal, J.A., J.E. Dick and J.A. Wright (1985) Membrane glycoprotein changes during the senescence of normal human diploid fibroblasts in culture, Mech. Ageing Dev., 30, 273-283. Boak, A.M., A.H. Bittles and P.J. Quinn (1983) Age-related ultrastructural changes in human embryonic lung fibroblasts, Exp. Gerontol., 18, 139-146. Bosmann, H.B., R.L. Gutheil Jr. and K.R. Case (1976) Loss of a critical neutral protease in aging WI-38 cells, Nature, 261,499-501. Bowman, P.D., and C.W. Daniel (1975) Aging of human fibroblasts in vitro: surface features and behavior of aging WI-38 cells, Mech. Ageing Dev., 4, 147-158. Chandrasekhar, S., and A.J.T. Millis (1980) Fibronectin from aged fibroblasts is defective in promoting cellular adhesion, J. Cell. Physiol., 103, 47-54. Cook, G.M.W., D.H. Heard and G.V.F. Seaman (1961) Sialic acids and the electrokinetic charge of human erythrocytes, Nature, 191, 44-47. Courtois, Y., and R.C. Hughes (1974) Glycoproteins of chick embryo fibroblasts in cultures with a finite life span, Eur. J. Biochem., 44, 131-138. Cristofalo, V.J. (1972) Animal cell cultures as a model system for the study of aging, Adv. Gerontol., 4, 45-79. Crusberg, T.C., B.B. Hoskins and R. Widdus (1979) Spreading behavior and surface characteristics of young and senescent W138 fibroblasts revealed by scanning electron microscopy, Exp. Cell Res., 118, 39-46. Edick, G.F., and A.J.T. Millis (1981) Fibronectin distribution of the surfaces of young and old human fibroblasts, Mech. Ageing Dev., 27, 249-256. Eylar, E.H., M.A. Madoff, O.V. Brody and J.L. Oncley (1962) The contribution of sialic acid to the surface charge of the erythrocyte, J. Biol. Chem., 237, 1992-2000. Folkman, F., and A. Moscona (1978) Role of cell shape in growth control, Nature, 273, 345-349. Goldstein, S., J.W. Littlefield and J.S. Solender (1969) Diabetes mellitus and aging: diminished plating efficiency of
cultured human fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 64, 155-160. Hall, M.D., K.S. Flickinger, M. Cutolo, L. Zardi and L.A. Culp (1988) Adhesion of human dermal reticular fibroblasts on complementary fragments of fibronectin: ageing in vivo or in vitro, Exp. Cell Res., 179, 115-136. Hayflick, L. (1965) The limited in vitro lifetime of human diploid cell strains, Exp. Cell Res., 37, 614-636. Hayflick, L. (1980a) Cell aging, Annu. Rev. Gerontol. Geriatr., 1, 26-67. Hayflick, L. (1980b) Recent advances in the cell biology of aging, Mech. Ageing Dev., 14, 59-79. Hayflick, L., and P.S. Moorhead (1961) The serial cultivation of human diploid cell strains, Exp. Cell Res., 25, 585-621. Hill, B.T., R.D.H. Whelan and S. Whatley (1978) Evidence that transcription changes in aging cultures are terminal events occurring after the expression of a reduced replicative potential, Mech. Ageing Dev., 8, 85-95. Holliday, R., and G.M. Tarrant (1972) Altered enzymes in ageing human fibroblasts, Nature, 238, 26-30. Jacobs, J.P., C.M. Jones and J.P. Baille (1970) Characteristics of a human diploid cell designated MRC-5, Nature, 227, 168-170. Jacobs, J.P., A.J. Garrett and J.P. Baille (1979) Characteristics of a serially propagated human diploid cell designated MRC-9, J. Biol. Standard., 7, 113-122. Kelley, R.O. (1976) Development of the aging cell surface: a freeze-fracture analysis of gap junctions between human embryo fibroblasts aging in culture. A brief note, Mech. Ageing Dev., 5, 339-345. Kelley, R.O., R. Azad and K.G. Vogel (1978) Development of the aging cell surface: concanavalin A-mediated intercellular binding and the distribution of binding sites with progressive subcultivation of human embryo fibroblasts, Mech. Ageing Dev., 8, 203-217. Kelley, R.O., J.A. Trotter, L.F. Marek, B.D. Perdue and C.B. Taylor (1980) Variation in cytoskeletal assembly during spreading of progressively subcultivated human embryo fibroblasts (IMR-90), Mech. Ageing Dev., 13, 127-141. Kelley, R.O., P.L. Mann, B.D. Perdue and L.F. Marek (1985) Reduction of filamin in late passage human diploid fibroblasts (IMR-90), Mech. Ageing Dev., 30, 79-98. Kihara, K., S. Ishida and H. Okumura (1981) Detection of mycoplasmal contaminations in sera, J. Biol. Standard., 9, 243 -251. Kondo, H., and K. Yamamoto (1981) Effects of in vitro aging and cell growth on the viability and recovery of human diploid fibroblasts, TIG-1, after freezing and thawing, Mech. Ageing Dev., 16, 117-126. Macieira-Coelho, A. (1983) Changes in membrane properties associated with cellular aging, Int. Rev. Cytol., 83, 183-220. Macieira-Coelho, A., and B. Azzarone (1982) Aging of human fibroblasts is a succession of subtle changes in the cell cycle and has a final short stage with abrupt events, Exp. Cell Res., 141,325-332. Macieira-Coelho, A., C. Diatloff and E. Malaise (1974) Concept of fibroblast aging in vitro: implications for cell biology, Gerontology, 23, 290-305.
