Nucleolar changes in senescing WI-38 cells

Mechanisms of Ageing and Development, 8 (1976) 417-427

417

© Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

N U C L E O L A R C H A N G E S IN S E N E S C I N G WI-38 C E L L S

PARASKEVI M. BEMILLER and LIEH-HAUH LEE School of Medicine, Southern Illinois University at Carbondale, Carbondale, Illinois 62901 (U.S.A.)

(Received January 4, 1978; in revised form June 13, 1978)

SUMMARY Changes in the area, dry mass and morphology of nucleoli were studied during in vitro aging of WI-38 cells. Interferometric methods were used for nucleolar dry mass determinations. The results show that there is (1)an increase in the fraction of cells with one large nucleolus per nucleus, 17% at population doubling 27.3 vs. 93% at population doubling 41.2, (2) an increase in mean nucleolar dry mass (583% at the last doubling), and (3) an increase in mean nucleolar area (236% at the last doubling) with in vitro senescence of WI-38 fibroblast cells. A strong correlation (r = 0.92) between nucleolar dry mass and nucleolar area was demonstrated.

INTRODUCTION Normal, human, diploid fibroblast cells display a limited lifespan in vitro and are, therefore, suggested as models for aging at the cellular level [ 1,2]. Aging manifestations include changes in cell organelles. Using WI-38 cells, Lipetz and Cristofalo [3] found that old cells are characterized by an increase in lysosomes and autophagic vacuoles, by prominent and active Golgi complexes, and by highly lobed nuclei. Recent studies in this laboratory [4] and that of Schneider [5] indicate that nuclear size increases with aging in vitro and that this increase is not exhibited uniformly by all cells in the population; rather, there is a gradual increase of the subpopulation of cells with larger nuclei with successive doublings. It has long been known that the nucleolus of a cell is involved in rRNA synthesis [6], and changes in the nucleolar structures have been correlated with their ability to synthesize RNA [7, 8-10]. Cristofalo and Sharf [11] observed that aging in vitro is characterized by a loss of proliferative capacity which can be monitored by [3H]-TdR autoradiography. Old cultures contain fewer cells which retain the ability to incorporate [3H]-TdR. Ryan and Crystofalo [12] showed a decrease in the chromatin template activity of cultures which were aged, and Bowman et al. [ 13 ] recently demonstrated that, in WI-38 cells, the age-related decline in RNA synthesis was entirely due to a decreased synthesis of nucleolar RNA, while nucleoplasmic synthesis was unchanged.

418 The present study is concerned with changes in the nucleolar area, dry mass and morphology of WI-38 cells with aging in vitro.

MATERIALS AND METHODS Cell cultures WI-38 cells obtained from the American Type Culture Collection Cell Repository at about the 14th passage (21.4 doublings) were cultured in Dulbecco's modification of Eagle's medium supplemented with calf serum to a concentration of 10%. Kimble Exax No. 2 glass coverslips were placed in 60 mm Falcon plastic Petri dishes and the dishes were inoculated with 5 × l0 s cells (1.77 × 104 cells/cm 2) approximately. The cultures were maintained as monolayers at 37 °C in an atmosphere of 5% CO2-95% air and were fed on alternate days. Coverslips containing attached cells were removed during the log phase of growth. Cultures were judged to be terminal when they failed to achieve confluent monolayers after 6 weeks with every other day refeeding. Measurement o f nucleolar area A previously reported procedure [4] was followed in measuring nucleolar areas (NoA). Briefly, the coverslips were removed, rinsed in phosphate-buffered saline (pH 5.7), fixed in a 3:1 v/v alcohol-acetic acid fixative for 15 min, and allowed to air dry. The nucleoli were photographed, and the circumference of each was traced. The area within the tracing was then determined with a planimeter (Kueffel and Esser Company, type A 620015, giving a direct reading in cm 2 with an accuracy of 0.1 cm2). Magnification was determined using a stage micrometer. Actual nucleolar area was calculated by dividing the planimeter-measured area by the square of linear magnification. The nucleolar areas of 35 randomly selected cells were measured at regular passage intervals. When more than one nucleolus per nucleus was present, the individual nucleolar areas were added to give a total nucleolar area per cell. Measurement o f nucleolar dry mass The nucleolar dry mass (NoDM) was determined using a Leitz transmitted-light, interference microscope calibrated with 546 nm radiation. The following equation was used : NoDM =

OPD × NoA

where NoDM = nucleolar dry mass in g, OPD = optical path difference expressed as a fraction of X, NoA = nucleolar area in cm 2, if= refractive index of protein (0.13 cma/g). Nucle'olar dry mass measurements represent an average of determinations in 35 ceils. As in the case of nucleolar areas, when more than one nucleolus existed per nucleus, the individual nucleolar dry masses were added to give a total nucleolar dry mass per cell.

