Cell surface changes accompanying aging in human diploid fibroblasts

Cell surface changes accompanying aging in human diploid fibroblasts

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Printed in Sweden Copyright @ 1980 by Academic F’re~, Inc. All nghtr of reproductmn in any form reserved 0014.4827/80/020297-07$02.00/O

Experimental

CELL

SURFACE CHANGES ACCOMPANYING HUMAN DIPLOID FIBROBLASTS

III. Division

SHINICHI ‘Nutrition

Cell Research 125 (1980) 297-303

AIZAWA,’

AGING

Age and Senescence Revealed by Concanavalin mediuted Red Blood Cell Adsorption YOUJI MITSUI,*

FUMIKO

KURIMOTO’

IN A-

and KOJI MATSUOKA’

Laboratory and ZBiochemical Pharmacology Laboratory, Tokyo Metropolitan of Gerontology, 35-2 Sakaecho, Itabushi-ku, Tokyo-173, Japan

Institute

SUMMARY The relationship between cell size, [3H]thymidine incorporation capacity, and cell surface property of human diploid tibroblasts was investigated using the concanavalin A (ConA)-mediated red blood cell (RBC) adsorption assay. Small cells in late passage populations adsorbed RBCs well with the RE%Ccoating method (in which ConA-coated RBCs are adsorbed to fibroblasts) as did large cells of this population, while small cells in early passage populations did not. The RBC adsorption capacity of rapidly dividing cells with this method differed among young, middle-aged and old cell populations. The results suggest that temporal cell size and [3H]thymidine incorporating capacity is not a measure of the division age of human diploid cells at the individual cell level. On the other hand, REX adsorption with the fibroblast coating method (in which RBCs are adsorbed to ConA-coated fibroblasts) occurred to non-dividing cells of the populations. Thus, the increase in RRC adsorption with this method is considered to be a reflection of the increase in non-dividing cells at phase III. Our results support the hypothesis that RBC adsorption with the RBC and fibroblast-coating methods represents a cell surface marker for division age and senescence of human diploid cells, respectively, at the individual cell level.

Normal human diploid fibroblasts have a limited doubling potential in culture, and have therefore been widely used as an in vitro model of aging at the cellular level [l-8]. During the past decade, extensive investigations have revealed age-related changes in various properties of these cells. Although changes in cell surfaces have also been suggested as participating in the mediation of in vitro cell aging [9-121, the relationship of cell surface changes to the proliferative decline of the diploid cells remains to be elucidated. To examine this relationship, new indices of cellular aging which can measure surface changes in individual cells are necessary. 20-791817

In the previous paper we reported that ConA-mediated REK adsorption with the REK and fibroblast coating methods reveals two types of age-related cell surface changes of human diploid tibroblasts, one which occurs from the early phases of cell passage up through cell senescence and the other which occurs with the entry into the senescent phase [13]. The change in RBC adsorption is not a simple reflection of the increase in cells being retained at a special phase of the cell cycle or of time spent in culture (metabolic time). Co-culturing of young cells with old cells also does not affect the RBC adsorption capacity of the respective cells. The greatest advantage of Exp Cell

Res 125 (1980)

298

Aizawa et al. coating method reflects cell surface changes associated with cell senescence. Our results suggest that small, rapidly dividing cells among young, middle-aged and old cell populations differ in their cell surface properties as revealed by RBC adsorption with the RBC coating method. That is, temporal cell size and [3H]thymidine incorporating capacity is not a measure of division age of human diploid cells at the individual cell level. On the other hand, REK adsorption occurred in non-dividing cells of the populations with the tibroblast coating method. The increase in RBC adsorption with this method is thought to reflect the increase in non-dividing cells at phase III. MATERIALS

I. Abscissa: surface area (X lo-’ pm*); ordinate: frequency (%). (a) PDL 14; (b) PDL 46; (c) PDL 64. Changes in surface area of TIC&l cells with aging. See Materials and Methods for the measurement of surface area. Frequency is given as percentage of cells having the indicated surface area over the total of cells measured.