175 Martin, G.M., C.A. Sprague and C.J. Epstein (1970) Replicarive life-span of cultivated human cells: effects of donor's age, tissue and genotype, Lab. Invest., 23, 86-92. Matsuo, M., K. Kaji, T. Utakoji and K. Hosoda (1982) Ploidy of human embryonic fibroblasts during in vitro aging, J. Gerontol., 37, 33-37. Matuoka, K., and Y. Mitsui (1981a) Changes in cell-surface glycosaminoglycans in human diploid fibroblasts during in vitro aging, Mech. Ageing Dev., 15, 153-163. Matuoka, K. and Y. Mitsui (1981b) Involvement of cell surface heparan sulfate in the density-dependent inhibition of cell proliferation, Cell Struct. Funct., 6, 23-33. Milo, G.E., and R.W. Hart (1976) Age-related alterations in plasma membrane glycoprotein content and scheduled or unscheduled DNA synthesis, Arch. Biochem. Biophys., 176, 324-333. Mitsui, Y., and E.L. Schneider (1976a) Relationship between cell replication and volume in senescent human diploid fibroblasts, Mech. Ageing Dev., 5, 45-56. Mitsui, Y., and E.L. Schneider (1976b) Characterization of fractionated human diploid fibroblast cell populations, Exp. Cell Res., 103, 23-30. Mitsui, Y., K. Yamamoto, M. Yamamoto and K. Matuoka (1985) Cell surface changes in senescent and Werner's syndrome fibroblasts: their role in cell proliferation, Adv. Exp. Med. Biol., 190, 567-585. Moley, J., and D.L. Engelhardt (1981) A comparison of surface antigens of senescent and presenescent human fibroblasts, J. Gerontol., 36, 136-141. Nichols, W.W., D.G. Murphy, V.J. Cristofalo, L.H. Toji, A.E. Greene and S.A. Dwight (1977) Characterization of a new human diploid cell strain, IMR-90, Science, 196, 60-63. Nichols, W.W., V.J. Cristofalo, L.H. Toji, A.E. Greene, M.M. Aronson, S. Dwight, R. Charpentier and E. Hoffman (1983) Characterization of a new human diploid cell line IMR-91, In Vitro, 19, 797-804. Ohashi, O., S. Aizawa, H. Ooka, T. Ohsawa, K. Kaji, H. Kondo, T. Kobayashi, T. Noumura, M. Matsuo, Y. Mitsui, S. Murota, K. Yamamoto, H. Ito, H. Shimada and T. Utakoji (1980) A new human diploid cell strain, TIG-1, for the research on cellular aging, Exp. Gerontol., 15, 121-133. Ohsawa, T., and Y. Nagai (1982) Ganglioside changes during cell aging in human diploid fibroblast TIG-1, Exp. Gerontol., 17, 287-293. Palumbo, M.E. (1979) The surface membrane proteins of the WI-38 fibroblasts in relation to transformation and aging, Age, 2, 1-4. Press, G.D., and J. Pitha (1974) Aging changes in uptake of polysaccharides by human diploid cells in culture, Mech. Ageing Dev., 3, 323-328. Schachtschabel, D.O., and J. Wever (1978) Age-related de-
cline in the synthesis of glycosaminoglycans by cultured human fibroblasts (WI-38), Mech. Ageing Dev., 8, 257-264. Schneider, E.L., and Y. Mitsui (1976) The relationship between in vitro cellular aging and in vivo human age, Proc. Natl. Acad. Sci. (U.S.A.), 73, 3584-3588. Schroeder, F., I. Goetz and E. Roberts (1984) Age-related alterations in cultured human fibroblast membrane structure and function, Mech. Ageing Dev., 25, 365-389. Sluke, G., D.O. Schachtschabel and J. Wever (1981) Age-related changes in the distribution pattern of glycosaminoglycans synthesized by cultured human diploid fibroblasts (WI-38), Mech. Ageing Dev., 16, 19-27. Sorrentino, J.A., and A.J.T. Millis (1984) Structural comparisons of fibronectins isolated from early and late passage cells, Mech. Ageing Dev., 28, 83-97. Stein, G.H., and L. Atkins (1986) Membrane-associated inhibitor of DNA synthesis in senescent human diploid fibroblasts: Characterization and comparison to quiescent cell inhibitor, Proc. Natl. Acad. Sci. (U.S.A.), 83, 90309034. Vogel, K.G., V.F. Kendall and R.E. Sapien (1981a) Glycosaminoglycan synthesis and composition in human fibroblasts during in vitro cellular aging, J. Cell. Physiol., 107, 271-281. Vogel, K.G., R.O. Kelley and C. Stewart (1981b) Loss of organized fibronectin matrix from the surface of aging diploid fibroblasts (IMR-90), Mech. Ageing Dev., 16, 295302. Wells, V., and L. Mallucci (1978) Determination of cell form in cultured fibroblasts. Role of surface components and cytokinetic elements, Exp. Cell. Res., 116, 301-312. Yamamoto, K., M. Yamamoto and H. Ooka (1977) Cell surface changes associated with aging of chick embryo fibroblasts in culture, Exp. Cell Res., 108, 87-93. Yamamoto, K., M. Yamamoto and H. Ooka (1988) Changes in negative surface charge of human diploid fibroblasts, TIG-1, during in vitro aging, Mech. Ageing Dev., 42, 183-195. Yamamoto, K., K. Kaji, H. Kondo, M. Matsuo, Y. Shibata, Y. Tasaki, T. Utakoji and H. Ooka (1991) A new human diploid cell strain, TIG-7: its age-related changes and comparison with a matched female TIG-1 cell strain, Exp. Gerontol., in press. Yamamoto, M., Y. Mitsui, H. Ooka and K. Yamamoto (1990) Appearance of the terminal senescent cell population in human diploid fibroblasts analyzed by flow cytometry, Mech. Ageing Dev., 51, 195-214. Zs.-Nagy, I. (1979) The role of membrane structure and function in cellular aging: a review, Mech. Ageing Dev., 9, 237-246.
