Cell surface changes accompanying aging in human diploid fibroblasts

Cell surface changes accompanying aging in human diploid fibroblasts

Printed in Sweden CopyrIght 0 1980 by Academic Pree. Inc All rights of reproduction in any form resned 0014.4827/80/0202X7-10S02.0010 E,uperimental ...

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Printed in Sweden CopyrIght 0 1980 by Academic Pree. Inc All rights of reproduction in any form resned 0014.4827/80/0202X7-10S02.0010

E,uperimental

CELL

SURFACE

CHANGES

IN HUMAN II.

Two Types

ACCOMPANYING

DIPLOID

AGING

FIBROBLASTS

qf Age-r-elated

SHINICHI ‘Nutrition

Cell Research 125 (1980) 287-296

Changes Revealed by Concana\~alin Red Blood Cell Adsorption

AIZAWA,’

YOUJI MITSUI’

and FUMIKO

A-mediated

KURIMOTO’

Luboratory and ‘Biochemical Phrtrmclcology Loboratory, Tokyo Merropolitrrn Instituie qf Gerontology, 35-2 Sakcrecho. Itahashi-ku. Tokyo-173, Jtrpcm

SUMMARY Age-related changes in cell surfaces of human diploid fibroblasts (TIC&l) were investigated using the concanavalin A (ConA)-mediated red blood cell (RBC) adsorption assay. When ConA-coated REICs were adsorbed to fibroblasts (RBC coating method), the amount of RBCs adsorbed per mg of fibroblast protein increased continuously from the early phases of cell passage up through cell senescence. On the other hand, when RBCs were adsorbed to ConA-coated fibroblasts (fibroblast coating method), REIC adsorption did not occur throughout phase II and increased with the advance of phase III. [3H]ConA binding to tibroblasts, however, did not change with aging to the extent that could explain the observed changes in RBC adsorption. These age-related characteristics in REX adsorption and [3H]ConA bindiig were also observed for WI38and IMR-90 cells. In addition, SV40- and 60Co-transformed WI38 cells showed a close resemblance in their RBC adsorption capacity to early phase III cells. RBC adsorption with both the REX and fibroblast coating methods was not a function of cell cycle phase and time spent in culture (metabolic time). Co-culturing of young cells with old or transformed cells did not affect the RBC adsorption capacity of respective cells. These results suggest that RBC adsorption with the REIC and fibroblast coating methods may represent cell surface markers for division age and senescence of aging human diploid cells.

Since the first description of the finite doubling potential of human diploid fibroblasts by Hayflick & Moorhead [ 11, these cells have been extensively studied as an in vitro model of aging at the cellular level [2-8]. Although considerable work has been reported on DNA, chromatin and enzyme changes accompanying the proliferative decline of the cells, relatively little is known about the change on the cell surfaces. Scanning electron microscopic (SEM) observations by Bowman & Daniel [9] and freeze fracture analyses by Kelley et al. [lo, 1 l] have suggested changes in cell surface topography and membrane organization. There are some reports on antigenic changes in

cell surfaces associated with cell senescence, although these reports sometimes conflict with each other [12, 13, 141. Selective agglutination of suspended cells by certain plant lectins has been a widely used criterion for cell surface alterations. Decrease in concanavalin A (ConA)-mediated agglutination has been reported in aged chick fibroblasts [ 151. However, the lectinmediated RBC adsorption assay of Furmanski et al. [16] has some unique advantages. The assay can be performed quantitatively on monolayered cells without fear of possible changes in cell surfaces accompanying suspension with trypsin or EDTA. In addition, examinations using this assay

288

Aizawa, Mitsui and Kurimoto

can be carried out on individual cells in connection with other properties of cells such as cell size and r3H]thymidine incorporating capacity. This advantage is important for clarifying the possible relationship of cell surface changes to the proliferative decline of the diploid cells. Hence, we have initiated a series of studies on cell surface alterations in aging human diploid fibroblasts using the ConA-mediated RBC adsorption assay. In the present paper, we report two types of age-related cell surface changes revealed by ConA-mediated RRC adsorption, with reference to surface changes associated with transformation. The effects of cell cycle phase, metabolic time and co-culturing of young cells with old or transformed cells were also investigated. Our results suggest that REX adsorption with the RRC and fibroblast coating methods can serve as new cell surface markers for division age and senescence of human diploid cells.

