Changes in negative surface charge of human diploid fibroblasts, TIG-1, during in vitro aging

Mechaniams of Ageing and Deveiopment, 48 (1988) 183--195

183

Elsevier Scientific Publishers Ireland Ltd.

CHANGES IN NEGATIVE SURFACE CHARGE OF HUMAN DIPLOID FIBROBLASTS, TIG-1, DURING IN VITRO AGING

KIYOTAKA YAMAMOTO*, MAR1 YAMAMOTO and HIROSHI OOKA Department of Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173 (Japan) (Received June 8th, 1987) (Revision received October 5th, 1987) SUMMARY

The electrophoretic mobility of human diploid fibroblasts, TIG-1, was studied at different passages. The net negative surface charge of the cells decreased from - 1.658 _+ 0.108 gin/s/V/era at an early passage (15 population doublings, PD) to -1.173 _+ 0.116 at the final passage (67 PD) in 1/15 M phosphate buffer supplemented with 5.4°?0 glucose. The decrease was slow at 15--45 PD, but was rapid at 45--67 PD. The net negative surface charge of small cells in the late passage populations was not different from that of larger cells in this population, and was significantly lower than that of small cells in the middle passage populations. The distribution of the mobilities of cells in each passage was independent of the size of the individual cells, and the mean value was distinct for the passage number. The viability of the cells was retained during the assay of electrophoretic mobility under these conditions. These results indicate that the net negative surface charge of human diploid fibroblasts represents a cell surface maker for in vitro cellular age in the population.

Key words: Human fibroblast; Cell surface charge; Cell electrophoresis; Cellular aging INTRODUCTION

Human diploid fibroblasts have a limited doubling capacity in vitro[I--3], and are widely used as a model for studying cellular aging [4--8]. In addition to changes in several biochemical parameters, changes in cell surface membranes associated with aging in vitro have recently attracted attention. Differences in intercellular *To whom all correspondence and reprint requests should be addressed. 0047-6374/88/$03.50 Printed and Published in Ireland

© 1988 Elsevier Scientific Publishers Ireland Ltd.

184

adhesion [9], agglutinability [10,11], concanavalin A-mediated red blood cell adsorption [12], surface proteins, glycoproteins and mucopolysaccharides [13--17], fibronectin [18,19], surface antigen [20], ultrastructure [21,22], membrane organization [23], and cell surface topography [24] between pre-senescent and senescent cells of human or chick embryo diploid fibroblasts have been reported. The role of membrane structure and function in cellular aging has been demonstrated [25,26]. Bosmann et al. [27] have determined the cell electrophoretic mobility in human diploid fibroblasts (WI-38 cells) in a 0.0145 M NaCI solution containing 4.5070 sorbitol and 0.6 mM NaHCO 3 (pH 7.2 _+ 0.1) at 25 _ 0.1 °C, and electrophoretic mobility of WI-38 cells decreases with the number of passages. The decrease is most pronounced at passages 25--28 and levels off at passages 42--44. In the present study, we have investigated changes in the electrophoretic motility in human diploid fibroblasts, TIG-1, during in vitro aging in 1/15 M phosphate buffer containing 5.407o glucose (pH 7.3) at 25 _+ 0.5 °C and found that the mobility decreased gradually in pre-senescent cells and decreased rapidly in senescent cells with the decline in proliferative capacity. MATERIALS AND METHODS

Cell culture Human diploid fetal lung fibroblasts, TIG-1, established at the Tokyo Metropolitan Institute of Gerontology [28], were used in the present study. The cells were cultured in 60-mm plastic dishes (Falcon, No. 3002) containing 5 ml of Eagle's basal medium (BME) supplemented with 10070 fetal bovine serum (FBS, GIBCO, lot No. R 472320), 100/~g of streptomycin/ml and 100 U of penicillin/ml at 37°C under humidified 5°70 CO2-95070 air. The cells were removed from surface of the dish by treatment with 0.25070 trypsin (Difco, 1:250) in Ca 2÷- and MgE+-free phosphate buffer (PBS), and subcultivated at a 1 : 4 split ratio every week except for the cells at 67 population doublings (PD), which were subcultivated at 2 weeks. The cells were judged mycoplasma-free in early and late passage by the method of Kihara et al. [29]. The cells were counted with a hemocytometer and their volume was measured with a Model ZBI Coulter counter (Coulter Electronics Inc.). The cells were tested for viability by staining with 0.2070 nigrosin in 0.9°70 NaCl solution. Measurement o f cell electrophoretic mobility Cells at 7 days after subcultivation (at nearly confluency) were washed with PBS, and then incubated with 0.5070 EDTA in PBS at 37°C for 10 min except when otherwise specified. The dissociated cells were washed once with PBS and then with an electrophoretic medium. 1/15 M phosphate buffer supplemented with 5.4070 glucose (pH 7.30; ionic strength, 0.167), and finally suspended in the electrophoretic

