ASSOCIATED WITH FEEDER EFFECTS. IN CULTURES OF CHINESE HAMSTER FIBROBLASTS. S. J. GAUNT and J. H. SUBAK-SHARPE. Institute of Virology.
Prrnted in Swden Copyright @ 1977 bg Academic Press, Inc. A// rights of reproducfbn in onyfirm reserved ISSN 00144827
Cell Research 109 (1977) 341-348
Experimental
CELL
CYCLE
VARIATION
IN CULTURES
ASSOCIATED
OF CHINESE
WITH
HAMSTER
FEEDER
EFFECTS
FIBROBLASTS
S. J. GAUNT and J. H. SUBAK-SHARPE Institute
of Virology.
Glasgow Cl1 5JR, Scotland
SUMMARY Sub-confluent monolayer cultures of an established line of Chinese hamster tibroblast (Don) are shown to exhibit a density-dependent stimulation of growth. Evidence is presented that both long and short range ‘feeder effects’ are involved. Using the technique of autoradiography, cell cycle parameters have been studied in sub-confluent cultures seeded at different densities to identify the source of this density-dependent variation in growth rate. The durations of S phase, G2, and mitosis are constant as indicated by “percentage labelled mitoses” curves. A simple procedure has been developed for measurement of the fraction of a cell population in the Gl state, and this fraction is shown to be inversely related to the density of the culture. It is concluded that regulation of cell growth associated with feeder effects in cultured Don cells occurs within the Gl state. The data obtained from “percentage labelled mitoses” curves are shown to be highly consistent with the predictions of the Transition Probability model for cell cycle regulation.
The concentration of cells growing in a culture has two distinct effects upon the population doubling rate. Density-dependent stimulation of growth is observed as a ‘feeder effect’ in both secondary cultures [l, 21 and established cell lines [3]; however, beyond a critical cell concentration there is a density-dependent inhibition of growth [4]. Other indications of a feeder effect are seen in the improved cloning efficiency of single cells when co-cultured with non-growing feeder cells [5], and in the stimulatory effect of conditioned medium upon sparse cultures [6]. Several distinct types of cellular interaction appear to contribute to the feeder effect. Some are only effective over short ranges. Stoker & Sussman [7] produced evidence that a cell is stimulated in growth by close proximity (up to approx. 1.5 mm) to other live cells. Rein & Rubin [2] were
able to observe such an effect over 0.5 mm distances, and suggested that the stimulant might be a poorly diffusible material forming a micro-environment around the growing cells. The importance of contact with secreted intercellular matrix has also been demonstrated [8,9]. A type of cellular interaction likely to produce stimulation over much longer ranges has been defined by Eagle & Piez [lo]. These workers produce evidence that cellular leakage of non-essential amino acids can prevent adequate intracellular concentrations for optimal growth. The rate of leakage slows as the concentration of these metabolic intermediates rises in the medium, but the ability to condition the medium in this way, and improve conditions for growth, is densitydependent. In this communication, sub-confluent monolayer cultures of the Don line of ChiExp
Cell
Res
109 (1977)
342
Gaunt and Subak-Sharpe
nese hamster tibroblast are shown to exhibit density-dependent stimulation of growth. Evidence is presented that both long and short-range feeder effects are involved. The main objective of the study is to account for this density-dependent variation in culture doubling times in terms of changes in cell cycle parameters. It would seem especially important that any such density-dependent effect should be analysed since it would demand stringent control during attempts to detect cell cycle variation due to other factors. Yaoi & Kanaseki [8] concluded that the block on cell growth in chick embryo fibroblasts growing at low density was in G2. The results now reported for Don cells indicate that the increased doubling rate of dense cultures is solely due to difference in the fate of cells within the Gl state, and this is readily interpreted as an increased probability for transition through a Gl restriction point.
placed in a common dish for seeding. The method of seeding was to set up serial dilutions of a cell suspension and inoculate an equal volume into all culture dishes. The dilutions and maximum seeding used varied between experiments, and are indicated in figure captions. In all cases, a maximum seeding density was chosen such that no culture reached confluence during the period of investigation. A modification of this procedure was used to seed cultures of different local density upon separate areas of the same slide. Such slides were first placed in Petri dishes under a layer of EFCIO. Cell suspensions of different density were then inoculated from a micropipette on to separate areas of the slides by touching drops from the pipette against the overlying surface of the culture medium. Provided the dish was not disturbed after seeding, the cells settled in a discrete area on the slide, and at a local density approximately proportional to the initial density of inoculum.