MATERIALS

AND METHODS

Cell strains TIG-I 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. A detailed characterization of TIC&l will be reported elsewhere. The WI38 strain was obtained from the National Institute on Aging, Baltimore, Md, through the courtesy of Dr E. L. Schneider, and the IMR90 strain from the Institute for Medical Research, Camden, N.Y., through the courtesy of Dr W. Nichols. The mean lifesnan of these cell strain was 68 population doubling levels (PDLs) for TIG-1, 52 PDLs for WI38 and 56 PDLs for IMR-90. The G°Co-transformed WI38 was the kind gift of Dr M. Namba, Kawasaki Medical University, Kurashiki-shi, Okayamaken, and its characterization has already been reported [17]. The SV40-transformed WI38 was purchased from Dainihon Seiyaku. All of the cell strains used were mycoplasm-free as determined by the microbiological method [ 181.

Cell cultures Cell cultures were grown in Eagle’s Basal Medium (Diploid, Gibco) supplemented with 10% fetal calf Exp CdRes

125(1980)

serum (FCS) (Gibco), penicillin (100 U/ml) and streptomvcin (100 &ml) in a 5 % CO, : 95 % air environment at ‘37°C. -Routine subcultivation consisted of harvesting confluent cell cultures with 0.25 % trvnsin (I : 250, Difco) in calcium- and magnesium-free phosphate-buffered saline (pH 7.4) and dividing the cells by a 1 : 4 split ratio into 75 cm* plastic flasks (Falcon). For the ConA-mediated RBC adsorption and [3H]ConA binding assays, approx. 8x 104cells were inoculated into 8 cm* plastic dishes (Falcon) and used on the third day after inoculation.

RBC adsorption Freshly drawn human blood (A, Rh+) was washed with 1 mM MgCI,-supplemented phosphate-buffered saline (PBS) and used within a few hours. ConA-coated RBCs were freshly prepared before use by incubating RBCs with ConA (3x recrystallized, Miles) at 25°C for 30 min in PBS. The RBCs were then washed three times with PBS and dispersed by pipette to give a single RBC suspension. Cell monolayers were rinsed with PBS five times and then incubated with ConAcoated RBCs at 25°C for 20 min (RBC coating method). Alternatively, rinsed cell monolayers were first treated with ConA at 25°C for 20 min, washed with PBS five times and then incubated with uncoated RBCs for 30 min (tibroblast coating method). RBCadsorbed fibroblasts were washed with PBS until free from unadsorbed RBCs, dissolved in 5 % SDS solution and later analysed spectrophotometrically for hemoglobin content at 418 nm. Unless otherwise indicated, RBCs were used at 2% and ConA at 100 pg/ml final concentrations. One of the problems with ConA-mediated RBC adsorption is the non-specific binding of RBCs to mastic substrates. This was ruled out in our experiments by appropriate shaking upon washing. In addition. the amount of non-snecific binding was checked qualitatively by simple microscopic obiervations, and quantitatively by determining the amount of unreleased RBCs after the cu-Me-mannoside treatment of RBC-adsorbed cells.

