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Alternative modes of population growth inhibition in a human lymphoid cell line growing in suspension
Printed in Sweden Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in onyform resewed ISSN 00144827
Experimental Cell Research 107 (1977) 325-341
ALTERNATIVE MODES OF POPULATION GROWTH INHIBITION IN A HUMAN LYMPHOID CELL LINE GROWING IN SUSPENSION A. YEN,’ J. FRIEDP and B. CLARKSON Laboratory
of Hematopoietic
Cell Kinetics, Memorial Sloan-Kettering New York, NY 10021, USA
Cancer Center,
SUMMARY Using a human lymphoid cell line grown under continuous culture conditions, two distinct plateau states were induced, either by lack of sufftcient medium-supplied nutrient, or by other unknown mechanisms dependent on cell density. Flow microfluorometric measurements show that growth arrest due to nutritional insufftciency results in an accumulation of cells with Gl DNA content. In contrast, growth arrest due to high cell density is not associated with an altered distribution of cells with respect to DNA content as the population progresses from exponential to plateau state growth. Cell size decreases with progression of the plateau state induced by either type of growth arrest. Cells in a plateau state induced by high cell density utilize glucose and incorporate exogenous amino acid into protein at approximately the same rate as exponential cells. Proliierating, high cell density, plateau state cells have cell cycle phase durations similar to exponential cells. The stable, plateau state cell density is maintained by cell loss. No stable, unbound growth inhibitory factor was found in the medium of density-inhibited plateau state cultures.
The typical proliferation pattern for mammalian cell lines consists of exponential growth for a limited time before growth inhibition occurs at high cell density. Cessation of population growth may be due to nutritional deficiency or may be induced by cell-cell interactions, mediated by unknown mechanisms. Both cell lines in culture and tumor cells maintained in animals have been extensively employed in studying cellular alterations associated with growth inhibition. In most of this work, no distinction has been made as to whether
the plateau state is due to a nutritional deficiency, such as exhaustion of some essential serum component, or to a cell-cell interaction associated with density inhibition of population growth. Making this distinction in practice is complicated. In substrate-attached cells in culture at advanced states of growth, cell crowding reduces the cell surface area interfacing medium and hampers any experimental approach to obviate the possibility of cell growth inhibition due to nutritional insufficiency. This problem is exacerbated in animal tumor cell systems where the cell population is depen1 Present address: Department of Cell Growth and dent on the animal host for nutritional Regulation. Sidney Farber Cancer Institute, Boston, supply. This is particularly in evidence in MA 02115;USA. solid tumors exhibiting internal necrosis. * To whom reprint requests should be addressed. Exp Cell Res 107 (1977)
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The problem this study addresses is the establishment of distinct plateau growth states due to either nutritional insufficiency or to social cell interactions at high cell density. The cells used in this study are from a human lymphoid line. To circumvent the nutritional problems described above, the cells are cultured in suspension in a commercially available spin filter, continuous culture device. In this culture system, used medium in the cellular environment is constantly exchanged for fresh medium without loss or disruption of cells in the culture vessel. Definition of a plateau growth state caused by cell-cell interaction at high cell density and independent of nutritional influences allows characterization of the cellular activities associated with proliferation regulation in density inhibition of growth. Although the absence of number increase in a density-inhibited cell population of high viability suggests that these cells may be “resting” or “quiescent”, it will be shown that these cells can be as metabolically active as exponentially proliferating cells as indexed by their glucose consumption from the medium and rate of incorporation of exogenously supplied amino acid into protein. The dynamics of cell size changes during population growth from exponential through nutritionally and density-induced plateau phases will be described. Variations in relative cell cycle phase transit times or cell cycle phase specific maturational blocks associated with nutritionally or density induced plateau growth states will be derived using flow microfluorometry and DNA fluorescent staining. The cell cycle kinetics of density-induced plateau state cells will be described. It will be shown that the stable cell density of the high cell density plateau state is maintained by cell death. Exp Cell Res 107 (1977)
MATERIALS
AND METHODS
Cells The cell line used, SK-L7, is a human lymphoid line which has been continuously cultured for over 10 years. Its properties and origins have been previously described [l-4]. Stock cultures were maintained in Falcon T-75 culture flasks (Falcon Plastics) in McCoy’s medium 5A (modified) (Gibco). The cells were growing exponentially when harvested from culture flasks for addition to the spin filter continuous culture device in preparation for an experiment. Cell densities were deteimiied by repeated cell counts using a hemacytometer. Cell viability was measured as the percentage of cells excluding 0.1% trypan-blue. Tests for the presence of mycoplasma were negative.
Determination
of cell size
The distribution of relative cell size in a population aliquot was measured with a Cytograf hiodel 6300 @o/Physics Systems, Inc.). The resulting output signals are accumulated in a multi-channel pulse-height analvzer (Model NS 602, Tracer-Northern Scientific, Inc.; Middleton, Wise.). The mean cell size was measured as the mean channel number of the resulting unimodal distribution accumulated by the multichannel analyzer. Measurement of standard polystyrene microspheres (Particle Information Se+&, -Inc., Grants Pass, Ore&) of a variety of diameters demonstrated a linear r&tionship between particle size and analyzer channel number. All measurements of cell size were made immediately after withdrawal of the cell sample from the culture vessel.
Determination of distribution in GI, S, and G2+M
of cells
The number of celIs with Gl, S, and G2 DNA content was determined by flow microfluorometry using a Cytofluorograf Model 4802 (BiofPhysics Systems, Inc.). An aliquot of 5x 1V cells in no more than 0.25 ml was mixed with 5 ml of a hvootonic solution of the fluorescent dye propidium iodide (0.05 mg/ml of prooidiumiodide in 0.1% sodium citrate) r51 and mainiained on ice for at least 10 min. Thd &ned nuclei are then analysed with the microfluorometer. The distribution of number of nuclei versus relative fluorescence intensity was analysed to yield the fraction of cells in the aliquot in Gl, S, and G2+M. The mathematical fitting method used for this analysis is described elsewhere [6, 7, 81. Briefly this method assumes that cellular DNA content is compartmentalized with values ranging from Gl DNA content to G2 DNA content. Each compartmentalized cellular DNA content is assumed to produce a normally distributed, emitted fluorescence intensity. The summed distribution of fluorescenceintensitiesis then fitted to the observed distribution to derive the number of cells with each DNA content. Experiments recently carried out with the SK-L7 cell line demonstrated the validity of the propidium iodide staining method and the mathematical method used for the analysis [9].
Population growth inhibition in lymphoid cell line
4
Determination of glucose concentration in culture jluid Glucose concentration in medium from the culture vessel was determined with a Technicon Autoanalyzer I using the potassium ferricyanide method, a modification of Hoffman’s method [IO]. Cell samples of 10 ml were withdrawn from the culture vessel. The cells were immediately pelleted. The supernatant was decanted and refrigerated until analysed for glucose concentration.
Measurement of rates of incorporation of exogenous amino acid into protein Relative rates of incorporation of exogenous amino acid into cellular protein at various population growth states were measured by the uptake of [sH]Ile into cold TCA-precipitable material. For each measurement. the cells in 10 ml of cell susnension withdrawn from’the culture vessel were pelleted by centrifugation (800 rpm, 160 g, for 5 min) (Model PR-6000 centrifuge, DammonlIEC Division), the supematant aspirated. and the cells resuspended in 10 ml of fresh medium at 37°C with 200 -pCi of [sH]Ile (spec. act. 83.4 CilmM. New E&and Nuclear). At this time and 5, 10 and 15 min late< 0.5 ml samples of cell suspension were withdrawn, brought to a final volume of 5 ml with ice-cold physiological (0.9 %) saline, and stored on ice until all four samples were taken. The cells were incubated at 37°C in 5 % CO* and 95 % air between samulina. All four samples were maniuulated simultaneously-after this point.i”ne cells were pelleted in a refrigerated centrifuge (3000 rpm, 2250 g, for 5 min) at 2”C, the supernatant decanted, and the cells resuspended in 5 ml ice-cold physiological saline. This wash was repeated twice more. On the final wash, the nellet was resusnended in 1 ml of ice-cold physiological saline and 1ml of ice-cold 25 % TCA was added. Following agitation, the precipitate was pelleted by centrifugation (3 000 rpm, 2 250 g, for 5 min at 2”C),
327
Fig. I. Schematic illustration of continuous culture device. I, 4 liter main medium reservoir: 2. intermediate reservoir maintained at 37°C: 3, oeristaltic oumn for medium input; 4, c&t& vessel maintained at 37°C; 5, air input; 6, peristaltic pump for removal of medium from culture vessel, regulated by level sensor; 7, level sensor regulator for peristaltic pump 6; 8, medium reservoir for medium removed from culture vessel. the supematant decanted and the precipitate resuspended in 2 ml of ice-cold 12.5% TCA. The above pelleting, decanting and resuspension were repeated twice more. The precipitate ias then collected on a GF/C glassfiber filter (Whatman) which was subsequently washed with 10 ml ice-cold 12.5% TCA and then 5 ml ice-cold ethanol. Radioactivity on the dry filters was counted using Aquasol liquid scintillation cocktail (New England Nuclear) in a Beckman LS2OOB scintillation counter. The same stock of medium supplemented with fetal calf serum and with 13H]Ile added was used in all measurements of [3H]Ile &&oration to be compared, obviating error induced by differences in specific activity of label due to possible variations in-serum or media concentrations of isoleucine.
Continuous culturing of cells In the experiments to be described, SK-L7 was grown in suspension using a spin filter culture device [ll, 121. The culture vessel-and its ancillary equipment are shown schematically in fig. 1. As used in this studv. the culture vessel (ADL Biosoin Culture Flask ModLculture System, V&is Co., G&diner, N.Y.) contained 450 ml of cell suspension accessed through eight ports and access tubes in the top of the vessel. Briefly, in this culture system, a spinning (650 rpm) cylindrical, glassfiber filter supported within the culture vessel performs the two functions of maintaining the cells in suspension and mixing the culture fluid. It allows medium to be exhausted from the culture vessel through the spinning filter which excludes cells. Bv virtue of the fluid, shear boundary at the surface of the spinning filter, retention of debris, e.g., cells, by the filter is minimized. In this way, cells are retained in the culture vessel while used medium is being removed and fresh medium influxed. In our hands, this system has been effective for up to approx. 230 h of continuous running prior to failure of the spin filter unit. As shown in fig. 1, fresh medium, Exp Cell Res 107 (1977)
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Yen, Fried and Clarkson Continuous
loo-
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Fig. 2. Abscissa: time after seeding spin filter flask (hoursl: ordinate: cell densitv X lob4 cells/ml. l - - -0, .~~ 0.2; O&, 1.0 ml/mitt medium intlux. Growth curve illustrating nutritionally limited population growth.
labelling
and autoradiograph:
Exponential cells at a cell density of 2.5 x lo5 cells/n at initiation of labelling were exposed to [3H]thymidin continuously for 16 h (spec. act. 6 Ci/mM, New Eng land Nuclear). Because of medium exchange durin continuous culturing, the concentration of label ranges from 0.05 &i/ml initiallv to 0.035 uCi/ml tinallv Plateau state cells at a cell density of approx. 6~ lc cells/ml were continuously labelled for 36 h after th plateau state had existed for 10 h. [3H]Thymidin (snec. act. 6 CilmM) was iniected into the cultur vessel every 6 ‘h maintaining the concentration c at 0.05 &i/ml to 0.023 &i/ml. A .13Hlthvmidine ., < each sampling time, 1.b ml of cell suspension wa withdrawn. Slides were made using a Cytocentrifug (Shandon Scientific Co., Limited, Sewickley, Pl loaded with 0.2 ml of cell suspension which had bee diluted with ice-cold medium-if necessary-t 250000 cells/ml per slide. Autoradiography was don with Kodak NTB2 emulsion. The exposure period wa 4 weeks. Cells with four or more grains over the m cleus were considered labelled. The background grai count was less than one per nucleus.