Measurement
of culture doubling rates
Cultures were grown in 90 mm plastic Petri dishes (Flow Laboratories, Irvine). Cells were seeded as indicated under fig. 1. The seeding densities and duration of culture were chosen such that cultures of each density grew over a different range of cell number for the duration of the experiment. Cells were removed from dishes in trypsin-versene, and counted using an electronic cell counter (Model D, Coulter Electronics Ltd.).
Labelling of cultures with r3H]TdR MATERIALS
AND METHODS
Cell culture and medium The cells used throughout this study were an established line of tibroblast (Don), derived from Chinese hamster lung by Hsu, and described as being diploid
Pll.
Cells were regularly examined for mycoplasma contamination by the method of Fogh & Fogh [12]. No evidence of such contamination was found. In routine passage, and in all the experiments described, cells were grown in monolayer in EFClO (Glasgow modification of Eagle’s medium, supplemented with 10% (v/v) foetal calf serum). Foetal calf serum was obtained from Gibco, Glasgow. Within each experiment, all medium to be used was first pooled, and all separate cultures were incubated together at 37°C. Cultures for estimation of population doubling rates were grown on plastic surfaces, but in all other experiments cells were grown on glass microscope slides. Slides were either used intact. or cut into onehalf or one-third pieces. Slides for cell culture were washed in Decon 75 (Decon Laboratories Ltd. Brighton), then running tap water for 3 days, prior to s&ilization. In most experiments, replicate cultures at each of 3-4 densities were used. Wherever possible, in the production of a set of replicate cultures, all slides were EXPCell
Rrs 109 11977)
[MethylJH]TdR was obtained from the Radiochemical Centre, Amersham. In all experiments where the fraction of unlabelled cells was to be measured, r3H]TdR was used at a concentration of 1 &i/ml. Carrier TdR (Sigma Chemical Co.. London) was added at a concentration nrooortional to the’length of time for which a culture was to be incubated. being 0.2~ 10m6M for a 1 h incubation. This procedure-was found to produce similar nuclear grain densities in autoradiograms over widely different periods of labelling. Checks on culture media after the completion of incubations indicated that in all cases there was retained capacity to label cells. In experiments where only the percentage of labelled mitoses were measured, cultures received a 30 min pulse of r3H]TdR (0.5 &i/ml; 4 CilmMol), followed by continued incubation in the presence of 5x 1O-6M TdR.
Fixation, staining and autoradiography of monolayers The monolayers on glass slides were washed gently in phosphate-buffered saline, pH 7.4, at 37°C; then fixed in methanol-acetic acid (3 : 1) vanour for 5 min followed by immersion in liquid fixative for 10 min. A different procedure was used in the case of monolayers for labelled mitoses estimation. These were immersed in 0.0175 M sodium citrate solution for 10 min to swell cells and facilitate observation of mitotic ti-
Feeder effects and the cell cycle
343
tude of this density-dependent stimulation was found to be very variable. “Percentage labelled mitoses” (PLM) curves
time after seeding (hours); ordinate: cell no./dish. n , Cells seeded at 3.5x103/cm2; 7-fold fewer cells seeded 0, than in W; 0, than in 0. Growth of Don cells over different ranges of culture density. Experiments ((I), (b) and (c) were carried out in the same way but at different times, and indicate the variation obtained between different batches of medium and serum. Doubling times (hours) are given in brackets.
Fig. 1. Abscissa:
gures. Fixation was then carried out by addition of an equal volume of methanol-acetic acid to the citrate solution and, after mixing, placing slides in 100% fixative for 10 min. After drying, all monolayers were washed in 10% trichloracetic acid (TCA) (w/v) at 4”C, and then tap water. Dried monolayers were lightly stained in acetoorcein, and then again washed in tap water. For autoradiography, slides were dipped in Ilford K2 emulsion diluted I : 2 (v/v) with distilled water, and at a temperature of 50°C. After exposures of 2-4 weeks at 4°C autoradiographs were developed in Kodak D19 developer, and fixed in ‘Amfix’. Measurements of the percentage of cells remaining unlabelled in any culture were made on 1000-3000 cells. For estimation of “percentage labelled mitoses”, IN-400 mitotic figures were observed.
