Age-related differences in intercellular adhesion for chick fibroblasts cultured in vitro

Printed in Sweden Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved

Experimental Cell Research 86 (1974) 69-74

AGE-RELATED DIFFERENCES IN INTERCELLULAR ADHESION FOR CHICK FIBROBLASTS CULTURED IN VITRO R. AZENCOTT

and Y. COURTOIS

Unite’ de Recherches Gkrontologiques, INSERM

U 118,29 rue Wilhem, 75016 Paris, France

SUMMARY Cell adhesiveness in chick embryo fibroblasts has been studied as a function of age of the culture. Using a modification of Roth’s assay, it has been shown that: 1. ‘Young’ cells adhere more frequently than old cells to young-cell aggregates. 2. This phenomenon is clearly due to a loss of intrinsic adhesiveness with ageing and not to gross differences of resynthesis of surface components. When the same analysis was used to compare exponential cells with confluent cells at the same passage, the former were leas adhesive than the latter. Combined data suggest cumulative modifications of membrane surface for in vitro ageing of chick fibroblasts.

The growth potential of chick embryo fibroblasts gradually decreasesduring serial cultivation in vitro, according to Hayflick’s model of limited lifespan in vitro [l]. Several age-related biochemical parameters have been investigated [14]. However, very little is known about modification related to age of the surface membrane. Recently several and contradictory results were published concerning changesin the surface antigens during ageing in vitro of human fibroblasts [5-71. It has been proposed that aged cells are more sensitive to contact inhibition of division than young cells [8]. Macieira-Coelho has shown that one of the first modifications occurring in chick fibroblasts showing senescence in culture is a decrease of the cell density at saturation following a decreasein protein synthesis [9]. A recent report [lo] has described in ageing cultures, an increase in cell loss as a result of detachment. The relationship between these parameters and cell adhesion has been described by several authors [l l-121.

In this study, we have compared the adhesion of young and old cells toward aggregates of young cells. We have shown that young cells, even after trypsinisation, are more adhesive than old cells. These results would suggest that modification at the level of the surface membrane takes place during in vitro ageing. MATERIAL

AND METHODS

Cell culture Chick fibroblasts were obtained from lea muscles of 12&y old chick embryos as described- by Hay & Strehler 1131.Cell culture and subculture were done with 0.25-%trypsin and a split ratio of 1: 2. Each subculture is quoted in this work as a ‘population doubling’. We used Minimum Essential Medium (MEM) with 10 % calf serum, and 100 units/ml penicillin and 100 pg/ml streptomycin.

Cell ageing After 19-23 population doublings in roller bottle, the chick fibroblasts cultures reached Hayflick’s phase III and the cells stopped dividing, in accordance with several authors [8, 131.

Preparation of label&d singIe cells Cells were labelled in monolayer culture for three generations with sH-thymidine (1 &i/ml). The radioExptl Cell Res 86 (1974)

70 Azencott and Courtois activity (cpm/cell) was determined by trypsinisation to disperse the cells followed by washing several times with Hanks’ balanced salt solution (BSS). Then a known number of single cells were collected and dried on Whatman filters. The filters were counted with an Intertechnique scintillation counter in 10 ml of scintillation mixture (4 g PPO, 0.1 g POPOPjl 1 toluene). The efficiency of incorporation ranged from 1 cpm/ 3 cells in young cultures to 1 cpm/l5 cells in old cultures. 3H-Thymidine (27 Ci/mmole spec. act.) was a gift from the French Commissariat a 1’Energie Atomique.

trypsinate”. These conditions did not produce more than 4 % of dead cells as judged by vital staining. The “extracellular material” and the “trypsinate” were dialysed extensively against distilled water. An aliquot of each was counted with 10 ml of scintillation mixture Instagel.