185

medium at approximately 1 × 106 cells/ml. The electrophoretic mobility o f the cells was measured at 25 _-_4-0 . 5 o c with a Cell Electrophoresis Microscope System (Model II V, Suglura L a b o r a t o r y Inc., Tokyo). The mobility o f cells was calculated in pro/ s / V / c m ( - S.D.) and each cellular mobility value as obtained by timing the movements o f at least 25 cells with reversal o f polarity after each measurement. For determination of the reproducibility o f the system, the electrophoretic mobility of the erythrocytes of normal rats (Wistar, 4 weeks) was measured whenever the mobility o f h u m a n diploid fibroblasts was measured. Under these conditions, the mean mobility o f rat erythrocytes from four independent experiments was - 1.099 _-4- 0.028 p x n / s / V / c m , with no significant differences a m o n g the four experiments (Table I), while Kojima and Maekawa [30] reported a value for the electrophoretic mobility of rat erythrocytes of - 1.100 _+ 0.040/~m/s/V/cm in the same phosphateglucose medium. When the electrophoretic mobility of rat erythrocytes was measured in 0.145 M NaC1 (pH 7.10; ionic strength, 0.145) and 0.0145 M NaCI supplemented with 4.507o sorbitol and 0.6 m M M a H C O 3 (pH 7.28; ionic strength, 0.015), the mean values o f the mobility in the four experiments were not constant and the standard deviation was large compared to the values obtained in the phosphate-glucose medium as shown in Table I. In the present experiments, therefore, the electrophoretic mobility o f human diploid fibroblasts was measured in the phosphate-glucose medium. In all cases populations o f the cells were homogeneous. TABLE I ELECTROPHORETIC MOBILITIES OF NORMAL MALE RAT (WISTAR, 4 WEEKS OLD) AND HUMAN ERYTHROCYTESMEASURED IN DIFFERENT ELECTROPHORETIC MEDIA Erythrocytes

Electrophoretic mobilities, mean ± S.D. (wn/s/V/cm) 1/15 Mphosphate buffer + 5.4% glucose (pH 7.30, ~a = 0.167)

O.145 M N a C I (pH 7.10, ~ = 0.145

O.0145 MNaCI + 4.5~b sorbitol + 0.6 m M NaHCOj (pH7.28, ~ = 0.015)

-1.099 -1.098 -1.098 -1.100 -1.099

-1.296 -1.285 -1.284 -1.290 -1.289

-2.683 -2.648 -2.661 -2.604 -2.649

Ra:

Expt. 1 Expt. 2 Expt. 3 Expt. 4 Average Humat:

± ± ± ± ±

0.028 0.022 0.029 0.030 0.028

-1.105 -*- 0.034

± 0.057 :e 0.052 ± 0.061 ± 0.059 ± 0.058

± ± ± ± ±

0.113 0.114 0.104 0.104 0.110

-2.786 + 0.079

~u, ionic strength. ~Each value of the mobilities was obtained by timing the movements of at least 25 cells with reversal of polarity after each measurement. ~Obtalnedby timing movementof 100 cells.