169
© 1991 Elsevier Science Publishers B.V. All rights reserved 0921-8734/91/$03.50
MUTAG1 00159
Changes in the cell surface of human diploid fibroblasts during cellular aging Kiyotaka Yamamoto and Mari Yamamoto Department of Cell Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173 (Japan) (Accepted 10 June 1991)
Keywords: Cell surface change; Human diploid fibroblasts; Cellular ageing
Summary The electrophoretic mobility of 13 human diploid cell strains, TIG-1, TIG-2, TIG-3, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, TIG-1H, TIG-1L, TIG-2M, TIG-2B, and TIG-3S, which were established from different tissues of human embryos, was studied at different passages. The net negative surface charge of the cells was characteristic for each cell strain and decreased significantly during the in vitro aging of the cells. The decrease in the net negative charge of the cells correlated well with the decrease in cell density throughout the life span of the cells. A strict linear correlation between the electrophoretic mobility and the number of cells harvested at each passage was obtained for all the human diploid cell strains. Moreover, almost the same linear regression coefficient of the cells was obtained among these cell strains. Therefore, the net negative surface charge of human diploid cell strains could serve as a cell surface marker for in vitro cellular aging.
The replicative capacity of normal human diploid fibroblasts decreases with advancing subcultivation; fibroblasts thus have a finite doubling potential (Hayflick and Moorhead, 1961; Jacobs et al., 1970, 1979; Nichols et al., 1977, 1983; Ohashi et al., 1980; Yamamoto et al., 1991). This progressive decrease might be regarded as a manifestation of senescence at the cellular level and therefore diploid cell cultures are widely used as model systems to study cellular aging (Cristofalo, 1972; Goldstein et al., 1969; Hayflick, 1965; Holli-
Correspondence: Dr. Kiyotaka Yamamoto, Department of Cell Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173 (Japan). Tel.: 03-3964-3241 ex. 3023; Fax: 03-3579-4776.
day and Tarrant, 1972; Martin et al., 1970; Macieira-Coelho et al., 1974; Schneider and Mitsui, 1976). During the past three decades, extensive investigations have revealed age-related changes in various properties (morphology, division pattern, cytogenetics and biochemistry) of these cells (Hayflick, 1980a, b; Yamamoto et al., 1990). Changes in cell-substratum adhesion and spreading (Chandrasekhar and Millis, 1980; Crusberg et al., 1979; Hall et al., 1988; Yamamoto et al., 1991), cell-cell adhesion (Azencoott and Courtois, 1974), agglutination (Kelley et al., 1978; Yamamoto et al., 1977), cell-ligand adhesion and transport (Aizawa and Mitsui, 1979; Press and Pitha, 1974), surface charge (Bosmann et al., 1976), surface proteins, glycoproteins, gangliosides, and glycosaminoglycans (Blondal et al.,
; Courtois and Hughes, 1974; Matuoka and Ji, 1981a; Ohsawa and Nagai, 1982; Palumbo, ; Schachtschabel and Wever, 1978; Sluke et .981; Stein and Atkins, 1986; Vogel et al., a), fibronectin (Edick and Millis, 1984; Sorno and Millis, 1984; Vogel et al., 1981b), ce antigens (Moley and Engelhardt, 1981), keleton (Kelley et al., 1980, 1985), and mem~ structure and function (Boak et al., 1983; man and Daniel, 1975; Kelley, 1976; 9eder et al., 1984) during the in vitro cellular ~of human diploid fibroblasts and chick emfibroblasts have been reported. Reviews deing aspects of membrane aging are already able (Macieira-Coelho, 1983; Zs.-Nagy, ). e have determined the electrophoretic moin human diploid fibroblasts (TIG-1 cells) in M phosphate buffer containing 5.4% glu(pH 7.3) at 25 + 0.5 ° C, and found that the lity of the cells decreased with the number ~ssages (Yamamoto et al., 1988). the present study, we investigated the ges in electrophoretic mobility of 13 human .id cell strains, which were established from lung, heart, liver, brain, muscle, and skin ~s, during in vitro aging in phosphate buffer, found that the mobility decreased with the ne in proliferative capacity. ~rials and m e t h o d s
:ulture firteen human diploid cell strains, TIG-1, 2, TIG-3, TIG-7, WI-38, IMR-90, MRC-5, 3-9, TIG-1H, TIG-1L, TIG-2M, TIG-3B, and 3S, were used in the present study. TIG-1 ~shi et al., 1980), TIG-3 (Matsuo et al., 1982), 7 (Yamamoto et al., 1991), IMR-90 (Nichols ., 1977), MRC-5 (Jacobs et al., 1970), and 3-9 (Jacobs et al., 1979) cells were estab:t from fetal lung tissues. TIG-2 cells were established from the lungs of a human fefetus obtained from a therapeutic abortion ~rmed on a Japanese female, as described ously (Ohashi et al., 1980). TIG-1L, TIG-1H, 2M, TIG-3B, and TIG-3S cells were estab:1 from fetal liver, heart, muscle, brain, and respectively, at the Tokyo Metropolitan In-
stitute of Gerontology. All cells were cultured in 5 ml of Eagle's minimal essential medium (MEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco, lot No. 34N1204) in plastic dishes (Falcon, 3002) at 37°C under humidified 5%-95% air. The cells were removed from dishes by treatment with 0.25% trypsin (Difco, 1:250)-0.02% EDTA in Ca2+-Mg2÷-free phosphate-buffered saline (PBS-) at 37°C for 10 min, and subcultured weekly at a 1 : 4 split ratio. The cells were counted with a hemocytometer after trypsin treatment. The cells were tested for viability by staining with 0.2% nigrosin in 0.9% NaCI solution. The cells were determined to be mycoplasma-free in early (or middle) and late passages by the method of Kihara et al. (1981).