419 RESULTS During the following presentation and discussion of the results reference to "early" and "late" in vitro doublings is made in terms of the lifespan of this particular culture after purchase. The earliest doubling in this study represents actual population doubling 27.3 (15th passage). The fibroblasts stopped proliferating after doubling 41.2 (23rd passage) and the latter was therefore considered to be the terminal one. The mean N o D M of WI-38 fibroblast cells increased with successive doublings in vitro; this increase was accompanied by a parallel increase in the mean NoA. The concomitant increases can be clearly seen in Fig. 1 where the two parameters are shown to change at about the same rate from the earliest to the last doublings. Figure 1 also shows an initial small decrease in both N o D M and N o A immediately after the cells were first passed in our laboratory. Table II presents results from the t-tests of the change in nucleolar area and dry mass across doublings. Test results for the 27.3-30.2 population doubling interval showed significant decreases in both measures. Approximately 34% of the N o D M and 16% of the N o A were lost (Table I). However, the 30.2-35.5 and 35.541.2 population doubling transitions showed significant increases in N o A and N o D M (Table II). The overall increase in N o D M across all doublings was 583% (Table I); the 70

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Population Doublings

Fig. I. Changes in the nucleolar areas ( e - - e ) and nucleolar dry mass (* successive doublings in vitro.

,A-) of W1-38 cells with

1.892 1.585 1.810 3.965 3.325 6.372

0.0 -16.0 -4.0 +109.6 +75.7 +236.8

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Mean

Mean

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NoA*

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% Change

*NoA (nucleolar area, 10- 7 cm2). **NoDM (nucleolar dry mass, 10 -12 g). ***The r value for combined doublings (n = 210) was equal to 0.92.

27.3 30.2 33.5 35.5 39.0 41.2

Population doublings

NUCLEOLAR AREA AND DRY MASS CHANGES WITH IN VITRO DOUBLINGS

TABLE I

0.569 0.634 0.877 1.750 1.229 2.440

NoA

3.467 3.305 5.030 18.795 10.790 35.073

NoDM

Standard deviation

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NoDM/NoA

0.76 0.81 0.88 0.89 0.82 0.83

r value for NoA and NoDM (n = 35)***

4~ to

421 TABLE II RESULTS OF t-TESTS OF THE CHANGE IN NUCLEOLAR AREA AND DRY MASS FROM EARLY TO LATE IN VITRO DOUBLINGS Population doublings

27.3-30.2 30.2-35.5 35.5-41.2

NoA

NoDM

Difference between means

t

Pt

Difference between means

t

Pt

-0.31 2.38 2.41

2.13 7.56 8.91

0.05 0.001 3.001

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4.08 8.12 4.95

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Fig. 2. Linear regression plot of the means for nucleolar dry mass and nucleolar area (r = 0.92) of W138 cells with successive doublings in vitro.

overall increase in mean NoA was 236% o f the value at the initial in vitro doubling (Table I), indicating that, although very significant, the increase in NoA was not as great as the increase in NoDM. In fact, the NoDM/NoA ratio increased twofold from the 27th to the 41 st doublings (Table I).

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Fig. 3. S a m p l e e x p e r i m e n t i l l u s t r a t i n g t h e c h a n g e in the n u c l e o l a r area profiles o f WI-38 cells w i t h successive p o p u l a t i o n d o u b l i n g s . (a) A t 27.3 d o u b l i n g s ; (b) at 30.2 d o u b l i n g s ; (c) at 33.5 d o u b l i n g s ; (d) at 35.5 d o u b l i n g s ; (e) at 4 1 . 2 d o u b l i n g s .