186

Flow cytometry analysis The cells at 7 days after subcultivation were rapidly rinsed twice with a cold 0.1 °70 sodium citrate solution and once with a cold propidium iodide (PI) solution (0.05 mg of PI [Calbiochem, San Diego, CA]/ml in 0.1070 sodium citrate containing 0.2070 Nonidet P-40 [BDH Chemicals Ltd.]) [13]. Then the cells were treated with 2 ml of the dye solution at 4°C for 10 min and the nuclei were isolated by pipetting. The nucleus suspension was incubated at 37°C for 15 min and filtered through a 37 lam nylon mesh. The distribution of the DNA content among the cells of a population was analyzed with a Fluorescence Activated Cell Sorter III (FACS III, BectonDickinson Electronics Laboratory) using an argon ion laser at 488 nm. Ploidy of the cells was analyzed in confluent monolayer cultures (14 days after transplantation). RESULTS

Measurement o f the electrophoretic mobility o f TIG-1 cells The density of TIG-1 cells at 7 days after subcultivation gradually decreased with population doublings (PD) throughout the lifespan of the culture. The lifespan was 67 PD as shown in Fig, la. Doubling time at the log phase of cell proliferation was slightly prolonged in the period from 15 to 55 PD, and then markedly increased to show a linear process (Fig. lb). The mean volume of TIG-1 cells slowly increased during 15--55 PD and rapidly increased at the late passages after 55 PD (Fig. lc). Table II shows the distribution of the cells among the phases of the cell cycle. Most of the cells in the 7-day culture were arrested in the G1 phase of the cell cycle throughout their lifespan in culture. The number of tetraploid cells, which was estimated by the measurement on the confluent culture, constituted the main part of the " G 2 + M " fraction in the 7-day culture. The tetraploid cells markedly increased at the final stage of cell senescence. The electrophoretic mobility of the cells at 7 days after subcultivation were measured in 1/15 M phosphate buffer supplemented with 5.4070 glucose (pH 7.30; ionic strength, 0.167). Two methods of cell dissociation for the meaurement of the electrophoretic mobility of TIG-I cells were compared. When the cells were scraped off with a rubber policeman, and suspended in phosphate-glucose medium, the mean values of the mobility of these cells were lower and the standard deviation of the mobility was larger at all of the three different PD examined than the values of the cells prepared by treatment with 0.05°70 EDTA in PBS as shown in Table III. It is presumed that cell damage was induced by the mechanical dissociation when a single-cell suspension was prepared by scraping with a rubber policeman. The electrophoretic mobility of TIG-I cells, therefore, was determined with the cells suspended in 1/15 M phosphate buffer with 5.4o7o glucose (pH 7.30) after treatment with 0.05°70 EDTA in PBS as described in Materials and Methods.

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Fig. 1. Changes in cell density (a), doubling time in the log phase (b), and mean cell volume (c) during the in vitro lifespan of human diploid fibroblasts (TIG-I). TIG-1 cells were subcultivated with 1 : 4 split ratio every week, and the cell number and the cell volume were measured as described in Materials and Methods.

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T A B L E II P E R C E N T A G E S OF TIG-I CELLS IN G I , S, A N D G2 + M F R A C T I O N S A F T E R P R O P I D I U M IODIDE STAINING DURING THE I N VITRO LIFESPAN

PD

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Fractions o f cell cycle in 7-day cultures

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1.2 1.4 1.9 1.6 1.0 1.0

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2.0 1.8 2.5 3.7 5.1 19.0

Changes in the net negative surface charge o f TIG-1 cells in the course of serial subcultivations Changes in electrophoretic mobility of TIG-1 cells during in vitro cellular aging are shown in Fig. 2. A minimum of four independent experiments were performed for each electrophoretic mobility determination, and all values are the means _ S.D. of at least 100 readings. The mean mobility of the cells gradually decreased in the period from 15 to 45 PD, and rapidly decreased in the senescent cells. The net negative surface charge decreased from - 1.658 _ 0.108/~m/s/V/cm (the maximal value determined herein) at the early passage (15 PD) to - 1.173 _+ 0.116 (the minimal value determined herein) at the final passage (67 PD). It should be noted here that the viability of cells is retained during assay of electrophoretic mobility. Figure 3 shows the frequency distribution of the electrophoretic mobilities in the period from 25 PD to 65 PD. A slight shift to lower mobility was observed in the period from 25 PD to 45 PD although changes in the mode of the mobility were not apparent. A shift to lower mobility was distinctly observed at a late passage (PD 55)

TABL E III C O M P A R I S O N B E T W E E N T H E M O B I L I T I E S OF TIG-I C E L L S D I S S O C I A T E D W I T H A R U B B ER P O L I C E M A N A N D W I T H 0.05% E D T A IN PBS

PD

Electrophoretic mobilities ~, mean +- S.D. (lam/s/V/cm) Rubber policeman

17 29 39

- 1.621 - 0.143 - 1 . 5 4 5 4- 0.149 - 1.526 ± 0.141

•Values were obtained by timing the m o v e m e n t s o f at least 50 ceils.