RESULTS stored in a 4 1 reservoir, flows into an intermediate reservoir maintained at 37°C before enterina the culture vessel. A precise intlow rate is mainb&ed by a peristaltic pump (Model 500 Automatic, Self-Regulating, Intravenous, Infusion Pump., Ivac Corp.). A liquid level sensor (LLSP-2 level sensing probe with carbon electrode and 57LC level controller, Virtis Co., Gardiner, N.Y.) within the culture vessel maintains a constant culture fluid volume by activating the exhaust pumping of medium (Model PP325 Peristaltic Pump, Virtis Co.) through the spin filter as fresh medium is continuously added. The total variation in volume of culture fluid over time is less than 10 ml. The DH of the culture fluid is monitored bv a DH electrode (Ingold Electrodes, Inc.) within the cult&e vessel. With cell growth and respiration, the medium became acidic. By influxing air into the culture vessel to purge it of CO1 and by titration with 5 N NaOH as needed, the pH of the culture fluid was routinely maintained at 7.0f0.3. Cell samples were withdrawn through one of the access tubes in the top of the culture vessel, using sterile hypodermic syringes. The culture vessel and intermediate medium reservoir were maintained at 37°C in a constant temperature water bath. The cells were at-own in McCov’s 5A medium (modified) supplemented with 30% fetal calf serum and 1% PSN (Penicillin, Streptomycin, Neomycin) Antibiotic Mixture (Gibco). Thetrypan blue excluding fraction in exponential and plateau state populations was routinely in excess of 99%. In most cases the duration of cell growth in this system was terminated by mechanical failure of the spin filter, which has a limited (and unpredictable) useful lifetime. Cell samples taken from different levels within the culture vessel were similar in cell density and absence of cell clumps, as expected of a uniformly dispersed suspension of cells. Exp Cell Res 107 (1977)
Plateau state population growth dependent on and independent of exogenous nutritional supply
In the continuous culture device employee in this study, fresh medium is continuousl! infused into a cell culture vessel as mediun from within the culture vessel is removed maintaining a constant volume of cuim fluid in the vessel. Cells are retained it suspension within the culture vessel as the medium is exchanged. If nutritional suppl! were the only growth limiting factor acting then the cell population could expand unti the rate of nutrition consumption by the ccl population was equal to the rate of nutri tion influx. If the rate of medium infusior were subsequently increased, then prolifer ation should resume. Fig. 2 shows the po pulation growth resulting from a fixed rate of medium infusion and the effect of sub sequently increasing the rate of infusion A medium infusion rate of 0.2 ml/min car responding to a medium fractional turnove: rate of l/38 (h-l), results in an exponentia increase in cell density to a plateau value
Population growth inhibition in lymphoid cell line
329
The initial cell density in this experiment was much lower than in that described by fig. 2 showing that the growth end point of 2.1 x lo6 cells/ml is independent of initial cell density as expected by nutritional considerations. Increasing the infusion rate to 0.4 ml/min resulted in population growth to a new plateau state with cell density of approx. 3.8~ lo6 cells/ml, nearly twice the maximum cell density with a 0.2 ml/min infusion rate. Increasing the infusion rate to 1.0 ml/min resulted in further population growth. The resulting maximum cell Figs 3, 4. Abscissa: time (hours); ordinate: X10+ density, however, was approx. 6~ 10” cells/ cells/ml. O---O, 0.2; O---O, 0.4; O-O, 1.0 ml/min ml. Based on the previous nutritional conmedium influx. Nutritionally and high cell density-limited populasiderations, the anticipated plateau state tion growth. Medium influx rate was increased to 0.4 ml/min at t = 132 h after a 16 h plateau phase and to cell density at 1.0 ml/min should have been 1.O ml/min at t= 180 h following a 12 h plateau phase. 9.5~10~ cells/ml, i.e. (1.0/0.4)X(3.8). Population growth therefore appeared to be inof approx. 2.1 x log cells/ml. The exponen- hibited by a mechanism or mechanisms tial doubling time was approx. 15.3 h. The other than nutritional deficiency. cell density then failed to increase for 20 h It can be confirmed that the plateau state until the medium infusion rate was in- at approx. 6x lo6 cells/ml is not dependent creased. Increasing the medium infusion on the previous occurrence of growth-inhirate at this time from 0.2 ml/min to 1.0 ml/ bited states. Fig. 4 shows the continuous min resulted in a resumption of population exponential growth and final plateau state growth. The implication is that a medium at approx. 6x lo6 cells/ml resulting from infusion rate of 0.2 ml/mm induces a plateau state of cell density 2.1 X lo6 cells/ml, through nutritional limitation. If the medium infusion rate were 0.4 ml/ min and nutritional supply were the only growth-limiting factor, then the anticipated plateau state cell density at this infusion rate is approx. 4x lo6 cells/ml. Likewise, any increase in infusion rate should result in a proportionately increased, plateau state cell density. Fig. 3 shows the population growth resulting from an initial medium infusion rate of 0.2 ml/min which was subsequently increased to 0.4 ml/min and finally to 1.0 ml/min. The infusion rate of 0.2 ml/ 4. Growth curve showing effect of changing inmin again resulted in a plateau state with Fig. flux rates prior to onset of nutrient-limited plateau cell density of approx. 2.1 x lo6 cells/ml. phase. Exp Cell Res 107(1977)
330
Yen, Fried and Clarkson
cessively from 0.2 ml/min to 0.4 ml/min to 1.O ml/min prior to the nutritionally deficient growth arrest anticipated at each infusion rate. When the population cell density was approx. 6~ lo6 cells/ml the previously observed maximum with a 1.0 ml/min infusion rate, the media infusion rate was increased from 1.0 ml/min to 2.0 ml/min. Nevertheless, the ensuing plateau state occurred almost immediately at a cell density of approx. 6.5-7~ lo6 cells/ml. This demonstrates that the growth-limiting mechanism 60 70 80 90 100 0 10 20 ;O 40 50 in the plateau state occurring at a cell denFig. 5. Abscissa: time (hours); ordinate: X lo-* cells/ sity of approx. 6x lo6 cells/ml is not priml. l - - -0,0.4; O-O, 1.Oml/min medium influx. marily depletion of the exogenous nutritioHigh cell density-limited population growth. nal supply, but some other process depensuccessively increasing the medium in- dent on cell density. Since the plateau fusion rate from 0.2 ml/min to 0.4 ml/min states observed at lower cell densities were and then to 1.0 ml/min prior to the occur- due to depletion of critical nutrients in the rence of growth arrest anticipated for each medium, these results demonstrate that two infusion rate. This observed growth and kinds of plateau states may be generated arrest are not dependent on the choice, in this cultured cell system. Although cell sequence and timing of infusion rates. The number density is stable during both types population growth resulting from an initial of plateau states, growth inhibition of the infusion rate of 0.4 ml/mm increased sub- population does not exclude the possibility sequently to 1.0 ml/min is shown in fig. 5. of ongoing cellular activity, including proThe 0.4 ml/min to 1.0 ml/min infusion rate liferation, in a steady state renewal process. increment occurs at a lower cell density Further references to “growth inhibition” than in the case of fig. 4. Nevertheless, in this report imply only the absence of inthe growth and plateau state shown in fig. 5 crease in number density of the population. are similar to that of fig. 3. The growth in- Certain cellular properties and activities hibition occurring at a cell density of ap- during each plateau state will now be disprox. 6~ lo6 cells/ml is, therefore, indepen- cussed. dent of the particular way the cell density Distribution of DNA contents in is generated in this culture system. If the plateau state with cell density of exponential; nutritionally inhibited and approx. 6~ lo6 cells/ml were due to nutri- density inhibited populations: Behavior tional depletion, then increasing the me- as a function of growth phase dium infusion rate to greater than 1.0 ml/ The fraction of a population with each of min prior to growth arrest should lead to a Gl , S, or G2+M DNA content at any time commensurate increase in the final plateau may be determined by flow microfluorostate cell density. Fig. 6 shows the result- metry as described earlier. It will be shown ing exponential population growth as the that monitored by this means, nutritionally medium infusion rate was increased suc- induced growth inhibition and density-in1000
I
/,/I 100 I
ExpCellRes 107(1977)
Population growth inhibition in lymphoid cell line
101 ’ 0 IO
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Fig. 6. Abscissa: time (hours); ordinate: x lo-’ cells/ ml. o---o, 0.2; o---o, 0.4; O-0, 1.0; o-e-0,
2.0 ml/min medium inkx. High cell density-limited population growth. Arrows designate sampling times for medium glucose concentration determinations.
duced growth inhibition are distinguishable. For the population described in fig. 2, a case of nutritionally induced growth inhibition, fig. 7 shows the percentage of cells with Gl, S, and G2+M DNA content as a function of time. During the initial 28 h of exponential growth, the distribution of cells with respect to DNA content is stable. The following 20 h of nutritionally deficient plateau state eventually result in an increased fraction of the population with Gl DNA content and a decreased proportion of cells with G2+M DNA content. During this time there is a transient increase in the relative number of S phase cells. The processes responsible for this redistribution during plateau state may involve retardation of cell maturation in Gl, selective cell death in S, G2 and M, or both. Increasing the nutritional supply after a 20 h plateau state resulted in a reduction in relative numbers of cells with Gl DNA content and increased percentages with G2+M DNA content. The results may be interpreted as a release from the altered cell cycle kinetics imposed by the plateau state. Growth inhi-
331
bition by nutritional insufficiency, therefore, is associated with a reversible accumulation of cells with Gl DNA content and depletion of cells with G2+M DNA content. Growth inhibition due to density inhibition independent of exogenous nutritional supply results in behavior distinct from the case of nutritionally induced growth inhibition. Fig. 8 shows the percentages of cells with Gl, S and G2+M DNA content as a function of time for the population undergoing density inhibition of growth described in fig. 5. The percentages of cells with Gl, S, and G2+M DNA content are approx. 40, 50 and lo%, respectively and remain stable as the population progresses from exponential into plateau phase. The distribution of cells with Gl , S and G2+M DNA content for the population described in fig. 6 is virtually identical to that shown in fig. 8 and is also independent of growth phase. The similar results in these two cases confirm that the two density-inhibited plateau states are alike independent of the differences in the ways in which they were obtained. The stability of the distribution of
20 IO 0
'-.~,,***--'.\..* E ---*________-c
I 0
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I 30
I 40
I 50
I 60
time (hours); ordinate: % of population in each cell cycle phase. (a) Gl; (b) S; (c)
Fig. 7. Abscissa: G2+M.