Cell binding of r3H]labeled

ConA

3H-labeled ConA was purchased from New England Nuclear (NET-452). Cells cultured under the same conditions as those for RBC adsorption were treated with 1 ml of 100 fig/ml r3H]ConA (1.2 pCi/mg) for 20 min at 25°C. The binding assay was performed according to the procedure of Marciani & Okazaki [ 191. In the assay, [3H]ConA bound to cells is the amount of [3H]ConA bound in the absence of cr-Me-mannoside minus the amount bound in the presence of 50 mM a-Me-mannoside. The amount of [3H]ConA bound in the presence of cY-Me-mannoside was approx. 10% of the amount bound in the absence of a-Me-mannoside, regardless of cell types (non-senescent, senescent or transformed cells). as it has been reported to be the case in chick fibroblasts by Marciani & Okazaki [ 191. The [3H]ConA-bound cells were solubilized in 1 ml of Soluene-350 (Packard), and the radioactivity was measured with a Beckman liquid scintillation counter (LS-lOOC), using a toluene scintillation solution (0.6% DPO, Dotite).

Cell surfaces

10

20

30

40

50

60

Fig. 1. Abscissa: PDL; ordinate: (I@) OD,,,/cell (x 106); (righr) pg [3H]ConA/cell (X 106). Changes in RBC adsorption and [3H]ConA binding to human diploid fibroblasts as a function of PDL. 0, [3H]ConA binding; A, RBC adsorption with the RBC coating method; W, RBC adsorption with the tibroblast coating method. S.D. did not exceed 10% of the mean. -

Cell incorporation of [3H]thymidine [3H]Thymidine was purchased from the Radiochemical Centre (5 $Zi/mmol). The cells were incubated with 0.05 $X/ml of [3H]thymidine for 1 h in culture medium at 37”C, washed with PBS, incubated for indicated intervals in culture medium at 37”C, washed with PBS, used for RBC adsorption, washed with PBS, fixed with 2.5 % glutaraldehyde, and processed for autoradiography as described previously [20]. The number of adsorbed RBCs was counted for labeled and unlabeled cells under a microscope. Cell number was measured with a Model G Coulter Counter. The protein content was determined by Lowry’s method in 5% SDS, using bovine serum albumin as a standard.

RESULTS Two types of age-related cell surface changes revealed by ConA-mediated RBC adsorption assay

The ConA-mediated RBC adsorption assay of monolayered cells was originally reported by Furmanski et al. [16]. However, the conditions for the assay have not been fully established. Details for optimizing the Furmanski assay to facilitate comparison of

and aging in vitro. II

289

RBC adsorption among cells with different PDLs will be reported elsewhere. Briefly, cells were used on the third day after inoculation of about 104 cells/cm’. ConA was used at 100 pug/ml. The incubation times for ConA and RBCs, ConA and fibroblasts, ConA-coated RBCs and fibroblasts, and RBCs and ConA-coated libroblasts are 30, 20, 20 and 30 min, respectively. All incubations were carried out at 25°C. The RBC adsorption was completely inhibited by (YMe-mannoside, and adsorbed RBCs were completely released by incubation with (YMe-mannoside. The non-specific binding of RBCs to plastic substrate could be ruled out. Under these conditions, changes in RBC adsorption were examined as a function of in vitro passage (fig. 1). Changes in the binding of [3H]ConA to fibroblasts are also given in fig. 1. The results are expressed as values/cell. RBC adsorption with the RBC and fibroblast coating methods and [3H]ConA binding increased with PDLs of the cells, especially at phase III. However, there are some differences in their patterns of increase. With the fibroblast coating method, RBC adsorption did not occur in phase II, non-senescent cells. The adsorption of ConA-coated RBCs was also negligible for PDL 10 cells, but increased from early phases of cell passage. The binding of [3H]ConA occurred even in PDL 10 cells and did not change significantly throughout the phase II period. It is known that the cell size of human diploid tibroblasts changes with aging, especially at phase III [21]. Thus, the proper choice of the unit to express the amounts of RBC adsorption and [3H]ConA binding is a problem in representing the net changes with aging. Fig. 2 presents the data of fig. 1 in terms of changes per mg of fibroblast protein. While the binding of [3H]ConA to fiExp CrllRr.s

125 (1980)