O.05 % E D TA in PBS - 1.634 ± 0.109 - 1 . 5 6 7 ± 0.108 - 1.544 + 0.108

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Fig. 2. Changes in the electrophoretic mobility of TIG-I cells during the in vitro lifespan. Electrophoretic mobilities of the cells at 7 days after subcultivation (at confluency) were measured in 1/15 M phosphate buffer with 5.4% glucose (pH 7.30, ionic strength, 0.167) at 25°C as described in Materials and Methods. Values represent mean ± S.D. for at least 100 independent cells at each passage. The viability of the cells was measured from an aliquot of the cell suspension after electrophoretic mobility assay. O, electrophoretic mobility; e , viability.

compared with the middle passage (45 PD). However, the most striking differences were observed when cultures at the senescent passage (65 PD) were compared with cultures at the middle or late passage. In addition to the shift to lower mobility, broader distribution of the mobilities was also seen.

Relationship between electrophoretic mobility and cell size It is well known that the mean size o f human diploid fibroblasts increases in the late passages [32]. The mean volume o f TIG-1 cells also increased at the late passages as shown in Fig. lc. The relationship between the shift to lower mobility and the increase in cell size in senescent cultures was examined. In Fig. 4, the frequency distribution o f cell sizes in a middle passage (PD 35) is presented with the electrophoretic mobility at each cell size. The cell sizes (diameter) were distributed in the range from 8 to 28.5/~m in the 200 cells measured, and 20~70 of the cells had the modal size of 17.1 /~n. On the other hand, the mean mobilities at all sizes were almost the same as each other and also as the mean mobility in the total cell populations at 35 PD, and they were distributed in the range of the standard deviation o f the mobility in the populations at 35 PD. A large increase in cell sizes up to 46.6/~n and a wide range in cell sizes from 9.1 to 46.6/~m were observed at 63 P D as shown in Fig. 5. The mean mobility in all

190

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fractions showed almost the same value as the value for the mobility of the cell populations at 63 PD of - 1.301 _ 0 . 1 1 4 / a m / s / V / c m . Even the largest cells in the 63 PD population did not show mobilities lower than those of smaller cells. Moreover, the mobility of smaller cells in senescent cultures was significantly lower than that of the cells of the same sizes in middle passage cultures.

Electrophoretic mobility and cell growth In order to examine the relationship between the electrophoretic mobility and the growth phase, the mobility of the cells in log phase (at 2 days after subcultivation) was measured. The net negative charge in the early passage (15 PD) was - 1.530 -+ 0.101 /am/s/V/cm, which was lower than the value ( - 1 . 6 5 8 ± 0.108) in the stationary phase (at 7 days), while in late passage (65 PD) the net negative surface charge was - 1.131 -+ 0.084, lower than the values in the stationary phase ( - 1.242

191

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Fig. 4. Relationship between cell size and electrophoretic mobility at the middle passage (35 PD). The mobility and cell size of 200 individual cells were determined on an accessory video monitor of Electrophoresis Microscope System. O, mean electropboretic mobility of each cell size; . . . . . and ........ , mean and S.D. of electrophoretic mobility in this population, respectively.

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192

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Fig. 6. The elec~rophoretic mobility of TIG-1 cells measured in NaCl-sorbitol medium. The mobility of the cells at 7 days after subcultivations were measured in 0.0145 M NaCl supplemented 4.5°70 sorbitoi and 0.6 mM NaHCO 3 (pH 7.28, ionic strength, 0.015) instead of in 1/15 M phosphate buffer with 5.4070 glucose. Values represent mean + S.D. for 100 independent cells at each passage. O, electrophoretic mobility; o , viability.