Distribution of cells with respect to cell cycle phase during exponential (O- - -0) and nutritionally limited plateau state (O-O) growth (see fig. 2). Exp Cell Res 107 (1977)
332 50 40 30
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Yen, Fried and Clarkson
These results suggest that density-in..--.-.*--*--)-----~--.--*---~ duced growth inhibition does not act at the E cellular level by simply restricting the upa
take of the same nutritional factor involved in the nutrient deficient plateau state. Were this the case, then a redistribution of cells with respect to Gl, S and G2+M DNA content similar to that induced by nutritional deficiency, i.e., as in fig. 7, would be expected; and this does not occur. It will be demonstrated later that, consistent with the lack of restriction of nutrient uptake, the rates of consumption of glucose from the culture medium and incorporation of exogenous amino acid into cellular protein are not reduced in density-inhibited plateau state cells.
a.-*--*-___ )--*-.--‘-__. : -_. EL I*----I I I 11I / I c
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Fig. 8. Abscissa: time (hours); ordinate: percentage of population in each cell cycle phase. (a) Gl; (b) S; (c) G2+M. O---a, Exponential growth; O-e, plateau state. Distribution of cells with respect to cell cycle phase during exponential and high cell density-limited population growth (see fig. 5).
cells with respect to DNA content as shown by flow microfluorometric analysis (fig. 9) is a characteristic of density-induced growth inhibition in this cell system. Although the distribution of cells among the phases of the cell cycle is the same in the plateau growth state as in the log phase, it is obvious that some of the cellular processes occurring under the two conditions must be different. If the action of a growth inhibitory process were simply to retard cells in a single cell cycle phase, then the occurrence of plateau state would be associated with an enhancement in the relative number of cells in that cell cycle phase. In the case of nutritionally induced growth inhibition, the results of fig. 7 are consistent with the existence of some Gl specific retardation of cell maturation, exerted during plateau state and released during growth resumption induced by increased nutritional supply. Density-induced growth inhibition, in contrast, acts as seen in fig. 8 with no simple cell cycle specificity of this character. Density-induced growth regulation, therefore, does not involve a process restricted to any single cell cycle phase. Exp CellRes 107 (1977)
Fig. 9. Abscissa: fluorescence intensity (channel no.); ordinate: cells x 103. (a) High cell density plateau; (b) exponential. Distribution of cells with respect to DNA content during exponential and high cell density plateau states as inferred from flow microfluorometric analysis. Lower diagrams show DNA histograms (data points shown as dots)-together with results of the mathematical analysis. Solid lines show the theoretical histogram corresponding to the solution and the calculated distributions of fluorescence intensities of the Gl, G2+M, and S phase populations. For clarity, the distribution of S phase cells is shown separately in the upper diagrams. The histograms describe the population shown in fig. 6 at 36 (exponential) and 80 (high cell density plateau) hours.
Population growth inhibition in lymphoid cell line Table 1. Glucose cone. in culturefluid during exponential (O-76 h) and plateau state (76-96 h) growth
m(t) -=coV
R -No z v+K
333
-I, eKt+De v
Cell
Growth state
Hour
density (cells/ml)
Glucose M8ho
:: 36 42 89 92
3.55ooo 525 ooo 769 000 1 165 000 6.9x 10“ 6.8X 106
2.24 2.12 1.81 1.60 2.39 1.22
Exponential
Plateau
Rates of glucose consumption and incorporation of exogenous amino acid into protein in exponential and density-inhibited cells During the period indicated by the population growth curve shown in fig. 6, the glucose concentration measured in the culture fluid is given in table 1. These data can be used to calculate the average rate of glucose uptake per cell per hour. The concentration of glucose in the culture fluid is described by the following differential condition based on conservation of total glucose:
dm(t),c -,mW ---Vn(t)R dt
’
V
where m(t) is the total glucose contained in the culture fluid as a function of time; co, the glucose concentration in fresh input medium; I, the rate of medium input; n(t), the cell density as a function of time; V, the volume of cell suspension in the culture vessel; R, the average rate of glucose uptake per cell per hour. In the case of exponential growth where: n(t)=N,efQ the solution is 22-771819
All the constants except R and D are known parameters of the experiment. K is derived by a least squares fit to cell densities during exponential growth. A fit to the glucose concentrations during exponential growth given in table 1 yields a value for R of 0.6~ lo-’ mg/hour-cell, i.e., approx. 3 x lo-lo mM/hour-cell. The resulting theoretical curve and experimental values for glucose concentration in the culture fluid are given in fig. 10. In the case of the plateau state, an analogous analysis, using n(t) equal to the plateau state average cell density, yields a value for R of 0.9~ lo+ mglhour-cell, i.e., approx. 5x lo-lo mM/hour-cell. The uncertainty in the derived values of R is equal to at least the difference between the values for exponential and plateau state cells. Furthermore, the theoretical curve of fig. 10 is insensitive to this degree of variation in the value of R. We conclude that exponentially proliferating cells and density-inhibited, plateau state cells consume glucose from the media at approximately the same rate.
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Fig. 10. Abscissa:
time (hours); ordinate: mg/ml. Concentration of glucose in medium during exponential growth. Dots, data points; solid line, theoretical concentration curve computed as described in text. Exp Cell Res 107 (1977)
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Yen, Fried and Clarkson
IOOO/r / I,./ IOO,/’ ,,” ,Y” -.,,*’ ,/’ /’ Ic 10: 0
IO
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GQ, \A)
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SO PO 100 110 120
Fig. Il. Abscissa: time (hours); ordinate: x lo-’ cells/ ml. o---o, 0.2; o---o, 0.4; O-0, 1.0; o-a-0, 0 ml/mm medium influx. High cell density-limited population growth showing effect of cessation of medium infhrx during plateau phase. (%), cell viability determined by trypan blue exclusion. Arrows, times of amino acid uptake measurements.
Consistent with this finding is the observation that terminating fresh medium input to the density-inhibited plateau population (fig. 11) results in extensive cell death. After the plateau state had been maintained for 28 h, medium influx (and efflux) was terminated. Approx. 25 h later the viable cell density had decreased to approx. 3.6~ lo6 cells/ml. Cell viability (as estimated by trypan blue exclusion) had decreased from 99% to55%. The rate of incorporation of exogenously supplied [3H]Ile into cellular protein was determined at three times during the population growth shown in fig. 11. The incorporation of r3H]Ile into cold TCA-precipitable radioactivity measured at 32 h (exponential growth), 68 h (early plateau state), and 74 h (plateau state) is shown in fig. 12. The incorporation observed in all three cases is similar. The absence of any lag in the observed uptake demonstrates that there was no pool effect which would’ Exp CellRes 107(1977)
have resulted in an initial dilution of label and a resulting lag in the onset of linear incorporation, Although the observed rate of incorporation is dependent on a variety of cellular processes such as transport, intra-cell precursor compartmentalization, etc., it is unlikely that all these processes would change at 3 different times in exact concert to yield the same observed incorporation rates. These results are consistent with the existence of similar rates of protein biosynthesis at the three times of observation. The results presented here demonstrate that the density-inhibited plateau state cells do not differ from exponential cells in their rates of glucose utilization or incorporation of exogenously supplied amino acid into cellular protein. Neither do they tend to accumulate in a portion of the cell cycle as under conditions of nutrient deficiency discussed earlier. The cells of the densityinhibited plateau state population are, therefore, apparently as metabolically active as exponential cells.
Cell size during exponential, nutritionally inhibited and density inhibited growth The cell size was monitored as described earlier by low angle light scattering. The population described in fig. 2 underwent
Fig. 12. Abscissa: time after addition of tracer (mm); ordinate: cpm/5x W cells. TCA-precipitable radioactivity during [WjIle labelling of (a) exponential; (b) early plateau; (c) plateau state cells. Samples were obtained at times (a) 32 h; (b) 68 h; (c) 74 h (see fig. 11).
Population growth inhibition in lymphoid cell line
335
causally related to resumption of cell proliferation.
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Fig. 13. Abscissa: time (hours); ordinate: channel. l - - -0, Exponential growth; O-O, plateau state. Rel. mean cell size during exponential growth and nutrient-limited plateau phase (see fig. 2).
.
growth inhibition by nutritional insuffkiency followed by resumption of growth following increase in medium intlux rate. Its relative mean cell size as a function of time is given in fig. 13. The mean cell size as well as the entire observed size distribution was stable during exponential growth. The onset of plateau phase was associated with an immediate progressive decrease in cell size. Population growth resumed at 48 h (fig. 2). The cell size, however, remained unchanged from 48 to 52 h (fig. 13). A lag of 4 h occurred following the resumption of growth before any change in cell size occurred. Cell size then increased at the same rate at which it had previously decreased. At all times the distributions of cell sizes were unimodal and similar in halfmaximum width and almost symmetric shape, while the means shifted as described. In the case of a population undergoing density inhibition of growth, fig. 14 shows the mean cell size as a function of time for the population described in fig. 5. Cell size was again stable during exponential growth and decreased progressively with the onset of plateau state. These results show that cells of both nutritionally and density-induced plateau states decrease progressively in size. The occurrence of a lag in the response of cell size to growth resumption shown in fig. 13 indicates that a cell size increase is not
Absence of a stable growth-inhibiting factor existing free in density-inhibited stationary state culturefluid The simplest mechanism by which a density-inhibited plateau state could be effected is by means of a growth-inhibiting factor in’the culture fluid of plateau state populations. It will be shown, however, that in this cell system no such stable factor exists in a free state. During the experiment shown in fig. 4, 3X 106plateau state cells in a volume of approx. 0.5 ml were removed from the culture vessel at 110 h, after 16 h of plateau state, and inoculated into 100 ml of cell-free culture fluid obtained at the same time from the culture vessel. (This was performed in duplicate.) In 35 h the cell density had increased S-fold with a doubling time of 15 h. This demonstrates that culture fluid from a density-inhibited plateau state culture can support cell growth, and that cells from a density-inhibited plateau state culture retain their proliferative capacity at lower cell densities. Confirming these results, it was also observed in separate experiments that both plateau state cells resuspended in 99.5 % fresh medium at low density and exponential cells inoculated in 95 % plateau state culture fluid proliferated exponenti) ______ * _____-. --------------.---
72
70 68 66
64 62
,
60I, ,o
20
, , 30 40
,
,
,
50
60
70
“:.. 80 90 100
Fig. 14. Abscissa: time (hours); ordinaie: channel. Rel. cell size during exponential or high cell densityinduced plateau phase. Differences in absolute channel number as compared with fig. 13 were due to differences in laser light output. Exp Cell Res 107(1977)
330
Yen, Fried and Clarkson in figs 4, 5, and 6, the plateau state cell density is not sensitive to the fractional turnover rate. The existence of a growthinhibiting factor is, therefore, not apparent in situ in the culture vessel.
7060-
P=
50-f 40-t 4 20: IOC
‘0
4
8
12
16
Fig. 15. Abscissa: time after beginning continuous exposure to rH]thymidine (hours): ordinate: (a) LI (%); (6) MI (%); (c) PLM (%). LI, MI, and PLM during continuous labelling of exponential cells. The 0 time sample was taken immediately after addition of r3H]thymidine to the cell cultures.