Measurement of cell electrophoretic mobility Cell electrophoretic mobility was measured according to the method described previously (Yamamoto et al., 1988). Briefly, cells on the seventh day after subcultivation (at confluence) were harvested by EDTA treatment (10 min at 37°C) from dishes and suspended in 1/15 M phosphate buffer with 5.4% glucose (pH 7.30; ionic strength, 0.167) at approximately 1 × 106 cells/ml. The electrophoretic mobility of the cells was measured at 25 + 0.5°C with a Cell Electophoresis Microscope System (Model II V, Sugiura Laboratory Inc., Tokyo). The mobility of cells was calculated i n / x m / s / V / c m . Each cellular mobility value was obtained by timing the movements of at least 100 cells with reversal of polarity after each measurement. For determination of the reproducibility of the system, the electrophoretic mobility of the erythrocytes of normal rats (Wistar, 4 weeks) was measured whenever the mobility of human diploid fibroblasts was measured. Under these conditions, the mean mobility of rat erythrocytes was - 1.099 _+ 0.028/xm/s/V/cm. Results
Changes in the net negative surface charge of human diploid embryonic fibroblasts during the in vitro life span Fig. 1 shows the changes in cell density of 13 human diploid embryonic cell strains on the sev-
171 Cell
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e n t h day a f t e r s u b c u l t i v a t i o n d u r i n g t h e life s p a n o f t h e culture. T h e cell strains h a d d i f f e r e n t cell growth characteristics. T a b l e I shows t h e life s p a n of e a c h cell strain. M o s t o f the cells in t h e 7-day c u l t u r e w e r e a r r e s t e d in t h e G l p h a s e o f t h e cell cycle t h r o u g h o u t t h e i r life s p a n in c u l t u r e ( d a t a n o t shown). Fig. 2 shows t h e c h a n g e s in the n e t n e g a t i v e s u r f a c e c h a r g e of h u m a n d i p l o i d cells d u r i n g in vitro c e l l u l a r aging. A d e c r e a s e in m e a n elec-
TABLE 1 DESCRIPTION OF THE 13 HUMAN DIPLOID CELL STRAINS Race a
Tissue
Sex
Life span (PDL)
M M M M C C C C M M M M M
lung lung lung lung lung lung lung lung heart liver brain muscle skin
F F M M F F M F F F F F M
67 71 83 77 51 57 62 60 51 39 45 79 75
a M, Mongoloid; C, Caucasian.
-_
• .~
Fig. 1. Changes in cell density during the in vitro life span of 13 human diploid cell strains. Cells were subcultured weekly at a 1:4 split ratio, and cells were counted with a hemocytometer.
TIG- 1 TIG-2 TIG-3 TIG-7 WI-38 IMR-90 MRC-5 MRC-9 TIG-1H TIG-1L TIG-2B TIG-2M TIG-3S
"~-~
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= r~-~
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TIG.1 TI~-~ ~G-~ ~G-? It~-~8 ~-~
/ I0
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$0
40
50
60
70
80
90
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Fig. 2. Changes in e]ectrophoretic mobility of 13 human diploid cell strains during the in vitro life s~an. Electrophoretic mobility of the cells on day 7 after subcultivation (at confluence)were measured in 1/15 M phosphate buffer at 25~C as described in Materials and methods.
t r o p h o r e t i c m o b i l i t y was a c h a r a c t e r i s t i c of e a c h cell strain. T h e m e a n m o b i l i t y o f T I G - 1 , T I G - 7 , I M R - 9 0 , T I G - 1 H , T I G - 1 L , a n d T I G - 2 B cells g r a d u a l l y d e c r e a s e d in the m i d d l e p a s s a g e s ( d u r ing t h e p e r i o d of p h a s e II), a n d r a p i d l y d e c r e a s e d in s e n e s c e n t cells. T h e m e a n m o b i l i t y of T I G - 2 a n d T I G - 2 M was stable d u r i n g the p e r i o d o f p h a s e II b u t d e c r e a s e d in early a n d late passages. T h e m e a n m o b i l i t y o f T I G - 3 , WI-38, M R C - 5 , a n d M R C - 9 cells was stable d u r i n g t h e p e r i o d o f p h a s e II a n d r a p i d l y d e c r e a s e d in s e n e s c e n t cells. T h e m e a n m o b i l i t y of T I G - 3 S cells c h a n g e d over f r a c t u a t i o n d u r i n g the p e r i o d o f p h a s e II a n d r a p i d l y d e c r e a s e d in s e n e s c e n t cells. T h e viability of the cells was h e l d c o n s t a n t at a b o u t 92% d u r i n g the assay of e l e c t r o p h o r e t i c m o b i l i t y in all cell strains. C h a n g e s in t h e m e a n m o b i l i t y o f the cell strains r e s e m b l e d t h o s e in t h e i r cell d e n s i t y t h r o u g h o u t the life s p a n in culture.