RESULTS Adhesion of single young or old cells to young cell aggregates

In Roth’s technique to compare cell adhesion, We used a slight modification of Roth’s assay [14-171 several parameters are difficult to control, for determination of the number of single radioactive particularly the size and the shape of the cells sticking to preformed aggregates of cells during aggregates. Several aggregates were selected a 4-h incubation. Aggregates of young cells from the first three randomly in adhesion assaysto minimize the passages were prepared by rotating at 70 rpm a 3 ml cell suspension (2 x 10’ cells/ml) in a 5 ml Falcon tube large influence of small differences in size of inclined at 45”. The incubation lasted 24 h at 37°C. the collecting aggregates on the surface A selection of aggregates according to their size (1.3 available to the single cells. After incubation mm diameter) was made with a calibrated Pasteur with labelled single cells, the collecting pipette. For the assay, 3 aggregates chosen randomly, were added in a 5 ml Falcon sterile tube with 3 ml of aggregateswere washed with the sameroutine adhesion medium (MEM supplemented with 10% heat-inactivated calf serum). Labelled single cells, technique by the same operator. The scatter freshly prepared, were pipetted into the tube at a in our results is of the same magnitude as cont. of 6 x lo5 cellsjtube. The tube was then incubated at 37°C for 4 h under the same conditions as those that of the dispersion obtained by Roth [15]. used for aggregate formation. During this time, no Since the cells were labelled with 3H-thymisizable self-aggregation of single cells occurred. At dine, there is little SH release from the cell the end of th~.in&bation, aggegates were collected, washed several times in PBS medium and dried on suspension during the assay and there is no Whatman filters. The radioactivity on the filters was counted as labelling of the preformed aggregates.Theredescribed previously for single cell suspension. fore the background was always very low The whole assay was carried out by the same operator and when two populations of cells were in our experiments. compared, the experiment was made for both at the Furthermore, 3H-thymidine was chosen as same time under the same conditions. a precursor in light of the finding [2] that Synthesis of membrane components chick fibroblasts cells were able to synthesize DNA until the very end of their life span. Cells synthesized new macromolecules during the adhesion assay. Thus the pattern of adhesion in old Table 1 summarizes the results obtained in and young cells could be due to differential rate of synthesis of newly formed material at their own sur- comparing the adhesion of old cells (19th face or excreted in the medium. This phenomenon population doublings) and young cells (4th was measured by labelling the cell suspension with xH-glucosamine and by counting the material re- population doublings) to young cell aggremoved throuah a second trwsin treatment. gates. To the celi suspension at‘ the same concentration When a non-parametric test (Mann-Whit(2 x lO’/ml) as that used in the adhesion assay, 20 Ci of aH-glucosamine (12 Ci/mmole) was added. ney’s Test) was used to check the data Incubation was as described previously. At different times. aliauots of the cells were cooked down and presented in table I a significant difference cent~fuged at 800 g for 4 min. They were washed between the two cell populations was obtwice with 2 ml of Hanks’ BSS. The medium and the wash were pooled together. This will be referred to as tained. For old and young cells the mean “the extra-cellular material”. The pellets were treated number of cells sticking to the aggregatesis with 0.25 % trypsin for 10 min at 37°C. The cells were respectively 62.0 and 476.7. The Mannpelleted and the supematants referred to as “the Cell adhesion assay

Exptl Cell Res 86 (1974)

Adhesion of ageing chick embryo jibroblasts

Table 1. Comparison of adhesion of populations of confluent young cells and old cells to young cell aggregates

Tube no. Old 1 2 3 4 5 6 7 8 1: Young :

No. of recovered cpm per aggregatesa aggregate

2 2 3 3 3 3 3

3 1

No. of adhesive cells/ aggregate

:3

:t 22 13 26 16 7:

45 135 150 480 330 195 390 240 1 140 90

3

42 55

495 378

3 3

2511 3 087 9 393

3 4 5 6

:

279 343 1 49

I

:

2;:

2 387 613

1:

3

324 158

21 916 422

a Out of three in each tube. Old (19th passage) and young (4th passage) were prepared from confluent cultures and carried a labelling of 1 cpm/l5 cells and 1 cpm/9 cells respectively.