A linear regression plot of the mean N o D M versus the mean N o A (Fig. 2) shows that there was a direct relationship between the changes in these two parameters with aging in vitro. At individual doublings, the r values ranged from 0.76 to 0.89 (Table I). However, when the r value for N o D M and N o A was calculated for all passages combined (n = 210), it was found to be equal to 0.92. Table I shows an increase in the standard deviation from the mean with successive doublings in vitro. This was true for both the N o D M and the NoA. This means that the populations of cells (1) became more heterogeneous with respect to mean N o D M and N o A and (2) that the subpopulation of cells with larger and heavier nucleoli increased with aging in vitro. Figures 3 and 4 further illustrate this. Microscopic observation of nucleoli as they aged in vitro revealed that there was a tendency for the number of nucleoli per nucleus to decrease with successive doublings. Table III summarizes these observations. Whereas at doubling 27.3 only 17% of the cells had a single large nucleolus per cell at doubling 41.2 this number increased to 66%. Concurrently, there was a decrease in the percentage of cells with two or more nucleoli per cell from 93 to 34. Typical WI-38 cells from early and late doublings are shown in Fig. 5(a) and (b). In young cultures (27-35 doublings), several nucleoli were seen

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Fig. 4. Sample experiments illustrating the change in the nucleolar dry mass profiles of WI-38 cells with successive population doublings. (a) At 27.3 doublings; (b) at 30.2 doublings; (c) at 33.5 doublings; (d) at 35.5 doublings; (e) at 41.2 doublings.

TABLE III DISTRIBUTION OF NUCLEOLAR COUNTS ACROSS IN VITRO DOUBLINGS Population doublings 27.3

30.5

33.5

35.5

39. 05

41.2

Cells with one nucleolus/cell (%)

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51

66

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93

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63

49

34

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scattered in each fibroblast nucleus. Their individual areas were small (Fig. 5a). As in vitro aging progressed ( 3 5 - 4 1 doublings), the prevailing m o r p h o l o g y was that o f a single large nucleolar mass in the center o f older cells (Fig. 5b). These nucleolar masses were n o t only larger b y as m u c h as 236%, b u t they were also 583% heavier at the last doubling (Table I).

424 DISCUSSION The results of this study show that nucleoli of WI-38 fibroblast cells aggregated and increased in area and dry mass during aging in vitro. The aggregation of nucleoli of these fetal lung fibroblast cells was similar to the aggregation of aging nucleolar of Paramecium aurelia [14] and Tetrahymena pyriformis [15] under different physiological conditions. Fused large nucleoli display reduced levels of incorporation of RNA precursors [16] and are, therefore, considered to b.e less active with regard to rRNA synthesis. Similarly, it is possible that the fused nucleoli of aged WI-38 cells observed here represent less active nucleoli with reference to rRNA synthesis. An age-related decline in nucleolar RNA synthesis was, in fact, recently demonstrated in WI-38 cells [13]. There was also an accumulation of nucleolar materials with aging of WI-38 cells in vitro as demonstrated here by the substantial increase in mean nucleolar dry mass with successive doublings (Table I). It was accompanied by a concomitant increase in nucleolar area. The increases in the two parameters were highly correlated as shown by a regression plot (Fig. 2).

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(b) Fig. 5. Sample photographs o f WI-38 cells at (a) early and (b) late doublings illustrating the change in nucleolar distribution with aging in vitro.

These seemingly contradictory observations, i.e., a decline in nucleolar RNA synthesis [13] and an increase in nucleolar dry mass with aging, may be explained as a cause-and-effect phenomenon. It is possible that rRNA synthesis increases initially with a;ging in vitro. If, however, there is an age-related defect in the transport process so that it is not transported, it would accumulate in the nucleolus and result in an increase in nucleolar dry mass and area. This in turn might cause fusion o f nucleoli into an aggregate incapable of further RNA synthesis. The following reports are offered in support of such a hypothesis. Schneider and Shorr [17] reported an increase in the rRNA of senescent WI-38 cells. They suggested that it may have been caused by an increased redundancy of

426 ribosomal DNA genes as had been shown to happen in certain tissues from aged animals [18]. It is possible that rRNA accumulates in the nucleoli either because o f age-related changes in nuclear membrane permeability or an age-related defect in the mechanism for rRNA transport involving carrier molecules [ 19]. The cause for nucleolar fusion is not clear. However, it has been shown to be associated with a decline in rRNA synthetic ability whereas nucleolar disaggregation was concurrent with increased rRNA synthesis in Tetrahymena [15]. Bowman et al. [13] also reported decreased synthesis o f nucleolar RNA in senescent WI-38 cells. Their results were consistent with those o f Ryan and Cristofalo [ 12]. These authors Suggested impaired accessibility of RNA polymerase to the chromatin template as an explanation of their findings. Accumulation of nucleolar material may be responsible for the impaired accessibility o f RNA polymerase to chromatin. Finally, Figs. 3 and 4 show that the populations o f cells were not synchronized with respect to nucleolar area and dry mass changes. A subpopulation of cells with aggregated, larger and heavier nucleoli increased with aging in vitro. This pattern is reminiscent o f that reported for increases in cell size [20] and nuclear size [4, 5] with aging in vitro. The initial decrease in nucleolar area and dry mass is also reminiscent of that reported for nuclear areas at the initial in vitro doublings [4] and may be due to adaptation o f cells to their new environment. Finally, even though it is reasonable to assume that the single, large, heavy nucleolar masses shown here to prevail in the older cultures o f WI-38 cells resulted from .the fusion o f several nucleoli, it is not clear how and when this occurred in relation to the mitotic cycle. These are important questions which must be investigated further.