Cell cycle phase durations of exponential cells Fig. 15 shows the percentage of cells labelled (LI), percentage of cells in mitosis (MI), and percentage of mitotic cells labelled (PLM) as a function of time during continuous [3H]TdR labelling of an exponentially growing population. The data are those typically associated with an exponential population. The age density function, n(a, t), of a population is the number of cells of age a to a +du at time t. For an exponentially growing population where the distribution of generation times is not broad, n(a, t) may be approximated by:
ally. The inferred result is that there is no factor which is stable and free in the culture n(0, 0)exp -F (u-t) ,O
Population growth inhibition in lymphoid cell line Table 2. Cell cycle phase durations (hours) Cell cycle phase
Cl
s
G2
M
Exponential cells ’ High cell density plateau state
4.8
8.0
2.5
0.5
4.9
8.3
2.0
0.7
where TG is the mean cellular generation time. A least squares fit of the growth curve to N,exp[(ln2/T,)t] yields TG= 15.9 h, assuming no cell loss. The duration of mitosis, TM, is derived from: 7,
MI=
s
n(a, t)da :“,-” c n(u, t)du
Jo
(2)
337
LIo is the fraction of cells which would be labelled in a pulse label and should represent the fraction of the population in S. The numerical value of LIo was derived from fig. 15 as the average of LI’s at 1.0, 1.5, and 2.0 h and is equal to 0.48. Eq (5) yields a Gl duration of 4.8 h. The duration of S is TG-TGI-TGP-TM and is equal to 8 h. In agreement with the observed distribution of cells with respect to DNA content, the fraction of cells with Gl DNA content calculated from TG, o s da, Oda TC n(u, t)du s0
as:
is 0.38. The fraction of cells with S DNA content estimated from the labelling index TM=% ln(MI+l.O) (3) is 0.49, and the fraction with G2+M DNA content is 0.13. The average mitotic index during the continuous labelling was 0.023. The duration of Continuous [3H]thymidine labelling of plateau state cells M is derived from eq (3) to be 0.5 h. The duration of G2 is equal to the time Fig. 16 shows the LI, MI, and PLM during required for cells labelled in late S to reach continuous labelling with [3H]thymidine of mitosis. From the PLM curve of fig. 15, density inhibited plateau state cells. The behavior of LI, MI, and PLM is similar to this time is approx. 2.5 h. The duration of Gl is derived from the that observed for exponentially growing equation cells (fig. 15) and indicates that cells in the plateau state population are traversing the TG-i-,-T,, cell cycle. Since the distribution of plateau n(u, t)da s LIo= ,;I * (4) state cells with respect to DNA content is the same as that of exponential cells, eq n(u, t)da (1) also approximates the age density func0 tion of plateau state cells. The transit times as of these cells through Gl, S, G2, and M were derived in the same manner as preTG1=-2ln { F+exp[-$f(TG-TM-TG2,]i viously. LI, was in this case 0.49. The average mitotic index was 0.03. From fig. 16 (5) the durations of the various phases were:
J”
Exp CeNRes 107 (1977)
338
Yen, Fried and Clarkson
liferate [21]. Stimulated cells also show alterations in chromatin structure detectable by ethidium bromide uptake and circular dichroism [ 191. A post-transcriptionally regulated increase in mRNA occurs in cells stimulated from quiescence [22]. Cells stimulated from quiescence have a posttranscriptionally regulated increasedpolyA(+)mRNA content which does not depend on the accompanying increased number of ribosomes [23]. Comparison of mRNA from proliferating and quiescent cells shows the majority of mRNA sequences in each case are common to both [24]. The occurrence of DISCUSSION quiescence is associated with a decreased Albeit growth inhibition has been observed endogenous mRNA mediated polypeptide in a variety of cell systems, fibroblastic synthesis capability of cytoplasmic extracts cells have been most extensively used to and polyribosome disaggregation [25] which investigate the differences between pro- is cyclohexamide sensitive [26]. Growth liferating and growth-arrested cells. Al- stimulation has resulted in an actinomycin though confluent, arrested cultures have D insensitive, increased polypeptide elonbeen called “contact” or “density” inhib- gation rate, as well as in alterations in riboited, it is well established that growth ar- some associated proteins [27]. Specific rest in many of these cases may be, in fact, tRNA nucleoside modifications have been induced by depletion of critical nutrients, found to be serum and cell density dependparticularly serum components [13, 141. ent [28]. Protein synthesis regulated at the The shift to a quiescent state of growth translational level has been found to be induced by suboptimal nutritional condi- obligatory for stimulation [29]. Increased tions is a growth regulatory process occur- transport of amino acids [30] as well as inring in Gl and has been called “restriction creased uridine and phosphate uptake [31] point control” [17]. Most of the widely have been observed associated with stimstudied cellular alterations associated with ulation. Increased cell surface glycolipids growth inhibition in confluent cultures are synthesis has been observed to occur at probably manifestations of this type of reg- confluency [32, 331. Cell size has been obulatory process. served to be reduced in confluent cultures Almost every level of cellular activity has [34, 351. In contrast to some of these findbeen studied in connection with this type ings, it has been observed that Chinese of growth regulation. Stimulation to prolifhamster cells fed daily did not show diferation to quiescent cells -has resulted in ferences in protein and RNA synthesis rates increased chromatin template activity and between exponential and stationary states in increased non-histone chromosomal pro- [36]. The apparent discrepancy may be retein synthesis [18, 19, 201. These activities solved if the differing responses are due to have been related to passage from GO to two different regulatory processes, restricGl by quiescent cells stimulated to pro- tion point growth inhibition due to subTG1=4.9 h, Ts=8.3 h, TG2=2.0 h, and TM=
0.7 h. These phase durations are comparable to the ones observed during exponential growth (table 2). We conclude that the stable cell number density of the plateau state is maintained by a process of cell death exactly cancelling the process of cell multiplication. In contrast to the gradually effected growth retardation by cell death observed in other systems [lS, 161,onset of growth retardation by cell death is apparently rapid in this cell system.
Exp CellRes 107 (1977)
Population growth inhibition in lymphoid cell line
optimal nutritional conditions and cell density-dependent, nutritionally independent growth inhibition. Fibroblastic cells grown to high cell density can detach from their substrate, presenting technical difficulties to attempts at establishing properties of high density cells. In the present study a lymphoid cell line grown in suspension under continuous culture conditions has been used to establish plateau states which are either nutritionally dependent or nutritionally independent. Both types of growth inhibition were thus examined. The results presented here demonstrate that growth-inhibited plateau states in the same cell system may be induced in two ways. A nutritionally dependent plateau state may be induced by limiting nutritional supply. Cells in this plateau state progressively accumulate in Gl or a state with Gl DNA content. Their cell size progressively decreases. These similarities to the case of arrested fibroblasts suggestthat cells in this state bear some correspondence to the GO cells arrested by nutritional factors discussed above. If nutritional sufficiency is insured, a higher cell density plateau state is generated. Dilution of the plateau state cells in plateau state media results in resumption of growth. In contrast to the case of nutritionally induced growth inhibition, there is no redistribution in the relative number of cells with respect to DNA content as growth becomes density inhibited. As discussed earlier, division control in density-inhibited cells is apparently not exercised by limitation of nutritional supply. The rates of incorporation of exogenous amino acid into cellular protein and glucose consumption are similar to those of exponentially proliferating cells. Cell size as determined by light scattering decreases during the plateau state. Although this is unexpected from the stability of amino acid
339
incorporation and glucose utilization rates, this decrease in cell size has been found consistently in several repetitions of the experiments. Proliferating (density-induced) plateau state cells have Gl, S, G2 and M phase durations similar to exponential cells. In order to maintain the constant cell density of plateau state, cell death must be occurring at a rate equal to the growth rate. This cell death process is not cell cycle phase specific in so far as the distribution of cells in Gl, S, G2 and M is the same for plateau state populations as for exponential populations. The simplest mode of cell loss consistent with the observed data is cell death occurring uniformly throughout the cell cycle. Cell lysis must rapidly follow cell death since the viability of the population, measured by trypan blue exclusion, is high. Because the transition from exponential to plateau state is abrupt, the control process effecting the cellular alterations described above must act rapidly. This suggests that control of these events is more likely to be exerted at the translational level than at the transcriptional or genomic level. No stable growth inhibitor could be found in the culture fluid of plateau state cells. It can be calculated, however, that at the onset of the density-inhibited plateau state, the averagecell-to-cell spacing is only approx. 3.5 cell diameters. Given that they probably are in close proximity of a neighbor a significant fraction of the time, it is possible to speculate that this degree of “contact” is sufficient to produce inhibition. Constant, physical contact is, however, certainly not required for inhibition. Except for these properties, the explicit mechanisms of SK-L7 population growth inhibition are still unknown. Cell volume is an index of cell maturation in the cell cycle and has been implicated as having a possible regulatory role Exp Cell Res 107 (1977)
340
Yen, Fried and Clarkson
in cell division. The relationship between cell size and cell cycle kinetics in this cell line has been investigated previously [3, 41. In an exponentially proliferating population heterogenous with respect to cell size (observed at mitosis) and generation time, smaller cells were found to have longer generation times due to protracted Gl durations. Differences in generation time were, therefore, associated with cell size heterogeneity. It may be asked whether the process of generation time or division regulation exercised is related to the processes at work maintaining nutritionally dependent or density-dependent plateau states. In both plateau states cell size decreases. If the regulatory processes responsible for generation time heterogeneity, i.e., the relatively slower proliferation of some cells in the population, were also responsible for growth inhibition in plateau states; then an increase in relative numbers of Gl cells would be associated with growth inhibition. Although this occurred with nutritionally inhibited cell populations, it did not with density-inhibited cells. It appears, therefore, that the regulatory processes involved in density-inhibited growth do not coincide with the regulatory processes involved in relative generation time retardation in a population with distributed generation times. Generation time heterogeneity is not a result of intercellular differences in the effect of the regulatory process responsible for density-induced growth inhibition. The accumulation in Gl of nutritionally arrested cells, however, suggests that generation time dilation in smaller cells may be induced by a minor relative nutritional deficiency due to or associated with a smaller cell size. The excellent technical assistance of Jean Campbell is gratefully acknowledged. We are grateful to Lana Exp CellRes 107(1977)
Hart for secretarial assistance in preparing this manuscript. This investigation was supported in part by Grant CA-16757, CA-08748 from the NCI, USPHS, Grants CH-6F and CH-6G from the ACS, and the United Leukemia Fund.
REFERENCES 1. Clarkson, B D, Strife, A & DeHarven, E, Cancer 20 (1%7) 926. 2. Clarkson, B D, Cancer res 27 (1%7) 2483. 3. Yen, A, Fried, J, Kitahara, T; Strife, A & Clarkson, B D, EXP cell res 95 (1975) 295. 4. - Ibid 95 (1975) 303. 5. Krishan, A, J cell bio166 (1975) 188. 6. Fried, J, Mathematical models in cell kinetics (ed A J Valleron) p. 68. European Press Medikon, Ghent, Belgium (1975). 7. - Computers biomed res 9 (1975) 263. 8. Fried, J, Yataganas, X, Kitahara, T, Perez, A, Ferguson, R, Sullivan, S & Clarkson, B, Computers biomed res 9 (1976) 277. 9. Fried, J, Perez, A Cl & Clarkson, B D, J cell biol 71 (1976) 172. 10. Hoffman, W S, J biol them 120 (1937) 51. 11. Thayer, P S, Tissue culture methods and applications (ed P F Kruse & M K Patterson) chap. 12. Academic Press, New York (1973). 12. Himmelfarb, P, Thayer, P S & Martin, H E, Science 164(1%9) 555. 13. Holley, R W & Kieman, J A, Proc natl acad sci US 60 (1968) 300. 14. Hohey, ‘R W, Control of proliferation in animal cells (ed B D Clarkson & R Baserga) p. 13. Cold Spring Harbor Laboratory (1974). 15. Steel. G G. Cell tissue kinet 1 (1968) 193. 16. Hartmann,’ N R & Dombemowsky,’ P, Cancer res 34 (1974) 32%. 17. Pardee, AB, Proc natl acad sci US 71(1974) 1286. 18. Rovera, G & Baserga, R, Exp cell res 78 (1973) 118. 19. ChZ;, N & Baserga, R, Biochemistry 14 (1975)
-_--.