71

(ii) this phenomenon seemsto be affected by the growth condition of the cells: i.e. the difference in cell adhesion is greater when young and old confluent cells were compared than when young and old exponential were compared. The low efficiency in collecting the cells can be explained by the fact that we used a rotating motor for the assay instead of a rotary shaker as used by others [15-181. Thus the frequency of collision between cells in suspension and an aggregate was lower. However, it can be argued that cells which stuck to aggregates were not representative of the real cell population. While this argument cannot be ruled out, it seemsunlikely in light of similar experiments made by others P71. The assay used to obtain these results involves a 4 h incubation at 37°C of trypsinised labelled cells. It could not be ruled out that the differences in adhesive properties of young or old cells was related to possible differences in the capacity of cells to resynthesize at different rate surface-associated or excreted materials involved in cell adhesion

Table 2. Differential Whitney’s treatment (U= 13) showed that the two populations are significantly different with a threshold of probability of PcO.02. P would be reduced to P
adhesion for confluent or exponential cells at several passages Expt no.

Passage

I

Confluent cells 4 22

II

III IV

2: Exponential cells 1 8 3 16

Mean number of cells per aggregate

1 880 500 1 590 90 519 231 272 154

In expts I, II and IV where the number of samples were numerous enough to utilise the Mann-Whitney’s test we calculated for U a value of zero and a p < 0.01 for each. Exptl Cell Rex 86 (1974)

12 Azencott and Courtois Comparison of adhesion between exponential or confluent cells

a

b Ip

I

I

0.2

2

4

I,

I

' 24 _

Fig. 1. Abscissa: time (hours); ordinate: 3H-glucosamine cpm/ml x 10S.(a) plot of “trypsinate”; (b) of “extracellular material”. Rate of synthesis of non-dialysable material from passage 3, young cells (0 0); and passage 23, old cells ( l 0); in suspension cultures. Bars represent duplicate experiments.

hypothesis, we made a comparative analysis of the rate at which new material was synthesized during the assay. [18]. In order

to check this

As the timing of the assay was relatively short, it was of interest to use it to compare exponential cells with confluent cells. It is a routine microscopic observation that, in exponential state, a proportion of cells which are in the process of dividing, moves out of the substratum, becomes refractile and more susceptible to be detached by mechanical stirring from the bottom of the Petri dish. Furthermore exponential cells move away from each other during growth and prefer to fill empty spaces at the bottom of the Petri dish than to pile up on top of each other. Finally if a clone of cells is allowed to grow, cells at the periphery have a higher mitotic activity and move faster than cells at the centre which are inhibited by contact. For these reasons it was predictable that exponential cells should be less adhesive toward other cells than confluent cells. The results shown in table 3 are in agreement with this prediction. Table 3. Differential

adhesion for confluent and exponential cells at the same passage

Expt no.

Comparison of the rate of resynthesis of membrane components at the surface of trypsinized young and old cells

Trypsin removes material from the surface of cells and it is well-known that as much as 20 his neededto resynthesizethesecompounds

Exptl Cell Res 86 (1974)

Exponential

Confluent

I

498 600 210

830 2400 600

II

40 40

210 340 225

III

loo0 540 1000

3000 4300 4200 3600 2900

WI. Fig. 1 shows that the rate of incorporation of radioactive glucosamine into non-dialysable material removed from cells by trypsin or excreted by these cells into the medium is the same for old and young cells.