Figs 2 and 3a indicate PLM curves produced from cultures growing at different densities, but under otherwise identical conditions. The durations of S phase (Ts), G2(T,,), and G2+M(TG2+M) were estimated from the PLM curves as indicated by Smith & Martin [ 131and shown in figs 2 and 3a. Ts is also given as the time from the initial rise to the onset of the fall in the first peak, but these points are not well defined in fig. 2. The values for TG2and TGZ+Mwere found not to vary over a 51-fold range of seeding
‘s -
0
2
4
RESULTS Doubling times of cultures growing over different ranges of density
Cultures seeded at different densities grew at different rates as indicated in fig. 1. Within all experiments, a lengthening of the doubling time was observed as cultures were seeded more sparsely. Between experiments, where different batches of medium and serum had been used, the magni-
6
8
10
iz
1‘
16
18
time after onset of [3H]TdR pulse (hours); ordinate: % labelled mitoses. m, Cells seeded at I x lW/cm2; 2.5fold fewer cells seeded 0, than in n ; 0, than in 0. “Percentage labelled mitoses” curves produced from cultures seeded over a 6.25fold range of density. Thirty-two hours after seeding, all cultures were pulse-labelled for 30 min with rH]TdR. The separate monolayers on one-half microscope slides were then washed with three changes of warm EFClO containing 5 x lo+ M TdR, and transferred to separate 50 mm Petri dishes each containing 6 ml EFCIO supplemented with SX~O-~ M TdR. At intervals, a monolayer at each of the three culture densities was fixed for estimation of ‘percentage labelled mitoses”. The first slides fixed after labelling were also scored for % labelled cells in the total population. Values obtained were W, 70%; 0,59% and 0.53%.
Fig. 2. Abscissa:
344
Gaunt and Subak-Sharpe
Ic TG2+M* 1OOr I--*
.
10
I
o. 0
1
2
3
4
$‘GZa
Fig. 3. Abscissa: time after addition of [3H]TdR (hours); ordinate: (a) % labelled mitoses; (b) % unlabelled cells. 0, Cells seeded at 2.6X 104 cells/cm’; 3.7-fold fewer cells seeded n , than in 0; 0, than in n ; q, than in 0. Data are shown for cultures seeded over a 51-fold range of density. Eighteen hours after seeding, the monolayers on whole microscope slides were transferred to separate 90 mm Petri dishes, each containing 18 ml EFClO. After a further 10 h of incubation r3H]TdR was added and, at intervals, a monolayer at each of the four culture densities was fixed for autoradiography. For each culture, estimates of the “percentage labelled mitoses” and the percentage of unlabelled cells were made from identical areas of the monolayer. In (b) figures in brackets are the percentages of cells in the Cl state for cultures at each of the four seeding densities. These values are estimated from the curves as the percentage of cells remaining unlabelled after TGZ+u.
density (fig. 3a), and for Ts over the 6.25 fold range investigated (fig. 2). The time interval between the beginning of the first and second peaks (TB) provides a measure of the minimum time needed by a cell to make one complete cycle [13] and this was found to be constant at all culture densities investigated (fig. 2). Fig. 2 provides a value for TB of approx. 8 h. A change in the form of the PLM curve with change in culture density was evident in the second peak (fig. 2). A fall in the height of this peak was observed as culExpCellRes 109(1977)
tures were seeded more sparsely. The errors inherent in the PLM technique limit the confidence which can be placed on the exact time of incubation at which these peaks reach their maxima. However, each value plotted in the region of the second peaks was obtained from observation on 400 mitoses, and all peaks were found to reach their maximum values at approximately the same time. In the experiment shown in fig. 2, cultures at each of the three densities were growing in separate dishes during the r3H]TdR pulse. In other experiments (not shown), the monolayers of different density were mixed in a common culture dish for the half-hour period of pulse labelling. This mixing was further to ensure that, at all densities, monolayers were exposed to identical labelling conditions. This modification of the experimental procedure did not affect the density-dependent variation observed in PLM curves. The constancies of TGZ+Mand Ts implied that the slower growth of sparser populations must be accounted for by delay in cell passage through the Gl state. The following experiments were carried out in an attempt to observe such an effect. Density-dependence of the delay in cell passage into S phase
Cultures of different density were exposed to continuous labelling with r3H]TdR, and the proportion of cells failing to have progressed into S phase at increasing times of incubation were observed as the percentage of unlabelled cells in autoradiograms (figs 3 b, 4). During a labelling period equal to T G?+M, the percentage of unlabelled cells can fall only due to the passage of Gl cells into S phase. At longer labelling times, the percentage of unlabelled cells will also fall due to doubling of labelled cells at mitosis.