20. Baserga, R, Bombik, B & Nicolini, C, The structure and function of chromatin, vol. 28 (new series), p. 269. Ciba Foundation Symposium (1975). 21. Augenlicht, L H & Baserga, R, Exp cell res 89 (1974) 255. 22. Johnson, L F, Williams, J G, Abelson, H T, Green, H &Penman, S, Cell 4 (1975) 69. 23. Johnson, L F, Penman, S & Green, H. J cell physio187 (1975) 141. 24. Williams, J G &Penman, S, Cell 6 (1975) 197. 25. Englehardt, D I & Samowski, J, J cell physio186 15 \--.-, *-.
f1979
26. Levine, E M, Jeng, D Y & Chang, Y, J cell physiol 84 (1974) 349. 27. Wheely, S M & Baserga, R, Biochim biophys acta 425 (1976) 234. 28. Katz, J R, Biochim biophys acta 383 (1975) 131. 29. Rovera, G, Farber, J &B~aserga, R, Proc natl acad sci US 68 (1971) 1725.
Population 30. Foster, D 0 & Pardee, A B, J biol them 244 (1969) 2675. 31. Cunningham, D D & Pardee, A B, Proc natl acad sci US 64 (1969) 1049. 32. Hirschberg, C B, Wolf, B A & Robbins, P W, J cell physiol85 (1974) 3 1, 33. Hakomori, S-I, Biochim biophys acta 417 (1975) 55.
growth inhibition
in lymphoid
cell line
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34. Kimball, R F, Perdue, SW, Chu, E Y H & Ortiz, J, Exp cell res 66 (1971) 17. 35. Meisler, A I, J cell sci 12 (1973) 847. 36. Hahn, G, Stewart, J R, Yang, S J & Parker, V, Exp cell res 49 (1968) 285. Received February 14, 1977 Accepted February 16, 1977
Exp Cell Res 107 (1977:
Experimental Cell Research 107 (1977) 325-341
ALTERNATIVE MODES OF POPULATION GROWTH INHIBITION IN A HUMAN LYMPHOID CELL LINE GROWING IN SUSPENSION A. YEN,’ J. FRIEDP and B. CLARKSON Laboratory
of Hematopoietic
Cell Kinetics, Memorial Sloan-Kettering New York, NY 10021, USA
Cancer Center,
SUMMARY Using a human lymphoid cell line grown under continuous culture conditions, two distinct plateau states were induced, either by lack of sufftcient medium-supplied nutrient, or by other unknown mechanisms dependent on cell density. Flow microfluorometric measurements show that growth arrest due to nutritional insufftciency results in an accumulation of cells with Gl DNA content. In contrast, growth arrest due to high cell density is not associated with an altered distribution of cells with respect to DNA content as the population progresses from exponential to plateau state growth. Cell size decreases with progression of the plateau state induced by either type of growth arrest. Cells in a plateau state induced by high cell density utilize glucose and incorporate exogenous amino acid into protein at approximately the same rate as exponential cells. Proliierating, high cell density, plateau state cells have cell cycle phase durations similar to exponential cells. The stable, plateau state cell density is maintained by cell loss. No stable, unbound growth inhibitory factor was found in the medium of density-inhibited plateau state cultures.
The typical proliferation pattern for mammalian cell lines consists of exponential growth for a limited time before growth inhibition occurs at high cell density. Cessation of population growth may be due to nutritional deficiency or may be induced by cell-cell interactions, mediated by unknown mechanisms. Both cell lines in culture and tumor cells maintained in animals have been extensively employed in studying cellular alterations associated with growth inhibition. In most of this work, no distinction has been made as to whether
the plateau state is due to a nutritional deficiency, such as exhaustion of some essential serum component, or to a cell-cell interaction associated with density inhibition of population growth. Making this distinction in practice is complicated. In substrate-attached cells in culture at advanced states of growth, cell crowding reduces the cell surface area interfacing medium and hampers any experimental approach to obviate the possibility of cell growth inhibition due to nutritional insufficiency. This problem is exacerbated in animal tumor cell systems where the cell population is depen1 Present address: Department of Cell Growth and dent on the animal host for nutritional Regulation. Sidney Farber Cancer Institute, Boston, supply. This is particularly in evidence in MA 02115;USA. solid tumors exhibiting internal necrosis. * To whom reprint requests should be addressed. Exp Cell Res 107 (1977)
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Yen, Fried and Clarkson
The problem this study addresses is the establishment of distinct plateau growth states due to either nutritional insufficiency or to social cell interactions at high cell density. The cells used in this study are from a human lymphoid line. To circumvent the nutritional problems described above, the cells are cultured in suspension in a commercially available spin filter, continuous culture device. In this culture system, used medium in the cellular environment is constantly exchanged for fresh medium without loss or disruption of cells in the culture vessel. Definition of a plateau growth state caused by cell-cell interaction at high cell density and independent of nutritional influences allows characterization of the cellular activities associated with proliferation regulation in density inhibition of growth. Although the absence of number increase in a density-inhibited cell population of high viability suggests that these cells may be “resting” or “quiescent”, it will be shown that these cells can be as metabolically active as exponentially proliferating cells as indexed by their glucose consumption from the medium and rate of incorporation of exogenously supplied amino acid into protein. The dynamics of cell size changes during population growth from exponential through nutritionally and density-induced plateau phases will be described. Variations in relative cell cycle phase transit times or cell cycle phase specific maturational blocks associated with nutritionally or density induced plateau growth states will be derived using flow microfluorometry and DNA fluorescent staining. The cell cycle kinetics of density-induced plateau state cells will be described. It will be shown that the stable cell density of the high cell density plateau state is maintained by cell death. Exp Cell Res 107 (1977)
MATERIALS
AND METHODS
Cells The cell line used, SK-L7, is a human lymphoid line which has been continuously cultured for over 10 years. Its properties and origins have been previously described [l-4]. Stock cultures were maintained in Falcon T-75 culture flasks (Falcon Plastics) in McCoy’s medium 5A (modified) (Gibco). The cells were growing exponentially when harvested from culture flasks for addition to the spin filter continuous culture device in preparation for an experiment. Cell densities were deteimiied by repeated cell counts using a hemacytometer. Cell viability was measured as the percentage of cells excluding 0.1% trypan-blue. Tests for the presence of mycoplasma were negative.
Determination
of cell size
The distribution of relative cell size in a population aliquot was measured with a Cytograf hiodel 6300 @o/Physics Systems, Inc.). The resulting output signals are accumulated in a multi-channel pulse-height analvzer (Model NS 602, Tracer-Northern Scientific, Inc.; Middleton, Wise.). The mean cell size was measured as the mean channel number of the resulting unimodal distribution accumulated by the multichannel analyzer. Measurement of standard polystyrene microspheres (Particle Information Se+&, -Inc., Grants Pass, Ore&) of a variety of diameters demonstrated a linear r&tionship between particle size and analyzer channel number. All measurements of cell size were made immediately after withdrawal of the cell sample from the culture vessel.
Determination of distribution in GI, S, and G2+M
of cells
The number of celIs with Gl, S, and G2 DNA content was determined by flow microfluorometry using a Cytofluorograf Model 4802 (BiofPhysics Systems, Inc.). An aliquot of 5x 1V cells in no more than 0.25 ml was mixed with 5 ml of a hvootonic solution of the fluorescent dye propidium iodide (0.05 mg/ml of prooidiumiodide in 0.1% sodium citrate) r51 and mainiained on ice for at least 10 min. Thd &ned nuclei are then analysed with the microfluorometer. The distribution of number of nuclei versus relative fluorescence intensity was analysed to yield the fraction of cells in the aliquot in Gl, S, and G2+M. The mathematical fitting method used for this analysis is described elsewhere [6, 7, 81. Briefly this method assumes that cellular DNA content is compartmentalized with values ranging from Gl DNA content to G2 DNA content. Each compartmentalized cellular DNA content is assumed to produce a normally distributed, emitted fluorescence intensity. The summed distribution of fluorescenceintensitiesis then fitted to the observed distribution to derive the number of cells with each DNA content. Experiments recently carried out with the SK-L7 cell line demonstrated the validity of the propidium iodide staining method and the mathematical method used for the analysis [9].
Population growth inhibition in lymphoid cell line
4
Determination of glucose concentration in culture jluid Glucose concentration in medium from the culture vessel was determined with a Technicon Autoanalyzer I using the potassium ferricyanide method, a modification of Hoffman’s method [IO]. Cell samples of 10 ml were withdrawn from the culture vessel. The cells were immediately pelleted. The supernatant was decanted and refrigerated until analysed for glucose concentration.
Measurement of rates of incorporation of exogenous amino acid into protein Relative rates of incorporation of exogenous amino acid into cellular protein at various population growth states were measured by the uptake of [sH]Ile into cold TCA-precipitable material. For each measurement. the cells in 10 ml of cell susnension withdrawn from’the culture vessel were pelleted by centrifugation (800 rpm, 160 g, for 5 min) (Model PR-6000 centrifuge, DammonlIEC Division), the supematant aspirated. and the cells resuspended in 10 ml of fresh medium at 37°C with 200 -pCi of [sH]Ile (spec. act. 83.4 CilmM. New E&and Nuclear). At this time and 5, 10 and 15 min late< 0.5 ml samples of cell suspension were withdrawn, brought to a final volume of 5 ml with ice-cold physiological (0.9 %) saline, and stored on ice until all four samples were taken. The cells were incubated at 37°C in 5 % CO* and 95 % air between samulina. All four samples were maniuulated simultaneously-after this point.i”ne cells were pelleted in a refrigerated centrifuge (3000 rpm, 2250 g, for 5 min) at 2”C, the supernatant decanted, and the cells resuspended in 5 ml ice-cold physiological saline. This wash was repeated twice more. On the final wash, the nellet was resusnended in 1 ml of ice-cold physiological saline and 1ml of ice-cold 25 % TCA was added. Following agitation, the precipitate was pelleted by centrifugation (3 000 rpm, 2 250 g, for 5 min at 2”C),
327
Fig. I. Schematic illustration of continuous culture device. I, 4 liter main medium reservoir: 2. intermediate reservoir maintained at 37°C: 3, oeristaltic oumn for medium input; 4, c&t& vessel maintained at 37°C; 5, air input; 6, peristaltic pump for removal of medium from culture vessel, regulated by level sensor; 7, level sensor regulator for peristaltic pump 6; 8, medium reservoir for medium removed from culture vessel. the supematant decanted and the precipitate resuspended in 2 ml of ice-cold 12.5% TCA. The above pelleting, decanting and resuspension were repeated twice more. The precipitate ias then collected on a GF/C glassfiber filter (Whatman) which was subsequently washed with 10 ml ice-cold 12.5% TCA and then 5 ml ice-cold ethanol. Radioactivity on the dry filters was counted using Aquasol liquid scintillation cocktail (New England Nuclear) in a Beckman LS2OOB scintillation counter. The same stock of medium supplemented with fetal calf serum and with 13H]Ile added was used in all measurements of [3H]Ile &&oration to be compared, obviating error induced by differences in specific activity of label due to possible variations in-serum or media concentrations of isoleucine.