290

Aizawa, Mitsui

and Kurimoto

a

b

4

10

20

30

40

50

60

70

10

20

30

40

50

60

70

Fig. 2. Abscissa: PDL; odinare: (a) OD,,,/mg of fibroblast protein; (b) pg [3H]ConA/mg of fibroblast protein. Changes in (a) REK adsorption; (b) [3H]ConA binding to human diploid tibroblasts as a function of PDL. The data for TIC-1 (0, 0) shown in fig. 1 are presented as values per mg of fibroblast protein. The re-

suits for WI38 (A, A) and IMR-90 n , 0) in table I are also shown. Note the continuous increase in RBC adsorption with the RBC coating method (closed symbols) from the early phases of cell passage up through cell senescence and the abrupt increase with the fibroblast coating method (open symbols) at late phases of cell passage. S.D. did not exceed 10% of the mean.

broblasts did not change significantly throughout the whole lifespan period, the adsorption of ConA-coated RBCs increased almost linearly with cell passage. The adsorption of RRCs to ConA-coated fibro-

blasts did not occur throughout the phase II period and occurred only to phase III cells. Table 1 presents RBC adsorption to WI38, IMR-90 and SV40- and 6oCo-trans-

Table 1. RBC adsorption and [3H]C~nA binding to normal diploid senescentfibroblasts, and their 6oCo- and SV40-transformed cells

non-senescent

REK adsorption” Cells

FCb

RC”

[3H]ConA bindingd

WI38 (32 PDLs) WI38 (37 PDLs) IMR-90 (20 PDLs) IMR-90 (45 PDLs) TIG-1 (10 PDLs) TIG-1 (45 PDLs) WI38 (51 PDLs) IMR-90 (54 PDLs) TIG-1 (58 PDLs) TIG- 1 (64 PDLs) G°Co-transformed WI38 SV40-transformed WI38

0.48 0.47 0.54 0.90 0.40 0.66 2.74 4.40 1.48 2.36 1.32 1.21

4.56 4.88 2.44 4.76 1.04 4.40 6.72 6.52 5.92 6.32 5.23 5.09

13.3

a OD,,,/mg of fibroblast protein. b REX adsorption with the fibroblast coating method. Exp

Cell

Kes

125 (1980)

12.5 11.5 13.6 14.1 13.1 13.4 15.0 10.1 9.2 c RBC adsorption with the RRC coating method. d pg/mg of tibroblast protein.

and

Cell surfaces and aging in vitro. II 30 -

20

60

40

-__ __--

20 -

10

20

uma r

10 -

0

o-I NC5

Fig. 3.

5SN
lOSN<15

15
no. of adsorbed RBCsltibroblast frequency (%). Cell cycle phases and REK adsorption with the REK coating method (I). PDL 30 cells were incubated with [“Hlthymidine for 1 h, used for REK adsorption with the RBC coating method and nrocessed for autoradiography as desc;ibed in Materials and Methods. The number of adsorbed REKs was counted for labeled (closed columns) and unlabeled (open columns) cells under a microscope. Frequency is given as the percentage of labeled and unlabeled cells from each fraction over the total cells counted from all fractions (285 cells). (N):

291

Abscissa: ordinate:

formed WI38 cells. Similar to TIG-1 cells, phase III cells adsorbed REKs with both the REK and fibroblast coating methods, while non-senescent cells showed less RBC adsorption in proportion to their PDLs with the REK coating method and did not show REK adsorption with the fibroblast coating method at all. Interestingly, with either method, the amount of RBCs adsorbed to transformed cells corresponds to the amount of RBCs adsorbed to early phase III cells, and is lower than the amount adsorbed to terminal phase III cells with either method. Table 1 also gives the changes in the amount of [3H]ConA binding. Observed differences in the amount of [“H]ConA bound to cells are of little significance to explain the changes in RlX adsorption with aging and transformation, indicating dif-

