Experimental Cell Research 100 (1976) 147-152

INCREASED

NUCLEAR DIPLOID

SIZES IN SENESCENT

FIBROBLAST

HUMAN

CULTURES

Y. MITSUI’ and E. L. SCHNEIDER Laboratory of Cellular and Comparative Physiology. Gerontology Research Center, National Institute on Aging, National Institutes of Health. USPHS. Department of Health, Education and Welfare, Bethesda and Baltimore City Hospitals, Baltimore, MD 21224, USA

SUMMARY Measurement of nuclear size in cultured human diploid fibroblasts (WI38) reveals a shift to larger cell nuclei as a function of in vitro passage. Examination of nuclear size distribution on the basis of replicative potential reveals that at all levels of in vitro passage, the rapidly replicating cell population have smaller nuclear sizes than comparable populations of slow or non-replicating cells. While the nuclear size distribution of the rabidly replicating cell population remains relatively constant, there is a marked shift in the nuclear size distribution of the slow or non-replicating cell population to larger sizes with increasing in vitro passage.

The limited in vitro lifespan of cultured technical difficulty of measuring both parahuman diploid fibroblasts is a well-docu- meters in the same cell population, since mented biological phenomenon [l-3]. Many fibroblast volume is best measured in cell investigators have utilized these early and suspensions [ 121 and replicative potential late passage cultured human fetal lung by autoradiographic analysis of fixed cell fibroblasts (WI38 cells) as a model system monolayers [ 111. In this report, the problem is approached for studying cellular aging in vitro [3-51. One of the earliest observed changes in by measuring nuclear size which has been these cell cultures as they progress through reported to be closely related to overall their in vitro lifespan is the appearance of cell size in mammalian cells [13-151. Since enlarged cells [6-81. It has been suggested replicative ability and nuclear size can be that these enlarged cells are non-repli- measured concomitantly on WI38 cells at cating cells [9, IO] and that the shift in cell different levels of in vitro passage, the relationship between these parameters can be volume distribution to larger sizes is due to the accumulation of non-replicating cells directly examined. with in vitro passage [1 11. Unfortunately, direct proof that the enlarged cells have altered replicative abilities is limited by the MATERIALS AND METHODS ’ Present address: Department of Pharmacology. Tokyo Metropolitan Institute of Gerontology. Tokyo. Japan.

Cell culture Human fetal lung fibroblasts (WI38 cells) obtained from Professor L. Hayflick at early, middle and late in

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vitro passage were cultured in Eagle’s minimal essential media (MEM), supplemented with nonessential amino acids, glutamine, 10% fetal calf serum and 50 pg/ml Aureomycin (Lederle). Mycoplasma screening was performed by previously described techniques [ 161.

Autoradiographic labeled nuclei

analysis of [3H]TdR

WI38 cells at 19, 35 and 45 cell uouulation doublinas (CPD) were inoculated at a concentration of 5~ l@ cells/cm2 into 35 mm plastic Petri dishes (Falcon) containing coverslips. Two days later, after cell attachment had occurred and the cell cultures were in logarithmic growth, [3H]TdR (1.9 CilmM, Schwartz) was added to a final concentration of 0.05 cLCi/ml. After 24, 48 and 72 h incubation with [3H]Tdk, the coverslips were removed from their Petri dishes, rinsed twice with Ca*+ and Mg *+ free phosphate buffered saline (CMFPBS), washed with 4% perchloric acid and fixed with methanol : acetic acid (3 : I). The coverslips were then mounted on slides, dipped in NTB-2 emulsion (Kodak), exposed for 7 days at 4”C, developed with D-19 developer (Kodak) and made permanent with Rapid Fixer (Kodak). Lastly, the slides were stained with Giemsa (Harleco) in phosphate buffer, pH 7.0. The slides at each level of cell passage and at each of the three time periods were analysed for the frequency of labeled and unlabeled nuclei. Labeled nuclei were defined as nuclei covered with more grains than an equivalent background region (usually .>5 grains/ nucleus).