Continuous culturing of cells In the experiments to be described, SK-L7 was grown in suspension using a spin filter culture device [ll, 121. The culture vessel-and its ancillary equipment are shown schematically in fig. 1. As used in this studv. the culture vessel (ADL Biosoin Culture Flask ModLculture System, V&is Co., G&diner, N.Y.) contained 450 ml of cell suspension accessed through eight ports and access tubes in the top of the vessel. Briefly, in this culture system, a spinning (650 rpm) cylindrical, glassfiber filter supported within the culture vessel performs the two functions of maintaining the cells in suspension and mixing the culture fluid. It allows medium to be exhausted from the culture vessel through the spinning filter which excludes cells. Bv virtue of the fluid, shear boundary at the surface of the spinning filter, retention of debris, e.g., cells, by the filter is minimized. In this way, cells are retained in the culture vessel while used medium is being removed and fresh medium influxed. In our hands, this system has been effective for up to approx. 230 h of continuous running prior to failure of the spin filter unit. As shown in fig. 1, fresh medium, Exp Cell Res 107 (1977)
328
Yen, Fried and Clarkson Continuous
loo-
IO
20
30
40
50
60
Fig. 2. Abscissa: time after seeding spin filter flask (hoursl: ordinate: cell densitv X lob4 cells/ml. l - - -0, .~~ 0.2; O&, 1.0 ml/mitt medium intlux. Growth curve illustrating nutritionally limited population growth.
labelling
and autoradiograph:
Exponential cells at a cell density of 2.5 x lo5 cells/n at initiation of labelling were exposed to [3H]thymidin continuously for 16 h (spec. act. 6 Ci/mM, New Eng land Nuclear). Because of medium exchange durin continuous culturing, the concentration of label ranges from 0.05 &i/ml initiallv to 0.035 uCi/ml tinallv Plateau state cells at a cell density of approx. 6~ lc cells/ml were continuously labelled for 36 h after th plateau state had existed for 10 h. [3H]Thymidin (snec. act. 6 CilmM) was iniected into the cultur vessel every 6 ‘h maintaining the concentration c at 0.05 &i/ml to 0.023 &i/ml. A .13Hlthvmidine ., < each sampling time, 1.b ml of cell suspension wa withdrawn. Slides were made using a Cytocentrifug (Shandon Scientific Co., Limited, Sewickley, Pl loaded with 0.2 ml of cell suspension which had bee diluted with ice-cold medium-if necessary-t 250000 cells/ml per slide. Autoradiography was don with Kodak NTB2 emulsion. The exposure period wa 4 weeks. Cells with four or more grains over the m cleus were considered labelled. The background grai count was less than one per nucleus.
RESULTS stored in a 4 1 reservoir, flows into an intermediate reservoir maintained at 37°C before enterina the culture vessel. A precise intlow rate is mainb&ed by a peristaltic pump (Model 500 Automatic, Self-Regulating, Intravenous, Infusion Pump., Ivac Corp.). A liquid level sensor (LLSP-2 level sensing probe with carbon electrode and 57LC level controller, Virtis Co., Gardiner, N.Y.) within the culture vessel maintains a constant culture fluid volume by activating the exhaust pumping of medium (Model PP325 Peristaltic Pump, Virtis Co.) through the spin filter as fresh medium is continuously added. The total variation in volume of culture fluid over time is less than 10 ml. The DH of the culture fluid is monitored bv a DH electrode (Ingold Electrodes, Inc.) within the cult&e vessel. With cell growth and respiration, the medium became acidic. By influxing air into the culture vessel to purge it of CO1 and by titration with 5 N NaOH as needed, the pH of the culture fluid was routinely maintained at 7.0f0.3. Cell samples were withdrawn through one of the access tubes in the top of the culture vessel, using sterile hypodermic syringes. The culture vessel and intermediate medium reservoir were maintained at 37°C in a constant temperature water bath. The cells were at-own in McCov’s 5A medium (modified) supplemented with 30% fetal calf serum and 1% PSN (Penicillin, Streptomycin, Neomycin) Antibiotic Mixture (Gibco). Thetrypan blue excluding fraction in exponential and plateau state populations was routinely in excess of 99%. In most cases the duration of cell growth in this system was terminated by mechanical failure of the spin filter, which has a limited (and unpredictable) useful lifetime. Cell samples taken from different levels within the culture vessel were similar in cell density and absence of cell clumps, as expected of a uniformly dispersed suspension of cells. Exp Cell Res 107 (1977)
Plateau state population growth dependent on and independent of exogenous nutritional supply
In the continuous culture device employee in this study, fresh medium is continuousl! infused into a cell culture vessel as mediun from within the culture vessel is removed maintaining a constant volume of cuim fluid in the vessel. Cells are retained it suspension within the culture vessel as the medium is exchanged. If nutritional suppl! were the only growth limiting factor acting then the cell population could expand unti the rate of nutrition consumption by the ccl population was equal to the rate of nutri tion influx. If the rate of medium infusior were subsequently increased, then prolifer ation should resume. Fig. 2 shows the po pulation growth resulting from a fixed rate of medium infusion and the effect of sub sequently increasing the rate of infusion A medium infusion rate of 0.2 ml/min car responding to a medium fractional turnove: rate of l/38 (h-l), results in an exponentia increase in cell density to a plateau value
Population growth inhibition in lymphoid cell line
329
The initial cell density in this experiment was much lower than in that described by fig. 2 showing that the growth end point of 2.1 x lo6 cells/ml is independent of initial cell density as expected by nutritional considerations. Increasing the infusion rate to 0.4 ml/min resulted in population growth to a new plateau state with cell density of approx. 3.8~ lo6 cells/ml, nearly twice the maximum cell density with a 0.2 ml/min infusion rate. Increasing the infusion rate to 1.0 ml/min resulted in further population growth. The resulting maximum cell Figs 3, 4. Abscissa: time (hours); ordinate: X10+ density, however, was approx. 6~ 10” cells/ cells/ml. O---O, 0.2; O---O, 0.4; O-O, 1.0 ml/min ml. Based on the previous nutritional conmedium influx. Nutritionally and high cell density-limited populasiderations, the anticipated plateau state tion growth. Medium influx rate was increased to 0.4 ml/min at t = 132 h after a 16 h plateau phase and to cell density at 1.0 ml/min should have been 1.O ml/min at t= 180 h following a 12 h plateau phase. 9.5~10~ cells/ml, i.e. (1.0/0.4)X(3.8). Population growth therefore appeared to be inof approx. 2.1 x log cells/ml. The exponen- hibited by a mechanism or mechanisms tial doubling time was approx. 15.3 h. The other than nutritional deficiency. cell density then failed to increase for 20 h It can be confirmed that the plateau state until the medium infusion rate was in- at approx. 6x lo6 cells/ml is not dependent creased. Increasing the medium infusion on the previous occurrence of growth-inhirate at this time from 0.2 ml/min to 1.0 ml/ bited states. Fig. 4 shows the continuous min resulted in a resumption of population exponential growth and final plateau state growth. The implication is that a medium at approx. 6x lo6 cells/ml resulting from infusion rate of 0.2 ml/mm induces a plateau state of cell density 2.1 X lo6 cells/ml, through nutritional limitation. If the medium infusion rate were 0.4 ml/ min and nutritional supply were the only growth-limiting factor, then the anticipated plateau state cell density at this infusion rate is approx. 4x lo6 cells/ml. Likewise, any increase in infusion rate should result in a proportionately increased, plateau state cell density. Fig. 3 shows the population growth resulting from an initial medium infusion rate of 0.2 ml/min which was subsequently increased to 0.4 ml/min and finally to 1.0 ml/min. The infusion rate of 0.2 ml/ 4. Growth curve showing effect of changing inmin again resulted in a plateau state with Fig. flux rates prior to onset of nutrient-limited plateau cell density of approx. 2.1 x lo6 cells/ml. phase. Exp Cell Res 107(1977)
330
Yen, Fried and Clarkson
cessively from 0.2 ml/min to 0.4 ml/min to 1.O ml/min prior to the nutritionally deficient growth arrest anticipated at each infusion rate. When the population cell density was approx. 6~ lo6 cells/ml the previously observed maximum with a 1.0 ml/min infusion rate, the media infusion rate was increased from 1.0 ml/min to 2.0 ml/min. Nevertheless, the ensuing plateau state occurred almost immediately at a cell density of approx. 6.5-7~ lo6 cells/ml. This demonstrates that the growth-limiting mechanism 60 70 80 90 100 0 10 20 ;O 40 50 in the plateau state occurring at a cell denFig. 5. Abscissa: time (hours); ordinate: X lo-* cells/ sity of approx. 6x lo6 cells/ml is not priml. l - - -0,0.4; O-O, 1.Oml/min medium influx. marily depletion of the exogenous nutritioHigh cell density-limited population growth. nal supply, but some other process depensuccessively increasing the medium in- dent on cell density. Since the plateau fusion rate from 0.2 ml/min to 0.4 ml/min states observed at lower cell densities were and then to 1.0 ml/min prior to the occur- due to depletion of critical nutrients in the rence of growth arrest anticipated for each medium, these results demonstrate that two infusion rate. This observed growth and kinds of plateau states may be generated arrest are not dependent on the choice, in this cultured cell system. Although cell sequence and timing of infusion rates. The number density is stable during both types population growth resulting from an initial of plateau states, growth inhibition of the infusion rate of 0.4 ml/mm increased sub- population does not exclude the possibility sequently to 1.0 ml/min is shown in fig. 5. of ongoing cellular activity, including proThe 0.4 ml/min to 1.0 ml/min infusion rate liferation, in a steady state renewal process. increment occurs at a lower cell density Further references to “growth inhibition” than in the case of fig. 4. Nevertheless, in this report imply only the absence of inthe growth and plateau state shown in fig. 5 crease in number density of the population. are similar to that of fig. 3. The growth in- Certain cellular properties and activities hibition occurring at a cell density of ap- during each plateau state will now be disprox. 6~ lo6 cells/ml is, therefore, indepen- cussed. dent of the particular way the cell density Distribution of DNA contents in is generated in this culture system. If the plateau state with cell density of exponential; nutritionally inhibited and approx. 6~ lo6 cells/ml were due to nutri- density inhibited populations: Behavior tional depletion, then increasing the me- as a function of growth phase dium infusion rate to greater than 1.0 ml/ The fraction of a population with each of min prior to growth arrest should lead to a Gl , S, or G2+M DNA content at any time commensurate increase in the final plateau may be determined by flow microfluorostate cell density. Fig. 6 shows the result- metry as described earlier. It will be shown ing exponential population growth as the that monitored by this means, nutritionally medium infusion rate was increased suc- induced growth inhibition and density-in1000
I
/,/I 100 I
ExpCellRes 107(1977)
Population growth inhibition in lymphoid cell line
101 ’ 0 IO
’
20
’
30
’
40
’
50
’
60
’
70
’
60
’
90
’
100
Fig. 6. Abscissa: time (hours); ordinate: x lo-’ cells/ ml. o---o, 0.2; o---o, 0.4; O-0, 1.0; o-e-0,
2.0 ml/min medium inkx. High cell density-limited population growth. Arrows designate sampling times for medium glucose concentration determinations.
duced growth inhibition are distinguishable. For the population described in fig. 2, a case of nutritionally induced growth inhibition, fig. 7 shows the percentage of cells with Gl, S, and G2+M DNA content as a function of time. During the initial 28 h of exponential growth, the distribution of cells with respect to DNA content is stable. The following 20 h of nutritionally deficient plateau state eventually result in an increased fraction of the population with Gl DNA content and a decreased proportion of cells with G2+M DNA content. During this time there is a transient increase in the relative number of S phase cells. The processes responsible for this redistribution during plateau state may involve retardation of cell maturation in Gl, selective cell death in S, G2 and M, or both. Increasing the nutritional supply after a 20 h plateau state resulted in a reduction in relative numbers of cells with Gl DNA content and increased percentages with G2+M DNA content. The results may be interpreted as a release from the altered cell cycle kinetics imposed by the plateau state. Growth inhi-
331
bition by nutritional insufficiency, therefore, is associated with a reversible accumulation of cells with Gl DNA content and depletion of cells with G2+M DNA content. Growth inhibition due to density inhibition independent of exogenous nutritional supply results in behavior distinct from the case of nutritionally induced growth inhibition. Fig. 8 shows the percentages of cells with Gl, S and G2+M DNA content as a function of time for the population undergoing density inhibition of growth described in fig. 5. The percentages of cells with Gl, S, and G2+M DNA content are approx. 40, 50 and lo%, respectively and remain stable as the population progresses from exponential into plateau phase. The distribution of cells with Gl , S and G2+M DNA content for the population described in fig. 6 is virtually identical to that shown in fig. 8 and is also independent of growth phase. The similar results in these two cases confirm that the two density-inhibited plateau states are alike independent of the differences in the ways in which they were obtained. The stability of the distribution of
20 IO 0
'-.~,,***--'.\..* E ---*________-c
I 0
I IO
I 20
I 30
I 40
I 50
I 60
time (hours); ordinate: % of population in each cell cycle phase. (a) Gl; (b) S; (c)
Fig. 7. Abscissa: G2+M.