ACKNOWLEDGEMENTS We are indebted to Song-Chiau Lee for his technical assistance and to C. C. Lindegren for the use of the Leitz Interference microscope.

REFERENCES 1 L. Hayflick and P. S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res., 25 (1961) 535-621. 2 L. HayfUck, The limited in vitro lifetime of human diploid cell strains, Exp. CellRes., 37 (1965) 614-636. 3 J. Lipetz and U. J. Cristofalo, Ultrastruetural changes accompanying the aging of human diploid cells in culture, J. Ultrastructure Res., 39 (1972) 43-56. 4 S. C. Lee, P. M. BeMiller, J. N. BeMiller and A. J. Pappelis, Nuclear area changes in senescing human diploid fibroblasts, Mech. Ageing Dev., (in press). 5 Y. Mitsui and E. L. Schneider, Increased nuclear sizes in senescent human diploid fibroblast cultures, Exp. Cell Res., 100 (1976) 147-152. 6 E. H. McConkey and J. W. Hopkins, The relationship of the nucleolus to the synthesis of ribosomal RNA in HeLa cells, Proc. Nat. Acad. Sci. U.S.A., 51 (1964) 1197. 7 J. R. Nilsson and V. Leiek, Nucleolar organization and ribosome formation in Tetrahyrnena pyriformis GL, Exp. Cell Res., 60 (1970) 361-372.

427 8 I. L. Cameron and E. E. Guile, Jr., Nucleolar and biochemical changes during unbalanced growth of Tetrahymena pyriformis, J. Cell Biol., 26 (1965) 845-855. 9 B. Satir and E. R. Dirksen, Nucleolar aging in Tetrahymena during the cultural growth cycle, J. CellBiol., 48 (1971) 143-154. 10 K. Smetna and H. Busch, The nucleolus and nucleolar DNA, in H. Busch (ed.), The CelINucleus, Academic Press, New York, 1974, pp. 75-141. 11 V. J. Cristofalo and B. B. Shaft, Cellular senescence and DNA synthesis, Exp. CellRes., 76 (1973) 419-427. 12 J. M. Ryan and V. J. Cristofalo, Chromatin template activity during aging in WI-38 cells, Exp. Cell Res., 90 (1975) 456-458. 13 P. D. Bowman, R. L. Meek and C. W. Daniel, Decreased synthesis of nucleolar RNA in aging human cells in vitro, Exp. Cell Res., 101 (1976) 434-437. 14 V. Sundararaman and D. J. Cummings, Morphological changes in aging cell lines of Paramecium aurelia. II. Macronuclear alterations, Mech. Ageing Dev., 5 (1976) 325-338. 15 I. L. Cameron and E. E. Guile, Jr., Nucleolar and biochemical changes during unbalanced growth of Tetrahymena pyriformis, J. Cell Biol., 26 (1965) 845-855. 16 M. R. Klass and J. Smith-Sonneborn, Studies on DNA content, RNA synthesis and DNA template activity in aging cells of Paramecium aurelia, Exp. Cell Res., 98 (1976) 63-72. 17 E. L. Schneider and S. S. Shorr, Alteration in cellular RNA's during the in vitro lifespan of cultured human diploid fibroblasts, Cell, 6 (1975) 179-185. 18 R. G. Cutler, Redundancy of information content in the genome of mammalian species as a protective mechanism determining aging rate, Mech. Ageing Dev., 2 (1974) 381-408. 19 T. M. Sonneborn, Tests and critique of some theories of aging, in B. L. Strehler (ed.), The Biology of Aging, Waverly Press, Baltimore, 1960, p. 281. 20 P. D. Bowman, R. L. Meek and C. W. Daniel, Aging of human fibroblasts in vitro correlation between DNA synthetic ability and cell size, Exp. Cell Res., 93 (1975) 184-190.