dli bed obcd otlc.3 0

16

20

24

Fig. 4. Abscissu: time (hours); ordinate: (kfl) frequency (%); (right) average no. of REKs adsorbed to labeled cells. Cell cycle phases and REK adsorption (II). PDL 30 cells were labeled as described in the caption to fig. 3 and cultured for indicated intervals in culture medium at 37°C. After REK adsorption with the RBC coating method and processing for autoradiography, the number of adsorbed RESCswas counted for labeled cells. Frequency is given as the percentage of labeled cells from each fraction over the total labeled cells counted from all fractions (300 cells) at the indicated culture interval. The number of adsorbed FU3Cs (N) in each fraction is as follows; a, N<5; h, 5
ferences in the surface ConA receptor modulating systems among non-senescent, senescent and transformed cells. Effects of cell cycle phase, metabolic time and co-culturing of young cells with old or transformed cells Many of the cell surface properties, especially those involving plant lectins, are known to change with cell cycle phase [22]. In addition, an increase in Gl phase time with in vitro aging has been reported [23, 241. Thus, a question arises about the possible dependency of REK adsorption capacity on cell cycle phases. Fig. 3 shows a comparison of RBC adsorption with the RBC coating method between labeled and unlabeled PDL 30 cells after incubation with [3H]thymidine for for 1 h. Most of the mitotic cells were detached from the subExp C-r// Rc., 125 (19801

292

Aizawa, Mitsui and Kurimoto

Table 2. Cell cycle phases and RBC adsorption (ZZZ)

Control 0.5 % FCSc Hydroxyuread

FC”

RCb

0.45 0.52 0.57

2.69 2.72 2.40

D RBC adsorption with the fibroblast coating method. b RBC adsorption with the RBC coating method. c Cells (PDL 30) were cultured for 48 h in culture medium containing 0.5 % FCS before the RBC adsorption assay. d Cells were cultured for 24 h in culture medium containing 76 pg/ml of hydroxyurea before the RBC adsorption assay.

strate during the RBC adsorption procedure. Thus, M phase cells are outside of the present consideration, and the data give the difference in RBC adsorption between S and Gl+G2 cells. There was no significant difference in REK adsorption between the S and Gl +G2 cells. With the fibroblast coating method, RBC adsorption to PDL 30 cells did not occur regardless of whether the cells are labeled or unlabeled under this condition. Fig. 4 gives the change in REK adsorption capacity of labeled PDL 30 cells with culture. The cells were labeled with [3H]thymidine for 1 h, cultured for indicated intervals and then subjected to REK adsorption assay. The number of adsorbed RBCs was counted under a microscope for labeled cells. As shown in fig. 4, with the RBC coating method, the average number of adsorbed REXs as well as the frequency distribution did not change with culture to a significant degree. With the fibroblast coating method, REK adsorption to labeled PDL 30 cells did not occur, regardless of the culture interval after labelling. In addition, the RBC adsorption capacity of cells arrested at the early Gl or GO phase by being maintained in the culture medium with 0.5% fetal calf serum (FCS) for 48 h Exp CeIlRes

125(1980)

was similar to that of growing control cells (table 2). Hydroxyurea-arrested cells at the S or Gl-S boundary also showed a similar amount of RBC adsorption. These results suggest that REK adsorption is not affected by cell cycle phase to a significant degree, and that the age-related changes in RBC adsorption with both the REK and fibroblast coating methods are not a simple reflection of the increase in cells being retained at special phases of the cell cycle. The lifespan of human diploid fibroblasts has been reported to be determined by the cumulative number of cell divisions and not by time spent in culture (metabolic time) [25, 261. To test whether the changes in RBC adsorption reflect surface changes brought about by cell division, the effect of prolonged maintenance of cells in culture on RBC adsorption was examined (table 3). PDL 18 cells were maintained at 37°C for 11 and 22 weeks without cell passage and then subcultivated twice by a 1 : 4 split ratio (the cells reached PDL 22) before REK adsorp-