Feeder effects and the cell cycle
345
pose of estimating the percentage of cells in the Cl state. The effect of more prolonged incubation upon the percentage of cells remaining unlabelled by [3H]TdR is shown in fig. 4. It appears that, at all densities, all cells in the population were contributing to culture growth; so that after 15 h, only 2% of cells remained unlabelled in even the sparsest culture. This also indicates that, within the range of culture conditions studied, any density-dependent effect upon cell viability can only be small. 4. Abscissa: time after addition of [3H]TdR (hours); or&are: % unlabelled cells. 0, Cells seeded at 2.4~ 1W/cm2; 5-fold fewer cells seeded n , than in 0; 0, than in W; q , than in 0. The percentage of cells remaining unlabelled during prolonged exposure to [aH]TdR. Data are shown for cultures seeded over a 125fold range of density. Eighteen hours after seeding, the monolayers on onehalf microscope slides were transferred to separate 50 mm Petri dishes, each containing 6 ml EFClO. After a further IO h of incubation, [3H]TdR was added to all dishes. At intervals after the addition of isotope, a monolayer at each of the four culture densities was fixed for estimation of percentage of unlabelled cells. Figures in brackets are the estimated percentage of cells in Gl in cultures at each of the four seeding densities.
Fig.
Density-dependent growth stimulation and culture confluence The main consideration in the choice of seeding density for the [3H]TdR labelling experiments was that no culture should reach confluence during labelling. Applying this limitation, no density dependent inhibition of growth was ever apparent. Thus, in
Table 1 % Unlabelled cells
Densely Sparsely This effect produced irregularities in the seeded area seeded area curves. The percentage of cells remaining unlabelled in a cell population following A 13.9 38.9 [3H]TdR labelling for a time equal to TGP+M Slide 1 2 12.8 42.0 will give an estimate of the percentage of Slide Slide 3 13.0 40.0 cells in the Cl state. In the experiment de- B 8.8 29.6 scribed under fig. 3, TGP+Mwas measured Slide 4 Slide 5 11.1 31.9 as 2.5 h (fig. 3a) and the percentage of un- Slide 6 10.6 28.6 labelled cells after exposure to C3H]TdR for were seeded at different densities on to adjacent this time was found to increase with reduc- Cells one-third divisions of whole microscope slides using tion in seeding density (fig. 3 b). TGZ+Mwas the drop technique described in Materials and MethThe inoculum used to seed the denser monofound to be consistently 2.5 h in several ods. layers was diluted 60-fold for seeding sparser monoseparate experiments where different layers. A. r3H]TdR added 13 h after seeding. batches of medium and serum had been B. [3H]TdR added 40 h after seeding. Cells were seeded at half the density used in A. used (data not shown). In other experiAfter 2.5 h exposure to [3H]TdR, all cultures were ments described in this communication, fixed, and autoradiograms prepared for estimation of TGP+Mis assumed to be 2.5 h for the pur- percentage unlabelled cells. Exp Cd/ Res 109 (1977)
346
Gaunt and Subak-Sharpe
fusible metabolites, the sparser cultures adopted a slower growth rate. This finding is evidence for a short-range feeder factor, not freely diffusible through the culture medium. The results shown in fig. 5 allow comparison of the Gl fractions of sparse cultures growing either with or without more densely seeded feeder layers within the same medium, but on separate slides. Due to the dimensions of slides and Petri dishes, cells in sparse cultures were separated from feeder cells by a distance of up to about 12 mm. Estimates of the percentage of unlabelled cells were made from the whole Fig. 5. Abscissa: time after addition of r3H]TdR (hours); ordinate: % unlabelled cells. n , Cells seeded of the area of sparse cultures. No local difat 1.7~ 104/cm2:Cl, 0, 64-fold fewer cells seeded than ferences in labelling were evident, to sugin 8. gest that proximity of cells to the flanking The effect of densely seeded feeder monolayers upon the r3H]TdR labelling of sparsely seeded cells. feeder slides was an important factor. The All monolayers were prepared on one-third microscope slides. Twentyone hours after seeding, each observed reduction in the Gl fraction of sparse culture was transferred to a 90 mm Petri dish sparse cultures growing in the presence of containing 30 ml EFCIO. In half of these (designated 0) a more densely seeded monolayer was placed as a feeder monolayers indicates a long-range feeder within the same medium and on each of the medium conditioning effect in contributing four sides of the sparse culture. The remaining half (designated 0) served as controls. After a further 18 to the density-dependent stimulation of h of incubation, [3H]TdR was added to all dishes, and, growth. so-
“b 2l M+j ,
at intervals, a monolayer at each of the three conditions was fixed for estimation of percentage of unlabelled cells. Figures in brackets are the estimated percentage of cells in GI in cultures at each of these conditions.