Fig.

the REK adsorption assay is that examinations can be carried out at individual cell level as well as at population level. In the present paper, the relationships between cell size, [3H]thymidine incorporating capacity and REK adsorption capacity with the REK and fibroblast coating methods were investigated in an attempt to elucidate whether RBC adsorption with the RBC coating method reflects cell surface changes accumulating with division, and whether RBC adsorption with the fibroblast Exp Ce//Rr.s

125 (IWO)

AND

METHODS

TIG-1 is a new human diploid fibroblast strain established at the Tokyo Metropolitan Institute of Gerontology from the lung of a 20-week-old female fetus. Its mean lifespan was 68 population doubling levels (PDLs). Cell culturing and RRC adsorption assay were performed as described in the previous paper [ 131. For determination of cell surface area, monolavercultured cells were fixed with 2.5% glutaraldehyde, stained with 0.1% crystal violet and photographed microscopically. The flattened cell surface area* was determined with a semi-automatic image analysing system (Leitz) to trace the cell circumference on enlareed photographs of the stained cells. For the determination of [3H]thymidine incorporating capacity, cells were incubated with 0.01 $X/ml of [3H]thymidine (5 Ci/mmol; The Radiochemical Centre, Amersham, U.K.) for 23 h or 7 days at 3PC in culture medium. Then, the cells were used for RRC adsorption, fixed with 2.5% glutaraldehyde and processed for autoradiography as described previously [16]. The number of adsorbed REICs was counted for labeled and unlabeled cells under a microscope. Y

RESULTS Cell size and RBC adsorption with the RBC coating method Human diploid tibroblast populations are heterogeneous in terms of cell size. As shown in fig. 1, not only mean cell size, but also heterogeneity increases with aging,

Cell surfaces

and aging

in vitro.

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2. RBC adsorption to (a) PDL 16; (h) PDL 64 cells with the RRC coating method. After RBC adsorption, the cells were fixed with 2.5% glutaraldehyde and stained with 0.5% crystal violet. Since the fields of vision were selected to show large and small cells simultaneously, the photographs do not show a typical distribution of cells in terms of size.

Fig.

especially at phase III [14-U]. Although few in number, large cells are always contained in early and small cells in late passage populations. In addition, a close correlation between cell size and proliferative capacity has been reported [16]. Thus, the question arises as to whether large cells in early passage populations are old and whether small cells in late passage populations are young. As shown in fig. 2, however, neither large nor small cells in the PDL 16-cell population adsorbed RBCs with the RBC coating method. On the other hand, small as well as large cells in the PDL 64-cell population adsorbed REKs well. To examine these re-

sults quantitatively, the number of adsorbed REXs was plotted against the surface area of individual cells. Fig. 3a illustrates the distribution for the PDL 16 population, and fig. 3 b, in which the shaded area shows the distribution for the PDL 16 popufor the lation of fig. 3a, the distribution PDL67 population. RBC adsorption to smaller cells in the PDL 67 population occurred to an extent similar to that of RBC adsorption to larger cells of the same population. In addition, smaller cells in the PDL

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3. Abscissu: surface area (pm*); ordinute: no. of adsorbed REWfibroblast. Relationship between cell size and RBC adsorption capacity of(u) PDL 16 and (b) PDL 67 cells with the RBC coating method. The number of RBCs adsorbed to and surface area of individual cells were determined on enlarged photographs. The shaded area in (b) shows the distribution of PDL 16 cells shown in (a).

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et al.

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67 population similar in size to PDL 16 cells adsorbed far more REKs than did PDL 16 cells. Thus, in old populations, small cells have the same surface properties as large cells and differ from small cells in young populations. [3H] Thymidine incorporating capacity and RBC adsorption with the RBC coating method

The relationship of RBC adsorption with the RBC coating method to the proliferative capacity of human diploid fibroblasts was examined by determining the number of RBCs adsorbed to cells labeled with [“HIthymidine for 24 h. Since heavy RBC adEA-p CdlRe,s