Table 3. Effects of time spent in culture (metabolic time) on RBC adsorption

Control cells (PDL 18+44) Maintained cells (PDL 18+22) Control cells (PDL 18+66) Maintained cells (PDL 18+22)

FC”

RCb

0.52

4.68

0.38

2.01

2.81

7.56

0.40

2.08

PDL 18 cells were maintained at 37°C in culture medium with medium change (twice a week) for 11 weeks (upper column) and 22 weeks (lower column) without cell passage. Then the cells were subcultivated twice by a 1 : 4 split ratio. Control cells were regularly subcultivated once a week by a 1 : 4 split ratio. The final PDLs reached by respective cells before RBC adsorption are indicated in parentheses. a RRC adsorption with the fibroblast coating method. b RRC adsorption with the REX coating method.

Cell surfaces and aging in vitro. ZZ Table 4. Effects of co-culturing adsorption capacity (Z)

young cells with old or transformed

293

cells on their RBC

09 (4 FC” PDL 18 cells PDL 64 cells ““Co-transformed cells RC” PDL 18 cells PDL 64 cells fi”Co-transformed cells

PDL 18 cells

PDL 64 cells

G°Co-transformed cells

0.38 2.41 1.20

0.42 2.18

0.36

1.64 6.59 5.13

1.58 6.60

1.78

1.08

5.01

Cells were inoculated on a coverslip at about lo4 cells/cm*. After 24 h, cells on a coverslip were co-cultured with cells on another coverslip for 48 h and then assayed for RBC adsorption. The amount of RBC adsorption is given as the values of the cells shown in the column (A) (which were co-cultured with the cells shown in the column (B)). a RBC adsorption with the fibroblast coating method. b RBC adsorption with the RBC coating method.

tion assay. Control cells were regularly subcultivated for 11 and 22 weeks, and reached PDL 44 and PDL 66, respectively, by this time. As shown in table 3, such maintenance of PDL 18 cells did not significantly enhance their RBC adsorption capacity. Considering some progress in cell division during the maintenance, it can be concluded

Table 5. Effects of co-culturing young cells with old or transformed cells on their RBC adsorption capacity (ZZ)

PDL PDL PDL PDL

18 cells 18 cells 64 cells 18 cells

fi”Co-transformed cells

PDL 18 cells PDL 64 cells PDL 64 cells ‘“Co-transformed cells ““Co-transformed cells

FC”

RC*

0.24 1.23 2.81 0.73

1.88 3.50 6.87 3.62

1.18

5.34

Cells on the first column were inoculated into 8 cm2 plastic dishes with an equal number (4x IO4 cells) of cells on the second column and co-cultured for 3 days. RBC adsorption to the mixed population was then assayed. a RBC adsorption with the fibroblast coating method. b RBC adsorption with the RBC coating method.

that RBC adsorption with both the RBC and fibroblast coating methods does not change by the metabolic time. Finally, to examine whether aging- and transformation-associated changes in RElC adsorption are intrinsic at the individual cell level, the effect of co-cultivating young cells with old or transformed cells was examined. In the first experiment, cells were inoculated on coverslips. After 24 h, young cells on a coverslip were co-cultured with old or transformed cells on another coverslip for 2 days. As shown in table 4, young cells remained low, and old or transformed cells high, in RBC adsorption capacity, even after such a co-cultivation. In the second experiment, young cells were co-inoculated with an equal number of old or transformed cells. After co-cultivation of three days, RE!C adsorption capacity of mixed cells was compared with their unmixed counterparts. As shown in table 5, mixed cell populations showed an amount of REIC adsorption intermediate to their unmixed counterparts. That is, young cells do not become old or transformed cells, and old or Exp Cd Rrs 125 (lY80)

294

Aizawa,

Mitsui

and Kurimoto

5 and fig. 5 strongly suggest that observed changes in RBC adsorption are intrinsic at the individual cell level.