Nuclear size measurement Nuclear size measurements were made on the premise that nuclear shape closely approximates a geometric ellipse. The larger and smaller diameters (d,, dJ of the cell nuclei were measured with a micrometric ocular attachment to a Zeiss photomicroscope and the nuclear area (n.a.) calculated by the equation:

Table 1. Cell population doubling time and per cent labeled nuclei of WI38 cells at early, middle and late passage In vitro passage level (cell population doublings)

Cell population doubling time (hours)

% labeled nuclei (after 24 h incubation with [3H]TdR)

19 35 45

15.2 19.9 26.0

97 80 62

Isoton (Coulter) solution and cell number and volume distribution measured in a Coulter model ZBI counter and channelizer. Cell replication rate was determined during the period of logarithmic growth, usually the first 3 days after inoculation.

Determination of nuclear size, cell volume and per cent rapidly replicating cells in fractionated cell populations Fractionation of late passage WI38 cells (47 CPD) by gravity sedimentation in fetal calf serum (FCS) gradients is described in detail in another renort (in preparation). In brief, after a 48-h incubation with r3H]TdR 1.3x IO6 cells were harvested from their monolayers, suspended in 2 ml of 0.1% methylcellulose in Ca2+ and Mg2+ free Eagle’s MEM and layered onto a 5-30% FCS gradient. After 90 min gravity sedimentation, 5 ml fractions were collected. Aliquots of cells from each fraction were taken for cell volume determinations and the remaining cells inoculated onto coverslips. After 16 h for cell attachment, the coverslips were treated as described above for measurement of nuclear size and for the presence or absence of r3HjTdR nuclear labeling.

rdd,d, n.a.= - 4 Measurement of unlabeled and labeled nuclei were alternated in random microscopic fields.

Measurement of cell population rate and cell volume distribution

replication

The techniques employed for measurement of cell volumes and cell population replication time are described in a previous report [8]. In brief, WI38 cells at 19, 35 and 45 CPD are inoculated at 5x IO3 cells/cm* into replicate 35 mm Petri dishes. On successive days, cell monolayers were washed with CMFPBS and cells detached with 0.05 % pronase. After additional washing to remove pronase, the cells were suspended in Exp Cc// Res 100 (IY76)

RESULTS Standardization of in vitro passage level for nuclear size determination

Since in vitro passage level has been defined in terms of cumulative cell population doublings (CPD) by some investigators [3] and as per cent rapidly replicating cells by others [4], nuclear labeling indices were determined at the three different in vitro passage levels chosen for the nuclear sizing measurements (table 1). At early in vitro

Nuclear size in senescent human fibroblasts

1

19

Increased nuclear sizes with increased in vitro passage

cm

45 CPD

loo

i-m 100

200

300

400

149

500

1 600

Fig. 1. Abscissa: nuclear size (pm2); ordinate: % of total cell no. Nuclear size distributions of WI38 cell cultures at early (19 CPD); middle (35 CPD); and late (45 CPD) in vitro passage. One hundred cell nuclei were measured at 19 and 35 CPD and 200 at 45 CPD.

Nuclear size distributions of WI38 cell cultures at early, middle and late in vitro passage are seen in fig. 1. For comparative purposes, cell volume distributions at the same levels of in vitro passage are seen in fig. 2. In both figures, a slight shift to larger cell and nuclear sizes is observed in middle passage (35 CPD) when compared to early passage cell cultures (19 CPD). However, the most striking differences are observed when late passage or senescent cell cultures (45 CPD) are compared with cultures at early or middle passage. In addition to a shift to larger cell and nuclear sizes, broader distributions are also seen. In the case of cell nuclei distribution, there is also a suggestion of bimodality in the cell nuclei distribution at 45 CPD.

20

passage (19 CPD), over 97 % of the cell population have labeled nuclei after a 24-h incubation time with r3H]TdR. By late in vitro passage, only 62% of the cells have labeled nuclei after this short incubation period. However, after 72 h exposure to [3H]TdR, this figure increases to 85 %. The unlabeled cells may be arrested non-replicating cells or merely cells with replication times longer than 24 or 72 h. Therefore, in this report these cells will be called “slow or non-replicating cells”. Measurements of nuclear size and replicative capacity were performed on cell populations after a 24 h incubation time with r3H]TdR since this time period revealed the greatest difference in per cent labeled nuclei between passage levels. Further standardization of in vitro passage level included measurement of cell population doubling times (table 1).