_+ 0.113 at 7 days and - 1.243 _+ 0.102 at 14 days). These results indicate that the decrease in surface negative charge associated with cellular aging is not due to the suppression o f cell growth. These results also indicate that the decrease o f negative surface charge in late passage is not due to the delayed proliferation o f senescent cells at 7 days, because the negative charge is low even in the confluent culture (at 14 days). Figure 6 shows the net negative surface charge o f TIG-1 cells in a 0.0145 M NaCl, 4.5% sorbitol and 0.6 m M N a H C O 3 medium (pH 7.28). The surface charge decreased linearly from - 1.893 _ 0.116 at 19 P D to - 1.431 _ 0.112 at 67 PD. The viability o f the cells during measurement in this medium rapidly declined with increase in the number o f passages. DISCUSSION

The net negative surface charge in human diploid fibroblasts WI-38 measured in a 0.0145 M NaCI, 4.5°7o sorbitol, and 0.6 m M N a H C O 3 medium (pH 7.2; ionic strength 0.015) rapidly decreased from - 2.82 _+ 0.04 at passage 22 to - 1.82 _ 0.02 at passage 42 in a previous study [27]. The disagreement between the previous and

193 present results may be due to differences in the cell lines, the medium for the measurement of electrophoretic mobility, or the systems for the measurement. Therefore, we measured the mobility of TIG-1 cells in the NaCI, sorbitol and NaHCO 3 medium described by Bosmann et ai. As shown in Fig. 6, the net negative surface charge in TIG-1 cells decreased linearly between 19 PD and 67 PD. However, a number of swollen cells were observed during the assay of electrophoretic mobility, and the viability of TIG-1 cells during the assay also decreased linearly with the increase in number of passages compared with a constant value at about 92W0 in the phosphate-glucose medium. Therefore, TIG-1 cells may suffer cell damage by swelling disruptively in the NaCl-sorbitol medium, and the damage may be somehow responsible for modulating the cell surface charge. In the present study, the standard deviations of electrophoretic mobilities of TIG-1 cells are larger in the cases of the measurements both in the phosphate-glucose medium and in the NaCl-sorbitol medium than that of WI-38 cells in the previous study [27]. Bosmann ef al. stated that the standard deviations are small because of the very large number of independent observations (more than 120). We made at least four determinations of the electrophoretic mobility of TIG-1 cells, and the values are means _ S.D. of at least 100 independent cells at each point. In the NaCl-sorbitol medium, the value for the mobility of human erythrocytes of - 2.786 _ 0.079/~m/ s/V/cm in our experiments agrees with - 2.78 _+ 0.08/~n/s/V/cm in the report by Bosmann [33] as shown in Table I, and the value for the mobility of rat erythrocytes in the phosphate-glucose medium also agrees with that reported by Kojima and Maekawa [30] as described in Materials and Methods. These results indicate the heterogeneity of the populations of TIG-I cells from the viewpoint of their electrophoretic mobility in the phosphate-glucose medium. After a late middle passage (51 PD) the net negative surface charge began to decline, possibly owing to changes in cell surface components. The rapid decrease in the net negative charge at 51 PD precedes the onset of phase III, because TIG-1 cells in the present study entered phase III after 55 PD as shown in Fig. lb. In the middle passage cell populations (35 PD) the cells were relatively small, and a shift to large cells and increased heterogeneity in size was observed in the late passage cell populations (63 PD) as described previously [32,34]. Mitsui and Schneider [35] have demonstrated a close relationship between cell size distribution and the cell population replication rate in human diploid fibroblasts, and cell populations with large volumes have lower replication rates than populations with small volumes. However, we have demonstrated that the net negative surface charge of small cells in the late passage populations was not different from that of larger cells in this population, and that it was significantly lower than that of small cells in the early passage populations; i.e. the surface negative charge of the cells in each passage population distributes around a value regardless of cell size. These results indicate that the cell electrophoretic mobility represents a cell surface marker for the population doublings of human diploid fibroblasts.

