Experimental Cell Research 101 (1976) 154-158
THE CELL CYCLE OF LOW PASSAGE AND HIGH PASSAGE HUMAN DIPLOID FIBROBLASTS L. N. KAPP’ and R. R. KLEVECZ Department
City of Hope National
CA 91010, USA
SUMMARY Using mitotic selection, synchronous populations of passage 25 and passage 50 WI38 cells were obtained. Thymidine incorporation, mitotic index, and labeling index were determined. No great differences could be seen in the organization of the cell cycle of low passage and high passage cells. Although labeling indices for passage 25 and passage 50 exponential cells were 83 % and 37 %, respectively, 75% of all mitotically selected cells were found to enter S phase, regardless of passage number. Thus, the loss of late passage diploid cell cultures does not occur from alteration of the cell cycle in the majority of rapidly proliferating cells.
Human diploid WI38 cells are considered to be a model for in vitro aging [ 11.Cultures of WI38 grow well to approximately passage 50, at which time the cells fail to grow and the culture deteriorates [2, 31. Many biochemical parameters have been investigated as a function of passage number, e.g., nucleic acid metabolism, respiration, lipid metabolism, protein and amino acid metabolism, and assays of various enzymes. However, there has been little correlation of most of these parameters with culture age PI. One obvious parameter which does vary with passage number is the percentage of cells which are capable of incorporating [3H]TdR. This has been shown to decline steadily with passage . Macieira-Coelho et al. [S] consider three reasons for decline of proliferative capacity ’ Present address: Laboratory of Radiobiology, University of California, San Francisco, CA 94143, USA. Exptl CellRes 101 (1976)
in phase III. (a) Only a small fraction of the cells retain an unaltered capacity to divide. (b) All cells have an increased interdivision time and by implication a specific site(s) where arrest is occurring. (c) The cell population has become heterogeneous and shows a spectrum between the two extremes. Making any of these assumptions, it is possible to generate equations and models which can describe the progressive loss of proliferative capacity in WI38 cell cultures [6, 7, 81. All the experiments described above have been performed on random exponential populations which were mixed with regard to proliferative capacity. A random exponential WI38 culture will be composed of (a) dividing cells with further proliferative capacity; (b) dividing cells without further proliferative capacity (terminally dividing cells); (c) non-dividing cells with further proliferative capacity (cells in confluent arrest) and; (d) non-dividing cells without fur-
Low passage and high passage cell cycle
Fig. 1. Abscissa: time after mitotic selection (hours); ordinate: r3H]TdR incorporation (% of maximum). [H3]TdR incorporation in synchronous cells. Cells were pulse labeled with rH]TdR (5 pCi/ml, 50 Ci/ mmole) for 60 min, and prepared for counting. Passage 25 graph is the average of three independent experiments and passage 50 graph is the average of two independent experiments.
ther capacity for division (senescent cells). It is unclear whether the decline in proliferative capacity is intrinsic to the individual cells or a consequence of interaction between cells and their immediate environment. An unambiguous resolution of the question would be facilitated by a pure dividing population. Although much cell cycle work has been done with established cell lines, there has been little work done on the cell cycle of untransformed diploid cultures. Using an automatic synchrony apparatus, passage 25 WI38’s were synchronized and their cell cycle described . Using the same approach, it is also possible to synchronize late passage WI38 and to examine some parameters of the passage 50 cell cycle.
roller speed was increased to 200 rpm for 5 min. The detached cells were then pumped into prescription bottles and new medium was pumped into the roller bottles. The process was repeated hourly until 20-24 samples had been collected. Mitotic indices were counted from cells which had been incubated with colcemid (0.06 pg/ml) for 2 h, trypsinized, fixed in aceto-orcein, spread on slides, and squashed. The per cent of labeled nuclei was measured by incubating cells with tritiated thymidine (6 Cilmmole, 1 &i/ml). Cells were then washed, trypsinized, and fixed for autoradiogranhv f91. After 2, 4 and 7 weeks’ exposure, slides were developed and scored for per cent labeled nuclei. DNA replication rate was measured by [3H]TdR incorporation. Cells were labeled in prescription bottles with [3H]TdR (50 Ci/mmole, 5 or 10 @i/ml) for 60 min. Cells were washed two times with Hanks’ balanced salt solution and trypsinized. Acid precipitable incorporation in early passage cells was determined using the extraction procedure of Steele et al. applied directly to synchronous cultures growing in scintillation vials as described previously . This method was compared with a second procedure in which cells were- sonicated, precipitated with 10% TCA, and collected on Whatman GF/C filters and was found to give identical results. The filters were then washed with 5% TCA. 1: 1 ethanol/ether, and then counted in Aquasol . Late passage cultures were analysed using only the second procedure.