100 I

111

11

IV

50

O-

l-1

a

b

c

a

b

Fig. 5. Abscissa:no. of (a, N-35; b, 25
I4 Ibc

c

a

b

adsorbed RBCslfibroblast c,

50~N);

ordinate:

c

(N) fre-

quency (%). RBC adsorption with the RBC coating method in the mixed population of young and old cells. Cells were inoculated as shown in table 5. On the third day after inoculation, the number of RBCs adsorbed with the REK coating method was determined on individual 200 cells of (I) PDL 17, (II) PDL 67 and (Ill) their mixed populations. The frequency data (IV) gives the composite of the data (I) and (II) in the 4.75 : 1 ratio. The ratio of cell number at the third day after inoculation was 4.75 : 1 between control PDL 17 and PDL 67 populations.

transformed cells do not become young cells in RBC adsorption capacity with either method by such co-cultivation. In the third experiment, frequency data are compared among young, old and mixed populations (fig. 5). Cells were inoculated as noted in table 5. On the third day after inoculation, the number of RBCs adsorbed with the RBC coating method was determined on individual 200 cells of each population. Fig. 5-I gives the frequency data of young cells, fig. 5-11 that of old cells, and fig. S-111 that of the mixed cells. By assuming that the ratio of young cells to old cells in the mixed culture on the third day after inoculation roughly coincides with the ratio of the cell number of control young cells to that of control old cells on the third day, the ratio could be obtained to be 4.75. Fig. 5-IV is the composite of fig. 5-I and 5-11 in this ratio, to which the frequency data of the mixed cells presented in fig. 5-111 coincides well. All these data presented in tables 4, E-Y/J Cdl

Re.\ 125 (IWO)

DISCUSSIONS The proper choice of the unit to express amounts of RBC adsorption is a problem in representing the net changes with aging, since a remarkable increase in cell size and protein content occurs at late passage [21, 271. Though the amount of [“H]ConA binding to fibroblasts per ceil increased at late passage (fig. l), the amount of [3H]ConA binding per mg of fibroblast protein did not significantly change throughout the lifespan of the fibroblasts. This indicates that there is a parallel increase in ConA receptor molecules and in the protein content of cells during aging. Although the cell surface area is one of the units which should be chosen, an estimate of ‘actual’ cell surface area is virtually impossible. In addition, our rough estimates of cell surface area with a semiautomatic image analysing system (Leitz) on enlarged photographs of the fixed and stained cells suggested that the change in cell surface area with aging is compatible with the change in protein content of tibroblasts (data not shown). Hence, RBC adsorption expressed as the amount per protein is considered to be most appropriate for expressing the net changes with aging. Although tissue and species dependency remain to be further investigated, and although female cell strains have a problem of being mosaic with respect to various Xlinked genes, our present results indicate that two types of age-related cell surface changes occur in human diploid fibroblasts from the lung of a female fetus. Changes in the various properties of diploid cells previously reported in association with aging can also be categorized into two types.

Cell surfaces

In one type, the change in cell properties is associated with the cell’s entering phase III, and this coincides with a change in the growth rate. Most of the cell properties reported have been of this type. The change in RBC adsorption with the fibroblast coating method belongs to this type and is obviously a phenomenon that occurs after the decline of proliferative capacity. In the second type, cell properties change continuously from the early phases of cell passage up through cell senescence. For the elucidation of the mechanisms of in vitro cell aging, this type of cellular change is very interesting. Only a few types of cellular alterations have been reported to follow this pattern. These include the activity of neutral protease [28], the electrophoretic mobility of cells [28], saturation density [29] and the colony formation capacity [30]. However, these properties have the disadvantage that their examination is either troublesome and time-consuming, or that they cannot be carried out on individual cells. To our knowledge, the ConA-mediated RBC adsorption capacity, using the RBC coating method reported here, is the only property of this type which can be easily examined on individual, monolayered cells in relation to other cell properties such as the [“Hlthymidine incorporating capacity. At first sight, it is curious that transformed cells and senescent cells show a close similarity in their cell surface properties, since they are opposite in their dividing capacity. However, it is questionable whether changes in cell surface properties are intrinsic to transformation [31-331. Although a high lectin-mediated agglutinability is known to be a characteristic of transformed cells, not all transformed cells necessarily show high agglutinability. This is also likely to apply to REK adsorption capacity. 3T3 cells, which are often called nor-