Relationship between trophoretic mobility
cell density
and
elec-
T h e r e l a t i o n s h i p b e t w e e n t h e shift to lower m o b i l i t y a n d t h e d e c r e a s e in cell d e n s i t y t h r o u g h o u t the life s p a n of h u m a n d i p l o i d f i b r o b l a s t s was e x a m i n e d . T h e e l e c t r o p h o r e t i c m o b i l i t y of T I G - 1 cells c o r r e l a t e d well with t h e cell d e n s i t y at e a c h p a s s a g e of t h e cells, a n d t h e r e l a t i o n s h i p b e t w e e n
172 107-
TABLE 2
o/
/
REGRESSION COEFFICIENTS AND CORRELATION COEFFICIENTS OF THE 13 HUMAN DIPLOID CELL STRAINS
~o
~°
/
N
'
10s.
~o
/ 1 0 s,
o
,
,
,
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,
0~
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- 1.0 E P M
,
.
,
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•
/
2.0
(/J,m/sec/V/cm)
Fig. 3. Relationship between electrophoretic mobility and cell number of TIG-1 cells at each passage. Electrophoretic mobility and cell number at harvesting time of each passage decreased throughout the life span of the cells. A linear correlation was found between the mobility and the cell number.
t he mobility an d t h e cell density f o l l o w e d a strict p o w e r law (Fig. 3). T h e l i n e a r r e g r e s s i o n coeffic ie n t o f t h e mobility on t h e cell density was 2.85, and the c o r r e l a t i o n coefficient, 0.987, was highly significant ( P < 0.001). This r e l a t i o n s h i p b e t w e e n mobility and cell density was o b s e r v e d in all the h u m a n e m b r y o n i c cell strains that we used in the
CeLL
No/21
I0 ~
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10;' o • • • ~
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TIG-3 TIG-7 W1-38
/
o o
6
~,~ o
10 -0.5
. . . . . . . . . . . . . . -1.0 -1.5 EPM
5 -2.0
Fig. 4. Linear correlation between electrophoretic mobility and cell number of 13 human diploid cell strains based on the data given in Figs. 1 and 2. A linear correlation was obtained for all the cell strains examined.
TIG-1 TIG-2 TIG-3 TIG-7 WI-38 IMR-90 MRC-5 MRC-9 TIG-1H TIG- 1L TIG-2B TIG-2M T1G-3S
Regression coefficient
Regression line
Correlation coefficient
2.38 2.27 1.90 2.02 1.81 1.80 2.08 2.07 2.30 2.09 2.35 2.12 1.99
y= y= y= y= y= y= y= y= y= y= y= y: y=
0.987 0.983 0.982 0.993 0.988 0.997 0.994 0.993 0.994 0.957 0.994 0.988 0.997
2.85 + 2.38x 3.07 + 2.27x 3.61 + 1.90x 3.48 + 2.02x 3.69+ 1.81x 3.73 + 1.80x 3.33 + 2.08x 3.35 + 2.07x 3.12+ 2.30x 3.26 + 2.09x 3.05 + 2.35x 3.33 + 2.12x 3.48 + 1.99x
p r e s e n t study, and m o r e o v e r the l i n e a r r e g r e s s i o n c o e f f i c i e n t of e a c h cell strain was similar to that o f T I G - 1 cells (Fig. 4). T h e c o r r e l a t i o n coefficients o f t h ese cells w e r e highly significant as shown in T a b l e 2.