DISCUSSION
Monolayer cultures of Don cells, growing over a wide range of culture densities, are the experiment described under fig. 4, the shown to exhibit a density-dependent stimmost densely seeded cultures covered ulation of growth. As previously found for about 80-90% of the substrate after the 15 h other cell types, both local feeder effects [7] and medium conditioning [6, lo] are inof incubation. volved. To account for the source of this Active range offeeder effects variation in doubling rate, cell cycle paraIn the experiment described under table 1, meters have been studied in cultures of difall cultures were exposed to r3H]TdR for ferent density, and the following observa2.5 h. Therefore the percentages of cells tions made. (i) At all densities, all cells appeared to remaining unlabelled allow a comparison of the approximate Gl fractions of sparse contribute to culture growth. Thus, in even and more densely seeded cultures after sparse cultures, after continuous exposure seeding within the same medium. In spite of to C3H]TdR for 15 h, only 2% of cells rethe common availability of all freely dif- mained unlabelled. Erp Cell Rrs 109 (1977)
Feeder effects and the cell cycle
......... .......... ..........
6. The effect of changes in transition probability upon the distribution of cells between A state and B phase. (a) Low; (6) high transition probability. The areas of squares and circles represent the proportion of the population in A state and B phase, respectively. The stippled area represents Gl. R, restriction point. Within B phase, the angle of any sector is proportional to the duration of cell passage through that state. (The relative areas of sectors are not intended to give a precise indication of the distribution of cells within B phase; with an increase in transition probability, the cells in S will account for a slightly greater proportion of those in B phase.) The exact relationship between transition probability and the distribution of cells between A state and B phase is given in equations derived by Smith & Martin [ 131.
Fig.
(ii) No differences could be detected in the duration of G2 or mitosis over a 51fold range of seeding density. Similarly, the duration of S phase was constant over the 6.25fold range of density investigated. (iii) The fraction of a population in the Gl state was found to fall with increasing seeding density. (iv) PLM curves produced from cultures of different density were identical for the first labelled peak. The second peaks were similar in the times of onset of rise and fall, but there was progressive damping of the peak height with reduction in seeding density. These findings are consistent with the concept that variation in culture doubling time is mainly due to differences occurring within the Gl state [ 151. The observed variation in the form of PLM curves allows consideration of the nature of this Gl difference. It has been assumed that the distance
347
between first and second PLM peaks is a measure of cell generation time [14]. However, in the present study, a change in culture growth rate produced variation in height, rather than position of the second PLM peak. Smith & Martin [13] showed that their Transition Probability model allows a simple interpretation of such a finding. Fig. 6 portrays the Transition Probability model, and makes clear its predictions for the form of PLM curves. The life of each cell is divided into two distinct parts, the A state, and the B phase. Cells proceed deterministically through B phase but, at a Gl restriction point, cease to progress further and are described as being in A state. The A state described a pool of uncommitted cells, all of which are of equal status regardless of time spent in this condition. Cell transition from A state to B phase is probabilistic. A change in culture growth rate is effected by a change in A-B transition probability, and results in a shift in the distribution of the total population between A state and B phase. The demonstration that cell generation times are exponentially distributed [ 13, 161 has provided good experimental evidence for such a probabilistic transition event. Changes in the height of the second peak of PLM curves under conditions which change the doubling rate of a cell population are predicted by the transition probability model: these are a consequence of the shift in the distribution of cells between A state and B phase. In the PLM technique, only those cells in a discrete sector of the cycle are labelled (S phase) and the progression of these labelled cells around the cycle is followed by observing their appearance and disappearance in the narrow window of mitosis. In moving through the first mitosis, the labelled cells have only to Exp