and aging in vitro. II

295

ma1 cells, but are actually an aneuploid, permanent cell line, and hence a transformant of normal diploid cells with a limited lifespan, do not adsorb RBCs with the fibroblast coating method and adsorb RBCs only to a limited degree with the REK coating method. SV40- and Py-transformed 3T3 cells have been reported to show a high RBC adsorption capacity with both methods [16, 341, but our line of SV40-3T3 did not (unpublished data). High lectin-mediated agglutinability and REK adsorption capacity are considered to be the phenotype which is frequently, but not always, expressed upon neoplastic transformation. Considering reports that ““Co- and SV40transformed cells originate from phase III populations [ 18, 351, possibly from early phase III cells, it is interesting that the RBC adsorption capacity of both fioC~- and SV40transformed cells resembles that of early phase III cells. The age-related changes in REK adsorption are not a reflection of the increase in cells being retained at a special phase of the cell cycle or of the advance of metabolic time. Co-culturing of young cells with old or transformed cells did not affect the RBC adsorption capacity of respective cells. Thus, REK adsorption with the REK and fibroblast coating methods may represent two types of cell surface markers, one revealing division age and the other cell senescence of aging human diploid fibroblasts, respectively. The greatest advantage of this assay is that examinations can be carried out easily on individual cells in relation to other properties of cells such as cell size and [3H]thymidine incorporating capacity. This is important in clarifying the possible relationship between cell surface changes and proliferative decline of cells in culture.

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We would like to thank Dr M. Namba (Kawasaki Medical University), Dr E. L. Schneider (National Institute on Aging) and Dr W. Nichols (Institute for Medical Research) for providing us with cells.

REFERENCES 1. Hayflick, L & Moorhead, P S, Exp cell res 25 (1961) 585. 2. Hayflick, L, Exp cell res 37 (1965) 614. 3. Cristofalo, V J, Adv geronto14 (1972) 45. 4. Martin, G M, Sprague, C A & Epstein, C J, Lab invest 23 (1970) 86. 5. Schneider, E L & Mitsui, Y, Proc natl acad sci US 73 (1976) 3584. 6. Holliday, R & Tarrant, G M, Nature 238 (1972) 26. 7. Macieira-Coelho, A, Diatloff, C & Malasie, E, Gerontology 23 (1977) 290. 8. Norwood, T H, Pendergrass, W R, Sprague, C A & Martin, G M, Proc natl acad sci US 71 (1974) 223. 9. Bowman, P D & Daniel, C W, Mech ageing dev 4 (1975) 147. 10. Kelley, R 0 & Shipper, B E, J ultrastruct res 59 (1977) 113. 11. Kelley, R 0, Mech ageing dev 5 (1976) 339. 12. Sasportes, M, Dehay, C & Fellous, M, Nature 233 (1971) 332. 13. I&au&-, C, Rayne, R & Hayflick, L, Exp cell res 75 (1972) 31. 14. Goldstein, S & Singal, D P, Exp cell res 75 (1972) 278. 15. Yamamoto, K, Yamamoto, M & Ooka, H, Exp cell res 108 (1977) 87. 16. Furmanski, P, Phillips, P G & Lubin, M, Proc sot exp biol med 140 (1972) 216. 17. Namba, M, Nishitani, K & Kimoto, T, Japan j exp med 48 (1978) 303.

Exp CeIIRes

125 (1980)

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