No. of cells per wgregate

1E

In expt III the Mann-Whitney test gave for U a due

of zero and P
Adhesion of ageing chick embryo fibroblasts

DISCUSSION In these experiments we showed that aged single cells are less adhesive toward aggregates of young cells than are young single cells. This phenomenon is in accordance with the observation that young cells are able to overlap to reach higher density of saturation. When cells become senescentthey enlarge by a factor of 25 % or more [2]. The reverse experiment-i.e. viewing the adhesivenessof young and old cells against aggregatesof old cells-could not be carried out as it was impossible to obtain good size aggregates with old cells, even in 24 h. This observation implies that the adhesivenessof old cells is permanently reduced after serial population doublings, because even a 24 h incubation does not permit a recovery of the original capacity for self-aggregation in young cells. The reason for this behaviour is not clear, especially because our findings showed old and young cells to incorporate the same amount of SH-glucosaminein the “trypsinate” and in the “extracellular material”. It is worth noting that the rate of incorporation of 3H-glucosamine in these fractions is 10 times higher for attached cells, in the same conditions, than that of those in suspension. However, several recent reports have shown that a glycoprotein synthesized by the cells is involved in the specific adhesion of cells [16, 18, 201. It is not unlikely that a small, undetected proportion of labelled material is synthesized differently or at a different rate in young and in old cells, during the adhesion assay. In a recent work [21] to be published elsewhere, a chemical analysis was made of the changes occurring with ageing at the cell surface of chick fibroblasts. It was found that mucopolysaccharides constitute a larger proportion of the glucosamine-labelled material from the surface of phase III cells than

73

from the surface of phase II cells. Conversely, phase II cells were found to be relatively rich in several glycopeptides when compared with phase III cells. The combined results of these two experiments are in favour of a model where the sites of adhesivenesswould be less and lessefficient with serial population doublings due to slight cumulative modifications of the plasma membrane. Another interpretation of the decreasein adhesiveness with age of cells in culture could be drawn from the observation that this phenomenon is less pronounced in exponential cells than in confluent cells. At least two modifications according to the state of growth in cell metabolism would be relevant to this phenomenon. First, there is little surface membrane turnover (review in [22]). Secondly the excretion of macromolecules in the medium is different during the growth phase. It has been shown recently [23] that the synthesis of collagen was impaired in old human fibroblast. While such studies have not been carried out in ageing chick fibroblasts, it is likely that the situation is similar. A second hypothesis would be that the material excreted by chick fibroblasts is different for young and for old cells. This hypothesis, together with the fact that chick cells in suspension are stopped in G2 and are required to synthesize some material to undergo mitosis [24], would explain why old cells have a longer G2 period [25]. The afore-mentioned would also be consistent with Good’s findings [IO] that there is an increase in cell detachment with ageing of the culture. In the second part of this study young exponential cells were shown to be less adhesive than confluent cells. This phenomenon could be explained likewise by two different hypotheses. The first is that the surface composition (or arrangement) is different for cells at Exptl Cell Res 86 (1974)

74 Azencott and Courtois

different points of the cell cycle. Cell cycledependent changesin membrane surface have been detected by Concanavalin A [26]. The second is that adhesivenessis related to material excreted by the cells and that cells which are highly dividing, do not produce the same material as resting cells. Such changes have been shown both for collagen synthesis [27] and for total proteins [28] in cultured fibroblasts. Finally the results obtained suggest that the adhesion assay is a good method for detecting minor differences due to ageing or to the growth state at the surface of chick fibroblasts cells. To clarify this last point however, synchronized cells should be used in the adhesion assay. The present data, combined with that of others [21] would suggestthat a great proportion of the behaviour of cells ageing in culture (such as decreasein cell density at saturation, loss of ability to overlap, increase in cell loss by detachment) could be the result of progressive accumulation of defects at the cell surface. But the results of experiments did not allow us to choose between the two current theories of ageing: stochastic accumulation of errors or genetic programming. According to the first theory, faulty protein synthesis [29] would be responsible for changes at the cell surface, but according to the second such changescould as well be programmed which is the case for some differentiating cells [30]. The authors are deeply grateful to Dr B. Pessac, C. Hughes and A. Macieira-Coelho for their fruitful advice, to Dr Poitrenaud for carrying the statistical analysis, and to Miss J. Tassin for her excellent technical assistance. This work was supported in part by grant (contract no. 7244157 INSERM).

Exptl Cell Res 86 (1974)

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