1SCPD

I lo t 2E 35CPD

lo o1 20 45CPD

,nI ‘” o 20’oo Fig. 2. Abscissa:

totalcell no.

cell volume (pm3); ordinate:

% of

Cell volume distributions of WI38 cell cultures at early (19 CPD); middle (35 CPD); and late (45 CPD) in vitro passage. E.rp Cell

Rrs

/OO (1976)

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CPD. However, by 45 CPD, a large increase in nuclear sizes in the slow or nonreplicating cell populations is observed. In addition to the increase in nuclear sizes, there also appears to be an increased variation in nuclear sizes present in senescent or late passage WI38 cell cultures.

30 20

10 0

C

Nuclear sizes, cell volumes and cell replicative capacities offractionated passage WI38 cell populations

late

Fractionation of late passage cells by velocity sedimentation yielded fractions with large differences in mean cell volumes (table 2). In all fractions, nuclear sizes correlated very closely with cell volumes. The 3 200 300 400 5600 Fig. 3. Abscissa: nuclear size (pm*); ordinate: % of fraction that possessed the largest cell total cell no. volumes and nuclear sizes had the highest Nuclear size distribution of the rapidly replicating cell population at (A) early (19 CPD); (B) middle (35 proportion of slow or non-replicating cells CPD); and (C) late (45 CPD) in vitro passage. One (36.3 %) while the fraction with the smallest hundred labeled nuclei (after a 24 h incubation with [3H]TdR) were measured for nuclear size at each level cell and nuclear sizes was almost entirely

;:I

of in vitro passage. Note that the distribution remains relatively constant in contrast to fig. 4.

A

Relationship between nuclear size and cell replicative capacity

Examination of nuclear size distributions on the basis of replicative potential reveals that at all levels of in vitro passage, the rapidly replicating cell populations (cells with labeled nuclei after a 24 h r3H]TdR pulse, fig. 3) have smaller nuclear sizes than comparable populations of slow or nonreplicating cells (fig. 4). The nuclear size distribution of the rapidly replicating cell populations remains relatively unchanged at all stages of the in vitro lifespan of WI38 cells with a modal nuclear size of approx. 125 pm2. In contrast to the stability of nuclear size in the rapidly replicating cell population, there is a definite shift in the nuclear size distribution of the slow or nonreplicating cell population to larger sizes. This shift is only minimal between 19 and 35 E.rp Cd/

Res

100 (1976)

B

1 ItIll,..,

(

0 100

200

Fig. 4. Abscissa:

300

400

500

T 600

nuclear size (pm*); ordinate: % of total cell no. Nuclear size distribution of the slow or nonreplicating cell population at (A) early (I9 CPD); (B) middle (35 CPD); and (C) late (45 CPD) in vitro passaee. One hundred unlabeled nuclei (after a 24 h incubation with [3H]TdR) were measured for nuclear size. Note the shift to larger nuclear sizes with increased in vitro passage.

Nuclear size in senescent human .fibroblasts

Table 2. Cell volume, nuclear size and per cent slow or non-replicating cells in fractionated late passage WI38 cell populations Fraction

Cell volume” km9

Nuclear size* km*)

% unlabeled nuclei’

I 3 5 7

7 5 3 2

294 234 159 119

36.3 11.4 2.2 1.3

080 863 580 550

a Mean of between 20 000 and 40 000 cells sampled by Coulter analysis. * Mean of 100 nuclei measured by micrometer attachment to Zeiss microscope. r After 48 h incubation period with [3H]TdR per cent cell nuclei with sbackground grain counts.

comprised of rapidly replicating cells. The mean nuclear size of this last fraction closely approximates the peak value for nuclear size of unfractionated rapidly replicating cells.