125 (1980)

sorption to phase III cells interferes with the identification of labeled cells, the incubation time of ConA-coated RBCs with fibroblasts was reduced to 12 min in these experiments. The percentages of labeled cells were about 82, 76 2nd 12 in the PDL 10, 30, and 67 populations, respectively. Labeled cells are under these conditions considered to be rapidly dividing cells, whereas slowly dividing and non-dividing cells are considered to be included in the unlabeled cell fraction [ 171. Most of the unlabeled cells in phase II populations may be slowly dividing, but a significant part of them in phase III populations may be nondividing cells. As shown in fig. 4, most of the PDL 10 cells did not adsorb RBCs, while most of the PDL 67 cells adsorbed RBCs well, regardless of whether they were labeled or unlabeled. On the other hand, PDL 40 cells were heterogeneous in terms of RBC adsorption capacity. The frequency data, however, indicate that in all cell populations the ratio of unlabeled to labeled cells was almost unchanged for each fraction with a different degree of RBC adsorption. That is, the percentage of labeled cells in each fraction roughly corresponds to that in each population. Therefore, although the distribution patterns differed among PDL 10, 40 and 67 cell populations, the distribution patterns of labeled cells were nearly identical with those of unlabeled cells in each cell population. These results indicate that rapidly dividing cells of a cell population resemble slowly dividing cells and/or non-dividing cells of the same population in cell surface properties revealed by RBC adsorption with the REW coating method. In addition, cell surface properties of rapidly dividing cells change from population to population with PDLs.

Cell surfaces and aging in vitro. III

301

100 I PDL

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5. Abscissa: no. of adsorbed RBCs/fibroblast (N) (a) N4; (b) XN
Fig.

[3H] Thymidine incorporating capacity and RBC adsorption with the fibroblast coating method Fig. 5 shows the relationship between RBC adsorption capacity with the fibroblast coating method and proliferative capacity of human diploid fibroblasts in PDL 10, 40 and 67 populations. In contrast to the RBC coating method, the distribution patterns of labeled and unlabeled cells differed in the frequency data shown. Most of the PDL 10 cells and most of the labeled cells in the PDL 40 and 67 population did not adsorb RBCs. In addition, most of the highly RBCadsorbant cells of the PDL 40 population, though few, were unlabeled cells and represented a minor part of the unlabeled cells of this population. On the other hand, a significant part of the unlabeled cells of the PDL 67 population adsorbed REKs well with the fibroblast coating method. These results suggest RBC adsorption with the fibroblast coating method to a certain subclass of unlabeled cells, i.e. non-dividing cells.

N-C5

5
Fig. 6. Abscissa: no. of adsorbed ordinate: frequency (%).

lO
15
RRCsltibroblast (N);

Relationship between proliferative capacity and RBC adsorption capacity of PDL 67 cells with the fibroblast coating method (II). Cells were incubated with [3H]thymidine for 7 days and processed as described in the caption to fig. 5.

To confirm the above assumption, PDL 67 cells were labeled with [3H]thymidine for 7 days to label all of the slowly dividing cells [ 171. Under these conditions unlabeled cells are considered to be non-dividing cells only, while the labeled cell fraction includes rapidly dividing, slowly dividing and nondividing cells (which were produced after the division of dividing cells). The percentage of unlabeled cells under these conditions was about 12%. As shown in fig. 6, all unlabeled cells adsorbed RBCs well, while a significant part of labeled cells did not adsorb REKs. In addition, the frequency distribution for the labeled cells was nearly the same as that for total PDL 67 cells shown in fig. 5. This indicates that dividing cells in the PDL 67 population produce cells with different REK adsorption capacity in a distribution similar to that of the PDL 67 population to which they belong. DISCUSSION Although there is an increase in the number of large cells with aging [14-151, and there is a close correlation between cell size and Exp Cd

Res 12.5 (1980)

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Aizawa et al.