Cells from 12 late passage confluent Falcon 75 cm2 flasks (6x 10’ cells) were plated into four 750 cm2 roller bottles. The rollers were then allowed to incubate until the cell layers were in middle to late log phase (approx. 108 cells/roller bottle) and were then used for synchrony. If the rollers were used too early or too late, the sparse or confluent monolayers gave smaller mitotic yields. For passage 25 cells, 4x lOa cells (4 roller bottles) gave a final yield of 250000 to 500 000 plated cells/sample. Under similar conditions 4~ lo8 passage 50 cells gave an average yield of only 25000 plated cells/ MATERIALS AND METHODS sample. This smaller sample greatly limited WI38 cells were obtained from Dr Leonard Haytlick the number of observations which could be (Stanford University) at the 12th passage and-were subcultured 1: 2 every 3 or 4 days in McCoy’s 5a made on the older cultures. medium supplemented-with 16% fetal calf serum. Synr3H]TdR incorporation was measured by chronous populations were obtained as described previously [9, lo]. Cells were grown in roller bottles pulse labeling for 60 min at hourly intervals and maintained at 0.5 rpm. To select mitotic cells, the throughout the cell cycle (fig. 1). The upper ExpCellRes
Kapp and Klevecz
Fig. 2. Abscissa: time after mitotic selection (hours); or&are: mitotic index. Mitotic index for synchronous populations. Cells were incubated with colcemid (0.06 pg/ml) for each 2 h period indicated by the bars in the graph. Cells were then trypsinized, fixed, spread on slides, and mitotic cells counted.
panel shows the results from passage 25 WI38 while the lower panel shows the pulse-labeling pattern from a passage 50 culture. Overall, the patterns appear very similar from the two different passages. In both figures, the maximum rate of DNA synthesis occurs at 12 h during the second peak, and at 17 h there is an additional smaller third peak. The first peak appears similar for passage 25 and 50, although it occurs at 8 h for passage 25 compared with 6 h for passage 50. This difference may be due to normal variation between synchronous cultures rather than an effect of culture age, since passage 25 cultures often display a first thymidine incorporation peak as early as 5 h and as late as 7 or 8 h into the cell cycle . In addition, in [H3]TdR incorporation patterns the peaks are somewhat sharper in passage 25 than in passage 50. From these results the early portion of S phase appears very similar in both passages with the possibility that some cells in late passage cultures require a longer time to complete S. To determine the generation time, cells were incubated in colcemid for 2 h, trypsinized, and mitotic indices determined (fig. 2). The upper panel shows the results Exp Cdl Res 101 (1976)
for passage 25 while the lower panel shows the results for passage 50. The passage 25 results gave a modal generation time of approx. 19-20 h, while the passage 50 figures gave a modal generation time of approx. 18 h. This difference is relatively small and again could be expected from normal differences between replicate cultures. The center of mass of the mitotic indices were 16.9 h and 16.8 h, for passage 25 and 50, respectively. Although there may be differences in generation times between high and low passage cells, fig. 2 does not support the idea of a gradually increasing generation time or cell cycle of the synchronous culture as a whole, since the passage 50 mitotic index peak is almost as sharp as the passage 25 peak, shows no skewing towards longer generation times, and shows a mode within 1 h of passage 25 cells. Fig. 3 shows the results of continuously exposing. cells to [3H]TdR from time of selection to various points in the cell cycle. At these times cells were trypsinized, slides prepared for autoradiography, and per cent
Fig. 3. Abscissa: time after mitotic selection (hours); ordinate: % labeled nuclei. Per cent labeled nuclei after continuous exposure to [JHJTdR. Mitotically selected cells were exposed to label immediately after selection and left in the presence of label until the indicated times. The cells were then trypsinized, fixed for autoradiography, and the per cent of tabekd nuclei determmed.
Table 1. Percentage of labeled nuclei
Passage 25 Passage 50
Log phase cells
Synchronously selected cells or log phase cells were placed on slides and labeled with [3H]TdR (6 Ci/ mmole, 1 &i/ml). Log phase cells were labeled for 24 h while synchronized cells were labeled for times from 1 h to 20 h (up to one generation time). The value used for synchronous cells was the average plateau value which represents the percentage of mitotically selected cells which enters S phase within one generation time of selection (see fig. 2). After labeling, the cells were fixed for autoradiography and the per cent of labeled nuclei determined.