Distribution of cells with respect to cell cycle phase during exponential (O- - -0) and nutritionally limited plateau state (O-O) growth (see fig. 2). Exp Cell Res 107 (1977)
332 50 40 30
Z0 IO 0
Yen, Fried and Clarkson
These results suggest that density-in..--.-.*--*--)-----~--.--*---~ duced growth inhibition does not act at the E cellular level by simply restricting the upa
take of the same nutritional factor involved in the nutrient deficient plateau state. Were this the case, then a redistribution of cells with respect to Gl, S and G2+M DNA content similar to that induced by nutritional deficiency, i.e., as in fig. 7, would be expected; and this does not occur. It will be demonstrated later that, consistent with the lack of restriction of nutrient uptake, the rates of consumption of glucose from the culture medium and incorporation of exogenous amino acid into cellular protein are not reduced in density-inhibited plateau state cells.
a.-*--*-___ )--*-.--‘-__. : -_. EL I*----I I I 11I / I c
10
20
30
40
50
6a
70
so
90
100
Fig. 8. Abscissa: time (hours); ordinate: percentage of population in each cell cycle phase. (a) Gl; (b) S; (c) G2+M. O---a, Exponential growth; O-e, plateau state. Distribution of cells with respect to cell cycle phase during exponential and high cell density-limited population growth (see fig. 5).
cells with respect to DNA content as shown by flow microfluorometric analysis (fig. 9) is a characteristic of density-induced growth inhibition in this cell system. Although the distribution of cells among the phases of the cell cycle is the same in the plateau growth state as in the log phase, it is obvious that some of the cellular processes occurring under the two conditions must be different. If the action of a growth inhibitory process were simply to retard cells in a single cell cycle phase, then the occurrence of plateau state would be associated with an enhancement in the relative number of cells in that cell cycle phase. In the case of nutritionally induced growth inhibition, the results of fig. 7 are consistent with the existence of some Gl specific retardation of cell maturation, exerted during plateau state and released during growth resumption induced by increased nutritional supply. Density-induced growth inhibition, in contrast, acts as seen in fig. 8 with no simple cell cycle specificity of this character. Density-induced growth regulation, therefore, does not involve a process restricted to any single cell cycle phase. Exp CellRes 107 (1977)
Fig. 9. Abscissa: fluorescence intensity (channel no.); ordinate: cells x 103. (a) High cell density plateau; (b) exponential. Distribution of cells with respect to DNA content during exponential and high cell density plateau states as inferred from flow microfluorometric analysis. Lower diagrams show DNA histograms (data points shown as dots)-together with results of the mathematical analysis. Solid lines show the theoretical histogram corresponding to the solution and the calculated distributions of fluorescence intensities of the Gl, G2+M, and S phase populations. For clarity, the distribution of S phase cells is shown separately in the upper diagrams. The histograms describe the population shown in fig. 6 at 36 (exponential) and 80 (high cell density plateau) hours.
Population growth inhibition in lymphoid cell line Table 1. Glucose cone. in culturefluid during exponential (O-76 h) and plateau state (76-96 h) growth
m(t) -=coV
R -No z v+K
333
-I, eKt+De v
Cell
Growth state
Hour
density (cells/ml)
Glucose M8ho
:: 36 42 89 92
3.55ooo 525 ooo 769 000 1 165 000 6.9x 10“ 6.8X 106
2.24 2.12 1.81 1.60 2.39 1.22
Exponential
Plateau
Rates of glucose consumption and incorporation of exogenous amino acid into protein in exponential and density-inhibited cells During the period indicated by the population growth curve shown in fig. 6, the glucose concentration measured in the culture fluid is given in table 1. These data can be used to calculate the average rate of glucose uptake per cell per hour. The concentration of glucose in the culture fluid is described by the following differential condition based on conservation of total glucose:
dm(t),c -,mW ---Vn(t)R dt
’
V
where m(t) is the total glucose contained in the culture fluid as a function of time; co, the glucose concentration in fresh input medium; I, the rate of medium input; n(t), the cell density as a function of time; V, the volume of cell suspension in the culture vessel; R, the average rate of glucose uptake per cell per hour. In the case of exponential growth where: n(t)=N,efQ the solution is 22-771819
All the constants except R and D are known parameters of the experiment. K is derived by a least squares fit to cell densities during exponential growth. A fit to the glucose concentrations during exponential growth given in table 1 yields a value for R of 0.6~ lo-’ mg/hour-cell, i.e., approx. 3 x lo-lo mM/hour-cell. The resulting theoretical curve and experimental values for glucose concentration in the culture fluid are given in fig. 10. In the case of the plateau state, an analogous analysis, using n(t) equal to the plateau state average cell density, yields a value for R of 0.9~ lo+ mglhour-cell, i.e., approx. 5x lo-lo mM/hour-cell. The uncertainty in the derived values of R is equal to at least the difference between the values for exponential and plateau state cells. Furthermore, the theoretical curve of fig. 10 is insensitive to this degree of variation in the value of R. We conclude that exponentially proliferating cells and density-inhibited, plateau state cells consume glucose from the media at approximately the same rate.
25
r
zo-
=b 0
IO
20
30
40
Fig. 10. Abscissa:
time (hours); ordinate: mg/ml. Concentration of glucose in medium during exponential growth. Dots, data points; solid line, theoretical concentration curve computed as described in text. Exp Cell Res 107 (1977)
334
Yen, Fried and Clarkson
IOOO/r / I,./ IOO,/’ ,,” ,Y” -.,,*’ ,/’ /’ Ic 10: 0
IO
20
30
40
50
60
GQ, \A)
70
SO PO 100 110 120
Fig. Il. Abscissa: time (hours); ordinate: x lo-’ cells/ ml. o---o, 0.2; o---o, 0.4; O-0, 1.0; o-a-0, 0 ml/mm medium influx. High cell density-limited population growth showing effect of cessation of medium infhrx during plateau phase. (%), cell viability determined by trypan blue exclusion. Arrows, times of amino acid uptake measurements.
Consistent with this finding is the observation that terminating fresh medium input to the density-inhibited plateau population (fig. 11) results in extensive cell death. After the plateau state had been maintained for 28 h, medium influx (and efflux) was terminated. Approx. 25 h later the viable cell density had decreased to approx. 3.6~ lo6 cells/ml. Cell viability (as estimated by trypan blue exclusion) had decreased from 99% to55%. The rate of incorporation of exogenously supplied [3H]Ile into cellular protein was determined at three times during the population growth shown in fig. 11. The incorporation of r3H]Ile into cold TCA-precipitable radioactivity measured at 32 h (exponential growth), 68 h (early plateau state), and 74 h (plateau state) is shown in fig. 12. The incorporation observed in all three cases is similar. The absence of any lag in the observed uptake demonstrates that there was no pool effect which would’ Exp CellRes 107(1977)
have resulted in an initial dilution of label and a resulting lag in the onset of linear incorporation, Although the observed rate of incorporation is dependent on a variety of cellular processes such as transport, intra-cell precursor compartmentalization, etc., it is unlikely that all these processes would change at 3 different times in exact concert to yield the same observed incorporation rates. These results are consistent with the existence of similar rates of protein biosynthesis at the three times of observation. The results presented here demonstrate that the density-inhibited plateau state cells do not differ from exponential cells in their rates of glucose utilization or incorporation of exogenously supplied amino acid into cellular protein. Neither do they tend to accumulate in a portion of the cell cycle as under conditions of nutrient deficiency discussed earlier. The cells of the densityinhibited plateau state population are, therefore, apparently as metabolically active as exponential cells.
Cell size during exponential, nutritionally inhibited and density inhibited growth The cell size was monitored as described earlier by low angle light scattering. The population described in fig. 2 underwent
Fig. 12. Abscissa: time after addition of tracer (mm); ordinate: cpm/5x W cells. TCA-precipitable radioactivity during [WjIle labelling of (a) exponential; (b) early plateau; (c) plateau state cells. Samples were obtained at times (a) 32 h; (b) 68 h; (c) 74 h (see fig. 11).
Population growth inhibition in lymphoid cell line
335
causally related to resumption of cell proliferation.
I
0
I
10
I
20
I
30
I
40
I
50
I
60
Fig. 13. Abscissa: time (hours); ordinate: channel. l - - -0, Exponential growth; O-O, plateau state. Rel. mean cell size during exponential growth and nutrient-limited plateau phase (see fig. 2).
.
growth inhibition by nutritional insuffkiency followed by resumption of growth following increase in medium intlux rate. Its relative mean cell size as a function of time is given in fig. 13. The mean cell size as well as the entire observed size distribution was stable during exponential growth. The onset of plateau phase was associated with an immediate progressive decrease in cell size. Population growth resumed at 48 h (fig. 2). The cell size, however, remained unchanged from 48 to 52 h (fig. 13). A lag of 4 h occurred following the resumption of growth before any change in cell size occurred. Cell size then increased at the same rate at which it had previously decreased. At all times the distributions of cell sizes were unimodal and similar in halfmaximum width and almost symmetric shape, while the means shifted as described. In the case of a population undergoing density inhibition of growth, fig. 14 shows the mean cell size as a function of time for the population described in fig. 5. Cell size was again stable during exponential growth and decreased progressively with the onset of plateau state. These results show that cells of both nutritionally and density-induced plateau states decrease progressively in size. The occurrence of a lag in the response of cell size to growth resumption shown in fig. 13 indicates that a cell size increase is not
Absence of a stable growth-inhibiting factor existing free in density-inhibited stationary state culturefluid The simplest mechanism by which a density-inhibited plateau state could be effected is by means of a growth-inhibiting factor in’the culture fluid of plateau state populations. It will be shown, however, that in this cell system no such stable factor exists in a free state. During the experiment shown in fig. 4, 3X 106plateau state cells in a volume of approx. 0.5 ml were removed from the culture vessel at 110 h, after 16 h of plateau state, and inoculated into 100 ml of cell-free culture fluid obtained at the same time from the culture vessel. (This was performed in duplicate.) In 35 h the cell density had increased S-fold with a doubling time of 15 h. This demonstrates that culture fluid from a density-inhibited plateau state culture can support cell growth, and that cells from a density-inhibited plateau state culture retain their proliferative capacity at lower cell densities. Confirming these results, it was also observed in separate experiments that both plateau state cells resuspended in 99.5 % fresh medium at low density and exponential cells inoculated in 95 % plateau state culture fluid proliferated exponenti) ______ * _____-. --------------.---
72
70 68 66
64 62
,
60I, ,o
20
, , 30 40
,
,
,
50
60
70
“:.. 80 90 100
Fig. 14. Abscissa: time (hours); ordinaie: channel. Rel. cell size during exponential or high cell densityinduced plateau phase. Differences in absolute channel number as compared with fig. 13 were due to differences in laser light output. Exp Cell Res 107(1977)
330
Yen, Fried and Clarkson in figs 4, 5, and 6, the plateau state cell density is not sensitive to the fractional turnover rate. The existence of a growthinhibiting factor is, therefore, not apparent in situ in the culture vessel.