Discussion M o s t functions of the p l a s m a m e m b r a n e dep e n d primarily on the a d h e s i o n o f t h ei r constituents to external molecules. Migration, cellto-cell c o m m u n i c a t i o n , r e s p o n s e to h o r m o n e s and g r o w t h factors, and cell division are all e x a m p l e s of m e m b r a n e functions d e p e n d e n t on i n t e r m o l e c ular forces of a t t r a c t i o n at the cell surface ( M a c i e i r a - C o e l h o , 1983). A g e d fibroblasts in vitro show d e c r e a s e d cell a t t a c h m e n t ( C h a n d r a s e k h a r and Millis, 1980; K o n d o and Y a m a m o t o , 1981). Cell a t t a c h m e n t d e p e n d s on t h e electric ch ar g es at the cell surface. In the p r e s e n t study, e x a m i n a tion of the n et n e g a t i v e surface c h a r g e o f h u m a n diploid cell strains d e r i v e d f r o m various embryonic tissues r e v e a l e d an a g e - r e l a t e d d e c r e a s e in all the cell strains e x a m i n e d (Fig. 2). I n t e r e s t ingly, t h e n et n e g a t i v e surface c h a r g e a p p e a r e d to d e c r e a s e with t h e d e c r e a s e in the rate of ent r a n c e into D N A synthesis that occurs t h r o u g h o u t the in vitro aging o f h u m a n diploid fibroblasts (Hill et al., 1978; M a c i e i r a - C o e h l o and A z z a r o n e ,
173
1982). The extent of flattening of the cells needed to enter the division cycle seems to be controlled in normal cells by a protein component of the cell surface (Wells and Mallucci, 1978). Cell spreading has been reported to be closely correlated with the initiation of DNA synthesis (Folkman and Moscona, 1978). Therefore, changes in surface charge may increase the time needed to reach the right amount of spreading necessary to initiate DNA synthesis. The decrease in the net negative surface charge during the in vitro aging of human diploid cells (WI-38) was first reported by Bosmann et al. (1976). They speculated that the age-dependent decrease is related to a loss of external surface N-acetyl-neuraminic acid on the basis of their work on other mammalian cells. This is in agreement with the decrease in senescent WI-38 cells of membrane-bound sialic acid (Milo and Hart, 1976). Indeed, the molecule is mainly responsible for the negative charge of mammalian red blood cells expressed at their surface (Cook et al., 1961; Eylar et al., 1962). However, we found that the net negative surface charge in human diploid fibroblasts contributes to cell surface glycosaminoglycans rather than sialic acid and that a decreased negative surface charge in aged cells is ascribed to decreases in the hyaluronic acid and chondroitin sulfates at the cell surface (Mitsui et al., 1985). It is known that cell surface glycosaminoglycans change in association with the cellular aging of human diploid fibroblasts (Matuoka and Mitsui, 1981a; Schachtschabel and Wever, 1978; Sluke et al., 1981; Vogel et al., 1981a) and that heparan sulfate on the cell surface is involved in the density-dependent inhibition of cell proliferation (Matuoka and Mitsui, 1981b). Therefore, cell surface glycosaminoglycans, characteristic of each cell strain, probably contribute to the age-related changes in cell surface charge and regulate the substratum adhesion and growth of aged cells. A strict linear relationship between the electrophoretic mobility and the cell number at confluence in several passages was found in all the human diploid cell strains (even TIG-3S cells which showed a fractually proliferative capacity) examined in the present study. Moreover, the regression coefficient of each cell strain (change
in cell number per change in electrophoretic mobility) was similar among the cell strains regardless of differences in genotype, sex, and original tissue. The higher the electrophoretic mobility, the higher the cell density, and the higher the proliferative activity, that is, the electrophoretic mobility implies the proliferative potential of the cells. Increased cell volume during in vitro aging has been reported to be directly related to a concomitant decline in cell replication rate (Mitsui and Schneider, 1976a) and reversion from large to small size seems possible (Mitsui and Schneider, 1976b). We found that the mean value of the negative surface charge is distinct for the passage number regardless of cell size, and that the negative surface charge is lower in the growing phase than in the stationary phase (Yamamoto et al., 1988). The electrophoretic mobility ( / z m / s / V / c m ) reflects the amount of negative charge per unit area of cell surface and decreases when cells are enlarged by hypotonic treatment. Therefore, determination of the mobility of different-sized cells in young and senescent cultures confirmed that the decrease in the negative surface charge in senescent cultures occurred even in the small cell populations and in the rapidly dividing cell populations of heterogeneous senescent cultures. This is in agreement with the findings reported by Aizawa et al. (1980), who found that small cells (even rapidly dividing cells) in late passage populations adsorbed concanavalin Amediated red blood cells just as well as did large cells of this population, while small cells in early passage populations did not. These findings indicate that small cells in late passage populations suffer age-dependent alterations in their surface structure, and differ from the cells in early passage populations. During cellular aging of human diploid fibroblasts, continuous changes in cell surface charge start early and progress throughout the life span of the populations and events at the levels of the membrane that lead to changes in the surface charge could be responsible for the retardation of the initiation of cell division through a delay in cell adhesion. Therefore, the cell electrophoretic mobility could serve as a cell surface marker for the population doubling levels of human diploid cell strains.
174
Acknowledgements We thank Drs. H. Okumura National Institute of Health,
and M. Kihara, Tokyo, for my-
coplasma testing of the cultures.