194 T h e results o b t a i n e d in the m e a s u r e m e n t o f e l e c t r o p h o r e t i c m o b i l i t y coincide well with t h e findings r e p o r t e d b y A i z a w a e t al. [36], w h o i n v e s t i g a t e d the r e l a t i o n s h i p b e t w e e n cell size a n d cell s u r f a c e p r o p e r t i e s o f h u m a n d i p l o i d f i b r o b l a s t s using the c o n c a n a v a l i n A ( C o n A ) - m e d i a t e d red b l o o d cell (RBC) a d s o r p t i o n assay. Small cells in late p a s s a g e p o p u l a t i o n s a d s o r b e d C o n A - R B C well as d i d large cells o f this p o p u l a t i o n , while small cells in e a r l y p a s s a g e p o p u l a t i o n s d i d not. T h e y have s h o w n , f u r t h e r m o r e , t h a t t h e R B C a d s o r p t i o n c a p a c i t y o f r a p i d l y d i v i d i n g cells differs a m o n g early, m i d d l e a n d o l d cell p o p u l a t i o n s , i.e. d i v i d i n g cells in late p a s s a g e p o p u l a t i o n s suffer a g e - d e p e n d e n t a l t e r a t i o n s in their s u r f a c e structure, a n d d i f f e r f r o m d i v i d i n g cells in e a r l y p a s s a g e p o p u l a t i o n s . B o s m a n n e t al. [27] h a v e s p e c u l a t e d t h a t the a g e - d e p e n d e n t decrease in the net negative c h a r g e is r e l a t e d to a loss o f e x t e r n a l s u r f a c e N - a c e t y l - n e u r a m i n i c a c i d o n t h e basis o f their w o r k o n o t h e r m a m m a l i a n cells. H o w e v e r , it is k n o w n t h a t cell surface m u c o p o l y s a c c h a r i d e s c h a n g e in a s s o c i a t i o n with cellular aging in c u l t u r e [10,14,16,17]. T h e r e f o r e cell s u r f a c e m u c o p o l y s a c c h a r i d e s p r o b a b l y c o n t r i b u t e to the a g e - r e l a t e d c h a n g e s in s u r f a c e charge. REFERENCES 1

2

L. HayflickandP.S. Moorhead, Theserialcultivationofhumandiploidcellstrains. Exp. CelIRes., 25 (1961) 585--621.

J.P. Jacobs, C.M. Jones and J.P. Baille, Characteristics of a human diploid cell designated MRC-5. Nature, 227(1970) 168--170.

3 4 5 6 7 8 9 10 11 12 13 14 15

W.W. Nichols, D.G. Murphy, V.J. Cristofalo, L.H. Toji, A.E. Greene and S.A. Dwight, Characterization of a new human diploid cell strain, IMR-90. Science, 196 (1977) 60--63. G.M. Martin, C.A. Sprague and C.J. Epstein, Replicative life-span of cultivated human cells: Effects of donor's age, tissue and genotype. Lab. Invest., 23 (1970) 86--92. V.J. Cristofalo, Animal cell cultures as a model system for the study of aging. Adv. Gerontol. Res., 4 (1972) 45--79. R. Holliday and G.M. Tarrant, Altered enzymes in ageing human fibroblasts. Nature, 238 (1972) 26 --30. E.L. Schneider and Y. Mitsui, The relationship between in vitro cellular aging and in vitro human age. Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 3584--3588. A. Macieira-Coelho, C. Diatloff and E. Malaise, Concept of fibroblast aging in vitro: Implications for cell biology. Gerontology, 23 (1977) 290--305. R. Azencott and Y. Courtois, Age-related differences in intercellular adhesion for chick fibroblasts cultured in vitro. Exp. Cell Res., 86 (1974)69--74. K. Yamamoto, M. Yamamoto and H. Ooka, Cell surface changes associated with aging of chick embryo fibroblasts in culture. Exp. CellRes., 108 (1977) 87--93. R.O. Kelley, R. Azad and K.G. Vogel, Development of the aging cell surface: concanavalin Amediated intercellular binding and the distribution of binding sites with progressive subcultivation of human embyro fibroblasts. Mech. Ageing Dev., 8 (1978) 203--217. S. Aizawa and Y. Mitsui, A new cell surface marker of aging in human diploid fibroblasts. J. Cell. Physiol., 108 (1979) 383--387. M.E. Palumbo, The surface membrane proteins of the WI-38 fibroblast in relation to transformation and aging. Age, 2 (1979) 1--4. Y. Courtois and R.C. Hughes, Glycoproteins of chick embryo fibroblasts in cultures with a finite life span. Eur. J. Biochem., 44 (1974) 131--138. J.A. Biondal, J.E. Dick and J.A. Wright, Membrane glycoprotein changes during the senescence of normal human diploid fibroblasts in culture. Mech. Ageing Dev., 30 (1985) 273--283.

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