dividing capacity of human diploid fibroblasts [ 161, our results suggest that the temporal cell size and r3H]thymidine incorporating capacity are not a measure of division age of human diploid tibroblasts at the individual cell level. That is, in terms of REK adsorption with the REK coating method, small cells in late passage populations have cell surface properties similar to those of large cells of the same populations and differ from small cells in early passage populations. In addition, rapidly dividing cells among young, middle-aged and old cell populations differ in their cell surface properties. Since RE3C adsorption with the REK coating method increases continuously with aging, small, rapidly dividing cells among young, middle-aged and old cell populations may differ in their division age. This is also supported by the result shown in fig. 6 with the fibroblast coating method. Dividing cells of a senescent population produce cells with different RRC adsorption capacity with this method in a distribution similar to that of the senescent population to which they belong. This suggests that dividing cells in late passage populations are old and differ from dividing cells in early passage populations. The result obtained in the RBC adsorption study mentioned above coincides well with the finding reported by Mitsui et al. [ 171 who fractioned cells into small cell and large cell groups by the sedimentation velocity method and examined the changes in cell size and proliferative capacity upon subsequent culturing. Fractioned cells soon become indistinguishable from the control unfractioned cells in terms of cell size and [3H]thymidine incorporating capacity. In addition, there was no difference in the remaining dividing capacity among the small, large and unfractioned cell groups [ 181. RBC adsorption with the fibroblast coatExp Cd

RPS 125 (1980)

ing methdd is, in contrast, thought to occur to non-dividing cells of the populations. With this method, RE3C adsorption to cell populations does not occur throughout the phase II period and increases with the advance of the phase III period. This suggests that the change in RBC adsorption with the fibroblast coating method reflects the increase in non-dividing cells at phase III. The results reported in the previous and present papers support the hypothesis that REK adsorption with the RBC coating method reflects cell surface changes which accumulate with division from early phases of cell passage, and that RBC adsorption with the fibroblast coating method reflects surface changes associated with cell senescence. Two types of aging index, one which can reveal division age and one which can reveal cell senescence, especially at the individual cell level, are important for the study of aging in human diploid tibroblasts. RBC adsorption with the REK and tibroblast coating methods appears to serve these functions. A problem encountered throughout the present study is the heterogeneity of cell populations in the extent of RBC adsorption, especially at middle passage populations (fig. 4). Elucidation of the causes of this heterogeneity might give us some important clues to an understanding of aging in human diploid fibroblasts. This line is currently being investigated in our laboratory in relation to the question of whether there is truly any difference in the remaining dividing capacity among cells with differing RBC adsorption capacities. REFERENCES 1. Hayflick, L, Exp cell res 37 (1965) 614. 2. Cristofalo, V J, Adv geronto14 (1972) 45. 3. Goldstein, S, Littlefield, J W & Solender, J S, Proc natl acad sci US 64 (1969) 155. 4. Holliday, R & Tarrant, GM, Nature 238 (1972) 26. 5. Macieira-Coelho, A, Diatloff, C & Malasise, E, Gerontology 23 (1977) 290.

Cell surfaces 6. Martin, G M, Sprague, C A & Epstein, C J, Lab invest 23 (1970) 86. 7. Norwood, T H, Pendergrass, W R, Sprague, C A & Martin. G M, Proc natl acad sci US 71 (1974) 223. 8. Schneider, E L & Mitsui, Y, Proc natl acad sci US 73 (1976) 3584. 9. Bowman, P D & Daniel, C W, Mech ageing dev 4 (1975) 147. 10. Kelley, R 0 & Skipper, B E, J ultrastruct res 59 (1977) 113. 11. Yamamoto, K, Yamamoto, M & Ooka, H, Exp cell res 108 (1977) 87. 12. Kelley, R 0, Azad, R & Vogel, K G, Mech ageing dev 8 (1978) 203.

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13. Aizawa, S, Mitsui, Y & Kurimoto, F, Exp cell res 125 (1980) 287. 14. Grove, G L & Cristofalo, V J, J cell physiol 90 (1976) 415. 15. Mitsui, Y & Schneider, E L, Mech ageing dev 5 (1976) 45. 16. - Exp cell res 100 (1976) 147. 17. - Ibid 103 (1976) 23. 18. Mitsui, Y, Smith, J R & Schneider, E L. In preparation. Received December 7, 1978 Revised version July 23, 1979 Accepted September 3, 1979

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