References Aizawa, S., and Y. Mitsui (1979) A new cell surface marker of aging in human diploid fibroblasts, J. Cell. Physiol., 100, 383-387. Aizawa, S., Y. Mitsui, F. Kurimoto and K. Matsuoka (1980) Cell surface changes accompanying aging in human diploid fibroblasts. III. Division age and senescence revealed by concanavalin A-mediated red blood cell adsorption, Exp. Cell Res., 125, 297-303. Azencott, R. and Y. Courtois (1974) Age-related differences in intercellular adhesion for chick fibroblasts cultured in vitro, Exp. Cell Res., 86, 69-74. Blondal, J.A., J.E. Dick and J.A. Wright (1985) Membrane glycoprotein changes during the senescence of normal human diploid fibroblasts in culture, Mech. Ageing Dev., 30, 273-283. Boak, A.M., A.H. Bittles and P.J. Quinn (1983) Age-related ultrastructural changes in human embryonic lung fibroblasts, Exp. Gerontol., 18, 139-146. Bosmann, H.B., R.L. Gutheil Jr. and K.R. Case (1976) Loss of a critical neutral protease in aging WI-38 cells, Nature, 261,499-501. Bowman, P.D., and C.W. Daniel (1975) Aging of human fibroblasts in vitro: surface features and behavior of aging WI-38 cells, Mech. Ageing Dev., 4, 147-158. Chandrasekhar, S., and A.J.T. Millis (1980) Fibronectin from aged fibroblasts is defective in promoting cellular adhesion, J. Cell. Physiol., 103, 47-54. Cook, G.M.W., D.H. Heard and G.V.F. Seaman (1961) Sialic acids and the electrokinetic charge of human erythrocytes, Nature, 191, 44-47. Courtois, Y., and R.C. Hughes (1974) Glycoproteins of chick embryo fibroblasts in cultures with a finite life span, Eur. J. Biochem., 44, 131-138. Cristofalo, V.J. (1972) Animal cell cultures as a model system for the study of aging, Adv. Gerontol., 4, 45-79. Crusberg, T.C., B.B. Hoskins and R. Widdus (1979) Spreading behavior and surface characteristics of young and senescent W138 fibroblasts revealed by scanning electron microscopy, Exp. Cell Res., 118, 39-46. Edick, G.F., and A.J.T. Millis (1981) Fibronectin distribution of the surfaces of young and old human fibroblasts, Mech. Ageing Dev., 27, 249-256. Eylar, E.H., M.A. Madoff, O.V. Brody and J.L. Oncley (1962) The contribution of sialic acid to the surface charge of the erythrocyte, J. Biol. Chem., 237, 1992-2000. Folkman, F., and A. Moscona (1978) Role of cell shape in growth control, Nature, 273, 345-349. Goldstein, S., J.W. Littlefield and J.S. Solender (1969) Diabetes mellitus and aging: diminished plating efficiency of
cultured human fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 64, 155-160. Hall, M.D., K.S. Flickinger, M. Cutolo, L. Zardi and L.A. Culp (1988) Adhesion of human dermal reticular fibroblasts on complementary fragments of fibronectin: ageing in vivo or in vitro, Exp. Cell Res., 179, 115-136. Hayflick, L. (1965) The limited in vitro lifetime of human diploid cell strains, Exp. Cell Res., 37, 614-636. Hayflick, L. (1980a) Cell aging, Annu. Rev. Gerontol. Geriatr., 1, 26-67. Hayflick, L. (1980b) Recent advances in the cell biology of aging, Mech. Ageing Dev., 14, 59-79. Hayflick, L., and P.S. Moorhead (1961) The serial cultivation of human diploid cell strains, Exp. Cell Res., 25, 585-621. Hill, B.T., R.D.H. Whelan and S. Whatley (1978) Evidence that transcription changes in aging cultures are terminal events occurring after the expression of a reduced replicative potential, Mech. Ageing Dev., 8, 85-95. Holliday, R., and G.M. Tarrant (1972) Altered enzymes in ageing human fibroblasts, Nature, 238, 26-30. Jacobs, J.P., C.M. Jones and J.P. Baille (1970) Characteristics of a human diploid cell designated MRC-5, Nature, 227, 168-170. Jacobs, J.P., A.J. Garrett and J.P. Baille (1979) Characteristics of a serially propagated human diploid cell designated MRC-9, J. Biol. Standard., 7, 113-122. Kelley, R.O. (1976) Development of the aging cell surface: a freeze-fracture analysis of gap junctions between human embryo fibroblasts aging in culture. A brief note, Mech. Ageing Dev., 5, 339-345. Kelley, R.O., R. Azad and K.G. Vogel (1978) Development of the aging cell surface: concanavalin A-mediated intercellular binding and the distribution of binding sites with progressive subcultivation of human embryo fibroblasts, Mech. Ageing Dev., 8, 203-217. Kelley, R.O., J.A. Trotter, L.F. Marek, B.D. Perdue and C.B. Taylor (1980) Variation in cytoskeletal assembly during spreading of progressively subcultivated human embryo fibroblasts (IMR-90), Mech. Ageing Dev., 13, 127-141. Kelley, R.O., P.L. Mann, B.D. Perdue and L.F. Marek (1985) Reduction of filamin in late passage human diploid fibroblasts (IMR-90), Mech. Ageing Dev., 30, 79-98. Kihara, K., S. Ishida and H. Okumura (1981) Detection of mycoplasmal contaminations in sera, J. Biol. Standard., 9, 243 -251. Kondo, H., and K. Yamamoto (1981) Effects of in vitro aging and cell growth on the viability and recovery of human diploid fibroblasts, TIG-1, after freezing and thawing, Mech. Ageing Dev., 16, 117-126. Macieira-Coelho, A. (1983) Changes in membrane properties associated with cellular aging, Int. Rev. Cytol., 83, 183-220. Macieira-Coelho, A., and B. Azzarone (1982) Aging of human fibroblasts is a succession of subtle changes in the cell cycle and has a final short stage with abrupt events, Exp. Cell Res., 141,325-332. Macieira-Coelho, A., C. Diatloff and E. Malaise (1974) Concept of fibroblast aging in vitro: implications for cell biology, Gerontology, 23, 290-305.