labeled nuclei scored. Fig. 3 shows the results from passage 25 and passage 50. In both cases, the per cent of labeled nuclei leveled off at approx. 75% indicating that roughly 75% of mitotically selected cells entered S phase, regardless of passage at selection. Since in other laboratories the per cent of cells labeled after exposure of exponential cultures to [3H]TdR declines as a function of passage, exponential passage 25 and 50 cells were exposed to r3H]TdR and the per cent labeled nuclei determined (table 1). Passage 25 gave a value of 83% and passage 50 gave a value of 37.5%. This compares with values of 82-83 % and 40-50% reported by Cristofalo & Sharf . Since synchronous passage 50 cells gave a value of 75% (table l), the relationship between per cent labeled nuclei in late passage synchronous and exponential cells was examined. Synchronous passage 50 cells were placed in a Falcon flask and allowed to grow to near confluent densities. The cells were then stimulated with fresh medium and fetal calf serum and exposed to [3H]TdR for 24 h, after which they were fixed for autoradiography and the per cent labeled
and high passage cell cycle
nuclei determined. Under these conditions, the per cent of labeled nuclei was 18%, about one-third that of passage 25 [ 111. DISCUSSION In comparing the cell cycle of passage 50 WI38 with that of passage 25 WI38, it was found that: (1) Generation times of the majority of the selected mitotic cells are the same, independent of passage number. (2) Although the structure of S phase and time of S phase was not detectably different, the possibility exists that some fraction of the population requires longer to complete S. The temporal structure of S appears somewhat different in early and late passage but the passage 50 S phase is still distinct from the unimodal pattern observed in established heteroploid lines. (3) The same proportion of mitotically selected synchronous cells (75%) enters S phase during the first cell cycle following selection. (4) In exponentially growing populations the per cent labeled nuclei was 83 % for passage 25 and 37.5% for passage 50. Hence, the heterogeneity in apparent cycle times may be due not to an increased Gl but to an altered ability to make the transition from the arrested to the proliferative state. Absher et al. [ 121have performed lineage studies using time lapse cinematography and have provided data which indicate that the mean generation time in WI38 increases linearly as the clone grows in size and that the variance in generation times is greater in passage 53 WI38 than it is in passage 20-30. They conclude that mean generation time and variance in generation time are both increased in late passage cells. While the increased variance in generation time may be an age-associated effect, the linear increase is most certainly due to parochial density effects since extrapolation of the increase Exp Cell RPS 101 (1976)
Kapp and Klevecz
from passage 33 cells onward would give a non-dividing culture in too short a time. Macieira-Coelho et al. [S] have shown an increased heterogeneity in the length of the cell cycle in late passage cells, which appeared to be due to increased lengths of G 1 and G2, or transient exits from the cycle in Gl and G2. We have repeated the results of Macieira-Coelho et al. showing an increase in apparent length of G2, or transient exit from G2, in approx. 15% of late passage cells (R. R. Klevecz, unpublished observation). The fact that a homogeneous generation time was obtained in synchronous cultures may seem at odds with work published by Macieira-Coelho et al.  and Absher et al. [ 121until one considers that all previous experiments on WI38 generation times were done in random cultures. When the cells were examined it was against a background of non-dividing and possibly senescent cells. Moreover, our results tend to emphasize the behavior of the majority of the selected population and would in all likelihood not reveal a difference in a minor fraction. It appears that the cell cycle of the majority of mitotically selected cells is essentially unchanged with increasing passage in W138,
Exp Cdl Rrs IOI (1976)
at least for the first cell cycle after mitotic selection, regardless of passage. Although there may be small differences in generation times or cell cycle substages, these cannot account for the differences in behavior between passage 25 and passage 50 log phase cells, and thus the loss of late passage cultures does not occur from alteration of the cell cycle in the actively proliferating cells. This work was supported by grant HD-04699 from the National Institutes of Child Health and Human Development.
REFERENCES 1. Cristofalo, V J, Adv geront res 4 (1972) 45. 2. Hayflick, L, Exp cell res 37 (1%5) 614. 3. Hayflick, L & Moorhead, P S, Exp cell res 25 (l%l) 585. 4. Cristofalo, V J & Sharf, B, Exp cell res 76 (1973) 419. 5. Macieira-Coelho, A, PontCn, J & Philipsson, L, Exp cell res 42 ( 1966)673. 6. Good, P I & Smith, J R, Biophys j 14 (1974) 8 I 1. I. Shakney, S E, J theor biol38 (1973) 305. 8. Smith, J A & Martin, L, Proc natl acad sci US 70 (1973) 1263. 9. Klevecz, R R & Kapp, L N, J cell biol 58 (1973) 564. 10. Klevecz, R R, Anal biochem 49 (1972) 407. II. Augenlicht, L H & Baserga, R, Exp cell res 89 (1964) 255. 12. Absher, P M, Absher, R G & Barnes, W D, Exp cell res 88 (1974) 95. Received December 2, 1975 Accepted March 16, 1976