7060-
P=
50-f 40-t 4 20: IOC
‘0
4
8
12
16
Fig. 15. Abscissa: time after beginning continuous exposure to rH]thymidine (hours): ordinate: (a) LI (%); (6) MI (%); (c) PLM (%). LI, MI, and PLM during continuous labelling of exponential cells. The 0 time sample was taken immediately after addition of r3H]thymidine to the cell cultures.
Cell cycle phase durations of exponential cells Fig. 15 shows the percentage of cells labelled (LI), percentage of cells in mitosis (MI), and percentage of mitotic cells labelled (PLM) as a function of time during continuous [3H]TdR labelling of an exponentially growing population. The data are those typically associated with an exponential population. The age density function, n(a, t), of a population is the number of cells of age a to a +du at time t. For an exponentially growing population where the distribution of generation times is not broad, n(a, t) may be approximated by:
ally. The inferred result is that there is no factor which is stable and free in the culture n(0, 0)exp -F (u-t) ,O
Population growth inhibition in lymphoid cell line Table 2. Cell cycle phase durations (hours) Cell cycle phase
Cl
s
G2
M
Exponential cells ’ High cell density plateau state
4.8
8.0
2.5
0.5
4.9
8.3
2.0
0.7
where TG is the mean cellular generation time. A least squares fit of the growth curve to N,exp[(ln2/T,)t] yields TG= 15.9 h, assuming no cell loss. The duration of mitosis, TM, is derived from: 7,
MI=
s
n(a, t)da :“,-” c n(u, t)du
Jo
(2)
337
LIo is the fraction of cells which would be labelled in a pulse label and should represent the fraction of the population in S. The numerical value of LIo was derived from fig. 15 as the average of LI’s at 1.0, 1.5, and 2.0 h and is equal to 0.48. Eq (5) yields a Gl duration of 4.8 h. The duration of S is TG-TGI-TGP-TM and is equal to 8 h. In agreement with the observed distribution of cells with respect to DNA content, the fraction of cells with Gl DNA content calculated from TG, o s da, Oda TC n(u, t)du s0
as:
is 0.38. The fraction of cells with S DNA content estimated from the labelling index TM=% ln(MI+l.O) (3) is 0.49, and the fraction with G2+M DNA content is 0.13. The average mitotic index during the continuous labelling was 0.023. The duration of Continuous [3H]thymidine labelling of plateau state cells M is derived from eq (3) to be 0.5 h. The duration of G2 is equal to the time Fig. 16 shows the LI, MI, and PLM during required for cells labelled in late S to reach continuous labelling with [3H]thymidine of mitosis. From the PLM curve of fig. 15, density inhibited plateau state cells. The behavior of LI, MI, and PLM is similar to this time is approx. 2.5 h. The duration of Gl is derived from the that observed for exponentially growing equation cells (fig. 15) and indicates that cells in the plateau state population are traversing the TG-i-,-T,, cell cycle. Since the distribution of plateau n(u, t)da s LIo= ,;I * (4) state cells with respect to DNA content is the same as that of exponential cells, eq n(u, t)da (1) also approximates the age density func0 tion of plateau state cells. The transit times as of these cells through Gl, S, G2, and M were derived in the same manner as preTG1=-2ln { F+exp[-$f(TG-TM-TG2,]i viously. LI, was in this case 0.49. The average mitotic index was 0.03. From fig. 16 (5) the durations of the various phases were:
J”
Exp CeNRes 107 (1977)
338
Yen, Fried and Clarkson
liferate [21]. Stimulated cells also show alterations in chromatin structure detectable by ethidium bromide uptake and circular dichroism [ 191. A post-transcriptionally regulated increase in mRNA occurs in cells stimulated from quiescence [22]. Cells stimulated from quiescence have a posttranscriptionally regulated increasedpolyA(+)mRNA content which does not depend on the accompanying increased number of ribosomes [23]. Comparison of mRNA from proliferating and quiescent cells shows the majority of mRNA sequences in each case are common to both [24]. The occurrence of DISCUSSION quiescence is associated with a decreased Albeit growth inhibition has been observed endogenous mRNA mediated polypeptide in a variety of cell systems, fibroblastic synthesis capability of cytoplasmic extracts cells have been most extensively used to and polyribosome disaggregation [25] which investigate the differences between pro- is cyclohexamide sensitive [26]. Growth liferating and growth-arrested cells. Al- stimulation has resulted in an actinomycin though confluent, arrested cultures have D insensitive, increased polypeptide elonbeen called “contact” or “density” inhib- gation rate, as well as in alterations in riboited, it is well established that growth ar- some associated proteins [27]. Specific rest in many of these cases may be, in fact, tRNA nucleoside modifications have been induced by depletion of critical nutrients, found to be serum and cell density dependparticularly serum components [13, 141. ent [28]. Protein synthesis regulated at the The shift to a quiescent state of growth translational level has been found to be induced by suboptimal nutritional condi- obligatory for stimulation [29]. Increased tions is a growth regulatory process occur- transport of amino acids [30] as well as inring in Gl and has been called “restriction creased uridine and phosphate uptake [31] point control” [17]. Most of the widely have been observed associated with stimstudied cellular alterations associated with ulation. Increased cell surface glycolipids growth inhibition in confluent cultures are synthesis has been observed to occur at probably manifestations of this type of reg- confluency [32, 331. Cell size has been obulatory process. served to be reduced in confluent cultures Almost every level of cellular activity has [34, 351. In contrast to some of these findbeen studied in connection with this type ings, it has been observed that Chinese of growth regulation. Stimulation to prolifhamster cells fed daily did not show diferation to quiescent cells -has resulted in ferences in protein and RNA synthesis rates increased chromatin template activity and between exponential and stationary states in increased non-histone chromosomal pro- [36]. The apparent discrepancy may be retein synthesis [18, 19, 201. These activities solved if the differing responses are due to have been related to passage from GO to two different regulatory processes, restricGl by quiescent cells stimulated to pro- tion point growth inhibition due to subTG1=4.9 h, Ts=8.3 h, TG2=2.0 h, and TM=
0.7 h. These phase durations are comparable to the ones observed during exponential growth (table 2). We conclude that the stable cell number density of the plateau state is maintained by a process of cell death exactly cancelling the process of cell multiplication. In contrast to the gradually effected growth retardation by cell death observed in other systems [lS, 161,onset of growth retardation by cell death is apparently rapid in this cell system.
Exp CellRes 107 (1977)
Population growth inhibition in lymphoid cell line
optimal nutritional conditions and cell density-dependent, nutritionally independent growth inhibition. Fibroblastic cells grown to high cell density can detach from their substrate, presenting technical difficulties to attempts at establishing properties of high density cells. In the present study a lymphoid cell line grown in suspension under continuous culture conditions has been used to establish plateau states which are either nutritionally dependent or nutritionally independent. Both types of growth inhibition were thus examined. The results presented here demonstrate that growth-inhibited plateau states in the same cell system may be induced in two ways. A nutritionally dependent plateau state may be induced by limiting nutritional supply. Cells in this plateau state progressively accumulate in Gl or a state with Gl DNA content. Their cell size progressively decreases. These similarities to the case of arrested fibroblasts suggestthat cells in this state bear some correspondence to the GO cells arrested by nutritional factors discussed above. If nutritional sufficiency is insured, a higher cell density plateau state is generated. Dilution of the plateau state cells in plateau state media results in resumption of growth. In contrast to the case of nutritionally induced growth inhibition, there is no redistribution in the relative number of cells with respect to DNA content as growth becomes density inhibited. As discussed earlier, division control in density-inhibited cells is apparently not exercised by limitation of nutritional supply. The rates of incorporation of exogenous amino acid into cellular protein and glucose consumption are similar to those of exponentially proliferating cells. Cell size as determined by light scattering decreases during the plateau state. Although this is unexpected from the stability of amino acid
339
incorporation and glucose utilization rates, this decrease in cell size has been found consistently in several repetitions of the experiments. Proliferating (density-induced) plateau state cells have Gl, S, G2 and M phase durations similar to exponential cells. In order to maintain the constant cell density of plateau state, cell death must be occurring at a rate equal to the growth rate. This cell death process is not cell cycle phase specific in so far as the distribution of cells in Gl, S, G2 and M is the same for plateau state populations as for exponential populations. The simplest mode of cell loss consistent with the observed data is cell death occurring uniformly throughout the cell cycle. Cell lysis must rapidly follow cell death since the viability of the population, measured by trypan blue exclusion, is high. Because the transition from exponential to plateau state is abrupt, the control process effecting the cellular alterations described above must act rapidly. This suggests that control of these events is more likely to be exerted at the translational level than at the transcriptional or genomic level. No stable growth inhibitor could be found in the culture fluid of plateau state cells. It can be calculated, however, that at the onset of the density-inhibited plateau state, the averagecell-to-cell spacing is only approx. 3.5 cell diameters. Given that they probably are in close proximity of a neighbor a significant fraction of the time, it is possible to speculate that this degree of “contact” is sufficient to produce inhibition. Constant, physical contact is, however, certainly not required for inhibition. Except for these properties, the explicit mechanisms of SK-L7 population growth inhibition are still unknown. Cell volume is an index of cell maturation in the cell cycle and has been implicated as having a possible regulatory role Exp Cell Res 107 (1977)
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in cell division. The relationship between cell size and cell cycle kinetics in this cell line has been investigated previously [3, 41. In an exponentially proliferating population heterogenous with respect to cell size (observed at mitosis) and generation time, smaller cells were found to have longer generation times due to protracted Gl durations. Differences in generation time were, therefore, associated with cell size heterogeneity. It may be asked whether the process of generation time or division regulation exercised is related to the processes at work maintaining nutritionally dependent or density-dependent plateau states. In both plateau states cell size decreases. If the regulatory processes responsible for generation time heterogeneity, i.e., the relatively slower proliferation of some cells in the population, were also responsible for growth inhibition in plateau states; then an increase in relative numbers of Gl cells would be associated with growth inhibition. Although this occurred with nutritionally inhibited cell populations, it did not with density-inhibited cells. It appears, therefore, that the regulatory processes involved in density-inhibited growth do not coincide with the regulatory processes involved in relative generation time retardation in a population with distributed generation times. Generation time heterogeneity is not a result of intercellular differences in the effect of the regulatory process responsible for density-induced growth inhibition. The accumulation in Gl of nutritionally arrested cells, however, suggests that generation time dilation in smaller cells may be induced by a minor relative nutritional deficiency due to or associated with a smaller cell size. The excellent technical assistance of Jean Campbell is gratefully acknowledged. We are grateful to Lana Exp CellRes 107(1977)
Hart for secretarial assistance in preparing this manuscript. This investigation was supported in part by Grant CA-16757, CA-08748 from the NCI, USPHS, Grants CH-6F and CH-6G from the ACS, and the United Leukemia Fund.
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