175 Martin, G.M., C.A. Sprague and C.J. Epstein (1970) Replicarive life-span of cultivated human cells: effects of donor's age, tissue and genotype, Lab. Invest., 23, 86-92. Matsuo, M., K. Kaji, T. Utakoji and K. Hosoda (1982) Ploidy of human embryonic fibroblasts during in vitro aging, J. Gerontol., 37, 33-37. Matuoka, K., and Y. Mitsui (1981a) Changes in cell-surface glycosaminoglycans in human diploid fibroblasts during in vitro aging, Mech. Ageing Dev., 15, 153-163. Matuoka, K. and Y. Mitsui (1981b) Involvement of cell surface heparan sulfate in the density-dependent inhibition of cell proliferation, Cell Struct. Funct., 6, 23-33. Milo, G.E., and R.W. Hart (1976) Age-related alterations in plasma membrane glycoprotein content and scheduled or unscheduled DNA synthesis, Arch. Biochem. Biophys., 176, 324-333. Mitsui, Y., and E.L. Schneider (1976a) Relationship between cell replication and volume in senescent human diploid fibroblasts, Mech. Ageing Dev., 5, 45-56. Mitsui, Y., and E.L. Schneider (1976b) Characterization of fractionated human diploid fibroblast cell populations, Exp. Cell Res., 103, 23-30. Mitsui, Y., K. Yamamoto, M. Yamamoto and K. Matuoka (1985) Cell surface changes in senescent and Werner's syndrome fibroblasts: their role in cell proliferation, Adv. Exp. Med. Biol., 190, 567-585. Moley, J., and D.L. Engelhardt (1981) A comparison of surface antigens of senescent and presenescent human fibroblasts, J. Gerontol., 36, 136-141. Nichols, W.W., D.G. Murphy, V.J. Cristofalo, L.H. Toji, A.E. Greene and S.A. Dwight (1977) Characterization of a new human diploid cell strain, IMR-90, Science, 196, 60-63. Nichols, W.W., V.J. Cristofalo, L.H. Toji, A.E. Greene, M.M. Aronson, S. Dwight, R. Charpentier and E. Hoffman (1983) Characterization of a new human diploid cell line IMR-91, In Vitro, 19, 797-804. Ohashi, O., S. Aizawa, H. Ooka, T. Ohsawa, K. Kaji, H. Kondo, T. Kobayashi, T. Noumura, M. Matsuo, Y. Mitsui, S. Murota, K. Yamamoto, H. Ito, H. Shimada and T. Utakoji (1980) A new human diploid cell strain, TIG-1, for the research on cellular aging, Exp. Gerontol., 15, 121-133. Ohsawa, T., and Y. Nagai (1982) Ganglioside changes during cell aging in human diploid fibroblast TIG-1, Exp. Gerontol., 17, 287-293. Palumbo, M.E. (1979) The surface membrane proteins of the WI-38 fibroblasts in relation to transformation and aging, Age, 2, 1-4. Press, G.D., and J. Pitha (1974) Aging changes in uptake of polysaccharides by human diploid cells in culture, Mech. Ageing Dev., 3, 323-328. Schachtschabel, D.O., and J. Wever (1978) Age-related de-
cline in the synthesis of glycosaminoglycans by cultured human fibroblasts (WI-38), Mech. Ageing Dev., 8, 257-264. Schneider, E.L., and Y. Mitsui (1976) The relationship between in vitro cellular aging and in vivo human age, Proc. Natl. Acad. Sci. (U.S.A.), 73, 3584-3588. Schroeder, F., I. Goetz and E. Roberts (1984) Age-related alterations in cultured human fibroblast membrane structure and function, Mech. Ageing Dev., 25, 365-389. Sluke, G., D.O. Schachtschabel and J. Wever (1981) Age-related changes in the distribution pattern of glycosaminoglycans synthesized by cultured human diploid fibroblasts (WI-38), Mech. Ageing Dev., 16, 19-27. Sorrentino, J.A., and A.J.T. Millis (1984) Structural comparisons of fibronectins isolated from early and late passage cells, Mech. Ageing Dev., 28, 83-97. Stein, G.H., and L. Atkins (1986) Membrane-associated inhibitor of DNA synthesis in senescent human diploid fibroblasts: Characterization and comparison to quiescent cell inhibitor, Proc. Natl. Acad. Sci. (U.S.A.), 83, 90309034. Vogel, K.G., V.F. Kendall and R.E. Sapien (1981a) Glycosaminoglycan synthesis and composition in human fibroblasts during in vitro cellular aging, J. Cell. Physiol., 107, 271-281. Vogel, K.G., R.O. Kelley and C. Stewart (1981b) Loss of organized fibronectin matrix from the surface of aging diploid fibroblasts (IMR-90), Mech. Ageing Dev., 16, 295302. Wells, V., and L. Mallucci (1978) Determination of cell form in cultured fibroblasts. Role of surface components and cytokinetic elements, Exp. Cell. Res., 116, 301-312. Yamamoto, K., M. Yamamoto and H. Ooka (1977) Cell surface changes associated with aging of chick embryo fibroblasts in culture, Exp. Cell Res., 108, 87-93. Yamamoto, K., M. Yamamoto and H. Ooka (1988) Changes in negative surface charge of human diploid fibroblasts, TIG-1, during in vitro aging, Mech. Ageing Dev., 42, 183-195. Yamamoto, K., K. Kaji, H. Kondo, M. Matsuo, Y. Shibata, Y. Tasaki, T. Utakoji and H. Ooka (1991) A new human diploid cell strain, TIG-7: its age-related changes and comparison with a matched female TIG-1 cell strain, Exp. Gerontol., in press. Yamamoto, M., Y. Mitsui, H. Ooka and K. Yamamoto (1990) Appearance of the terminal senescent cell population in human diploid fibroblasts analyzed by flow cytometry, Mech. Ageing Dev., 51, 195-214. Zs.-Nagy, I. (1979) The role of membrane structure and function in cellular aging: a review, Mech. Ageing Dev., 9, 237-246.