DISCUSSION The increase in nuclear sizes observed in WI38 cells as a function of in vitro passage closely parallels the shift to larger cell volume distributions. This confirms the correlation between nuclear and cell sizes which has been reported by investigators in other cell systems [ 13-151. Nuclear and cell sizes have been shown to be directly related to ploidy in mammalian cells with polyploid cells having proportional increases in these parameters [14, 151.Although increased polyploidy has been reported in senescent WI38 cells [17, 181,it was not of sufficient magnitude to account for the shift in cell and nuclear sizes observed in these cell cultures. In addition, if the increased cell and nuclear sizes were due solely to polyploidy, one might expect peaks at intervals proportional to 2C, 4C and 8C DNA contents rather than the

151

gradual shift to larger sizes that is observed. By identifying rapidly and slowly or nonreplicating cells, we were able to demonstrate that the increase in nuclear sizes with in vitro passage was the result of an accumulation of slow or non-replicating cells with enlarged nuclei. Since cell volume and nuclear size appear to be closely related in WI38 cells, one could also attribute the observed shift to larger cell volumes with increased in vitro passage to the evolution of a population of enlarged slow or nonreplicating cells. This relationship between increased cell and nuclear sizes and decreased replicative ability is further strengthened by the results of cell fractionation which revealed shifts to larger nuclear and cell sizes in fractions that contained increased numbers of slow or nonreplicating cells. Despite the shift of slow or non-replicating cell nuclear size distributions to larger sizes as a function of in vitro passage, the nuclear sizes of the rapidly dividing cell population remained unchanged. This finding of relatively constant nuclear size is similar to the observation of Bowman et al. [lo] that the cell volume distribution of isolated mitotic cell populations remained constant throughout the in vitro lifespan of WI38 cells. Since the proportion of slow or non-replicating cells increases as a function of in vitro passage, it is tempting to attribute in vitro aging simply to the accumulation of these cells and an elimination of a “normal” or “young” rapidly dividing cell population. However, Smith & Hayflick [19] have demonstrated by cloning middle and late passage cells that no cells were present with the in vitro lifespan potential seen in cloned early passage cells. Small cell populations isolated from late passage cultures by cell fractionation have also dem-

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onstrated a reduced cell population replication rate when compared with comparably sized early passage cells (in preparation). Therefore, although morphologically similar at all levels of in vitro passage, the small rapidly replicating cell population at late passage appears to be functionally different from comparably sized populations at early passages. We thank Kin-Sing Au and Karen Braunschweiger for excellent technical assistance. WI38 cell packs/starter cultures used in these studies were obtained through Contract NO]-HD-4-2828 from the National Institute of Child Health and Human Development to Dr Leonard Hayflick of the Stanford University School of Medicine.

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(1969) 313. 8. Schneider, E L & Mitsui, Y, Proc 10th int con-

gress aeront. vol. 1. D. 69 (1975). 9. Martin, G M, Sprague, C A, ‘Norwood, T H & Penderarass, W R. Am i nathol74 (1974) 137. 10. Bowman, P D, Meek, RL & Daniel, C W, Exp cell

res 93 (1975) 184. 11. Cristofalo, V J & Sharf, B B, Exp cell res 76 (1973) 419. 12. Ben-Sasson, S, Patinkin, D, Grover, N B & Dol-

janski, F, J cell physiol205 (1974). 13. Schindler, P D, Acta anat 44 (1961) 273. 14. Epstein, C J & Gatens, E A, Nature 214 (1967) 1050.

15. Epstein, C J, Proc natl acad sci US 57 (1967) 327. 16. Schneider, E L, Stanbridge, E J & Epstein, C J, Exp cell res 84 (1974) 3 11. 17. Saksela, E & Moorhead, P S, Proc natl acad sci US 50 (1963) 390. 18. Yanishevsky, R, Mendelsohn, M L, Mayall, B H & Cristofalo, V J, J cell physio184 (1974) 165. 19. Smith, J R & Hayflick, L, J cell bio162 (1974) 48. Received October 13, 1975 Accepted December 16, 1975