Cell
Rrs
109 (1977)
348
Gaunt and Subak-Sharpe
travel through B phase. All cells travel through this phase at an equal rate, and thus the labelled sector retains its integrity and can totally saturate mitosis over several hours to produce the plateau of the first labelled peak. After the leading edge of the labelled sector has reached the restriction point, then for a time equal to the duration of S phase, the labelling index of the cells moving into the pool of A state cells will be 100%. In consequence, the concentration of labelled cells within the A state pool will rise over a period equal to the duration of S, then, as unlabelled cells follow into the pool, it will fall. In the Transition Probability model, this changing labelling index of the A state pool determines the form of the second peak in PLM curves. This follows because the labelling index of cells moving out of A state and into B phase in any unit of time is identical to the concurrent labelling index in the A state pool. Sequential changes in labelling index of cells emerging from A state will be maintained on progression through B phase since all cells move in this phase at a constant rate. There are three important features of the sequential changes in labelling index of the A state pool, and all are subsequently observed in the form of the second labelled mitotic peak: (i) For a given cell type, the labelling index will begin to rise after a constant interval following the r3H]TdR pulse. It will continue to rise for duration of S phase, and will then fall off. (ii) At any transition probability ~1, the labelling index will never reach 100 % since there will always be unlabelled cells remaining in the pool to dilute the labelled cells. (iii) At low population growth rates, the maximum labelling index reached will al-
Exp Cell Res 109 (1977)
ways be less than the maximum achieved at high growth rates. This is a consequence of the greater dilution of labelled cells by unlabelled cells and is evident upon comparison of fig. 6a and 6. Although PLM analyses do not provide clear proof of an exponential distribution of Gl times, and thus of the validity of a transition probability: Gl distributions in our cultures must, at least, be nearly exponential in order to produce PLM curves so consistent with the three predictions given above. We conclude that the density-dependent stimulation of growth, which we have observed in cultures of Don cells, is entirely accountable to Gl changes. Our results are highly consistent with the interpretation that these are changes in transition probability.
REFERENCES 1. Todaro, G J & Green, H, J cell biol 17 (1%3) 299. 2. Rein, A & Rubin, H, Exp cell res 49 (1968) 666.
3. Earle, W R, Sanford, K K, Evans, V J, Watlz, H K & Shannon, J E, J natl cancer inst 12 (1951) 133. 4. Stoker, M G P & Rubin, H, Nature 215 (1%7) 171. 5. Puck, T T, Marcus, PI & Cieciura, S J, J exp med 103 (1956) 273. Rubin, H, Exp cell res 41 (1%6) 138. 4: Stoker, M G P & Sussman, M, Exp cell res 38 (1%5) 645. 8. Yaoi, Y & Kanaseki, T, Nature 237 (1972) 283. 9. Weiss, L, Poste. G. MacKeamin. A & Willett. , K. J cell biol64 (1975) i35. Eagle, H & Piez, K, J exp med 116 (1962) 29. f :: Hsu, T C & Zenzes, M T, J natl cancer inst 32 (1964) 857. 12. Fogh, J & Fogh, H, Proc soc exp biol med 117 (1964) 899. 13. Smith, J A & Martin, L, Proc natl acad sci US 70 (1973) 1263. 14. Quastler, H & Sherman, F G, Exp cell res 17 (1959) 420. 15. Defendi, V & Manson, LA, Nature 198 (1%3) 359. 16. Minor, P D & Smith, J A, Nature 248 (1973) 241. Received March 28, 1977 Revised version received May 27, 1977 Accepted June 2, 1977
